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High-level resistance to ceftazidime conferred by a novel enzyme, CTX-M-32, derived from CTX-M-1 through a single Asp240-Gly substitution.

ABSTRACT: A clinical strain of Escherichia coli isolated from pleural liquid with high levels of resistance to cefotaxime, ceftazidime, and aztreonam harbors a novel CTX-M gene (bla(CTX-M-32)) whose amino acid sequence differs from that of CTX-M-1 by a single Asp240-Gly substitution. Moreover, by site-directed mutagenesis we demonstrated that this replacement is a key event in ceftazidime hydrolysis

SUBMITTER: Cartelle M

PROVIDER: S-EPMC415568 | biostudies-literature | 2004 Jun

REPOSITORIES: biostudies-literature

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High-level resistance to ceftazidime conferred by a novel enzyme, CTX-M-32, derived from CTX-M-1 through a single Asp240-Gly substitution.

Cartelle Monica M del Mar Tomas Maria M Molina Francisca F Moure Rita R Villanueva Rosa R Bou German G

Antimicrobial agents and chemotherapy 20040601 6

A clinical strain of Escherichia coli isolated from pleural liquid with high levels of resistance to cefotaxime, ceftazidime, and aztreonam harbors a novel CTX-M gene (bla(CTX-M-32)) whose amino acid sequence differs from that of CTX-M-1 by a single Asp240-Gly substitution. Moreover, by site-directed mutagenesis we demonstrated that this replacement is a key event in ceftazidime hydrolysis ...[more]

PMID: 15155242

Similar Datasets

CTX-M-type extended-spectrum beta-lactamase that hydrolyzes ceftazidime through a single amino acid substitution in the omega loop.

Project description:Escherichia coli ILT-1, Klebsiella pneumoniae ILT-2, and K. pneumoniae ILT-3 were isolated in May 1999 in Paris, France, from a rectal swab of a hospitalized 5-month-old girl. These isolates had a clavulanic acid-inhibited substrate profile that included expanded-spectrum cephalosporins. The MICs of cefotaxime were higher for E. coli ILT-1 and K. pneumoniae ILT-2 than for K. pneumoniae ILT-3, while the opposite was found for the MICs of ceftazidime. Genetic and biochemical analyses revealed that E. coli ILT-1 and K. pneumoniae ILT-2 produced the CTX-M-18 beta-lactamase, while K. pneumoniae ILT-3 produced the CTX-M-19 beta-lactamase. The amino acid sequence of the CTX-M-18 beta-lactamase differed from that of the CTX-M-9 beta-lactamase by an Ala-to-Val change at position 231, while CTX-M-19 possessed an additional Pro-to-Ser change at position 167 in the omega loop of Ambler class A enzymes. The latter amino acid substitution may explain the CTX-M-19-mediated hydrolysis of ceftazidime, which has not been reported for other CTX-M-type enzymes. The bla(CTX-M-18) and bla(CTX-M-19) genes were located on transferable plasmids that varied in size (ca. 60 and 50 kb, respectively) but that showed similar restriction patterns.

| S-EPMC90837 | biostudies-literature

Novel cefotaximase (CTX-M-16) with increased catalytic efficiency due to substitution Asp-240-->Gly.

Project description:Three clinical strains (Escherichia coli Rio-6, E. coli Rio-7, and Enterobacter cloacae Rio-9) collected in 1996 and 1999 from hospitals in Rio de Janeiro (Brazil) were resistant to broad-spectrum cephalosporins and gave a positive double-disk synergy test. Two bla(CTX-M) genes encoding beta-lactamases of pl 7.9 and 8.2 were implicated in this resistance: the bla(CTX-M-9) gene observed in E. coli Rio-7 and E. cloacae Rio-9 and a novel CTX-M-encoding gene, designated bla(CTX-M-16), observed in E. coli strain Rio-6. The deduced amino acid sequence of CTX-M-16 differed from CTX-M-9 only by the substitution Asp-240-->Gly. The CTX-M-16-producing E. coli transformant exhibited the same level of resistance to cefotaxime (MIC, 16 microg/ml) but had a higher MIC of ceftazidime (MIC, 8 versus 1 microg/ml) than the CTX-M-9-producing transformant. Enzymatic studies revealed that CTX-M-16 had a 13-fold higher affinity for aztreonam and a 7.5-fold higher k(cat) for ceftazidime than CTX-M-9, thereby showing that the residue in position 240 can modulate the enzymatic properties of CTX-M enzymes. The two bla(CTX-M-9) genes and the bla(CTX-M-16) gene were located on different plasmids, suggesting the presence of mobile elements associated with CTX-M-encoding genes. CTX-M-2 and CTX-M-8 enzymes were found in Brazil in 1996, and two other CTX-M beta-lactamases, CTX-M-9 and CTX-M-16, were subsequently observed. These reports are evidence of the diversity of CTX-M-type extended-spectrum beta-lactamases in Brazil.

