Characterization of the plasmid genes blaT-4 and blaT-5 which encode the broad-spectrum beta-lactamases TEM-4 and TEM-5 in enterobacteriaceae

We have determined the nucleotide sequence of the plasmid genes blaT-4 and blaT-5 which encode the broad-substrate-range beta-lactamases TEM-4 and TEM-5, respectively. The TEM-4 enzyme, which confers high-level resistance to cefotaxime (Ctx) and ceftazidime (Caz), differed from the TEM-1 penicillinase by four amino acid substitutions. Two of the mutations are identical to those responsible for the wide substrate range of the TEM-3 beta-lactamase which hydrolyses Ctx and Caz. The amino acid sequence of TEM-5, which confers higher levels of resistance to Caz than to other recently developed cephalosporins, differed from that of TEM-1 by three mutations distinct from those of TEM-4. Analysis of the location of the mutations in the primary and tertiary structures of class A beta-lactamases suggests that interactions between the substituted residues and beta-lactam antibiotics non-hydrolysable by TEM-1 and TEM-2 allow TEM-4 and TEM-5 to hydrolyse efficiently novel broad-spectrum cephalosporins such as Ctx and Caz.     

The loop between β-strands β2 and β3 and its interaction with the N-terminal α-helix is essential for biogenesis of α7 nicotinic receptors

Recently, we have shown that the α-helix present at the N-termini of α7 nicotinic acetylcholine receptors plays a crucial role in their biogenesis. Structural data suggest that this helix interacts with the loop linking β-strands β2 and β3 (loop 3). We studied the role of this loop as well as its interaction with the helix in membrane receptor expression. Residues from Asp62 to Val75 in loop 3 were mutated. Mutations of conserved amino acids, such as Asp62, Leu65 and Trp67 abolished membrane receptor expression in Xenopus oocytes. Others mutations, at residues Asn68, Ala69, Ser70, Tyr72, Gly74, and Val 75 were less harmful although still produced significant expression decreases. Steady state levels of wild-type and mutant α7 receptors (L65A, W67A, and Y72A) were similar but the formation of pentameric receptors was impaired in the latter (W67A). Mutation of critical residues in subunits of heteromeric nicotinic acetylcholine receptors (α3β4) also abolished their membrane expression. Complementarity between the helix and loop 3 was evidenced by studying the expression of chimeric α7 receptors in which these domains were substituted by homologous sequences from other subunits. We conclude that loop 3 and its docking to the α-helix is an important requirement for receptor assembly.     

β-Lactam resistance in Streptococcus pneumoniae: penicillin-binding proteins and non-penicillin-binding proteins

The beta-lactams are by far the most widely used and efficacious of all antibiotics. Over the past few decades, however, widespread resistance has evolved among most common pathogens. Streptococcus pneumoniae has become a paradigm for understanding the evolution of resistance mechanisms, the simplest of which, by far, is the production of beta-lactamases. As these enzymes are frequently plasmid encoded, resistance can readily be transmitted between bacteria. Despite the fact that pneumococci are naturally transformable organisms, no beta-lactamase-producing strain has yet been described. A much more complex resistance mechanism has evolved in S. pneumoniae that is mediated by a sophisticated restructuring of the targets of the beta-lactams, the penicillin-binding proteins (PBPs); however, this may not be the whole story. Recently, a third level of resistance mechanisms has been identified in laboratory mutants, wherein non-PBP genes are mutated and resistance development is accompanied by deficiency in genetic transformation. Two such non-PBP genes have been described: a putative glycosyltransferase, CpoA, and a histidine protein kinase, CiaH. We propose that these non-PBP genes are involved in the biosynthesis of cell wall components at a step prior to the biosynthetic functions of PBPs, and that the mutations selected during beta-lactam treatment counteract the effects caused by the inhibition of penicillin-binding proteins.     

Separation of plasmid-mediated extended spectrum β-lactamases by fast protein liquid chromatography (FPLC system)

We have devised a reliable procedure for the separation of three beta-lactamases of isoelectric focusing points (pI), 5.4, 6.5, and 7.9 by Fast Protein Liquid Chromatography (FPLC System). All of these enzymes were transferable and originated from a ceftazidime and cefotaxime resistant Klebsiella pneumoniae isolated in Bombay, India. The complete separation of the enzymes, achievable by this method, allowed each of the different individual beta-lactamases to be characterized biochemically. This analysis revealed that the enzymes of pI 6.5 and pI 7.9 hydrolysed ceftazidime and cefotaxime, and were responsible for the resistance of K. pneumoniae, and its Escherichia coli J53-2 transconjugant to third generation cephalosporins. The enzyme of pI 5.4 was the TEM-1 beta-lactamase. The beta-lactamase of pI 7.9 appears quite different from any previously reported third generation cephalosporin hydrolysing beta-lactamase, and consequently given the preliminary designation DJP-1. This is also the first example of extended spectrum hydrolysing beta-lactamases found in Asia.     

