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Serine active-site β-lactamases hydrolyze β-lactam antibiotics through the formation of a covalent acyl-enzyme intermediate followed by deacylation via an activated water molecule. Carbapenem antibiotics are poorly hydrolyzed by most β-lactamases owing to slow hydrolysis of the acyl-enzyme intermediate. However, the emergence of the KPC-2 carbapenemase has resulted in widespread resistance to these drugs, suggesting it operates more efficiently. Here, we investigated the unusual features of KPC-2 that enable this resistance. We show that KPC-2 has a 20,000-fold increased deacylation rate compared with the common TEM-1 β-lactamase. Furthermore, kinetic analysis of active site alanine mutants indicates that carbapenem hydrolysis is a concerted effort involving multiple residues. Substitution of Asn170 greatly decreases the deacylation rate, but this residue is conserved in both KPC-2 and non-carbapenemase β-lactamases, suggesting it promotes carbapenem hydrolysis only in the context of KPC-2. X-ray structure determination of the N170A enzyme in complex with hydrolyzed imipenem suggests Asn170 may prevent the inactivation of the deacylating water by the 6α-hydroxyethyl substituent of carbapenems. In addition, the Thr235 residue, which interacts with the C3 carboxylate of carbapenems, also contributes strongly to the deacylation reaction. In contrast, mutation of the Arg220 and Thr237 residues decreases the acylation rate and, paradoxically, improves binding affinity for carbapenems. Thus, the role of these residues may be ground state destabilization of the enzyme-substrate complex or, alternatively, to ensure proper alignment of the substrate with key catalytic residues to facilitate acylation. These findings suggest modifications of the carbapenem scaffold to avoid hydrolysis by KPC-2 β-lactamase., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

In 2016, we identified a new class A carbapenemase, VCC-1, in a nontoxigenic Vibrio cholerae strain that had been isolated from retail shrimp imported into Canada for human consumption. Shortly thereafter, seven additional VCC-1-producing V. cholerae isolates were recovered along the German coastline. These isolates appear to have acquired the VCC-1 gene ( bla VCC-1 ) independently from the Canadian isolate, suggesting that bla VCC-1 is mobile and widely distributed. VCC-1 hydrolyzes penicillins, cephalothin, aztreonam, and carbapenems and, like the broadly disseminated class A carbapenemase KPC-2, is only weakly inhibited by clavulanic acid or tazobactam. Although VCC-1 has yet to be observed in the clinic, its encroachment into aquaculture and other areas with human activity suggests that the enzyme may be emerging as a public health threat. To preemptively address this threat, we examined the structural and functional biology of VCC-1 against the FDA-approved non-β-lactam-based inhibitor avibactam. We found that avibactam restored the in vitro sensitivity of V. cholerae to meropenem, imipenem, and ertapenem. The acylation efficiency was lower for VCC-1 than for KPC-2 and akin to that of Pseudomonas aeruginosa PAO1 AmpC ( k 2 / K i  = 3.0 × 10 3 M -1  s -1 ). The tertiary structure of VCC-1 is similar to that of KPC-2, and they bind avibactam similarly; however, our analyses suggest that VCC-1 may be unable to degrade avibactam, as has been found for KPC-2. Based on our prior genomics-based surveillance, we were able to target VCC-1 for detailed molecular studies to gain early insights that could be used to combat this carbapenemase in the future., (Copyright © 2019 American Society for Microbiology.)

A widely applicable free energy contribution analysis (FECA) method based on the quantum mechanical/molecular mechanical (QM/MM) approximation using response kernel approaches has been proposed to investigate the influences of environmental residues and/or atoms in the QM region on the free energy profile. This method can evaluate atomic contributions to the free energy along the reaction path including polarization effects on the QM region within a dramatically reduced computational time. The rate-limiting step in the deactivation of the β-lactam antibiotic cefalotin (CLS) by β-lactamase was studied using this method. The experimentally observed activation barrier was successfully reproduced by free energy perturbation calculations along the optimized reaction path that involved activation by the carboxylate moiety in CLS. It was found that the free energy profile in the QM region was slightly higher than the isolated energy and that two residues, Lys67 and Lys315, as well as water molecules deeply influenced the QM atoms associated with the bond alternation reaction in the acyl-enzyme intermediate. These facts suggested that the surrounding residues are favorable for the reactant complex and prevent the intermediate from being too stabilized to proceed to the following deacylation reaction. We have demonstrated that the free energy contribution analysis should be a useful method to investigate enzyme catalysis and to facilitate intelligent molecular design.

Understanding the molecular details of antibiotic resistance by the bacterial enzymes β-lactamases is vital for the development of novel antibiotics and inhibitors. In this spirit, the detailed mechanism of deacylation of the acyl-enzyme complex formed by cephalothin and class C β-lactamase is investigated here using hybrid quantum-mechanical/molecular-mechanical molecular dynamics methods. The roles of various active-site residues and substrate in the deacylation reaction are elucidated. We identify the base that activates the hydrolyzing water molecule and the residue that protonates the catalytic serine (Ser64). Conformational changes in the active sites and proton transfers that potentiate the efficiency of the deacylation reaction are presented. We have also characterized the oxyanion holes and other H-bonding interactions that stabilize the reaction intermediates. Together with the kinetic and mechanistic details of the acylation reaction, we analyze the complete mechanism and the overall kinetics of the drug hydrolysis. Finally, the apparent rate-determining step in the drug hydrolysis is scrutinized.

