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The use of β-lactam/β-lactamase inhibitors constitutes an important strategy to counteract β-lactamases in multidrug-resistant (MDR) Gram-negative bacteria. Recent reports have described ceftazidime-/avibactam-resistant isolates producing CTX-M variants with different amino acid substitutions (e.g., P167S, L169Q, and S130G). Relebactam (REL) combined with imipenem has proved very effective against Enterobacterales producing ESBLs, serine-carbapenemases, and AmpCs. Herein, we evaluated the inhibitory efficacy of REL against CTX-M-96, a CTX-M-15-type variant. The CTX-M-96 structure was obtained in complex with REL at 1.03 Å resolution (PDB 8EHH). REL was covalently bound to the S70-Oγ atom upon cleavage of the C7-N6 bond. Compared with apo CTX-M-96, binding of REL forces a slight displacement of the deacylating water inwards the active site (0.81 Å), making the E166 and N170 side chains shift to create a proper hydrogen bonding network. Binding of REL also disturbs the hydrophobic patch formed by Y105, P107, and Y129, likely due to the piperidine ring of REL that creates clashes with these residues. Also, a remarkable change in the positioning of the N104 sidechain is also affected by the piperidine ring. Therefore, differences in the kinetic behavior of REL against class A β-lactamases seem to rely, at least in part, on differences in the residues being involved in the association and stabilization of the inhibitor before hydrolysis. Our data provide the biochemical and structural basis for REL effectiveness against CTX-M-producing Gram-negative pathogens and essential details for further DBO design. Imipenem/REL remains an important choice for dealing with isolates co-producing CTX-M with other β-lactamases., Competing Interests: The authors declare no conflict of interest.

The amino acid substitutions in Klebsiella pneumoniae carbapenemase 2 (KPC-2) that have arisen in the clinic are observed to lead to the development of resistance to ceftazidime-avibactam, a preferred treatment for KPC bearing Gram-negative bacteria. Specific substitutions in the omega loop (R164-D179) result in changes in the structure and function of the enzyme, leading to alterations in substrate specificity, decreased stability, and more recently observed, increased resistance to ceftazidime/avibactam. Using accelerated rare-event sampling well-tempered metadynamics simulations, we explored in detail the structural role of R164 and D179 variants that are described to confer ceftazidime/avibactam resistance. The buried conformation of D179 substitutions produce a pronounced structural disorder in the omega loop - more than R164 mutants, where the crystallographic omega loop structure remains mostly intact. Our findings also reveal that the conformation of N170 plays an underappreciated role impacting drug binding and restricting deacylation. The results further support the hypothesis that KPC-2 D179 variants employ substrate-assisted catalysis for ceftazidime hydrolysis, involving the ring amine of the aminothiazole group to promote deacylation and catalytic turnover. Moreover, the shift in the WT conformation of N170 contributes to reduced deacylation and an altered spectrum of enzymatic activity., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Mycobacterium abscessus is a highly drug-resistant nontuberculous mycobacterium (NTM). Efforts to discover new treatments for M. abscessus infections are accelerating, with a focus on cell wall synthesis proteins ( M. abscessus l,d-transpeptidases 1 to 5 [Ldt Mab1 to Ldt Mab5 ] and d,d-carboxypeptidase) that are targeted by β-lactam antibiotics. A challenge to this approach is the presence of chromosomally encoded β-lactamase (Bla Mab ). Using a mechanism-based approach, we found that a novel ceftaroline-imipenem combination effectively lowered the MICs of M. abscessus isolates (MIC 50 ≤ 0.25 μg/ml; MIC 90 ≤ 0.5 μg/ml). Combining ceftaroline and imipenem with a β-lactamase inhibitor, i.e., relebactam or avibactam, demonstrated only a modest effect on susceptibility compared to each of the β-lactams alone. In steady-state kinetic assays, Bla Mab exhibited a lower K i  app (0.30 ± 0.03 μM for avibactam and 136 ± 14 μM for relebactam) and a higher acylation rate for avibactam ( k 2 / K  = 3.4 × 10 5 ± 0.4 × 10 5 M -1 s -1 for avibactam and 6 × 10 2 ± 0.6 × 10 2 M -1 s -1 for relebactam). The k cat / K m was nearly 10-fold lower for ceftaroline fosamil (0.007 ± 0.001 μM -1 s -1 ) than for imipenem (0.056 ± 0.006 μM -1 s -1 ). Timed mass spectrometry captured complexes of avibactam and Bla Mab , Ldt Mab1 , Ldt Mab2 , Ldt Mab4 , and d,d-carboxypeptidase, whereas relebactam bound only Bla Mab , Ldt Mab1 , and Ldt Mab2 Interestingly, Ldt Mab1 , Ldt Mab2 , Ldt Mab4 , Ldt Mab5 , and d,d-carboxypeptidase bound only to imipenem when incubated with imipenem and ceftaroline fosamil. We next determined the binding constants of imipenem and ceftaroline fosamil for Ldt Mab1 , Ldt Mab2 , Ldt Mab4 , and Ldt Mab5 and showed that imipenem bound >100-fold more avidly than ceftaroline fosamil to Ldt Mab1 and Ldt Mab2 (e.g., K i  app or K m of Ldt Mab1  = 0.01 ± 0.01 μM for imipenem versus 0.73 ± 0.08 μM for ceftaroline fosamil). Molecular modeling indicates that Ldt Mab2 readily accommodates imipenem, but the active site must widen to ≥8 Å for ceftaroline to enter. Our analysis demonstrates that ceftaroline and imipenem binding to multiple targets (l,d-transpeptidases and d,d-carboxypeptidase) and provides a mechanistic rationale for the effectiveness of this dual β-lactam combination in M. abscessus infections., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