| S-EPMC90641 | biostudies-literature

A new SHV-derived extended-spectrum beta-lactamase (SHV-24) that hydrolyzes ceftazidime through a single-amino-acid substitution (D179G) in the -loop.

Project description:A new SHV-derived extended-spectrum beta-lactamase (SHV-24) conferring high-level resistance to ceftazidime but not cefotaxime and cefazolin was identified in Japan. This enzyme was encoded by a transferable 150-kb plasmid from an Escherichia coli clinical isolate. The pI and K(m) for CAZ of this enzyme were 7.5 and 30 microM, respectively. SHV-24 was found to have a D179G substitution in the Omega-loop of the enzyme.

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Diastereoselective DNA cleavage recognition by Ni(II) x Gly-Gly-His-derived metallopeptides.

Project description:Site-selective DNA cleavage by diastereoisomers of Ni(II) x Gly-Gly-His-derived metallopeptides was investigated through high-resolution gel analyses and molecular dynamics simulations. Ni(II) x L-Arg-Gly-His and Ni(II) x D-Arg-Gly-His (and their respective Lys analogues) targeted A/T-rich regions; however, the L-isomers consistently modified a subset of available nucleotides within a given minor groove site, while the D-isomers differed in both their sites of preference and their ability to target individual nucleotides within some sites. In comparison, Ni(II) x L-Pro-Gly-His and Ni(II) x D-Pro-Gly-His were unable to exhibit a similar diastereoselectivity. Simulations of the above systems, along with Ni(II) x Gly-Gly-His, indicated that the stereochemistry of the amino-terminal amino acid produces either an isohelical metallopeptide that associates stably at individual DNA sites (L-Arg or L-Lys) or, with D-Arg and D-Lys, a noncomplementary metallopeptide structure that cannot fully employ its side chain nor amino-terminal amine as positional stabilizing moieties. In contrast, amino-terminal Pro-containing metallopeptides of either stereochemistry, lacking an extended side chain directed toward the minor groove, did not exhibit a similar diastereoselectivity. While the identity and stereochemistry of amino acids located in the amino-terminal peptide position influenced DNA cleavage, metallopeptide diastereoisomers containing L- and D-Arg (or Lys) within the second peptide position did not exhibit diastereoselective DNA cleavage patterns; simulations indicated that a positively charged amino acid in this location alters the interaction of the metallopeptide equatorial plane and the minor groove leading to an interaction similar to Ni(II) x Gly-Gly-His.

| S-EPMC2538425 | biostudies-literature

A new TEM-derived extended-spectrum beta-lactamase (TEM-91) with an R164C substitution at the omega-loop confers ceftazidime resistance.

Project description:A new plasmid-mediated TEM-derived extended-spectrum beta-lactamase, TEM-91, was identified in a ceftazidime-resistant (MIC, >128 microg per ml) Escherichia coli strain isolated in 1996 in Japan. TEM-91 has three amino acid substitutions, R164C, M184T, and E240K, compared with TEM-1 penicillinase. The isoelectric point (pI), K(m), and k(cat) of TEM-91 for ceftazidime were 5.7, 179 microM, and 29.0 s(-1), respectively. The K(i) of clavulanic acid for ceftazidime hydrolysis was 30.3 nM.

| S-EPMC182605 | biostudies-literature

Ceftazidime-Avibactam Resistance Mediated by the N346Y Substitution in Various AmpC ?-Lactamases.

