Ambler Numbering Research Articles

Erratum for Mangat et al., Characterization of VCC-1, a Novel Ambler Class A Carbapenemase from Vibrio cholerae Isolated from Imported Retail Shrimp Sold in Canada.

Volume 60, no. 3, p. [1819–1825][1], 2016. Page 1823, [Figure 3][2]: It was brought to our attention that the Ambler numbering in this figure was incorrect. [Figure 3][2] and its legend should appear as shown below, with corrected Ambler coordinates. We sincerely thank Alain Philippon for his

A Structure-Based Classification of Class A β-Lactamases, a Broadly Diverse Family of Enzymes.

For medical biologists, sequencing has become a commonplace technique to support diagnosis. Rapid changes in this field have led to the generation of large amounts of data, which are not always correctly listed in databases. This is particularly true for data concerning class A β-lactamases, a group of key antibiotic resistance enzymes produced by bacteria. Many genomes have been reported to contain putative β-lactamase genes, which can be compared with representative types. We analyzed several hundred amino acid sequences of class A β-lactamase enzymes for phylogenic relationships, the presence of specific residues, and cluster patterns. A clear distinction was first made between dd-peptidases and class A enzymes based on a small number of residues (S70, K73, P107, 130SDN132, G144, E166, 234K/R, 235T/S, and 236G [Ambler numbering]). Other residues clearly separated two main branches, which we named subclasses A1 and A2. Various clusters were identified on the major branch (subclass A1) on the basis of signature residues associated with catalytic properties (e.g., limited-spectrum β-lactamases, extended-spectrum β-lactamases, and carbapenemases). For subclass A2 enzymes (e.g., CfxA, CIA-1, CME-1, PER-1, and VEB-1), 43 conserved residues were characterized, and several significant insertions were detected. This diversity in the amino acid sequences of β-lactamases must be taken into account to ensure that new enzymes are accurately identified. However, with the exception of PER types, this diversity is poorly represented in existing X-ray crystallographic data.

On 3LEZ, a Deep‐Sea Halophilic Protein with in vitro Class‐A β‐Lactamase Activity: Molecular‐Dynamics, Docking, and Reactivity Simulations

This work shows that a deep-sea protein, 3LEZ, with known in vitro β-lactamase activity, proved stable, substantially in the conformation detected by X-ray diffraction of the crystal, when subjected to molecular-dynamics (MD) simulations under conditions compatible with shallow seas. Docking simulations showed that the β-lactamase active site S85 of 3LEZ (S70 in Ambler numbering) is the preferential binding pocket for not only β-lactam antibiotics and inhibitors, but, surprisingly, also for a wide variety of other biologically active compounds in various chemical classes, including marine metabolites. In line with the in vitro β-lactamase activity, a) affinities on docking β-lactam antibiotics and inhibitors onto 3LEZ were found to roughly parallel published K(m) and K(i) values, obtained from MichaelisMenten kinetics under room conditions, and b) DFT calculations agreed with experiments that the irreversible reaction of the β-lactamase inhibitor clavulanic acid with the whole S85 catalytic center of 3LEZ is spontaneous. These observations must be viewed in the light that a) the compounds in other chemical classes showed comparable affinities, and, in some cases, even higher than β-lactams, for the S85 active site, b) K(m) and K(i) data are not available at the high hydrostatic pressure of the deep sea, where 3LEZ is believed to have evolved, c) an inverse order of affinities for the β-lactams, with respect to both experimentation and simulations at room conditions, was observed from comparative docking simulations with 3LEZ derived from MD under high hydrostatic pressure. Although MD requires a general assessment for high hydrostatic pressure before c) above is given the same weight as all other observations, this work questions the conclusion that the in vitro determined β-lactamase activity represents the ecological role of 3LEZ.