Impeding, as well as reducing, the burden of antimicrobial resistance in Gram-negative pathogens is an urgent public health endeavor. Our current antibiotic armamentarium is dwindling, while major resistance determinants (e.g., extended-spectrum β-lactamases [ESBLs]) continue to evolve and disseminate around the world. One approach to attack this problem is to develop novel therapies that will protect our current agents. AAI101 is a novel penicillanic acid sulfone β-lactamase inhibitor similar in structure to tazobactam, with one important difference. AAI101 possesses a strategically placed methyl group that gives the inhibitor a net neutral charge, enhancing bacterial cell penetration. AAI101 paired with cefepime, also a zwitterion, is in phase III of clinical development for the treatment of serious Gram-negative infections. Here, AAI101 was found to restore the activity of cefepime against class A ESBLs (e.g., CTX-M-15) and demonstrated increased potency compared to that of piperacillin-tazobactam when tested against an established isogenic panel. The enzymological properties of AAI101 further revealed that AAI101 possessed a unique mechanism of β-lactamase inhibition compared to that of tazobactam. Additionally, upon reaction with AAI101, CTX-M-15 was modified to an inactive state. Notably, the in vivo efficacy of cefepime-AAI101 was demonstrated using a mouse septicemia model, indicating the ability of AAI101 to bolster significantly the therapeutic efficacy of cefepime in vivo The combination of AAI101 with cefepime represents a potential carbapenem-sparing treatment regimen for infections suspected to be caused by Enterobacteriaceae expressing ESBLs., (Copyright © 2019 American Society for Microbiology.)

Currently, CTX-M β-lactamases are among the most prevalent and most heterogeneous extended-spectrum β-lactamases (ESBLs). In general, CTX-M enzymes are susceptible to inhibition by β-lactamase inhibitors. However, it is unknown if the pathway to inhibition by β-lactamase inhibitors for CTX-M ESBLs is similar to TEM and SHV β-lactamases and why bacteria possessing only CTX-M ESBLs are so susceptible to carbapenems. Here, we have performed a kinetic analysis and timed electrospray ionization mass spectrometry (ESI-MS) studies to reveal the intermediates of inhibition of CTX-M-9, an ESBL representative of this family of enzymes. CTX-M-9 β-lactamase was inactivated by sulbactam, tazobactam, clavulanate, meropenem, doripenem, ertapenem, and a 6-methylidene penem, penem 1. K(i) values ranged from 1.6 ± 0.3 μM (mean ± standard error) for tazobactam to 0.02 ± 0.01 μM for penem 1. Before and after tryptic digestion of the CTX-M-9 β-lactamase apo-enzyme and CTX-M-9 inactivation by inhibitors (meropenem, clavulanate, sulbactam, tazobactam, and penem 1), ESI-MS and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identified different adducts attached to the peptide containing the active site Ser70 (+52, 70, 88, and 156 ± 3 atomic mass units). This study shows that a multistep inhibition pathway results from modification or fragmentation with clavulanate, sulbactam, and tazobactam, while a single acyl enzyme intermediate is detected when meropenem and penem 1 inactivate CTX-M-9 β-lactamase. More generally, we propose that Arg276 in CTX-M-9 plays an essential role in the recognition of the C(3) carboxylate of inhibitors and that the localization of this positive charge to a "region of the active site" rather than a specific residue represents an important evolutionary strategy used by β-lactamases.