Project description:Chromosomal and plasmid-borne AmpC cephalosporinases are a major resistance mechanism to ?-lactams in Enterobacteriaceae and Pseudomonas aeruginosa The new ?-lactamase inhibitor avibactam effectively inhibits class C enzymes and can fully restore ceftazidime susceptibility. The conserved amino acid residue Asn346 of AmpC cephalosporinases directly interacts with the avibactam sulfonate. Disruption of this interaction caused by the N346Y amino acid substitution in Citrobacter freundii AmpC was previously shown to confer resistance to the ceftazidime-avibactam combination (CAZ-AVI). The aim of this study was to phenotypically and biochemically characterize the consequences of the N346Y substitution in various AmpC backgrounds. Introduction of N346Y into Enterobacter cloacae AmpC (AmpCcloacae), plasmid-mediated DHA-1, and P. aeruginosa PDC-5 led to 270-, 12,000-, and 79-fold decreases in the inhibitory efficacy (k 2/Ki ) of avibactam, respectively. The kinetic parameters of AmpCcloacae and DHA-1 for ceftazidime hydrolysis were moderately affected by the substitution. Accordingly, AmpCcloacae and DHA-1 harboring N346Y conferred CAZ-AVI resistance (MIC of ceftazidime of 16??g/ml in the presence of 4??g/ml of avibactam). In contrast, production of PDC-5 N346Y was associated with a lower MIC (4??g/ml) since this ?-lactamase retained a higher inactivation efficacy by avibactam in comparison to AmpCcloacae N346Y. For FOX-3, the I346Y substitution did not reduce the inactivation efficacy of avibactam and the substitution was highly deleterious for ?-lactam hydrolysis, including ceftazidime, preventing CAZ-AVI resistance. Since AmpCcloacae and DHA-1 display substantial sequence diversity, our results suggest that loss of hydrogen interaction between Asn346 and avibactam could be a common mechanism of acquisition of CAZ-AVI resistance.

| S-EPMC7269493 | biostudies-literature

Defining Substrate Specificity in the CTX-M Family: the Role of Asp240 in Ceftazidime Hydrolysis.

Project description:The natural diversification of CTX-M ?-lactamases led to the emergence of Asp240Gly variants in the clinic that confer reduced susceptibility to ceftazidime (CAZ). In this study, we compared the impact of this substitution on CAZ and ceftazidime-avibactam (CZA) MICs against isogenic Escherichia coli strains with different porin deficiencies. Our results show a noticeable increase in CAZ resistance in clones expressing Asp240Gly-harboring CTX-M when combined with OmpF porin deficiency. Kinetic analysis revealed that the kcat/Km for CAZ was 5- to 15-fold higher for all Asp240Gly variants but remained 200- to 725-fold lower than that for cefotaxime (CTX). In vitro selection of CAZ-resistant clones yielded nonsusceptible CTX-M producers (MIC of >16 ?g/ml) only after overnight incubation; the addition of avibactam (AVI) decreased MICs to a susceptible range against these variants. In contrast, the use of CZA as a selective agent did not yield resistant clones. AVI inactivated both CTX-M-12 and CTX-M-96, with an apparent inhibition constant comparable to that of SHV-2 and 1,000-fold greater than that of PER-2 and CMY-2, and k2/K for CTX-M-12 was 24- and 35-fold higher than that for CTX-M-96 and CTX-M-15, respectively. Molecular modeling suggests that AVI interacts similarly with CTX-M-96 and CTX-M-15. We conclude that the impact of Asp240Gly in resistance may arise when other mechanisms are also present (i.e., OmpF deficiency). Additionally, CAZ selection could favor the emergence of CAZ-resistant subpopulations. These results define the role of Asp240 and the impact of the -Gly substitution and allow us to hypothesize that the use of CZA is an effective preventive strategy to delay the development of resistance in this family of extended-spectrum ?-lactamases.

| S-EPMC5971610 | biostudies-literature

Novel chimeric beta-lactamase CTX-M-64, a hybrid of CTX-M-15-like and CTX-M-14 beta-lactamases, found in a Shigella sonnei strain resistant to various oxyimino-cephalosporins, including ceftazidime.