TEM-168, a Heretofore Laboratory-Derived TEM β-Lactamase Variant Found in an Escherichia coli Clinical Isolate

There have been over 160 variants of the TEM-1 and TEM-2 penicillinases described, most of which also exhibit activity against extended-spectrum cephalosporins (www.lahey.org/Studies/). It was noted in 1994 that a T265M mutation (Ambler numbering) was often associated with active-site mutations E104K, R164S, G238S, and E240K in some TEM variants (4). Comparison of MICs and kinetic studies of TEM-1 variants with the amino acid substitutions T265M, G238S, G238S:T265M, G238S:E240K, and G238S:G240K:T265M constructed by in vitro mutagenesis showed that the T265M mutation had no measurable effect on the TEM-1 wild type or on the G238S or G238S:240K variant. To date, no naturally occurring T265M variant of TEM-1 has been reported, though currently, 22 TEM variants contain an M265 residue along with other residue changes (www.lahey.org/Studies/). Escherichia coli N08-1503 obtained by the National Microbiology Laboratory for advanced testing was resistant to ampicillin, cefazolin, and ampicillin-sulbactam, as determined by the submitting laboratory. Antimicrobial testing was carried out by disk diffusion and broth microdilution according to CLSI guidelines and Etests (AB Biodisk) according to manufacturer's instructions (Table ​(Table1)1) (1). The CLSI methods indicated that E. coli N08-1503 did not harbor an extended-spectrum β-lactamase (ESBL), though a reduced susceptibility to ceftazidime was noted. Analysis using the ESBL Etest strips yielded similar results. Interestingly, the cefepime ESBL Etest strip yielded a positive result. This strip is recommended outside of the United States when testing a strain with an inducible ampC gene (e.g., Enterobacter) or when a nondeterminable result is obtained. Broth microdilution confirmed the reduced susceptibility to cefepime and synergy with clavulanic acid, though the cefepime MICs were one or two doubling dilutions higher than the Etest results were. Resistance to amoxicillin-clavulanic acid and piperacillin-tazobactam and intermediate susceptibility to cefoxitin were also noted for E. coli N08-1503 and DH10B transformed with a plasmid (pT168) harboring blaTEM-168 (see below). TABLE 1. Antimicrobial data for E. coli N08-1503 and the transformants harboring pT168a PCR analysis results for β-lactamase genes were positive for TEM and negative for SHV, CTX-M, OXA-1, and CMY-2 types. Sequence analysis of the TEM amplicon revealed a translation product with a single amino acid change from TEM-1, a T261M change (T265M by Ambler numbering) caused by a C782T transition. This variant has been assigned the name TEM-168 (www.lahey.org/Studies). In the blaTEM-168 promoter region, we detected the G162T change that defines the strong P4 promoter (3, 5). Plasmid analysis showed that an ∼13-kb plasmid, pT168, harboring blaTEM-168 could be transformed into E. coli DH10B. Isoelectric focusing of crude extracts from E. coli N08-1503 and the DH10B transformant identified a single band of β-lactamase activity with a pI of 5.4. The resistance to β-lactam inhibitor combinations was likely due to the hyperproduction of plasmidic TEM-168 expressed from the P4 promoter, similar to what has been shown for TEM-1 (5, 6). Since the TEM-168 did not test positive with standard ESBL tests, it is easy to understand why this variant has not been described previously in clinical isolates. It was the unusual result with the cefepime ESBL Etest strip that made us continue our investigation to further characterize the TEM harbored by E. coli N08-1503.

First Characterization in Tunisia of a TEM-15, Extended-Spectrumβ-Lactamase-ProducingKlebsiella pneumoniaeIsolate

Klebsiella pneumoniae CH0905 strain exhibiting high-level cefotaxime resistance was isolated from a stool culture in the intensive care unit. The resistance gene responsible was shown to be located on a conjugative 60-kb plasmid designated pCH0905. The minimum inhibitory concentration (MIC) values for cefotaxime and ceftazidime of the original isolate and the transconjugates were 256 mug/ml. Isoelectric focusing of a protein preparation from the K. pneumoniae strain showed beta-lactamases with the pI values of 7.6 and 6.3. A 1,080-bp fragment amplified with PCR was cloned into the pGEM-T Easy vector. The nucleotide sequence of the complete 1,080 bp was determined. Sequence analysis revealed that the bla(TEM) gene of pCH0905 differed from bla(TEM-1) by two mutations, leading to the following amino acid substitutions: the glutamic acid residue at position 104 by lysine and the glycine residue at position 238 by serine (Ambler numbering). The association of these two mutations was described previously in TEM-15 beta-lactamase, but this is the first detection of this enzyme in Tunisia.