VIM-39, a VIM-1-like metallo-β-lactamase variant (VIM-1 Thr33Ala His224Leu) was identified in a clinical isolate of Klebsiella pneumoniae belonging to sequence type 147. VIM-39 hydrolyzed ampicillin, cephalothin, and imipenem more efficiently than did VIM-1 and VIM-26 (a VIM-1 variant with the His224Leu substitution) because of higher turnover rates., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Here we describe the presence of two very similar but unusual variants of AmpC cephalosporinase in each Cronobacter sakazakii and C. malonaticus isolates conferring resistance exclusively to first generation cephalosporins. During a survey on the antibiotic resistance patterns of C. sakazakii and C. malonaticus strains isolated from a milk powder production facility, originally two different phenotypes regarding the susceptibility/resistance for the two beta-lactam antibiotics ampicillin (amp) and cephalothin (ceph) were observed: (i) isolates being susceptible for both antibiotics (amp(S)/ceph(S)), and (ii) strains exhibiting susceptibility to ampicillin but resistance to cephalothin (amp(S)/ceph(R)). The latter phenotype (amp(S)/ceph(R)) was observed in the majority of the environmental strains from the facility. Analysis of whole genome sequences of C. sakazakii revealed a gene putatively coding for an AmpC beta-lactamase. Consequently, the ampC genes from both species and both phenotypes were subjected to a cloning approach. Surprisingly, when expressed in Escherichia coli, all transformants exhibited the amp(S)/ceph(R) phenotype regardless of (i) the phenotypic backgrounds or (ii) the AmpC amino acid sequences of the original strains from which the clones were derived. The novel AmpC beta-lactamases were designated CSA-1 and CSA-2 (from C. sakazakii) and CMA-1 and CMA-2 (from C. malonaticus). The observed variations in the minimum inhibitory concentration (MIC) levels for cephalothin (wt compared to transformants) suggest that this feature is a target of a yet unknown regulatory mechanism present in the natural Cronobacter background but absent in the neutral E. coli host.

An understanding of antibiotic hydrolysis rates is important for predicting their environmental persistence. Hydrolysis rates and Arrhenius constants were determined as a function of pH and temperature for three common β-lactam antibiotics, ampicillin, cefalotin, and cefoxitin. Antibiotic hydrolysis rates at pH4-9 at 25 °C, 50 °C, and 60 °C were quantified, and degradation products were identified. The three antibiotics hydrolyzed under ambient conditions (pH7 and 25 °C); half-lives ranged from 5.3 to 27 d. Base-catalyzed hydrolysis rates were significantly greater than acid-catalyzed and neutral pH hydrolysis rates. Hydrolysis rates increased 2.5- to 3.9-fold for a 10 °C increase in temperature. Based on the degradation product masses found, the likely functional groups that underwent hydrolysis were lactam, ester, carbamate, and amide moieties. Many of the proposed products resulting from the hydrolysis of ampicillin, cefalotin, and cefoxitin likely have reduced antimicrobial activity because many products contained a hydrated lactam ring. The results of this research demonstrate that β-lactam antibiotics hydrolyze under ambient pH and temperature conditions. Degradation of β-lactam antibiotics will likely occur over several weeks in most surface waters and over several days in more alkaline systems., (Copyright © 2013 Elsevier B.V. All rights reserved.)

EstU1 is a unique family VIII carboxylesterase that displays hydrolytic activity toward the amide bond of clinically used β-lactam antibiotics as well as the ester bond of p-nitrophenyl esters. EstU1 assumes a β-lactamase-like modular architecture and contains the residues Ser100, Lys103, and Tyr218, which correspond to the three catalytic residues (Ser64, Lys67, and Tyr150, respectively) of class C β-lactamases. The structure of the EstU1/cephalothin complex demonstrates that the active site of EstU1 is not ideally tailored to perform an efficient deacylation reaction during the hydrolysis of β-lactam antibiotics. This result explains the weak β-lactamase activity of EstU1 compared with class C β-lactamases. Finally, structural and sequential comparison of EstU1 with other family VIII carboxylesterases elucidates an operative molecular strategy used by family VIII carboxylesterases to extend their substrate spectrum., (Copyright © 2013 Wiley Periodicals, Inc.)

Bacteria that cause most of the hospital-acquired infections make use of class C β-lactamase (CBL) among other enzymes to resist a wide spectrum of modern antibiotics and pose a major public health concern. Other than the general features, details of the defensive mechanism by CBL, leading to the hydrolysis of drug molecules, remain a matter of debate, in particular the identification of the general base and role of the active site residues and substrate. In an attempt to unravel the detailed molecular mechanism, we carried out extensive hybrid quantum mechanical/molecular mechanical Car-Parrinello molecular dynamics simulation of the reaction with the aid of the metadynamics technique. On this basis, we report here the mechanism of the formation of the acyl-enzyme complex from the Henry-Michaelis complex formed by β-lactam antibiotics and CBL. We considered two β-lactam antibiotics, namely, cephalothin and aztreonam, belonging to two different subfamilies. A general mechanism for the formation of a β-lactam antibiotic-CBL acyl-enzyme complex is elicited, and the individual roles of the active site residues and substrate are probed. The general base in the acylation step has been identified as Lys67, while Tyr150 aids the protonation of the β-lactam nitrogen through either the substrate carboxylate group or a water molecule.

Acinetobacter species show high levels of intrinsic resistance to many antibiotics. The major protein species in the outer membrane of Acinetobacter baumannii does not belong to the high-permeability trimeric porin family, which includes Escherichia coli OmpF/OmpC, and instead is a close homolog of E. coli OmpA and Pseudomonas aeruginosa OprF. We characterized the pore-forming function of this OmpA homolog, OmpA(Ab), by a reconstitution assay. OmpA(Ab) produced very low pore-forming activity, about 70-fold lower than that of OmpF and an activity similar to that of E. coli OmpA and P. aeruginosa OprF. The pore size of the OmpA(Ab) channel was similar to that of OprF, i.e., about 2 nm in diameter. The low permeability of OmpA(Ab) is not due to the inactivation of this protein during purification, because the permeability of the whole A. baumannii outer membrane was also very low. Furthermore, the outer membrane permeability to cephalothin and cephaloridine, measured in intact cells, was about 100-fold lower than that of E. coli K-12. The permeability of cephalothin and cephaloridine in A. baumannii was decreased 2- to 3-fold when the ompA(Ab) gene was deleted. These results show that OmpA(Ab) is the major nonspecific channel in A. baumannii. The low permeability of this porin, together with the presence of constitutive β-lactamases and multidrug efflux pumps, such as AdeABC and AdeIJK, appears to be essential for the high levels of intrinsic resistance to a number of antibiotics.