Project description:The plasmid-mediated novel beta-lactamase CTX-M-64 was first identified in Shigella sonnei strain UIH-1, which exhibited resistance to cefotaxime (MIC, 1,024 microg/ml) and ceftazidime (MIC, 32 microg/ml). The amino acid sequence of CTX-M-64 showed a chimeric structure of a CTX-M-15-like beta-lactamase (N- and C-terminal moieties) and a CTX-M-14-like beta-lactamase (central portion, amino acids 63 to 226), suggesting that it originated by homologous recombination between the corresponding genes. The introduction of a recombinant plasmid carrying bla(CTX-M-64) conferred resistance to cefotaxime in Escherichia coli, and the activities of cefotaxime and ceftazidime were restored in the presence of clavulanic acid. Of note, CTX-M-64 production could also confer consistent resistance to ceftazidime, which differs from the majority of CTX-M-type enzymes, which poorly hydrolyze ceftazidime. These results were consistent with the kinetic parameters determined with the purified CTX-M-64 enzyme. The bla(CTX-M-64) gene was flanked upstream by an ISEcp1 sequence and downstream by an orf477 sequence. The sequence of the 45-bp spacer region between the right inverted repeat (IRR) of ISEcp1 and bla(CTX-M-64) was exactly identical to that of ISEcp1-bla(CTX-M-15-like). Moreover, the presence of a putative IRR of ISEcp1 at the right end of truncated orf477 is indicative of an ISEcp1-mediated transposition event in the bla(CTX-M-64) gene. The emergence of CTX-M-64 by probable homologous recombination would suggest the natural potential of an alternative mechanism for the diversification of CTX-M-type beta-lactamases.

| S-EPMC2612187 | biostudies-literature

Combination of Amino Acid Substitutions Leading to CTX-M-15-Mediated Resistance to the Ceftazidime-Avibactam Combination.

Project description:Single amino acid substitutions in the ? loop of KPC ?-lactamases are known to lead to resistance to the ceftazidime-avibactam combination. Here, we investigate this mechanism of resistance in CTX-M enzymes, which are the most widely spread extended-spectrum ?-lactamases worldwide. Nine single amino acid polymorphisms were identified in the ? loop of the 172 CTX-M sequences present in the Lahey database of ?-lactamases. The corresponding modifications were introduced in CTX-M-15 by site-directed mutagenesis. None of the nine substitutions was associated with ceftazidime-avibactam resistance in Escherichia coli TOP10. However, two substitutions led to 4-fold (P167S) and 16-fold (L169Q) increases in the MIC of ceftazidime. We determined whether these substitutions favor the in vitro selection of mutants resistant to ceftazidime-avibactam. The selection provided mutants for the L169Q substitution but not for the P167S substitution or for the parental enzyme CTX-M-15. Resistance to the drug combination (MIC of ceftazidime, 16 ?g/ml in the presence of 4 ?g/ml of avibactam) resulted from the acquisition of the S130G substitution by CTX-M-15 L169Q. Purified CTX-M-15 with the two substitutions, L169Q and S130G, was only partially inhibited by avibactam at concentrations as high as 50,000 ?M but retained ceftazidime hydrolysis activity with partially compensatory decreases in kcat and Km These results indicate that emergence of resistance to the ceftazidime-avibactam combination requires more than one mutation in most CTX-M-encoding genes. Acquisition of resistance could be restricted to rare variants harboring predisposing polymorphisms such as Q at position 169 detected in a single naturally occurring CTX-M enzyme (CTX-M-93).

| S-EPMC6125495 | biostudies-literature

The enzyme mechanism of nitrite reductase studied at single-molecule level.

Project description:A generic method is described for the fluorescence "readout" of the activity of single redox enzyme molecules based on Förster resonance energy transfer from a fluorescent label to the enzyme cofactor. The method is applied to the study of copper-containing nitrite reductase from Alcaligenes faecalis S-6 immobilized on a glass surface. The parameters extracted from the single-molecule fluorescence time traces can be connected to and agree with the macroscopic ensemble averaged kinetic constants. The rates of the electron transfer from the type 1 to the type 2 center and back during turnover exhibit a distribution related to disorder in the catalytic site. The described approach opens the door to single-molecule mechanistic studies of a wide range of redox enzymes and the precise investigation of their internal workings.

| S-EPMC2265120 | biostudies-literature

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