Structural and Computational Characterization of the SHV-1 β-Lactamase-β-Lactamase Inhibitor Protein Interface

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

CTX-M β-Lactamases: An Update

During recent years, new extended-spectrum β-lactamase-type enzymes (ESBL) have emerged worldwide. The new enzymes, called CTX-M-type enzymes, belong to Ambler class A β-lactamases, and possess a serine in the active center of the molecule. The family of CTX-M enzymes is grouped, on the basis of similarities in amino acid sequences, into five major phylogenetic categories: the CTX-M-1 group, the CTX-M-2 group, the CTX-M-8 group, the CTX-M-25 and the CTX-M-9 group. The designation “CTX-M” refers to potent activity against cefotaxime, an aminotiazolil cephalosporin with a methoximino group; these enzymes have only remanent activity toward ceftazidime, another aminotiazolil cephalosporin with a 2-carboxy-2-oxypropaneimino group instead of the methoximino group. The substrate specificity suggests the existence of an active centre in the enzymes able to provide the amino acids and structural requirements of this selective antibiotic hydrolytic profile. As regards the crystalline structure of the CTX-M enzymes, only that of Toho-1 has so far been resolved. There have recently been increasing numbers of reports in the medical literature describing the appearance of new CTXM- type enzymes that are able to hydrolyze ceftazidime, and indicating that the bacterial strains harboring these enzymes show high levels of resistance to ceftazidime. So far, it has been shown that only two unique amino acid replacements are required to confer this peculiar phenotype of ceftazidime hydrolysis (Asp240-Gly and Pro167-Ser, following the criteria for Ambler numbering). As regards cefotaxime hydrolysis, Ser130-Gly replacement in CTX-M-9 and Arg276-Asn in CTX-M-4 decreases hydrolytic activity against cefotaxime. However, no further studies have been carried out to elucidate which new amino acids are necessary for cefotaxime incorporation and hydrolysis. Our research team is currently investigating this, and we have recently discovered new amino acids with critical activity against cefotaxime. In summary, we report here up-to-date information about structural/functional studies of CTX-M- enzymes as well as a description of the enzymes and their phylogenetic relationships. Keywords: ctx-m enzymes, structure-function

Biochemical analysis of the ceftazidime-hydrolysing extended-spectrum beta-lactamase CTX-M-15 and of its structurally related beta-lactamase CTX-M-3.

The extended-spectrum beta-lactamase CTX-M-15 confers resistance to ceftazidime, unlike the majority of CTX-M-type enzymes. Kinetic parameters were determined from purified CTX-M-15 and CTX-M-3, which differ by the single amino acid substitution Asp-240 to Gly, according to the Ambler numbering of class A beta-lactamases. Relative molecular masses of CTX-M-15 and CTX-M-3 were approximately 29 kDa and pI values were 8.9 and 8.4, respectively. CTX-M-15 had higher affinities for beta-lactams (lower K(m) values) than those of CTX-M-3 but catalytic efficiency (k(cat)/K(m) values) was variable depending on the beta-lactam substrate. Only CTX-M-15 showed a measurable catalytic efficiency for ceftazidime. Clavulanic acid and tazobactam were good inhibitors of both enzymes. MICs of beta-lactams for Escherichia coli reference strains expressing cloned beta-lactamase genes in the same genetic background were similar except for ceftazidime. This work underlines the fact that some CTX-M enzymes may hydrolyse ceftazidime and thus confer resistance to this expanded-spectrum cephalosporin in Enterobacteriaceae.

