In vivo evolution of GES β-lactamases driven by ceftazidme/avibactam treatment of Pseudomonas aeruginosa infections
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB44930 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR5922336_1.fastq.gz | Fastqsanger.gz | |||
| ERR5922336_2.fastq.gz | Fastqsanger.gz | |||
| ERR5922337_1.fastq.gz | Fastqsanger.gz | |||
| ERR5922337_2.fastq.gz | Fastqsanger.gz |
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Similar Datasets
Project description:Mechanisms leading to In vivo ceftazidime/avibactam resistance development during treatment of GES-5-producing Pseudomonas aeruginosa infections
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Project description:GES-1 is a class A extended-spectrum ?-lactamase conferring resistance to penicillins, narrow- and expanded-spectrum cephalosporins, and ceftazidime. However, GES-1 poorly hydrolyzes aztreonam and cephamycins and exhibits very low k(cat) values for carbapenems. Twenty-two GES variants have been discovered thus far, differing from each other by 1 to 3 amino acid substitutions that affect substrate specificity. GES-11 possesses a Gly243Ala substitution which seems to confer to this variant an increased activity against aztreonam and ceftazidime. GES-12 differs from GES-11 by a single Thr237Ala substitution, while GES-14 differs from GES-11 by the Gly170Ser mutation, which is known to confer increased carbapenemase activity. GES-11 and GES-12 were kinetically characterized and compared to GES-1 and GES-14. Purified GES-11 and GES-12 showed strong activities against most tested ?-lactams, with the exception of temocillin, cefoxitin, and carbapenems. Both variants showed a significantly increased rate of hydrolysis of cefotaxime, ceftazidime, and aztreonam. On the other hand, GES-11 and GES-12 (and GES-14) variants all containing Ala243 exhibited increased susceptibility to classical inhibitors. The crystallographic structures of the GES-11 and GES-14 ?-lactamases were solved. The overall structures of GES-11 and GES-14 are similar to that of GES-1. The Gly243Ala substitution caused only subtle local rearrangements, notably in the typical carbapenemase disulfide bond. The active sites of GES-14 and GES-11 are very similar, with the Gly170Ser substitution leading only to the formation of additional hydrogen bonds of the Ser residue with hydrolytic water and the Glu166 residue.
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Project description:Homo sapiens strain:GES-1 Raw sequence reads
| PRJNA874328 | ENA
Project description:GES-type ?-lactamases are a group of enzymes that have evolved their hydrolytic activity against carbapenems. In this study, the role of residue 174 inside the ?-loop of GES-1 and GES-5 was investigated. GES-1P174E and GES-5P174E mutants, selected by site saturation mutagenesis, were purified and kinetically characterized. In comparison with GES-1 and GES-5 wild-type enzymes, GES-1P174E and GES-5P174E mutants exhibited lower kcat and kcat/Km values for cephalosporins and penicillins. Concerning carbapenems, GES-1P174E shared higher kcat values but lower Km values than those calculated for GES-1. The GES-1P174E and GES-5P174E mutants showed high hydrolytic efficiency for imipenem, with kcat/Km values 100- and 660-fold higher, respectively, than those of GES-1. Clavulanic acid and tazobactam are good inhibitors for both GES-1P174E and GES-5P174E Molecular dynamic (MD) simulations carried out for GES-1, GES-5, GES-1P174E, and GES-5P174E complexed with imipenem and meropenem have shown that mutation at position 174 induces a drastic increase of enzyme flexibility, in particular in the ?-loop. The circular dichroism (CD) spectroscopy spectra of the four enzymes indicate that the P174E substitution in GES-1 and GES-5 does not affect the secondary structural content of the enzymes.