Among Gram-negative bacteria, resistance to β-lactams is mediated primarily by β-lactamases (EC 3.2.6.5), periplasmic enzymes that inactivate β-lactam antibiotics. Substitutions at critical amino acid positions in the class A β-lactamase families result in enzymes that can hydrolyze extended-spectrum cephalosporins, thus demonstrating an "extended-spectrum" β-lactamase (ESBL) phenotype. Using SHV ESBLs with substitutions in the Ω loop (R164H and R164S) as target enzymes to understand this enhanced biochemical capability and to serve as a basis for novel β-lactamase inhibitor development, we determined the spectra of activity and crystal structures of these variants. We also studied the inactivation of the R164H and R164S mutants with tazobactam and SA2-13, a unique β-lactamase inhibitor that undergoes a distinctive reaction chemistry in the active site. We noted that the reduced Ki values for the R164H and R164S mutants with SA2-13 are comparable to those with tazobactam (submicromolar). The apo enzyme crystal structures of the R164H and R164S SHV variants revealed an ordered Ω loop architecture that became disordered when SA2-13 was bound. Important structural alterations that result from the binding of SA2-13 explain the enhanced susceptibility of these ESBL enzymes to this inhibitor and highlight ligand-dependent Ω loop flexibility as a mechanism for accommodating and hydrolyzing β-lactam substrates.

Boronic acid transition state inhibitors (BATSIs) are potent class A and C β-lactamase inactivators and are of particular interest due to their reversible nature mimicking the transition state. Here, we present structural and kinetic data describing the inhibition of the SHV-1 β-lactamase, a clinically important enzyme found in Klebsiella pneumoniae, by BATSI compounds possessing the R1 side chains of ceftazidime and cefoperazone and designed variants of the latter, compounds 1 and 2. The ceftazidime and cefoperazone BATSI compounds inhibit the SHV-1 β-lactamase with micromolar affinity that is considerably weaker than their inhibition of other β-lactamases. The solved crystal structures of these two BATSIs in complex with SHV-1 reveal a possible reason for SHV-1's relative resistance to inhibition, as the BATSIs adopt a deacylation transition state conformation compared to the usual acylation transition state conformation when complexed to other β-lactamases. Active-site comparison suggests that these conformational differences might be attributed to a subtle shift of residue A237 in SHV-1. The ceftazidime BATSI structure revealed that the carboxyl-dimethyl moiety is positioned in SHV-1's carboxyl binding pocket. In contrast, the cefoperazone BATSI has its R1 group pointing away from the active site such that its phenol moiety moves residue Y105 from the active site via end-on stacking interactions. To work toward improving the affinity of the cefoperazone BATSI, we synthesized two variants in which either one or two extra carbons were added to the phenol linker. Both variants yielded improved affinity against SHV-1, possibly as a consequence of releasing the strain of its interaction with the unusual Y105 conformation.

OXA beta-lactamases are largely responsible for beta-lactam resistance in Acinetobacter spp. and Pseudomonas aeruginosa, two of the most difficult-to-treat nosocomial pathogens. In general, the beta-lactamase inhibitors used in clinical practice (clavulanic acid, sulbactam, and tazobactam) demonstrate poor activity against class D beta-lactamases. To overcome this challenge, we explored the abilities of beta-lactamase inhibitors of the C-2- and C-3-substituted penicillin and cephalosporin sulfone families against OXA-1, extended-spectrum (OXA-10, OXA-14, and OXA-17), and carbapenemase-type (OXA-24/40) class D beta-lactamases. Three C-2-substituted penicillin sulfone compounds (JDB/LN-1-255, JDB/LN-III-26, and JDB/ASR-II-292) showed low K(i) values for the OXA-1 beta-lactamase (0.70 +/- 0.14 --> 1.60 +/- 0.30 microM) and demonstrated significant K(i) improvements compared to the C-3-substituted cephalosporin sulfone (JDB/DVR-II-214), tazobactam, and clavulanic acid. The C-2-substituted penicillin sulfones JDB/ASR-II-292 and JDB/LN-1-255 also demonstrated low K(i)s for the OXA-10, -14, -17, and -24/40 beta-lactamases (0.20 +/- 0.04 --> 17 +/- 4 microM). Furthermore, JDB/LN-1-255 displayed stoichiometric inactivation of OXA-1 (the turnover number, i.e., the partitioning of the initial enzyme inhibitor complex between hydrolysis and enzyme inactivation [t(n)] = 0) and t(n)s ranging from 5 to 8 for the other OXA enzymes. Using mass spectroscopy to study the intermediates in the inactivation pathway, we determined that JDB/LN-1-255 inhibited OXA beta-lactamases by forming covalent adducts that do not fragment. On the basis of the substrate and inhibitor kinetics of OXA-1, we constructed a model showing that the C-3 carboxylate of JDB/LN-1-255 interacts with Ser115 and Thr213, the R-2 group at C-2 fits between the space created by the long B9 and B10 beta strands, and stabilizing hydrophobic interactions are formed between the pyridyl ring of JDB/LN-1-255 and Val116 and Leu161. By exploiting conserved structural and mechanistic features, JDB/LN-1-255 is a promising lead compound in the quest for effective inhibitors of OXA-type beta-lactamases.