OHIO-1 β-lactamase mutants: the Arg 244Ser mutant and resistance to β-lactams and β-lactamase inhibitors

Mutations at residue 244 (Ambler numbering system) in the class A TEM β-lactamase confer resistance to inactivation by β-lactamase inhibitors and result in diminished turnover of β-lactam substrates. The Arg 244Ser mutant of the OHIO-1 β-lactamase, an SHV family enzyme, demonstrates variable susceptibilities to β-lactamase inhibitors and has significantly reduced catalytic efficiency. The minimum inhibitory concentrations (MICs) for Escherichia coli DH5α expressing the Arg 244Ser β-lactamase were reduced when compared to the strain bearing the OHIO-1 β-lactamase: ampicillin, 512 vs. 8192 μg ml −1; cephaloridine, 4 vs. 32 μg ml −1, respectively. The MICs for the β-lactam β-lactamase inhibitor combinations demonstrated resistance only to ampicillin–clavulanate, 16/8 vs. 8/4 μg ml −1 respectively. In contrast, there was increased susceptibility to ampicillin–sulbactam, ampicillin–tazobactam, and piperacillin–tazobactam. When compared to the OHIO-1 β-lactamase homogenous preparations of the Arg 244Ser β-lactamase enzyme demonstrated increased K m and decreased k cat values for benzylpenicillin ( K m=17 vs. 50 μM, k cat=345 vs. 234 s −1) and cephaloridine ( K m=97 vs. 202 μM, k cat=1023 vs. 202 s −1). Although the K i and IC 50 values were increased for each inhibitor when compared to OHIO-1 β-lactamase, the turnover numbers ( t n) required for inactivation were increased only for clavulanate. For the Arg 244Ser mutant enzyme of OHIO-1, the increased K i, decreased t n for the sulfones, and different partition ratio ( k cat/ k inact) support the notion that not all class A enzymes are inactivated in the same manner, and that certain class A β-lactamase enzymes may react differently with identical substitutions in structurally conserved amino acids. The resistance phenotype of a specific mutations can vary depending on the enzyme.

Aspartic acid for asparagine substitution at position 276 reduces susceptibility to mechanism-based inhibitors in SHV-1 and SHV-5 beta-lactamases.

In SHV-type beta-actamases, position 276 (in Ambler's numbering scheme) is occupied by an asparagine (Asn) residue. The effect on SHV-1 beta-lactamase and its extended-spectrum derivative SHV-5 of substituting an aspartic acid (Asp) residue for Asn276 was studied. Mutations were introduced by a PCR-based site-directed mutagenesis procedure. Wild-type SHV-1 and -5 beta-lactamases and their respective Asn276-->Asp mutants were expressed under isogenic conditions by cloning the respective bla genes into the pBCSK(+) plasmid and transforming Escherichia coli DH5alpha. Determination of IC50 showed that SHV-1(Asn276-->Asp), compared with SHV-1, was inhibited by 8- and 8.8-fold higher concentrations of clavulanate and tazobactam respectively. Replacement of Asn276 by Asp in SHV-5 beta-lactamase caused a ten-fold increase in the IC50 of clavulanate; the increases in the IC50s of tazobactam and sulbactam were 10- and 5.5-fold, respectively. Beta-lactam susceptibility testing showed that both Asn276-->Asp mutant enzymes, compared with the parental beta-lactamases, conferred slightly lower levels of resistance to penicillins (amoxycillin, ticarcillin and piperacillin), cephalosporins (cephalothin and cefprozil) and some of the expanded-spectrum oxyimino beta-lactams tested (cefotaxime, ceftriaxone and aztreonam). The MICs of ceftazidime remained unaltered, while those of cefepime and cefpirome were slightly elevated in the clones producing the mutant beta-lactamases. The latter clones were also less susceptible to penicillin-inhibitor combinations. Asn276-->Asp mutation was associated with changes in the substrate profiles of SHV-1 and SHV-5 enzymes. Based on the structure of TEM-1 beta-lactamase, the potential effects of the introduced mutation on SHV-1 and SHV-5 are discussed.