In Pseudomonas aeruginosa, the chromosomally encoded class C cephalosporinase (AmpC β-lactamase) is often responsible for high-level resistance to β-lactam antibiotics. Despite years of study of these important β-lactamases, knowledge regarding how amino acid sequence dictates function of the AmpC Pseudomonas-derived cephalosporinase (PDC) remains scarce. Insights into structure-function relationships are crucial to the design of both β-lactams and high-affinity inhibitors. In order to understand how PDC recognizes the C₃/C₄ carboxylate of β-lactams, we first examined a molecular model of a P. aeruginosa AmpC β-lactamase, PDC-3, in complex with a boronate inhibitor that possesses a side chain that mimics the thiazolidine/dihydrothiazine ring and the C₃/C₄ carboxylate characteristic of β-lactam substrates. We next tested the hypothesis generated by our model, i.e. that more than one amino acid residue is involved in recognition of the C₃/C₄ β-lactam carboxylate, and engineered alanine variants at three putative carboxylate binding amino acids. Antimicrobial susceptibility testing showed that the PDC-3 β-lactamase maintains a high level of activity despite the substitution of C₃/C₄ β-lactam carboxylate recognition residues. Enzyme kinetics were determined for a panel of nine penicillin and cephalosporin analog boronates synthesized as active site probes of the PDC-3 enzyme and the Arg349Ala variant. Our examination of the PDC-3 active site revealed that more than one residue could serve to interact with the C₃/C₄ carboxylate of the β-lactam. This functional versatility has implications for novel drug design, protein evolution, and resistance profile of this enzyme., (Copyright © 2011 The Protein Society.)

The metallo-β-lactamase VIM-4, mainly found in Pseudomonas aeruginosa or Acinetobacter baumannii, was produced in Escherichia coli and characterized by biochemical and X-ray techniques. A detailed kinetic study performed in the presence of Zn²+ at concentrations ranging from 0.4 to 100 μM showed that VIM-4 exhibits a kinetic profile similar to the profiles of VIM-2 and VIM-1. However, VIM-4 is more active than VIM-1 against benzylpenicillin, cephalothin, nitrocefin, and imipenem and is less active than VIM-2 against ampicillin and meropenem. The crystal structure of the dizinc form of VIM-4 was solved at 1.9 Å. The sole difference between VIM-4 and VIM-1 is found at residue 228, which is Ser in VIM-1 and Arg in VIM-4. This substitution has a major impact on the VIM-4 catalytic efficiency compared to that of VIM-1. In contrast, the differences between VIM-2 and VIM-4 seem to be due to a different position of the flapping loop and two substitutions in loop 2. Study of the thermal stability and the activity of the holo- and apo-VIM-4 enzymes revealed that Zn²+ ions have a pronounced stabilizing effect on the enzyme and are necessary for preserving the structure.

A Swedish patient of Indian origin traveled to New Delhi, India, and acquired a urinary tract infection caused by a carbapenem-resistant Klebsiella pneumoniae strain that typed to the sequence type 14 complex. The isolate, Klebsiella pneumoniae 05-506, was shown to possess a metallo-beta-lactamase (MBL) but was negative for previously known MBL genes. Gene libraries and amplification of class 1 integrons revealed three resistance-conferring regions; the first contained bla(CMY-4) flanked by ISEcP1 and blc. The second region of 4.8 kb contained a complex class 1 integron with the gene cassettes arr-2, a new erythromycin esterase gene; ereC; aadA1; and cmlA7. An intact ISCR1 element was shown to be downstream from the qac/sul genes. The third region consisted of a new MBL gene, designated bla(NDM-1), flanked on one side by K. pneumoniae DNA and a truncated IS26 element on its other side. The last two regions lie adjacent to one another, and all three regions are found on a 180-kb region that is easily transferable to recipient strains and that confers resistance to all antibiotics except fluoroquinolones and colistin. NDM-1 shares very little identity with other MBLs, with the most similar MBLs being VIM-1/VIM-2, with which it has only 32.4% identity. As well as possessing unique residues near the active site, NDM-1 also has an additional insert between positions 162 and 166 not present in other MBLs. NDM-1 has a molecular mass of 28 kDa, is monomeric, and can hydrolyze all beta-lactams except aztreonam. Compared to VIM-2, NDM-1 displays tighter binding to most cephalosporins, in particular, cefuroxime, cefotaxime, and cephalothin (cefalotin), and also to the penicillins. NDM-1 does not bind to the carbapenems as tightly as IMP-1 or VIM-2 and turns over the carbapenems at a rate similar to that of VIM-2. In addition to K. pneumoniae 05-506, bla(NDM-1) was found on a 140-kb plasmid in an Escherichia coli strain isolated from the patient's feces, inferring the possibility of in vivo conjugation. The broad resistance carried on these plasmids is a further worrying development for India, which already has high levels of antibiotic resistance.

TEM-1 beta-lactamase is the most common plasmid-encoded beta-lactamase in Gram-negative bacteria and is a model class A enzyme. The active site of class A beta-lactamases share several conserved residues including Ser(70), Glu(166), and Asn(170) that coordinate a hydrolytic water involved in deacylation. Unlike Ser(70) and Glu(166), the functional significance of residue Asn(170) is not well understood even though it forms hydrogen bonds with both Glu(166) and the hydrolytic water. The goal of this study was to examine the importance of Asn(170) for catalysis and substrate specificity of beta-lactam antibiotic hydrolysis. The codon for position 170 was randomized to create a library containing all 20 possible amino acids. The random library was introduced into Escherichia coli, and functional clones were selected on agar plates containing ampicillin. DNA sequencing of the functional clones revealed that only asparagine (wild type) and glycine at this position are consistent with wild-type function. The determination of kinetic parameters for several substrates revealed that the N170G mutant is very efficient at hydrolyzing substrates that contain a primary amine in the antibiotic R-group that would be close to the Asn(170) side chain in the acyl-intermediate. In addition, the x-ray structure of the N170G enzyme indicated that the position of an active site water important for deacylation is altered compared with the wild-type enzyme. Taken together, the results suggest the N170G TEM-1 enzyme hydrolyzes ampicillin efficiently because of substrate-assisted catalysis where the primary amine of the ampicillin R-group positions the hydrolytic water and allows for efficient deacylation.