As resistance determinants, KPC beta-lactamases demonstrate a wide substrate spectrum that includes carbapenems, oxyimino-cephalosporins, and cephamycins. In addition, clinical strains harboring KPC-type beta-lactamases are often identified as resistant to standard beta-lactam-beta-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 beta-lactamase inhibitors by determining that clavulanic acid, sulbactam, and tazobactam are hydrolyzed by KPC-2 with partition ratios (kcat/kinact ratios, where kinact 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: Km of penem 1, 0.06+/-0.01 microM, and Km of penem 2, 0.006+/-0.001 microM. We also demonstrate that penems 1 and 2 are mechanism-based inactivators, having partition ratios (kcat/kinact 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 (kinact/Km=6.5+/-0.6 microM(-1) s(-1)). We conclude that KPC-2 is unique among class A beta-lactamases in being able to readily hydrolyze clavulanic acid, sulbactam, and tazobactam. In contrast, penem-type beta-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 beta-lactamase inhibitors.

Background: beta-lactam resistance in Gram-negative bacteria is a significant clinical problem in the community, long-term care facilities, and hospitals. In these organisms, beta-lactam resistance most commonly results from the production of beta-lactamases. In Gram-negative bacilli, TEM-, SHV-, and CTX-M-type beta-lactamases predominate. Therefore, new and accurate detection methods for these beta-lactamase producing isolates are needed., Results: E. coli DH10B cells producing SHV-1 beta-lactamase and a clinical isolate of K. pneumoniae producing SHV-5 beta-lactamase were rendered membrane permeable, fixed and adhered to poly-L-lysine coated slides, and stained with purified polyclonal anti-SHV antibodies that were fluorescein labeled. E. coli DH10B cells without a blaSHV gene were used as a negative control. The procedure generated a fluorescence signal from those slides containing cells expressing SHV beta-lactamase that was sufficient for direct imaging., Conclusion: We developed a rapid and accurate method of visualizing the SHV family of enzymes in clinical samples containing Gram-negative bacilli using a fluorescein-labeled polyclonal antibody.

In an effort to devise strategies for overcoming bacterial beta-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 beta-lactamase inhibitors. LN-1-255 combined with piperacillin was more potent against Escherichia coli DH10B strains bearing bla(SHV) extended-spectrum and inhibitor-resistant beta-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 beta-lactamases with nm affinity (K(I) = 110 +/- 10 and 100 +/- 10 nm, respectively). When LN-1-255 inactivated SHV beta-lactamases, a single intermediate was detected by mass spectrometry. The crystal structure of LN-1-255 in complex with SHV-1 was determined at 1.55A resolution. Interestingly, this novel inhibitor forms a bicyclic aromatic intermediate with its carbonyl oxygen pointing out of the oxyanion hole and forming hydrogen bonds with Lys-234 and Ser-130 in the active site. Electron density for the "tail" of LN-1-255 is less ordered and modeled in two conformations. Both conformations have the LN-1-255 carboxyl group interacting with Arg-244, yet the remaining tails of the two conformations diverge. The observed presence of the bicyclic aromatic intermediate with its carbonyl oxygen positioned outside of the oxyanion hole provides a rationale for the stability of this inhibitory intermediate. The 2'-substituted penicillin sulfone, LN-1-255, is proving to be an important lead compound for novel beta-lactamase inhibitor design.

The partnering of a beta-lactam with a beta-lactamase inhibitor is a highly effective strategy that can be used to combat bacterial resistance to beta-lactam antibiotics mediated by serine beta-lactamases (EC 3.2.5.6). To this end, we tested two novel penem inhibitors against OXA-1, a class D beta-lactamase that is resistant to inactivation by tazobactam. The K(i) of each penem inhibitor for OXA-1 was in the nM range (K(i) of penem 1, 45 +/- 8 nM; K(i) of penem 2, 12 +/- 2 nM). The first-order rate constant for enzyme and inhibitor complex inactivation of penems 1 and 2 for OXA-1 beta-lactamase were 0.13 +/- 0.01 s(-1) and 0.11 +/- 0.01 s(-1), respectively. By using an inhibitor-to-enzyme ratio of 1:1, 100% inactivation was achieved in

The presence of plasmid-mediated quinolone resistance genes [i.e., qnrA, qnrB, qnrS, aac(6')-Ib-cr, and qepA] was evaluated among 42 bla(KPC)-containing Klebsiella pneumoniae isolates collected in the eastern United States. One isolate carried the bla(KPC-3) and qnrB19 genes on the same conjugative plasmid, whereas another carried the bla(KPC-3) and qnrA1 genes on separate plasmids.