Substitution of Thr for Ala-237 in TEM-17, TEM-12 and TEM-26: alterations in β-lactam resistance conferred on Escherichia coli

Non-naturally occurring mutants of TEM-17 (E104K), TEM-12 (R164S) and TEM-26 (E104K:R164S) extended-spectrum (ES) beta-lactamases bearing threonine at position 237 were constructed by site-specific mutagenesis and expressed under isogenic conditions in Escherichia coli. Quantification of beta-lactamase activities and immunoblotting indicated that Ala-237-->Thr did not significantly affect expression levels of these ES enzymes. Minimum inhibitory concentrations of beta-lactam antibiotics showed that the presence of threonine at position 237 exerted a dominant effect increasing the enzymes' preference for various early generation cephalosporins over penicillins. Activity against broad-spectrum oxyimino-beta-lactams was also changed. The effect of Ala-237-->Thr on the activity against ceftazidime, aztreonam, cefepime and cefpirome of all three ES TEM enzymes was detrimental. Introduction of Thr-237 improved activity against cefotaxime and ceftriaxone in TEM-12 and TEM-26, but not in TEM-17.     

Bacterial TEM-Type Serine Beta-Lactamases: Structure and Analysis of Mutations

Beta-lactamases (EC 3.5.2.6) represent a superfamily containing more than 2000 members: it includes genetically and functionally different bacterial enzymes capable to degrade the beta-lactam antibiotics. Beta-lactamases of molecular class A with serine residue in the active center are the most common ones. In the context of studies of the mechanisms underlying of evolution of the resistance, TEM type beta-lactamases are of particular interest due to their broad polymorphism. To date, more than 200 sequences of TEM type beta-lactamases have been described and more than 60 structures of different mutant forms of these enzymes have been presented in the Protein Data Bank. We have considered here the main structural features of the enzymes of this type with particular attention to the analysis of key mutations determining drug resistance and the secondary mutations, their location relative to the active center and the surface of the protein globule. We have developed a BlaSIDB database (www.blasidb.org) which is an open information resource combining available data on 3D structures, amino acid sequences and nomenclature of the TEM type beta-lactamases.     

Structures of ceftazidime and its transition-state analogue in complex with AmpC beta-lactamase: implications for resistance mutations and inhibitor design

Third-generation cephalosporins are widely used beta-lactam antibiotics that resist hydrolysis by beta-lactamases. Recently, mutant beta-lactamases that rapidly inactivate these drugs have emerged. To investigate why third-generation cephalosporins are relatively stable to wild-type class C beta-lactamases and how mutant enzymes might overcome this, the structures of the class C beta-lactamase AmpC in complex with the third-generation cephalosporin ceftazidime and with a transition-state analogue of ceftazidime were determined by X-ray crystallography to 2.0 and 2.3 A resolution, respectively. Comparison of the acyl-enzyme structures of ceftazidime and loracarbef, a beta-lactam substrate, reveals that the conformation of ceftazidime in the active site differs from that of substrates. Comparison of the structures of the acyl-enzyme intermediate and the transition-state analogue suggests that ceftazidime blocks formation of the tetrahedral transition state, explaining why it is an inhibitor of AmpC. Ceftazidime cannot adopt a conformation competent for catalysis due to steric clashes that would occur with conserved residues Val211 and Tyr221. The X-ray crystal structure of the mutant beta-lactamase GC1, which has improved activity against third-generation cephalosporins, suggests that a tandem tripeptide insertion in the Omega loop, which contains Val211, has caused a shift of this residue and also of Tyr221 that would allow ceftazidime and other third-generation cephalosporins to adopt a more catalytically competent conformation. These structural differences may explain the extended spectrum activity of GC1 against this class of cephalosporins. In addition, the complexed structure of the transition-state analogue inhibitor (K(i) 20 nM) with AmpC reveals potential opportunities for further inhibitor design.     