The genus Chryseobacterium and other genera belonging to the family Flavobacteriaceae include organisms that can behave as human pathogens and are known to cause different kinds of infections. Several species of Flavobacteriaceae, including Chryseobacterium indologenes, are naturally resistant to beta-lactam antibiotics (including carbapenems), due to the production of a resident metallo-beta-lactamase. Although C. indologenes presently constitutes a limited clinical threat, the incidence of infections caused by this organism is increasing in some settings, where isolates that exhibit multidrug resistance phenotypes (including resistance to aminoglycosides and quinolones) have been detected. Here, we report the identification and characterization of a new IND-type variant from a C. indologenes isolate from Burkina Faso that is resistant to beta-lactams and aminoglycosides. The levels of sequence identity of the new variant to other IND-type metallo-beta-lactamases range between 72 and 90% (for IND-4 and IND-5, respectively). The purified enzyme exhibited N-terminal heterogeneity and a posttranslational modification consisting of the presence of a pyroglutamate residue at the N terminus. IND-6 shows a broad substrate profile, with overall higher turnover rates than IND-5 and higher activities than IND-2 and IND-5 against ceftazidime and cefepime.

Multidrug efflux transporters, especially those that belong to the resistance-nodulation-division (RND) family, often show very broad substrate specificity and play a major role both in the intrinsic antibiotic resistance and, with increased levels of expression, in the elevated resistance of Gram-negative bacteria. However, it has not been possible to determine the kinetic behavior of these important pumps so far. This is partly because these pumps form a tripartite complex traversing both the cytoplasmic and outer membranes, with an outer membrane channel and a periplasmic adaptor protein, and it is uncertain if the behavior of an isolated component protein reflects that of the protein in this multiprotein complex. Here we use intact cells of Escherichia coli containing the intact multiprotein complex AcrB-AcrA-TolC, and measure the kinetic constants for various cephalosporins, by assessing the periplasmic concentration of the drug from their rate of hydrolysis by periplasmic beta-lactamase and the rate of efflux as the difference between the influx rate and the hydrolysis rate. Nitrocefin efflux showed a K(m) of about 5 microM with little sign of cooperativity. For other compounds (cephalothin, cefamandole, and cephaloridine) that showed lower affinity to the pump, however, kinetics showed strong positive cooperativity, which is consistent with the rotating catalysis model of this trimeric pump. For the very hydrophilic cefazolin there was little sign of efflux.

Objectives: In order to assess whether or not the Arg-276 of CTX-M-type enzymes is equivalent to the Arg-244 of IRT-TEM-derivative enzymes, we replaced the former with six different amino acids, some of them previously described as involved in resistance to beta-lactamase inhibitors in TEM-IRT derivatives. We also investigated the role of Arg276 in cefotaxime hydrolysis., Methods: By site-directed mutagenesis and by use of the bla(CTX-M-1) gene as template, Arg-276 was replaced with six different amino acids (Trp, His, Cys, Asn, Gly and Ser). MICs of beta-lactams alone and in combination with beta-lactamase inhibitors were established. The seven enzymes (CTX-M-1 wild-type and six derived mutants) were purified by affinity chromatography, and kinetic parameters (k(cat), K(m), k(cat)/K(m)) towards cefalotin and cefotaxime were determined. Clavulanic acid IC(50) values were also assessed with all enzymes., Results: No increase in MICs of beta-lactam/beta-lactamase inhibitor combination was detected with any of the six CTX-M-1-derived mutants, in agreement with the clavulanic acid IC(50) values. The MICs of cefotaxime were clearly lower for the Escherichia coli harbouring the Trp, Cys, Ser and Gly CTX-M-1 mutant enzymes than for CTX-M-1, and these enzymes showed a clearly reduced catalytic efficiency towards cefotaxime. As regards cefalotin, there was a moderate reduction in catalytic efficiency for Cys and His., Conclusions: Arg-276 in CTX-M-type beta-lactamases is not equivalent to Arg-244 in IRT-type enzymes. Position Arg-276 appears to be important for cefotaxime hydrolysis in CTX-M-type enzymes, although different effects were obtained regarding the replaced amino acid.

Seventy-six (75.2%) of 101 Escherichia coli isolates, collected over a 2-month period in 2002 at the Children's Hospital of Tunis (Tunisia), were resistant to at least one beta-lactam. They produced beta-lactamases which were further characterized by spectrophotometry, isoelectric focusing (IEF) and PCR. Seventy-five isolates had a pI 5.4 beta-lactamase and one isolate expressed a pI 5.6 beta-lactamase. These beta-lactamases were active against penicillins and cephalothin. Nineteen of 76 isolates were resistant to ticarcillin-clavulanic acid combination. The bla(TEM) gene was detected in 71 isolates, all expressed a pI 5.4 beta-lactamase that was assumed to be TEM-1 or inhibitor-resistant TEM (IRT). Thirty-eight of 76 isolates showed one weak band on IEF with pIs ranging from 8.2 to >9, suggesting low level expression of the chromosomal AmpC beta-lactamase. Five of 76 isolates produced extended-spectrum beta-lactamases (ESBLs), with a basic pI of 7.9 or 8.7, active on penicillins, extended-spectrum cephalosporins, but not on cefoxitin. bla(SHV) genes were detected in three isolates producing pI 7.9 ESBLs but not in two isolates expressing pI 8.7 ESBLs. These latter showed strong cefotaxime-hydrolyzing activities. Hence, they might be CTX-M-type ESBLs.

