Hydrolysis of third-generation cephalosporins by class C beta-lactamases. Structures of a transition state analog of cefotoxamine in wild-type and extended spectrum enzymes

Bacterial resistance to the third-generation cephalosporins is an issue of great concern in current antibiotic therapeutics. An important source of this resistance is from production of extended-spectrum (ES) beta-lactamases by bacteria. The Enterobacter cloacae GC1 enzyme is an example of a class C ES beta-lactamase. Unlike wild-type (WT) forms, such as the E. cloacae P99 and Citrobacter freundii enzymes, the ES GC1 beta-lactamase is able to rapidly hydrolyze third-generation cephalosporins such as cefotaxime and ceftazidime. To understand the basis for this ES activity, m-nitrophenyl 2-(2-aminothiazol-4-yl)-2-[(Z)-methoxyimino]acetylaminomethyl phosphonate has been synthesized and characterized. This phosphonate was designed to generate a transition state analog for turnover of cefotaxime. The crystal structures of complexes of the phosphonate with both ES GC1 and WT C. freundii GN346 beta-lactamases have been determined to high resolution (1.4-1.5 Angstroms). The serine-bound analog of the tetrahedral transition state for deacylation exhibits a very different binding geometry in each enzyme. In the WT beta-lactamase the cefotaxime-like side chain is crowded against the Omega loop and must protrude from the binding site with its methyloxime branch exposed. In the ES enzyme, a mutated Omega loop adopts an alternate conformation allowing the side chain to be much more buried. During the binding and turnover of the cefotaxime substrate by this ES enzyme, it is proposed that ligand-protein contacts and intra-ligand contacts are considerably relieved relative to WT, facilitating positioning and activation of the hydrolytic water molecule. The ES beta-lactamase is thus able to efficiently inactivate third-generation cephalosporins.     

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.     

Molecular characterization of TEM-type beta-lactamases identified in cold-seep sediments of Edison Seamount (south of Lihir Island, Papua New Guinea)

To determine the prevalence and genotypes of beta-lactamases among clones of a metagenomic library from the cold-seep sediments of Edison seamount (10,000 years old), we performed pulse-field gel electrophoresis, antibiotic susceptibility testing, pI determination, and DNA sequencing analysis. Among the 8,823 clones of the library, thirty clones produced beta-lactamases and had high levels of genetic diversity. Consistent with minimum inhibitory concentration patterns, we found that five (16.7%) of thirty clones produced an extended-spectrum beta-lactamase. 837- and 259-bp fragments specific to blaTEM genes were amplified, as determined by banding patterns of PCR amplification with designed primers. TEM-1 was the most prevalent beta-lactamase and conferred resistance to ampicillin, piperacillin, and cephalothin. TEM-116 had a spectrum that was extended to ceftazidime, cefotaxime, and aztreonam. The resistance levels conferred by the pre-antibiotic era alleles of TEM-type beta-lactamases were essentially the same as the resistance levels conferred by the TEM-type alleles which had been isolated from clinically resistant strains of bacteria of the antibiotic era. Our first report on TEM-type beta-lactamases of the pre-antibiotic era indicates that TEM-type beta-lactamases paint a picture in which most of the diversity of the enzymes may not be the result of recent evolution, but that of ancient evolution.     

Atomic resolution structures of CTX-M beta-lactamases: extended spectrum activities from increased mobility and decreased stability