Effect of substitution of Asn for Arg-276 in the cefotaxime-hydrolyzing class A β-lactamase CTX-M-4

The effect of substitution of asparagine for arginine at position 276 (Ambler's numbering) on the properties of the extended-spectrum β-lactamase CTX-M-4 was studied. Compared with CTX-M-4, the mutant β-lactamase CTX-M-4(R276N) conferred lower levels of resistance to cefotaxime, ceftriaxone and aztreonam while the levels of resistance to penicillins and penicillin-inhibitor combinations were similar. Arg-276→Asn substitution rendered CTX-M-4 slightly less susceptible to inhibition by clavulanate and tazobactam. It also caused a three-fold reduction in the relative rate of hydrolysis of cefotaxime. These results indicate that Arg-276 in CTX-M-type β-lactamases may be implicated in hydrolysis of oxyimino-β-lactams; they do not, however, support the hypothesis that Arg-276 is the functional equivalent of Arg-244 found in other class A β-lactamases.

A novel extended-spectrum TEM-type beta-lactamase (TEM-52) associated with decreased susceptibility to moxalactam in Klebsiella pneumoniae.

Klebsiella pneumoniae NEM865 was isolated from the culture of a stool sample from a patient previously treated with ceftazidime (CAZ). Analysis of this strain by the disk diffusion test revealed synergies between amoxicillin-clavulanate (AMX-CA) and CAZ, AMX-CA and cefotaxime (CTX), AMX-CA and aztreonam (ATM), and more surprisingly, AMX-CA and moxalactam (MOX). Clavulanic acid (CA) decreased the MICs of CAZ, CTX, and MOX, which suggested that NEM865 produced a novel extended-spectrum beta-lactamase. Genetic, restriction endonuclease, and Southern blot analyses revealed that the resistance phenotype was due to the presence in NEM865 of a 13.5-kb mobilizable plasmid, designated pNEC865, harboring a Tn3-like element. Sequence analysis revealed that the blaT gene of pNEC865 differed from blaTEM-1 by three mutations leading to the following amino acid substitutions: Glu104-->Lys, Met182-->Thr, and Gly238-->Ser (Ambler numbering). The association of these three mutations has thus far never been described, and the blaT gene carried by pNEC865 was therefore designated blaTEM-52. The enzymatic parameters of TEM-52 and TEM-3 were found to be very similar except for those for MOX, for which the affinity of TEM-52 (Ki, 0.16 microM) was 10-fold higher than that of TEM-3 (Ki, 1.9 microM). Allelic replacement analysis revealed that the combination of Lys104, Thr182, and Ser238 was responsible for the increase in the MICs of MOX for the TEM-52 producers.

Detection of extended-spectrum plasmid-mediated beta-lactamases by disk diffusion.

Detection of extended-spectrum plasmid-mediated beta-lactamases by disk diffusion.

A TEM-derived extended-spectrum beta-lactamase in Pseudomonas aeruginosa.

A clinical strain of Pseudomonas aeruginosa, PAe1100, was found to be resistant to all antipseudomonal beta-lactam antibiotics and to aminoglycosides, including gentamicin, amikacin, and isepamicin. PAe1100 produced two beta-lactamases, TEM-2 (pI 5.6) and a novel, TEM-derived extended-spectrum beta-lactamase called TEM-42 (pI 5.8), susceptible to inhibition by clavulanate, sulbactam, and tazobactam. Both enzymes, as well as the aminoglycoside resistance which resulted from AAC(3)-IIa and AAC(6')-I production, were encoded by an 18-kb nonconjugative plasmid, pLRM1, that could be transferred to Escherichia coli by transformation. The gene coding for TEM-42 had four mutations that led to as many amino acid substitutions with respect to TEM-2: Val for Ala at position 42 (Ala42), Ser for Gly238, Lys for Glu240, and Met for Thr265 (Ambler numbering). The double mutation Ser for Gly238 and Lys for Glu240, which has so far only been described in SHV-type but not TEM-type enzymes, conferred concomitant high-level resistance to cefotaxime and ceftazidime. The novel, TEM-derived extended-spectrum beta-lactamase appears to be the first of its class to be described in P. aeruginosa.