临床分离的革兰阴性细菌的耐药谱及耐药机制的研究

目的 了解前临床上分离G^－细菌的药敏状况和耐药机制及提供合理使用抗生素的依据。方法 主要使用MICROSCAN WALKAWAY／－40全自动微生物分析仪对1999年3月－2000年3月全院住院病人的尿、痰、腹水、脓液、创面、前列腺液、血液等培养呈阳性的标本进行细菌鉴定和药敏试验,结果共检出G^-菌1152株包括27个菌属80个菌种,觉细菌是大肠埃希菌（16.1％）、铜绿假单胞菌（6.5％）、肺炎克雷伯菌（5.3％）等。G^-杆菌（除不动杆菌外）对第三代头孢霉素敏感率已降到（3.0%-76.1%)、对亚胺培南（80.7%-92%)、头孢哌酮／舒巴坦（58.8％－100％）、阿米卡星（41.4%-93.2%)、环丙沙星（30.5％－67.3％）较敏感;对第三代头孢霉素产生超广谱β－内酰胺酶（ESBLs）,肺炎克雷伯菌高达35.0%-36.9%,大肠5埃希菌达21.8％－23％。对常用β-内酰胺类抗生素产诱导酶（IB）,铜绿假单胸菌高达51％－60.9％,弗劳地枸橼酸菌达4.5％－63.6％,阴沟肠杆菌达8.7%-35.3％。结论 目前G^－杆菌对β-内酰胺类抗生素药的主要机制是产生ESBLs和IB0G^-杆菌引起的感染首选亚胺培南单用或第三代浆孢霉素复合制剂（头孢哌酮／舒巴坦）联合阿米卡星或氟喹酮类,第三代头孢霉素除非药敏提示否则不宜选用。     

Ultrahigh resolution structure of a class A beta-lactamase: on the mechanism and specificity of the extended-spectrum SHV-2 enzyme

Bacterial beta-lactamases hydrolyze beta-lactam antibiotics such as penicillins and cephalosporins. The TEM-type class A beta-lactamase SHV-2 is a natural variant that exhibits activity against third-generation cephalosporins normally resistant to hydrolysis by class A enzymes. SHV-2 contains a single Gly238Ser change relative to the wild-type enzyme SHV-1. Crystallographic refinement of a model including hydrogen atoms gave R and R(free) of 12.4% and 15.0% for data to 0.91 A resolution. The hydrogen atom on the O(gamma) atom of the reactive Ser70 is clearly seen for the first time, bridging to the water molecule activated by Glu166. Though hydrogen atoms on the nearby Lys73 are not seen, this observation of the Ser70 hydrogen atom and the hydrogen bonding pattern around Lys73 indicate that Lys73 is protonated. These findings support a role for the Glu166-water couple, rather than Lys73, as the general base in the deprotonation of Ser70 in the acylation process of class A beta-lactamases. Overlay of SHV-2 with SHV-1 shows a significant 1-3 A displacement in the 238-242 beta-strand-turn segment, making the beta-lactam binding site more open to newer cephalosporins with large C7 substituents and thereby expanding the substrate spectrum of the variant enzyme. The OH group of the buried Ser238 side-chain hydrogen bonds to the main-chain CO of Asn170 on the Omega loop, that is unaltered in position relative to SHV-1. This structural role for Ser238 in protein-protein binding makes less likely its hydrogen bonding to oximino cephalosporins such as cefotaxime or ceftazidime.     

Acyl-intermediate structures of the extended-spectrum class A beta-lactamase,Toho-1, in complex with cefotaxime,cephalothin, and benzylpenicillin

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.     

Degradation of beta-lactam antibiotics in the presence of Zn2+ and 2-amino-2-hydroxymethylpropane-1,3-diol (Tris). A hypothetical non-enzymic model of beta-lactamases.

The system composed of 2-amino-2-hydroxymethylpropane-1,3-diol (Tris) and Zn2+ catalyses the degradation of cephalosporins. The beta-lactam opening fits to a first-order process, with a constant directly proportional to the zinc ion concentration. The pH and Tris concentration dependency displayed by the first-order constant, as well as the nature of the degradation products point to a mechanism that can be considered as an extension of that proposed for the benzylpenicillin degradation. The mechanism proposed here, and the values of the kinetic constants calculated, as compared with those of beta-lactamases, lead to the conclusion that the Tris-Zn2+ system simulates the catalytic action of the serine beta-lactamases rather than the action of the Zn(2+)-dependent type of enzymes.     