The OXA-1 beta-lactamase is one of the few class D enzymes that has an aspartate residue at position 66, a position that is proximal to the active-site residue Ser(67). In class A beta-lactamases, such as TEM-1 and SHV-1, residues adjacent to the active-site serine residue play a crucial role in inhibitor resistance and substrate selectivity. To probe the role of Asp(66) in substrate affinity and catalysis, we performed site-saturation mutagenesis at this position. Ampicillin MIC (minimum inhibitory concentration) values for the full set of Asp(66) mutants expressed in Escherichia coli DH10B ranged from < or =8 microg/ml for cysteine, proline and the basic amino acids to > or =256 microg/ml for asparagine, leucine and the wild-type aspartate. Replacement of aspartic acid by asparagine at position 66 also led to a moderate enhancement of extended-spectrum cephalosporin resistance. OXA-1 shares with other class D enzymes a carboxylated residue, Lys(70), that acts as a general base in the catalytic mechanism. The addition of 25 mM bicarbonate to Luria-Bertani-broth agar resulted in a > or =16-fold increase in MICs for most OXA-1 variants with amino acid replacements at position 66 when expressed in E. coli. Because Asp(66) forms hydrogen bonds with several other residues in the OXA-1 active site, we propose that this residue plays a role in stabilizing the CO2 bound to Lys(70) and thereby profoundly affects substrate turnover.

A 6-alkylidiene penam sulfone, SA-1-204, is an efficient inhibitor of both SHV-1 and OXA-1 beta-lactamases with K(I) = 42 +/- 4 nm and 1.0 +/- 0.1 microm, respectively. To gain insight into the reaction chemistry of SA-1-204, the reactions between this inhibitor and SHV-1 and OXA-1 were studied by Raman spectroscopy in single crystals and in solution. Raman signatures characteristic of the unreacted beta-lactam ring show that in both phases the inhibitor binds as a noncovalent Michaelis-like complex. This complex is present as the major population for periods of up to an hour. On longer time scales, the Raman data show that beta-lactam ring opening eventually leads to a complex mixture of reaction products. However, the data clearly demonstrate that the key species for inhibition on the time scale of bacterial half-lives is the noncovalent complex preceding acylation.

Beta-lactamases inactivate beta-lactam antibiotics and are a major cause of antibiotic resistance. The recent outbreaks of Klebsiella pneumoniae carbapenem resistant (KPC) infections mediated by KPC type beta-lactamases are creating a serious threat to our "last resort" antibiotics, the carbapenems. KPC beta-lactamases are serine carbapenemases and are a subclass of class A beta-lactamases that have evolved to efficiently hydrolyze carbapenems and cephamycins which contain substitutions at the alpha-position proximal to the carbonyl group that normally render these beta-lactams resistant to hydrolysis. To investigate the molecular basis of this carbapenemase activity, we have determined the structure of KPC-2 at 1.85 A resolution. The active site of KPC-2 reveals the presence of a bicine buffer molecule which interacts via its carboxyl group with conserved active site residues S130, K234, T235, and T237; these likely resemble the interactions the beta-lactam carboxyl moiety makes in the Michaelis-Menten complex. Comparison of the KPC-2 structure with non-carbapenemases and previously determined NMC-A and SME-1 carbapenemase structures shows several active site alterations that are unique among carbapenemases. An outward shift of the catalytic S70 residue renders the active sites of the carbapenemases more shallow, likely allowing easier access of the bulkier substrates. Further space for the alpha-substituents is potentially provided by shifts in N132 and N170 in addition to concerted movements in the postulated carboxyl binding pocket that might allow the substrates to bind at a slightly different angle to accommodate these alpha-substituents. The structure of KPC-2 provides key insights into the carbapenemase activity of emerging class A beta-lactamases.

Boronic acid transition state inhibitors (BATSIs) with R1 side chains of cefotaxime and ceftazidime were assayed against SHV-1, SHV-2, SHV-5, D104K, and D104K G238S beta-lactamases. The D104K variant was the most susceptible to inhibition by the ceftazidime BATSI (Ki, 730+/-80 nM), while the D104K G238S variant was the most susceptible to the cefotaxime BATSI (Ki, 1.1+/-0.2 microM).