The development of beta-lactam antibiotics has been a continuous battle of the design of new compounds to withstand inactivation by the ever-increasing diversity of beta-lactamases. Semisynthetic cephalosporins like cephalothin were synthesized from 7-amino-cephalosporanic acid (7-ACA), and thiophene-2-acetic acid using cephalosporin-C acylase enzyme was studied. The production of cephalosporin-C acylase by Pseudomonas diminuta was used and the growth kinetics studied. The optimum condition of enzyme activity was determined by using response surface methodology. A 2(3) full-factorial composite design was employed for experimental design and the result analyzed. The pH value and temperature for optimum activity were 6.5 and 32 degrees C, respectively. The structural analog compound similar to the side-chain of semisynthetic cephalosporins, e.g., thiophene-2-acetic acid, was added. HPLC data analysis indicate that the concentration of cephalothin was 1.6 mg/mL.

A beta-lactamase-free penicillin amidase from Alcaligenes sp. active against various beta-lactams was purified to homogeneity. The enzyme can hydrolyze penicillin G to 6-amino penicillanic acid (6-APA) and furnish penicillin G from 6-APA and phenyl acetic acid by condensation. The penicillin amidase is a heterodimer of subunit masses of 63 kDa and 22 kDa, respectively. Its isoelectric point is at pH 8.5. Cephalothin was found to be the best substrate. This is a novel type II penicillin amidase which shares the properties of both type II and type III enzymes. It is thermostable and, unlike penicillin amidase from A. faecalis, its stability remains unperturbed even in presence of reductant. An inhibition study by 2-hydroxy-5-nitro benzylbromide indicated the involvement of tryptophan in catalysis by the enzyme.

The origin of the substantial difference in deacylation rates for acyl-enzyme intermediates in penicillin-binding proteins (PBPs) and beta-lactamases has remained an unsolved puzzle whose solution is of great importance to understanding bacterial antibiotic resistance. In this work, accurate, large-scale mixed ab initio quantum mechanical/molecular mechanical (QM/MM) calculations have been used to study the hydrolysis of acyl-enzyme intermediates formed between cephalothin and the dd-peptidase of Streptomyces sp. R61, a PBP, and the Enterobacter cloacae P99 cephalosporinase, a class C beta-lactamase. Qualitative and, in the case of P99, quantitative agreement was achieved with experimental kinetics. The faster rate of deacylation in the beta-lactamase is attributed to a more favorable electrostatic environment around Tyr150 in P99 (as compared to that for Tyr159 in R61) which facilitates this residue's function as the general base. This is found to be in large part accomplished by the ability of P99 to covalently bind the ligand without concurrent elimination of hydrogen bonds to Tyr150, which proves not to be the case with Tyr159 in R61. This work provides an essential foundation for further work in this area, such as selecting mutations capable of converting the PBP into a beta-lactamase.

Molecular models for the Henry Michaelis complexes of Enterobacter cloacae, a class C beta-lactamase, with penicillin G and cephalotin have been constructed by using molecular mechanic calculations, based on the AMBER force field, to examine the molecular differentiation mechanisms between cephalosporins and penicillins in beta-lactamases. Ser318Ala and Thr316Ala mutations in both complexes and Asn346Ala and Thr316Ala/Asn346Ala double mutation in penicillin G complex have also been studied. Results confirm that Thr316, Ser318, and Asn346 play a crucial role in the substrate recognition, via their interactions with one of the oxygens of the antibiotic carboxyl group. Both mutation Ser318Ala and Thr316Ala strongly affect the correct binding of cephalotin to P99, the first mainly by precluding the discriminating salt bridge between carboxyl and serine OH groups, and the second one by the Ser318, Lys315, and Tyr150 spatial rearrangements. On the other hand, Ser318Ala mutation has little effect on penicillin G binding, but the Thr316Ala/Asn346Ala double mutation causes the departure of the antibiotic from the oxyanion hole. Molecular dynamic simulations allow us to interpret the experimental results of some class C and A beta-lactamases., (Copyright 2003 Wiley-Liss, Inc.)

Bacterial resistance to beta-lactam antibiotics is a serious problem limiting current clinical therapy. The most common form of resistance is the production of beta-lactamases that inactivate beta-lactam antibiotics. Toho-1 is an extended-spectrum beta-lactamase that has acquired efficient activity not only to penicillins but also to cephalosporins including the expanded-spectrum cephalosporins that were developed to be stable in former beta-lactamases. We present the acyl-intermediate structures of Toho-1 in complex with cefotaxime (expanded-spectrum cephalosporin), cephalothin (non-expanded-spectrum cephalosporin), and benzylpenicillin at 1.8-, 2.0-, and 2.1-A resolutions, respectively. These structures reveal distinct features that can explain the ability of Toho-1 to hydrolyze expanded-spectrum cephalosporins. First, the Omega-loop of Toho-1 is displaced to avoid the steric contacts with the bulky side chain of cefotaxime. Second, the conserved residues Asn(104) and Asp(240) form unique interactions with the bulky side chain of cefotaxime to fix it tightly. Finally, the unique interaction between the conserved Ser(237) and cephalosporins probably helps to bring the beta-lactam carbonyl group to the suitable position in the oxyanion hole, thus increasing the cephalosporinase activity.

Beta-lactamases hydrolyze beta-lactam antibiotics and are the leading cause of bacterial resistance to these drugs. Although beta-lactamases have been extensively studied, structures of the substrate-enzyme and product-enzyme complexes have proven elusive. Here, the structure of a mutant AmpC in complex with the beta-lactam cephalothin in its substrate and product forms was determined by X-ray crystallography to 1.53 A resolution. The acyl-enzyme intermediate between AmpC and cephalothin was determined to 2.06 A resolution. The ligand undergoes a dramatic conformational change as the reaction progresses, with the characteristic six-membered dihydrothiazine ring of cephalothin rotating by 109 degrees. These structures correspond to all three intermediates along the reaction path and provide insight into substrate recognition, catalysis, and product expulsion.