Susceptibility and molecular mechanisms of resistance to cephalosporins of Escherichia coli strains isolated from patients with nosocomial infections

Resistance to wide spectrum of antibiotics was studied and most widespread genetic determinants of resistance to beta-lactam antibiotics were revealed. Susceptibility testing was performed using serial broth microdilution method. Detection of class A expanded spectrum beta-lactamases genes (TEM, SHV, CTX) by polymerase-chain reaction method was performed in 90 strains. Carbapenems remained the most active antibacterial agents with respect to studied E. coli strains. Among the 3rd generation cephalosporins the lowest minimal inhibitory concentrations were observed for inhibition-protected combined agents (ceftazidime/clavulanic acid and cefoperazone/ sulbactam). Alone or in various combinations TEM, SHV, and CTX types of beta-lactamases were found in 58.9%, 14.4%, and 77.8% of strains. Combinations of 2 determinants were detected in 55.6% of the isolates, and all 3 determinants--in 5.6%. Most often E. coli was isolated in patients with urinary tract infections. Carbapenems and inhibition-protected combined 3rd generation cephalosporins are the most active agents against E. coli.     

Analysis of site-directed mutations in the α-and β-subunits of Klebsiella pneumoniae nitrogenase

Using directed mutagenesis, amino acid substitutions have been made in the alpha- and beta-subunits of the klebsiella pneumoniae nitrogenase component 1 at positions normally occupied by conserved cysteine or tyrosine residues. Nif+, Nif- and intermediate phenotypes have been obtained. To extend our earlier biochemical characterization (Kent et al., 1989) the electrophoretic mobility of component 1 of the mutant and wild-type nitrogenases has been analysed by non-denaturing gel electrophoresis. The major and minor forms of component 1 separated by this methodology have been probed for by using both polyclonal and monoclonal antibodies. All Nif+ mutants exhibited a distribution of electrophoretic forms of component 1 comparable to the wild type, and the abundance of the major form found in purified nitrogenase correlated approximately with the specific activity of the extract. In contrast, after electrophoresis, component 1 from Nif- mutants exhibited either a major low-mobility form or a fast-moving form. Analysis of nitrogenase polypeptides synthesized in the absence of co-factor (FeMoco) allowed us to conclude that changing cysteine 275 to alanine in the alpha-subunit produces component 1 defective in its interaction with FeMoco. Substitution of other conserved cysteine residues by alanine appears to prevent early steps in nitrogenase assembly or to promote degradation. Two single mutations (cysteine 89 to alanine in the alpha-subunit and cysteine 94 to alanine in the beta-subunit) which are tightly Nif- can be combined to produce a weakly active nitrogenase, indicating regions involved in the interaction between subunits.     

Two novel transferable extended-spectrum β-lactamases from Klebsiella pneumoniae in Tunisia

Two novel beta-lactamases conferring multiresistance to antibiotics including oxyimino beta-lactams have been identified in two nosocomial K. pneumoniae strains isolated in Tunis in 1986 and 1988. Both enzymes were encoded by ca. 150-kilobase plasmids. Donor and transconjugant strains producing these enzymes exhibited highly similar pattern of resistance (CTX phenotype) to beta-lactams including penicillins and oxyimino beta-lactams e.g. cefotaxime, ceftriaxone, ceftazidime, and aztreonam. High and variable synergy (16 to 1066-fold) was obtained when combined to 0.1 microgram/ml of clavulanate (beta-lactamase inhibitor). The isoelectric points of these two enzymes were 5.4 and 6.4. These beta-lactamases differed from TEM types by hydrolysis for cefotaxime or ceftriaxone but were inhibited by clavulanate and cloxacillin. DNA hybridization studies suggested that that the genes of these enzymes may be derived from genes encoding TEM-type enzymes.     