Among the TEM-type extended-spectrum beta-lactamases (ESBLs), an amino acid change at Ambler position 104 (Glu to Lys) results in increased resistance to ceftazidime and cefotaxime when found with other substitutions (e.g., Gly238Ser and Arg164Ser). To examine the role of Asp104 in SHV beta-lactamases, site saturation mutagenesis was performed. Our goal was to investigate the properties of amino acid residues at this position that affect resistance to penicillins and oxyimino-cephalosporins. Unexpectedly, 58% of amino acid variants at position 104 in SHV expressed in Escherichia coli DH10B resulted in beta-lactamases with lowered resistance to ampicillin. In contrast, increased resistance to cefotaxime was demonstrated only for the Asp104Arg and Asp104Lys beta-lactamases. When all 19 substitutions were introduced into the SHV-2 (Gly238Ser) ESBL, the most significant increases in cefotaxime and ceftazidime resistance were noted for both the doubly substituted Asp104Lys Gly238Ser and the doubly substituted Asp104Arg Gly238Ser beta-lactamases. Correspondingly, the overall catalytic efficiency (kcat/Km) of hydrolysis for cefotaxime was increased from 0.60 +/- 0.07 microM(-1) s(-1) (mean +/- standard deviation) for Gly238Ser to 1.70 +/- 0.01 microM(-1) s(-1) for the Asp104Lys and Gly238Ser beta-lactamase (threefold increase). We also showed that (i) k3 was the rate-limiting step for the hydrolysis of cefotaxime by Asp104Lys, (ii) the Km for cefotaxime of the doubly substituted Asp104Lys Gly238Ser variant approached that of the Gly238Ser beta-lactamase as pH increased, and (iii) Lys at position 104 functions in an energetically additive manner with the Gly238Ser substitution to enhance catalysis of cephalothin. Based on this analysis, we propose that the amino acid at Ambler position 104 in SHV-1 beta-lactamase plays a major role in substrate binding and recognition of oxyimino-cephalosporins and influences the interactions of Tyr105 with penicillins.

beta-Lactamases are one of the major causes of antibiotic resistance in Gram negative bacteria. The continuing evolution of beta-lactamases that are capable of hydrolyzing our most potent beta-lactams presents a vexing clinical problem, in particular since a number of them are resistant to inhibitors. The efficient inhibition of these enzymes is therefore of great clinical importance. Building upon our previous structural studies that examined tazobactam trapped as a trans-enamine intermediate in a deacylation deficient SHV variant, we designed a novel penam sulfone derivative that forms a more stable trans-enamine intermediate. We report here the 1.28 A resolution crystal structure of wt SHV-1 in complex with a rationally designed penam sulfone, SA2-13. The compound is covalently bound to the active site of wt SHV-1 similar to tazobactam yet forms an additional salt-bridge with K234 and hydrogen bonds with S130 and T235 to stabilize the trans-enamine intermediate. Kinetic measurements show that SA2-13, once reacted with SHV-1 beta-lactamase, is about 10-fold slower at being released from the enzyme compared to tazobactam. Stabilizing the trans-enamine intermediate represents a novel strategy for the rational design of mechanism-based class A beta-lactamase inhibitors.

Beta-lactamase inhibitor protein (BLIP) binds a variety of class A beta-lactamases with affinities ranging from micromolar to picomolar. Whereas the TEM-1 and SHV-1 beta-lactamases are almost structurally identical, BLIP binds TEM-1 approximately 1000-fold tighter than SHV-1. Determining the underlying source of this affinity difference is important for understanding the molecular basis of beta-lactamase inhibition and mechanisms of protein-protein interface specificity and affinity. Here we present the 1.6A resolution crystal structure of SHV-1.BLIP. In addition, a point mutation was identified, SHV D104E, that increases SHV.BLIP binding affinity from micromolar to nanomolar. Comparison of the SHV-1.BLIP structure with the published TEM-1.BLIP structure suggests that the increased volume of Glu-104 stabilizes a key binding loop in the interface. Solution of the 1.8A SHV D104K.BLIP crystal structure identifies a novel conformation in which this binding loop is removed from the interface. Using these structural data, we evaluated the ability of EGAD, a program developed for computational protein design, to calculate changes in the stability of mutant beta-lactamase.BLIP complexes. Changes in binding affinity were calculated within an error of 1.6 kcal/mol of the experimental values for 112 mutations at the TEM-1.BLIP interface and within an error of 2.2 kcal/mol for 24 mutations at the SHV-1.BLIP interface. The reasonable success of EGAD in predicting changes in interface stability is a promising step toward understanding the stability of the beta-lactamase.BLIP complexes and computationally assisted design of tight binding BLIP variants.