Photosensitization reaction of drugs leading to the formation of reactive oxygen species (ROS) cause tissue injury causing damage to various cellular macromolecules. The aim of this study was to determine the superoxide anion (O2-) generation potential of commonly used antibiotics so that due precaution could be exercised to minimize their photosensitizing action and oxidative stress potential. The selected antibiotics were examined for their ability to produce (O2-) under sunlight and artificial UVA (320-400 nm) and UVB (290-320 nm). Lincomycin, cephalothin and erythromycin generated significant amount of O2- under various irradiation conditions. Superoxide dismutase (SOD), an enzymatic quencher of O2- inhibited O2- production in all drugs tested. The results suggest that due precaution are necessary to avoid ultraviolet radiation (UVR) after the intake of photoreactive drugs, especially in tropical and sub tropical countries.

Kinetic schemes are established for degradation catalysed by Cd2+ ions in methanolic medium for penicillin G, penicillin V and cephalothin, a cephalosporin. Methanolysis of penicillin V and cephalothin occurs with the formation of a single substrate-metal ion intermediate complex, SM, while degradation of penicillin G occurs with the initial formation of two complexes with different stoichiometry, SM and S2M. In each case. degradation is of first order with respect to SM with rate constant values equal to 0.079 min(-1), 0.120 min(-1) and 0.166 min(-1) at 20, 25 and 30 degrees C, respectively, for penicillin G; 0.061 min(-1) at 20 degrees C for penicillin V; and 2.0 x 10(-3) min(-1) at 20 degrees C for cephalothin. Activation energy for the decomposition process of the SM intermediate for penicillin G was calculated to be about 5.5 x 10(4) J/mol. Equilibrium constant values between SM compound and S2M at 20 degrees C (77.1 l/mol), 25 degrees C (45.3 l/mol) and at 30 degrees C (25.7 l/mol) were also calculated as well as the normal enthalpy of this equilibrium. With respect to the reaction products there is evidence that Cd2+ becomes part of their structure, forming complexes between Cd2+ and the product resulting from antibiotic methanolysis (L). Some characteristics of these complexes are discussed.

Two clinically-important beta-lactam antibiotics, cephalothin and cefotaxime, have been observed by X-ray crystallography bound to the reactive Ser62 of the D-alanyl-D-alanine carboxypeptidase/transpeptidase of Streptomyces sp. R61. Refinement of the two crystal structures produced R factors for 3 sigma (F) data of 0.166 (to 1.8 A) and 0.170 (to 2.0 A) for the cephalothin and cefotaxime complexes, respectively. In each complex, a water molecule is within 3.1 and 3.6 A of the acylated beta-lactam carbonyl carbon atom, but is poorly activated by active site residues for nucleophilic attack and deacylation. This apparent lack of good stereochemistry for facile hydrolysis is in accord with the long half-lives of cephalosporin intermediates in solution (20-40 h) and the efficacy of these beta-lactams as inhibitors of bacterial cell wall synthesis. Different hydrogen binding patterns of the two cephalosporins to Thr301 are consistent with the low cefotaxime affinity of an altered penicillin-binding protein, PBP-2x, reported in cefotaxime-resistant strains of Streptococcus pneumoniae, and with the ability of mutant class A beta-lactamases to hydrolyze third-generation cephalosporins.

The kinetics of beta-lactam hydrolysis by wild-type AmpC beta-lactamase from Escherichia coli and three mutant proteins created by substitution of tyrosine 150 have been examined. The catalytic efficiency was decreased 10- to 1000-fold according to the substrate and mutant being studied. The effect of the mutation was much stronger with rapidly hydrolyzed substrates (e.g., cephalothin) than it was with slowly hydrolyzed substrates (e.g., ceftriaxone). With the latter substrates, the mutagenesis had a much stronger effect on apparent affinity than it did on rates of catalysis. Indeed, the enzyme appeared to be more reactive toward certain of the slowly hydrolyzed substances (e.g., methicillin, aztreonam, and ceftriaxone). These observations were not compatible with an obligatory role of tyrosine 150 in catalysis. The analysis of the effects of the mutation on activity was complicated by the observation of at least two, kinetically distinct, forms of the enzymes. It appeared that mutation of tyrosine 150 influenced the kinetic properties of one state and that this residue is involved in the partitioning of the enzyme between the different reactive states.

A beta-lactamase was purified 430-fold from the culture supernatant of Acinetobacter calcoaceticus by ion exchange chromatography on CM-Sephadex and affinity chromatography on phenylboronic-acid-agarose. The purified enzyme was homogeneous as judged by SDS-PAGE, and was characterized with respect to molecular mass (38 and 41 kDa by gel filtration on Sephadex G-75 and SDS-PAGE, respectively), pH optimum (pH 7.0), temperature optimum (45 degrees C) and isoelectric point (9.3). The beta-lactamase showed mainly cephalosporinase activity. It was inhibited by cloxacillin, carbenicillin, penicillanic acid sulphone (sulbactam) and aztreonam. It was not inhibited by clavulanic acid up to a concentration of 0.25 mM. Neither EDTA nor p-chlormercuribenzoate, up to concentrations of 1 or 100 mM, respectively, affected activity. According to these characteristics, it is a typical CEP-N cephalosporinase.

In order to analyze the mutual displacement effects on the protein binding of beta-lactam antibiotics, binding experiments with the human serum albumin (HSA) were performed for cephalothin (CET) and phenoxymethylpenicillin (PCV) by using the centrifugal ultrafiltration method. The numbers of primary and secondary binding sites, n1 and n2, and the affinity constants for the primary and secondary binding sites, K1 and K2 were determined for CET to be 1.00 +/- 0.06 (mean +/- S.D.) and 4.54 +/- 0.12 and 2.59 x 10(3) +/- 0.10 x 10(3) (M-1) and 2.59 x 10(2) +/- 0.16 x 10(2) (M-1), respectively, and for PCV to be 0.94 +/- 0.10 and 5.41 +/- 0.40 and 3.52 x 10(3) +/- 0.25 x 10(3) (M-1) and 4.07 x 10(2) +/- 0.54 x 10(2) (M-1), respectively. Using the predicted optimum unbound concentration of PCV, i.e., 4.6 x 10(-4) M, the displacement effect of PCV to the binding of CET at the primary site has been demonstrated, while no significant effect was observed at the secondary binding site. Moreover, a competitive displacement effect of CET was also demonstrated for the binding of PCV to HSA at the primary binding site, suggesting that CET and PCV bound to HSA at the same primary binding site.