Signalling proteins in enterobacterial AmpC β-lactamase regulation

The cloned Citrobacter freundii ampC beta-lactamase is inducible in the presence of its regulatory gene ampR in Escherichia coli (Lindberg et al., 1985). The basal level of expression and inducibility are affected by two E. coli proteins encoded by the closely linked ampD and ampE genes. Deletion of both genes led to constitutive ampR-dependent overproduction of beta-lactamase, whereas an out-of-frame deletion in AmpD caused the basal expression to increase two-fold. This ampD1 mutant was inducible at lower beta-lactam concentrations than the wild type. An IS1 insertion in ampD was polar on ampE expression and increased basal beta-lactamase expression 30-fold while mediating a semi-constitutive phenotype. AmpE expressed from a recombinant plasmid in an ampD-ampE deletion mutant reduced basal beta-lactamase expression to wild-type levels but did not markedly reduce beta-lactam resistance since the cells became hyperinducible. In the absence of AmpD, increasing levels of AmpE therefore decrease the basal expression of AmpC beta-lactamase in an AmpR-dependent manner. AmpD modulated the response exerted on beta-lactamase expression by AmpE. The ampD gene encodes a 20.5kD cytoplasmic protein while the 32.1kD ampE gene product is an integral membrane protein with a likely ATP-binding site between the second and third putative transmembrane region. Since neither AmpD nor AmpE are needed for beta-lactam induction and since these proteins could not be covalently labelled by benzylpenicillin, they are not thought to act as beta-lactam-binding sensory transducers. Instead it is suggested that AmpD and AmpE sense the effect of beta-lactam action on peptidoglycan biosynthesis and relay this signal to AmpR.     

Correlation Between Hydrolysis of the β-Lactam Bond of the Cephalosporin Nucleus and Expulsion of the 3-Substituent

The hydrolysis of two cephalosporins by three different beta-lactamases has been studied. Each enzyme caused a decrease in ultraviolet absorption, a loss of biological activity, and the release of the leaving group from the 3-position. The changes occurred at the same rate and to the same extent with each enzyme, and it is inferred that the loss of the leaving group is a consequence of, and not a prerequisite for, hydrolysis of the beta-lactam ring.     

Inhibition of AmpC beta-lactamase through a destabilizing interaction in the active site.

Beta-lactamases hydrolyze beta-lactam antibiotics, including penicillins and cephalosporins; these enzymes are the most widespread resistance mechanism to these drugs and pose a growing threat to public health. beta-Lactams that contain a bulky 6(7)alpha substituent, such as imipenem and moxalactam, actually inhibit serine beta-lactamases and are widely used for this reason. Although mutant serine beta-lactamases have arisen that hydrolyze beta-lactamase resistant beta-lactams (e.g., ceftazidime) or avoid mechanism-based inhibitors (e.g., clavulanate), mutant serine beta-lactamases have not yet arisen in the clinic with imipenemase or moxalactamase activity. Structural and thermodynamic studies suggest that the 6(7)alpha substituents of these inhibitors form destabilizing contacts within the covalent adduct with the conserved Asn152 in class C beta-lactamases (Asn132 in class A beta-lactamases). This unfavorable interaction may be crucial to inhibition. To test this destabilization hypothesis, we replaced Asn152 with Ala in the class C beta-lactamase AmpC from Escherichia coli and examined the mutant enzyme's thermodynamic stability in complex with imipenem and moxalactam. Consistent with the hypothesis, the Asn152 --> Ala substitution relieved 0.44 and 1.10 kcal/mol of strain introduced by imipenem and moxalactam, respectively, relative to the wild-type complexes. However, the kinetic efficiency of AmpC N152A was reduced by 6300-fold relative to that of the wild-type enzyme. To further investigate the inhibitor's interaction with the mutant enzyme, the X-ray crystal structure of moxalactam in complex with N152A was determined to a resolution of 1.83 A. Moxalactam in the mutant complex is significantly displaced from its orientation in the wild-type complex; however, moxalactam does not adopt an orientation that would restore competence for hydrolysis. Although Asn152 forces beta-lactams with 6(7)alpha substituents out of a catalytically competent configuration, making them inhibitors, the residue is essential for orienting beta-lactam substrates and cannot simply be replaced with a much smaller residue to restore catalytic activity. Designing beta-lactam inhibitors that interact unfavorably with this conserved residue when in the covalent adduct merits further investigation.     

Beta-lactamase of Lysobacter enzymogenes: induction, purification and characterization

Lysobacter enzymogenes produces an inducible beta-lactamase and induction with 100 micrograms ampicillin ml-1 resulted in an increase of more than 100-fold in enzyme activity. Various other beta-lactam antibiotics also served as effective inducers. The enzyme was obtained from cells by osmotic shocking to release periplasmic components and it was purified primarily by ion-exchange chromatography and PAGE. The beta-lactamase consists of one polypeptide with a molecular mass of about 28 kDa and an isoelectric point greater than 9.6. It is strongly inhibited by p-chloromercuribenzoate and clavulanic acid but not by EDTA. The enzyme readily hydrolyses several penicillins and cephalosporins, but not oxacillin or cloxacillin. The enzyme therefore belongs to group 2b of the bacterial beta-lactamases.