Clavulanic acid is a potent mechanism-based inhibitor of TEM-1 and SHV-1beta-lactamases, enzymes that confer resistance to beta-lactams in many gram-negative pathogens. This compound has enjoyed widespread clinical use as part of beta-lactam beta-lactamase inhibitor therapy directed against penicillin-resistant pathogens. Unfortunately, the emergence of clavulanic acid-resistant variants of TEM-1 and SHV-1 beta-lactamase significantly compromise the efficacy of this combination. A single amino acid change at Ambler position Ser130 (Ser --> Gly) results in resistance to inactivation by clavulanate in the SHV-1 and TEM-1beta-lactamases. Herein, we investigated the inactivation of SHV-1 and the inhibitor-resistant S130G variant beta-lactamases by clavulanate. Using liquid chromatography electrospray ionization mass spectrometry, we detected multiple modified proteins when SHV-1 beta-lactamase is inactivated by clavulanate. Matrix-assisted laser desorption ionization-time of flight mass spectrometry was used to study tryptic digests of SHV-1 and S130Gbeta-lactamases (+/- inactivation with clavulanate) and identified peptides modified at the active site Ser70. Ultraviolet (UV) difference spectral studies comparing SHV-1 and S130Gbeta-lactamases inactivated by clavulanate showed that the formation of reaction intermediates with absorption maxima at 227 and 280 nm are diminished and delayed when S130Gbeta-lactamase is inactivated. We conclude that the clavulanic acid inhibition of the S130G beta-lactamase must follow a branch of the normal inactivation pathway. These findings highlight the importance of understanding the intermediates formed in the inactivation process of inhibitor-resistant beta-lactamases and suggest how strategic chemical design can lead to novel ways to inhibit beta-lactamases.

Acinetobacter spp. are emerging as opportunistic hospital pathogens that demonstrate resistance to many classes of antibiotics. In a metropolitan hospital in Cleveland, a clinical isolate of Acinetobacter baumannii that tested resistant to cefepime and ceftazidime (MIC = 32 microg/ml) was identified. Herein, we sought to determine the molecular basis for the extended-spectrum-cephalosporin resistance. Using analytical isoelectric focusing, a beta-lactamase with a pI of > or = 9.2 was detected. PCR amplification with specific A. baumannii cephalosporinase primers yielded a 1,152-bp product which, when sequenced, identified a novel 383-amino-acid class C enzyme. Expressed in Escherichia coli DH10B, this beta-lactamase demonstrated greater resistance against ceftazidime and cefotaxime than cefepime (4.0 microg/ml versus 0.06 microg/ml). The kinetic characteristics of this beta-lactamase were similar to other cephalosporinases found in Acinetobacter spp. In addition, this cephalosporinase was inhibited by meropenem, imipenem, ertapenem, and sulopenem (K(i) < 40 microM). The amino acid compositions of this novel enzyme and other class C beta-lactamases thus far described for A. baumannii, Acinetobacter genomic species 3, and Oligella urethralis in Europe and South Africa suggest that this cephalosporinase defines a unique family of class C enzymes. We propose a uniform designation for this family of cephalosporinases (Acinetobacter-derived cephalosporinases [ADC]) found in Acinetobacter spp. and identify this enzyme as ADC-7 beta-lactamase. The coalescence of Acinetobacter ampC beta-lactamases into a single common ancestor and the substantial phylogenetic distance separating them from other ampC genes support the logical value of developing a system of nomenclature for these Acinetobacter cephalosporinase genes.

Knowledge of the amino acids that define recognition of anti-beta-lactamase antibodies is critical to the interpretation of sensitivity and specificity of these antibodies when they are used in a clinical or research setting. To this end, we mapped the epitopes of the CMY-2 and SHV-1 beta-lactamases by using the SPOT synthesis method. Eight linear epitopes in SHV-1 and seven linear epitopes in CMY-2 were identified by using anti-SHV-1 and anti-CMY-2 polyclonal antibodies, respectively. The epitopes of SHV-1 were mapped to amino acids at the Ambler positions ABL 28 to 38, 42 to 54, 88 to 100, 102 to 114, 170 to 182, 186 to 194, 202 to 210, and 276 to 288. In the epitope spanning amino acids 102 to 114, alanine and X-Scan analysis demonstrated that D104, Y105, P107, and S109 are essential residues for antibody recognition. In the epitope containing amino acids 170 to 182, N170, L173, P174, G175, and D176 were immunodominant. In CMY-2 beta-lactamase, amino acids 4 to 16, 70 to 79, 211 to 223, 274 to 286, 289 to 298, 322 to 334, and 343 to 358 of the mature enzyme defined the major linear epitopes. A detailed analysis of the recognition sites that are located in an area analogous to the omega loop of class A beta-lactamases (V211 to V223) showed that the amino acids Q215 to E219 are important in antibody binding. Incubation of CMY-2 beta-lactamase with a 10-fold molar excess of anti-CMY-2 antibody for 60 min resulted in greater than 80% inhibition of nitrocefin hydrolysis. A 10-fold molar excess of anti-SHV-1 antibody reduced the activity of SHV-1 by 69%. Analysis of the CMY-2 and SHV-1 structures suggest that this reduction of hydrolytic activity may be due in part to the direct binding of antibodies to the omega loop, thereby hindering access of substrate to the active site.