Klebsiella pneumoniae strain L 164 produces a penicillinase whose isoelectric point is 8.1, an unusual figure for this bacterial species. This strain exhibits resistance to conventional penicillins and a synergistic effect is seen with clavulanic acid. In contrast, susceptibility to cephalosporins is marked, as shown by the low minimum inhibitory concentrations (MICs). This phenotype is characteristic of strains with no acquired resistance. A first mutant with MICs for cephalothin and cefotaxime 8-fold to 16-fold those of the initial strain was obtained spontaneously. This mutant's MICs for the other beta-lactams were not substantially changed. In addition to the same penicillinase as the one produced by the parent strain, this mutant produced an acetyl-esterase capable of hydrolyzing the cephalosporins with an acetoxyl side-chain, i.e., cefalothin and cefotaxime, to deacetylated derivates which retain substantial antibacterial activity. Another mutant selected on an amoxicillin gradient produced ten times more penicillinase than the parent strain but no esterase. This second mutant exhibited very high MICs for penicillins and first and second generation cephalosporins. The MIC for cefotaxime was comparable to that seen with the esterase-producing mutant. Among the antimicrobials tested, only third generation cephalosporins and cefoxitin showed adequate activity.

This study was employed to investigate whether the serum protein binding of various cephems [cefpiramide (CPM), cefalotin (CET), latamoxef (LMOX)] differ among healthy subjects and patients with chronic renal failure (CRF) by means of in vitro equilibrium dialysis. The protein binding capacities of cephems in patients with CRF (hemodialysis, continuous ambulatory peritoneal dialysis, non-dialysis) decreased significantly compared to those in healthy subjects. The binding capacities correlated directly with total protein, albumin concentration and correlated inversely with blood urea nitrogen and serum creatinine concentration. In the study of protein binding during and after hemodialysis, the binding capacities of CPM and LMOX decreased immediately after dialysis and then increased with the time. However, the binding capacities of CET increased immediately after dialysis and then decreased. The binding capacities of CPM and LMOX correlated inversely with non-esterified fatty acids (NEFA) and those of CET correlated directly with NEFA. In the study of protein binding in pooled sera from healthy subjects with or without palmitic acid (PA), the binding capacities of CPM and LMOX decreased by increasing the concentration of PA, while those of CET increased by increasing PA up to 3 mM. The changes in binding capacity of cephems during and after hemodialysis have been possibly caused by increase of NEFA due to activation of lipase in use of heparin as an anticoagulant. In conclusion, changes in protein binding capacity of cephems in sera from CRF, which should be taken into consideration to avoid possible side effects.

A mutant with decreased susceptibility to imipenem (IpR) was selected in vitro from a susceptible clinical isolate of Listeria monocytogenes (IpS). IpR exhibited decreased susceptibility to penicillin G (4 x MIC) and imipenem (16 x MIC) and increased susceptibility to cefotaxime (0.25 x MIC). Electrophoretic profiles of membrane proteins and penicillin-binding proteins (PBPs) were identical in the two strains; each strain had five PBPs with molecular masses of ca. 97, 83.3, 81, 77.1, and 42.6 kilodaltons. A decreased affinity of PBP 3 for penicillin G and imipenem (10-fold) was observed in IpR. In contrast, the affinity of PBP 3 for cefotaxime in IpR was increased twofold and correlated with the decreased MIC of this drug. From these findings and competition experiments with different beta-lactam antibiotics, we conclude that the alteration of PBP 3 is responsible for the decreased susceptibility of IpR to penicillin and imipenem and that PBP 3 might be an essential target of beta-lactam antibiotics in L. monocytogenes.

The presence of an additional penicillin-binding protein (PBP) was demonstrated in methicillin-resistant strains of Staphylococcus epidermidis, S. haemolyticus, S. hominis, and S. simulans. In these four species, the apparent molecular mass of this protein was analogous to that of PBP 2' of methicillin-resistant S. aureus SR 1550-9. It exhibited a low affinity for methicillin, cephalothin, and cefamandole; and its synthesis was methicillin inducible. Peptide mapping of this PBP from the four species yielded identical results that were analogous to those obtained with S. aureus SR 1550-9. These results suggest that this protein is similar to, if not the same as, PBP 2' of S. aureus and that it is involved in methicillin resistance in the four species studied.

The objective of this study was to determine the efficacy of a hypotensive anesthetic in enhancing tissue perfusion while simultaneously controlling surgical blood loss. One hundred ten patients undergoing 126 total hip arthroplasties were administered either cephalothin or cefamandole on a double-blind basis. Hypotensive anesthesia was administered to 64 patients; 46 received a normotensive anesthetic. Degree of tissue perfusion was measured by the concentration of cephalosporin found in samplings of serum, soft tissue, and bone. These samples, obtained during the course of total joint arthroplasty, indicate that the hypotensive anesthetic sodium nitroprusside did not significantly alter the concentrations of cefamandole or cephalothin from those found in the normotensive group.

Perioperative antibiotic administration to produce systemic effects reduces the frequency of operative wound infection in selected procedures. Controlled clinical trials now indicate that cephalothin sodium may not be effective in this role, whereas cephaloridine and cefazolin sodium have been beneficial. Review of estimates of incisional antibiotic concentrations taken during prospective patient studies display patterns compatible with these data. EAch drug reaches acceptable incisional concentrations, which are relatively well maintained for cephaloridine and cefazolin; cephalothin disappears from human incisions so rapidly as to provide little wound protection in operations lasting more than one hour.