Extended spectrum beta-lactamases (ESBLs) confer bacterial resistance to third-generation cephalosporins, such as cefotaxime and ceftazidime, increasing hospital mortality rates. Whereas these antibiotics are almost impervious to classic beta-lactamases, such as TEM-1, ESBLs have one to four orders greater activity against them. The origins of this activity have been widely studied for the TEM and SHV-type ESBLs, but have received less attention for the CTX-M beta-lactamases, an emerging family that is now the dominant ESBL in several regions. To understand how CTX-M beta-lactamases achieve their remarkable activity, biophysical and structural studies were undertaken. Using reversible, two-state thermal denaturation, it was found that as these enzymes evolve a broader substrate range, they sacrifice stability. Thus, the mutant enzyme CTX-M-16 is eightfold more active against ceftazidime than the pseudo-wild-type CTX-M-14 but is 1.9 kcal/mol less stable. This is consistent with a "stability-activity tradeoff," similar to that observed in the evolution of other resistance enzymes. To investigate the structural basis of enzyme activity and stability, the structures of four CTX-M enzymes were determined by X-ray crystallography. The structures of CTX-M-14, CTX-M-27, CTX-M-9 and CTX-M-16 were determined to 1.10 Angstroms, 1.20 Angstroms, 0.98 Angstroms and 1.74 Angstroms resolution, respectively. The enzyme active sites resemble those of the narrow-spectrum TEM-1 and SHV-1, and not the enlarged sites typical of ESBL mutants such as TEM-52 and TEM-64. Instead, point substitutions leading to specific interactions may be responsible for the improved activity against ceftazidime and cefotaxime, consistent with observations first made for the related Toho-1 enzyme. The broadened substrate range of CTX-M-16 may result from coupled defects in the enzyme's B3 strand, which lines the active site. Substitutions Val231-->Ala and Asp240-->Gly, which convert CTX-M-14 into CTX-M-16, occur at either end of this strand. These defects appear to increase the mobility of B3 based on anisotropic B-factor analyses at ultrahigh resolution, consistent with stability loss and activity gain. The unusually high resolution of these structures that makes such analyses possible also makes them good templates for inhibitor discovery.     

Construction by polymerase chain reaction and intragenic DNA probes for three main types of transferable β-lactamases (TEM, SHV, CARB)

Intragenic DNA probes were synthesized by polymerase chain reaction using fragments of the genes of three major types of beta-lactamases (TEM, SHV, CARB) as templates. The TEM probe hybridized with the genes encoding TEM-1, TEM-2 and six extended-spectrum related enzymes (TEM-3 to TEM-7, TEM-2O) in colony hybridizations and Southern-blot analysis. The SHV probe hybridized with the genes for SHV-1, OHIO-1 and four derived extended-spectrum beta-lactamases (SHV-2, SHV-3, SHV-4 and SHV-5). The CARB probe hybridized with the genes for PSE-1 (CARB-2), PSE-4 (CARB-1), CARB-3 and CARB-4. None of the probes hybridized with genes for any of eight oxacillin-hydrolysing enzymes, PSE-2, OXA-1 to OXA-7, ROB-1 and chromosomal beta-lactamases of various Enterobacteriaceae (except Klebsiella pneumoniae) and Pseudomonas aeruginosa. Investigations of Escherichia coli clinical isolates using these probes indicate the presence of a novel type of extended-spectrum, transferable beta-lactamase.     

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.     

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.     

Treatment options for extended-spectrum beta-lactamase-producers

A review of antibiotic options for the treatment of infections caused by extended-spectrum beta-lactamase-producing isolates is presented. The use of the third-generation cephalosporin, cefotaxime, for infections caused by isolates producing ceftazidimase-type extended-spectrum beta-lactamases is controversial, despite in vitro susceptibility to the antibiotic in many instances. The fourth-generation cephalosporin, cefipime, although active against most extended-spectrum beta-lactamases, is reported to show a marked inoculum effect. The cephamycins, such as cefoxitin. are generally effective against Enterobacteriaceae producing TEM- and SHV-derived extended-spectrum beta-lactamases, but Klebsella pneumoniae strains are prone to cephamycin resistance as a result of porin loss. The use of beta-lactamase inhibitor combinations is variable. Sulbactam is less effective than clavulanate for the inhibition of SHV-derived extended-spectrum beta-lactamases and a marked inoculum effect has been noted, while the efficacy of tazobactam against SHV-derived extended-spectrum beta-lactamase producers is controversial. Furthermore, extended-spectrum beta-lactamases are often encoded by multi-resistant plasmids carrying genes conferring resistance to aminoglycosides, chloramphenicol, sulfonamides, trimethoprim and other antimicrobials, severely limiting even alternative therapies. Extensive susceptibility testing before the institution of antibiotic therapy is thus vital.     