Geriatric patients frequently are cared for in long term care facilities (LTCFs), which are now a major component of our health care delivery system. Nearly half of the 2.2 million people who turned 65 years old in 1990 will enter an LTCF at least once before they die. Infections are one of the principal causes of morbidity and mortality in LTCFs. Because LTCFs are a less costly alternative to hospitalization, clinicians are treating many serious infections in the nursing home. As a result of antibiotic use, LTCFs will increasingly be recognized as sources of organisms resistant to multiple antibiotics. b-Lactams are a valuable class of potent antimicrobials with broad-spectrum activity against Gram-negative and Gram-positive organisms. The safety and efficacy of this class of antibiotics make them easy choices for empiric treatment of infections in the elderly. Unfortunately, excessive use of these antibiotics has created serious threats to our therapeutic armamentarium: the emergence of methicillin-resistant Staphylococcus aureus and of Gram-negative pathogens resistant to third-generation cephalosporins such as cefotaxime, ceftazidime, and ceftriaxone. Of these third-generation cephalosporins, resistance to ceftazidime is most frequently recognized. The major mechanism responsible for ceftazidime resistance in Gram-negative bacteria is the production of b-lactamases. This article summarizes the diversity of b-lactamases, highlights the important enzymes that confer ceftazidime resistance in LTCFs, and details some methods used to identify and characterize these enzymes. A clear challenge is to apply these techniques to epidemiologic and molecular studies conducted in LTCFs.

The increasing number of bacteria resistant to combinations of beta-lactam and beta-lactamase inhibitors is creating great difficulties in the treatment of serious hospital-acquired infections. Understanding the mechanisms and structural basis for the inactivation of these inhibitor-resistant beta-lactamases provides a rationale for the design of novel compounds. In the present work, SHV-1 and the Ser(130) --> Gly inhibitor-resistant variant of SHV-1 beta-lactamase were inactivated with tazobactam, a potent class A beta-lactamase inhibitor. Apoenzymes and inhibited beta-lactamases were analyzed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), digested with trypsin, and the products resolved using LC-ESI/MS and matrix-assisted laser desorption ionization-time of flight mass spectrometry. The mass increases observed for SHV-1 and Ser(130) --> Gly (+ Delta 88 Da and + Delta 70 Da, respectively) suggest that fragmentation of tazobactam readily occurs in the inhibitor-resistant variant to yield an inactive beta-lactamase. These two mass increments are consistent with the formation of an aldehyde (+ Delta 70 Da) and a hydrated aldehyde (+ Delta 88 Da) as stable products of inhibition. Our results reveal that the Ser --> Gly substitution at amino acid position 130 is not essential for enzyme inactivation. By examining the inhibitor-resistant Ser(130) --> Gly beta-lactamase, our data are the first to show that tazobactam undergoes fragmentation while still attached to the active site Ser(70) in this enzyme. After acylation of tazobactam by Ser(130) --> Gly, inactivation proceeds independent of any additional covalent interactions.

Thirteen clinical isolates of Escherichia coli resistant to ceftazidime that possessed an AmpC and other (beta-lactamases were identified. The effectiveness of different formulations of piperacillin/tazobactam to other beta-lactams was compared. Antibiotic susceptibility testing, polymerase chain reaction, amplification of blaTEM, blaSHV and blaAmpC, and enzyme-linked immunosorbent assays to identify AmpC beta-lactamases were performed. Hydrolysis rates were obtained and residual enzymatic activity was determined. Cefepime and ertapenem were more active than piperacillin/tazobactam. In contrast, increasing the relative proportion of tazobactam improved susceptibility testing. Twenty micromolar tazobactam inhibited total beta-lactamase activity (as measured by nitrocefin hydrolysis rates) by greater than 75% against all isolates tested: in 11 of 13 E. coli isolates, total beta-lactamase activity was inhibited by 90%. The observed differences between MIC determinations and susceptibility to enzymatic inactivation by tazobactam against E. coli containing AmpC and other -lactamases may be due to the final tazobactam concentration achieved in the periplasmic space. Factors determining this are critical considerations in assessing beta-lactamase inhibitor potency.

Bacterial resistance to beta-lactam/beta-lactamase inhibitor combinations by single amino acid mutations in class A beta-lactamases threatens our most potent clinical antibiotics. In TEM-1 and SHV-1, the common class A beta-lactamases, alterations at Ser-130 confer resistance to inactivation by the beta-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 beta-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 beta-lactamase expressed in Escherichia coli. Kinetic analysis of the S130G beta-lactamase demonstrated a significant elevation in apparent Km and a reduction in kcat/Km for ampicillin. Marked increases in the dissociation constant for the preacylation complex, KI, of clavulanic acid (SHV-1, 0.14 microm; S130G, 46.5 microm) and tazobactam (SHV-1, 0.07 microm; S130G, 4.2 microm) 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 beta-lactams and beta-lactamase inhibitors. These results underscore the mechanistic versatility of class A beta-lactamases and have implications for the design of novel beta-lactamase inhibitors.