The pharmacokinetics of cephalothin and its deacetyl metabolite were studied with use of a chemical assay involving high-pressure liquid chromatography. In seven patients with normal renal function, the half-life of cephalothin was about 28 min and concentrations of deacetylcephalothin in serum were low. In five patients with uremia, the half-life of cephalothin averaged 221 min and there was a continuous accumulation of the deacetyl metabolite in serum during treatment. Recovery of the drug in urine was quantitative when renal function was normal, whereas the amount of drug excreted was very low in cases of uremia. The results offer some explanation of the complex pharmacokinetics of cephalothin seen with microbiological tests and illustrate the usefulness of specific chemical assays, especially in the monitoring of serum concentrations of mixtures of antibiotic compounds that differ in antibacterial activity.

The cephalosporins have been available for clinical use for nearly 20 years and a large number is presently marketed, including drugs with a wide range of different pharmacokinetic and microbiologic properties. While some of these agents have certain specific uses in which they excel, the cephalosporins have not replaced older antibiotics but do provide the physician with a broader range of choices for the treatment of many infections, allowing greater individualization of therapy.

beta-Lactamase from strains of Bacteroides melaninogenicus and Bacteroides oralis hydrolyzed penicillin more rapidly than ampicillin or carbenicillin. Cephalothin and a chromogenic cephalosporin (87/312) were also hydrolyzed by the enzyme. Activity was found only in beta-lactam-resistant strains, but there was considerable variation in activity among strains having the same minimal inhibitory concentrations of antibiotic. beta-Lactamase activity was cell bound and appeared to be tightly associated with the cell envelope since detergents were required to elute this activity.

Methicillin-resistant clinical isolates of Staphylococcus aureus are intrinsically resistant to beta-lactam antibiotics in that the resistance mechanism is unrelated to the possession of beta-lactamases. We have demonstrated that a new, high-molecular-mass penicillin-binding protein (PBP) is present in these strains with a low affinity for beta-lactams and that its amount is regulated by the growth conditions. The new PBP from all strains that have been examined has an identical mobility on SDS gel electrophoresis and is the only PBP still present in an uncomplexed state with beta-lactams (and therefore the only functional PBP when these strains are grown in media containing concentrations of beta-lactam antibiotics sufficient to kill sensitive strains.

Antibiotics should be administered so as to maintain effective therapeutic levels in target organs. In routine clinical treatment, however, doses of antibiotics are determined on the basis of their achievable concentrations in blood. To determine the optimum doses of antibiotics against obstetric and gynecologic infections, the authors gave a single intravenous dose of 2 gm of cefazolin or cephalothin to 51 patients before hysterectomy and measured the levels of the antibiotics in uterine tissues and compared them with corresponding serum levels. The maximum tissue levels of cefazolin were generally about 6 to 7 times higher than those of cephalothin: In tthe perimetrium, levels were 154 microgram/gm for cefazolin and 24.1 microgram/gm for cephalothin; in the myometrium, 109.5 microgram/gm for cefazolin, and 15.5 microgram/gm for cephalothin; and in the endometrium, 98.9 microgram/gm for cefazolin and 10.4 microgram/gm for cephalothin. In the tissues, however, cephalothin reached peak levels more rapidly than cefazolin. Comparisons of the tissue levels and MIC of cefazolin and cephalothin revealed that cefazolin was maintained at over 12.5 microgram/gm for at least 150 minutes in the perimetrium, myometrium, and endometrium, whereas cephalothin was maintained at these levels for 40 to 50 minutes only in the perimetrium and myometrium. The ratio of tissue levels to serum levels was 40-70 to 100 for cefazolin and about 30 or less to 100 for cephalothin.

Thirty-two pediatric patients having open-heart operation received a single dose of either cephalothin or cephapirin intravenously, 30 mg per kilogram of body weight, in the operating room, for prophylaxis before the chest cavity was opened. Samples of right atrial appendage, pericardial fluid, muscle, fat, and plasma were obtained at various time intervals after injection of the antibiotics, and assayed for cephalosporin concentration. The concentration-time profiles of cephalothin and cephapirin in atrial appendage, muscle, fat, and plasma were identical. Cephapirin produced higher total and free concentrations in pericardial fluid compared with cephalothin. This presumably was due to the lower protein binding of cephapirin. Antibiotic concentrations above the minimal inhibitory concentration for Staphylococcus aureus and S. epidermidis were present in myocardial tissue for at least 90 minutes after the dose was administered. These data support the need to administer these antibiotics shortly before surgical intervention and, if the operation is prolonged, the need to administer a second dose of antibiotic.

Using an open one- and two-compartment model, the authors studied the kinetics of cephalothin during pregnancy and in the course of labour as well as in a group of non-pregnant women. They found that the parenteral application of cephalothin leads to therapeutic levels in the serum, tissues and urine also during pregnancy and in the course of labour. The bearing of pregnancy and labour on the kinetics of cephalothin is but insignificant; consequently, no modification of the pharmacotherapy will be necessary.

Cefazolin was more susceptible than cephaloridine and cephalothin to in vitro inactivation by coagulase-positive, penicillinase-producing strains of Staphylococcus aureus. Inactivation (which was greater with methicillin-resistant than with methicillin-sensitive strains) was demonstrated by assay of the antibiotics in broth cultures with simultaneous colony counts and by exposure of the antibiotics to penicillinase powders extracted from S. aureus. Cefazolin was destroyed to a greater extent than was cephaloridine, whereas cephalothin underwent little, if any, destruction. The clinical implications of this degradation, thought by some to be of importance for cephaloridine, might also apply to cefazolin.

When the antibacterial substances of cephalothin and cephapirin in the serum and urine after intramuscular injection were separated and assayed, desacetyl metabolities of both drugs were detected. These tendencies were especially apparent in the tissue concentrations. When both the drugs were given intravenously to healthy volunteers, the amounts of their desacetyl metabolites were not greater in man than in rats.