Structure of an extended-spectrum class A beta-lactamase from Proteus vulgaris K1

The structure of a chromosomal extended-spectrum beta-lactamase (ESBL) having the ability to hydrolyze cephalosporins including cefuroxime and ceftazidime has been determined by X-ray crystallography to 1.75 A resolution. The species-specific class A beta-lactamase from Proteus vulgaris K1 was crystallized at pH 6.25 and its structure solved by molecular replacement. Refinement of the model resulted in crystallographic R and R(free) of 16.9 % and 19.3 %, respectively. The folding of the K1 enzyme is broadly similar to that of non-ESBL TEM-type beta-lactamases (2 A rmsd for C(alpha)) and differs by only 0.35 A for all atoms of six conserved residues in the catalytic site. Other residues promoting extended-spectrum activity in K1 include the side-chains of atypical residues Ser237 and Lys276. These side-chains are linked by two water molecules, one of which lies in the position normally filled by the guanidinium group of Arg244, present in most non-ESBL enzymes but absent from K1. The ammonium group of Lys276, ca 3.5 A from the virtual Arg244 guanidinium position, may interact with polar R2 substitutents on the dihydrothiazene ring of cephalosporins.     

Structural Insights into Substrate Recognition and Product Expulsion in CTX-M Enzymes

β-Lactamase-mediated resistance to β-lactam antibiotics poses a major threat to our antibiotic armamentarium. Among β-lactamases, a significant threat comes from enzymes that hydrolyze extended-spectrum cephalosporins such as cefotaxime. Among the enzymes that exhibit this phenotype, the CTX-M family is found worldwide. These enzymes have a small active site, which makes it difficult to explain how they hydrolyze the bulky extended-spectrum cephalosporins into the binding site. We investigated noncovalent substrate recognition and product release in CTX-M enzymes using steered molecular dynamics simulation and X-ray diffraction. An arginine residue located far from the binding site favors the capture and tracking of substrates during entrance into the catalytic pocket. We show that the accommodation of extended-spectrum cephalosporins by CTX-M enzymes induced subtle changes in the active site and established a high density of electrostatic interactions. Interestingly, the product of the catalytic reaction initiates its own release because of steric hindrances and electrostatic repulsions. This suggests that there exists a general mechanism for product release for all members of the β-lactamase family and probably for most carboxypeptidases.     

Diversity of CTX-M beta-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals

Nine clinical isolates of Enterobacteriaceae (six Escherichia coli and three Proteus mirabilis) isolated in three Parisian hospitals between 1989 and 2000 showed a particular extended-spectrum cephalosporin-resistance profile characterized by resistance to cefotaxime and aztreonam but not to ceftazidime. CTX-M-1, CTX-M-2, CTX-M-9, CTX-M-14 and two novel plasmid-mediated CTX-M beta-lactamases (CTX-M-20, and CTX-M-21) were identified by polymerase chain reaction and isoelectric focusing (pI>8) and were associated in eight cases with TEM-1 (pI=5.4) or TEM-2 (pI=5.6) beta-lactamases. We used internal ISEcp1 and IS26 forward primers and the CTX-M consensus reverse primer to characterize the CTX-M beta-lactamase promoter regions and showed their high degree of structure diversity. We found upstream of some bla(CTX-M) genes, a 266-bp sequence 100% identical to the sequence upstream of the Kluyvera ascorbata beta-lactamase gene, suggesting that this chromosomal enzyme is the progenitor of the CTX-M-2/5 cluster.     

Antibiotic susceptibility and occurrence of ESBL in Pseudomonas aeruginosa strains isolated from different clinical specimens

The aim of this study was to evaluate the drug susceptibility of 132 P. aeruginosa strains isolated from patients hospitalized in SPSK University Hospital in Bialystok. The isolates were obtained from clinical specimens over an 11-month period in 2001 and 2002. All the strains were identified in automatic ATB system using API 20 NE strips, and their susceptibility to antibiotics was tested by standard disc-diffusion method and agar dilution method. The minimal inhibitory concentration (MIC) was determined for five antibiotics: piperacillin, amikacin, ceftazidime, imipenem and ciprofloxacin. The majority of strains were susceptible to ceftazidime (91.7%), piperacillin combined with tazobactam (85.6%), amikacin (80.3%), meropenem and imipenem (81.8%). Many of our strains were resistant to cefotaxime (73.5%), ticarcillin (53%) and ciprofloxacin (48.5%). Also, the trial was undertaken to detect strains producing extended-spectrum beta-lactamases (ESBL) and inducible beta-lactamases (IBL) among P. aeruginosa rods isolated from different specimens. ESBL-producing strains were detected with double disc test (DDST) and combination double disc (CD) test. Clavulanate was applied as the inhibitor of these beta-lactamases. Strains producing ESBL were not found. On the other hand, as many as 127 P. aeruginosa strains (96.2%) produced inducible beta-lactamases (IBL).     