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Project description:β-Lactamase enzymes (EC 3.5.2.6) are a significant threat to the continued use of β-lactam antibiotics to treat infections. A novel non-β-lactam β-lactamase inhibitor with activity against many class A and C and some class D β-lactamase variants, avibactam, is now available in the clinic in partnership with ceftazidime. Here, we explored the activity of avibactam against a variety of characterized isogenic laboratory constructs of β-lactamase inhibitor-resistant variants of the class A enzyme SHV (M69I/L/V, S130G, K234R, R244S, and N276D). We discovered that the S130G variant of SHV-1 shows the most significant resistance to inhibition by avibactam, based on both microbiological and biochemical characterizations. Using a constant concentration of 4 mg/liter of avibactam as a β-lactamase inhibitor in combination with ampicillin, the MIC increased from 1 mg/liter for blaSHV-1 to 256 mg/liter for blaSHV S130G expressed in Escherichia coli DH10B. At steady state, the k2/K value of the S130G variant when inactivated by avibactam was 1.3 M(-1) s(-1), versus 60,300 M(-1) s(-1) for the SHV-1 β-lactamase. Under timed inactivation conditions, we found that an approximately 1,700-fold-higher avibactam concentration was required to inhibit SHV S130G than the concentration that inhibited SHV-1. Molecular modeling suggested that the positioning of amino acids in the active site of SHV may result in an alternative pathway of inactivation when complexed with avibactam, compared to the structure of CTX-M-15-avibactam, and that S130 plays a role in the acylation of avibactam as a general acid/base. In addition, S130 may play a role in recyclization. As a result, we advance that the lack of a hydroxyl group at position 130 in the S130G variant of SHV-1 substantially slows carbamylation of the β-lactamase by avibactam by (i) removing an important proton acceptor and donator in catalysis and (ii) decreasing the number of H bonds. In addition, recyclization is most likely also slow due to the lack of a general base to initiate the process. Considering other inhibitor-resistant mechanisms among class A β-lactamases, S130 may be the most important amino acid for the inhibition of class A β-lactamases, perhaps even for the novel diazabicyclooctane class of β-lactamase inhibitors.
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Project description:Mecillinam/avibactam synergy
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Project description:During a PCR-based surveillance study of ?-lactam resistance, 125 multidrug-resistant (MDR) Acinetobacter baumannii isolates were obtained from 18 hospitals in Belgium from January 2008 to December 2009. Nine GES-positive A. baumannii isolates were detected at 6 Belgian hospitals. DNA sequencing of the bla(GES) genes identified GES-11, GES-12, and a novel variant GES-14, which differs from GES-11 by a single amino acid substitution (Gly170Ser). All index isolates were travel associated and originated from patients transferred from Turkey (n = 2), Egypt (n = 2), and Palestinian territories (Gaza) (n = 2). A nosocomial outbreak involving three additional patients occurred in a burn unit at a single hospital. No clonal relatedness could be established between the 6 index isolates by pulsed-field gel electrophoresis (PFGE) analysis. Three different alleles (the plasmid-located bla(GES-11) and bla(GES-12) and a likely chromosomally located novel variant bla(GES-14)) were detected as part of a class 1 integron, also including the aac6'Ib and dfrA7 genes. Restriction analysis of plasmids suggests a common origin for the plasmids bearing bla(GES-11) and bla(GES-12). Cloning of the bla(GES) genes in Escherichia coli identified GES-14 as hydrolyzing imipenem, while GES-12 showed the highest specific activity against ceftazidime. This report highlights the emergence of various bla(GES-like) genes, especially those conferring carbapenem resistance in A. baumannii and its importation in Western Europe from Middle Eastern countries.
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Project description:GES-1 gastric epithelial cells were used for co-culturing with H.pylori with high and low Trx1 expression, and RNA sequencing was performed on the co-cultured cells to explore transcriptome differences between the two groups, so as to further explore the pathogenic mechanism of Trx1 in H.pylori.
2022-08-31 | GSE186902 | GEO
Project description:Helicobacter pylori (H.pylori) infection is an important factor in the occurrence of human gastric diseases, but its pathogenic mechanism is not clear. N6-methyladenosine (m6A) is the most prevalent reversible methylation modification in mammalian RNA and it plays a crucial role in controlling many biological processes. We used MeRIP-seq technology to sequence the GES-1 cells infected with Helicobacter pylori(H. pylori) for 48 h.
2023-05-11 | GSE230869 | GEO
Project description:?-Lactamases are the most important mechanisms of resistance to the ?-lactam antibacterials. There are two mechanistic classes of ?-lactamases: the serine ?-lactamases (SBLs) and the zinc-dependent metallo-?-lactamases (MBLs). Avibactam, the first clinically useful non-?-lactam ?-lactamase inhibitor, is a broad-spectrum SBL inhibitor, which is used in combination with a cephalosporin antibiotic (ceftazidime). There are multiple reports on the interaction of avibactam with SBLs but few such studies with MBLs. We report biochemical and biophysical studies on the binding and reactivity of avibactam with representatives from all 3 MBL subfamilies (B1, B2, and B3). Avibactam has only limited or no activity versus MBL-mediated resistance in pathogens. Avibactam does not inhibit MBLs and binds only weakly to most of the MBLs tested; in some cases, avibactam undergoes slow hydrolysis of one of its urea N-CO bonds followed by loss of CO2, in a process different from that observed with the SBLs studied. The results suggest that while the evolution of MBLs that more efficiently catalyze avibactam hydrolysis should be anticipated, pursuing the development of dual-action SBL and MBL inhibitors based on the diazabicyclooctane core of avibactam may be productive.
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