The diversity of the catalytic properties of class A beta-lactamases.

The catalytic properties of four class A beta-lactamases were studied with 24 different substrates. They exhibit a wide range of variation. Similarly, the amino acid sequences are also quite different. However, no relationships were found between the sequence similarities and the substrate profiles. Lags and bursts were observed with various compounds containing a large sterically hindered side chain. As a group, the enzymes could be distinguished from the class C beta-lactamases on the basis of the kappa cat. values for several substrates, particularly oxacillin, cloxacillin and carbenicillin. Surprisingly, that distinction was impossible with the kappa cat./Km values, which represent the rates of acylation of the active-site serine residue by the beta-lactam. For several cephalosporin substrates (e.g. cefuroxime and cefotaxime) class A enzymes consistently exhibited higher kappa cat. values than class C enzymes, thus belying the usual distinction between 'penicillinases' and 'cephalosporinases'. The problem of the repartition of class A beta-lactamases into sub-classes is discussed.     

An epidemic of plasmids? Dissemination of extended-spectrum cephalosporinases among Salmonella and other Enterobacteriaceae

CTX-M- and AmpC-type beta-lactamases comprise the two most rapidly growing populations among the extended-spectrum cephalosporinases. The evolution and dissemination of resistance genes encoding these enzymes occur mostly through the transmission of plasmids. The high prevalence of clinical isolates of Enterobacteriaceae producing the plasmid-mediated extended-spectrum cephalosporinases resembles an epidemic of plasmids, and has generated serious therapeutic problems. This review describes the emergence and worldwide spread of various classes of plasmid-mediated extended-spectrum cephalosporinases in Salmonella and other Enterobacteriaceae, the transfer mechanism of the plasmids, detection methods, and therapeutic choices.     

Carbapenemases: molecular diversity and clinical consequences.

Carbapenemases are beta-lactamases that hydrolyze most beta-lactams including carbapenems. Carbapenemases are classified in four molecular classes; those belonging to class A are the chromosomally-encoded and clavulanic acid-inhibited IMI, NMC-A and SME, identified in Enterobacter cloacae and Serratia marcescens; the plasmid-encoded KPC enzymes identified in Enterobacteriaceae (and rarely in Pseudomonas aeruginosa); and the GES-type enzymes identified in Enterobacteriaceae and P. aeruginosa. The class B enzymes are the most clinically-significant carbapenemases; they are metallo-beta-lactamases, mostly of the IMP and the VIM series. They have been reported worldwide and their genes are plasmid- and integron-located, hydrolyzing all beta-lactams with the exception of aztreonam. One single plasmid-mediated AmpC beta-lactamase, CMY-10, identified in an Enterobacter aerogenes isolate, has been shown to be a cephaslosporinase with some carbapenemase properties. Finally, the class D carbapenemases are being increasingly reported, mostly in Acinetobacter baumannii, and they compromise the efficacy of imipenem and meropenem significantly.     

New perspectives on the endo-beta-glucanases of glycosyl hydrolase Family 17

Isozymes of glycosyl hydrolase Family 17 hydrolyze 1,3-beta-glucan polysaccharides found in the cell wall matrix of plants and fungi, enabling these plant enzymes to serve diverse roles in plant defense and plant development. Fourteen genes from Family 17 have been characterized in the genome of rice. A sequence dendrogram analysis divided these genes into four subfamilies. The recombinant GNS1 enzyme from subfamily B had 1,3;1,4-beta-glucanase activity, suggesting a role for this isozyme in plant development.     

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.     

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.     

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.