1,721,126 research outputs found
Antibiotico-resistenza e Batteri ambientali
No full textLa diffusione di patogeni batterici altamente resistenti alle maggiori classi di farmaci antibatterici rappresenta un importante problema per la sanità pubblica mondiale. La straordinaria capacità dei microrganismi a sviluppare meccanismi di resistenza si è sempre tradotta, di seguito all’introduzione di nuovi farmaci antibatterici, nell’emergenza e nella diffusione di isolati clinici resistenti. In questa prospettiva, lo studio complessivo del resistoma, mediante genomica e metagenomica, di specie batteriche prevalentemente ambientali ha rivelato la presenza di numerosi determinanti di resistenza agli antibiotici, talvolta capaci di conferire resistenza ai farmaci antibatterici di più recente sviluppo (e. g. carbapenemi, daptomicina). Lo studio molecolare di tali determinanti di resistenza, oltre ad offrire un interessante paradigma per la comprensione dei meccanismi alla base dell’evoluzione e della diversificazione della funzione delle proteine, rivelano altri aspetti interessanti. In particolare, è stato osservato che alcuni fattori di resistenza prodotti da batteri ambientali sarebbero enzimi apparentemente dotati di attività promiscua, indicando che la loro funzione primaria potrebbe essere diversa dalla resistenza agli antibiotici e che i meccanismi di resistenza potrebbe anche essere il frutto di evoluzione convergente. Rimane controverso l’impatto dell’utilizzo di composti antibatterici legato ad attività umane sull’evoluzione e sulla presenza di meccanismi di resistenza in batteri ambientali. Tuttavia, è stata dimostrata l’origine ambientale di alcuni determinanti di resistenza recentemente comparsi in isolati clinici, sottolineando l’importanza di batteri ambientali nell’evoluzione complessiva del resistoma di patogeni umani. Una maggiore conoscenza del resistoma ambientale e della sua evoluzione è quindi importante anche in vista dello sviluppo di nuovi farmaci antibatterici
Structure and function of metallo-β-lactamases. Meet-the-Experts session “Structure, Function and Genetics of Metallo-β-Lactamases”.
No full textMetallo-β-lactamases (Ambler’s class B, Bush-Jacoby’s classification group 3) are zinc-dependent enzymes able to hydrolyze a wide variety of β-lactam antibiotics, including oxyimino cephalosporins and carbapenems, while they are not inhibited by conventional β-lactamase inhibitors (e. g. clavulanic acid, tazobactam). Although resident metallo-β-lactamases have been reported in various bacterial species of limited clinical relevance, acquired metallo-β-lactamases (such as IMP- and VIM-type enzymes) begun to emerge in clinically-relevant opportunistic pathogens in the 90’s. Nowadays, metallo-β-lactamase-producing clinical isolates have been identified virtually worldwide, while new enzymes are still appearing in the clinical setting (e. g. NDM-1), consolidating the relevance of metallo-β-lactamases as a serious public health issue and emphasizing the need for specific clinically-useful inhibitors.
The current knowledge on the functional and structural properties of metallo-β-lactamases will be reviewed, with a particular focus on the clinically-relevant enzymes such as IMP-1, VIM-2 and NDM-1. The latest data on the structure-function relationships of these enzymes, and their potential in the identification of metallo-β-lactamase inhibitors, will also be discussed
Clinically-relevant β-lactamases: Structure-function relationships and inhibition.
No full textβ-lactamases are important resistance factors to β-lactam antibiotics, especially in Gram-negative bacteria, that show an extraordinary diversity in terms of structure, mechanism of hydrolysis and substrate profile and susceptibility to inhibitors. Two families of β-lactamases are currently known: (a) the active site serine β-lactamases which perform hydrolysis of β-lactams via an acylation-deacylation mechanism and (b) the metallo-β-lactamases, which require one or two zinc ion(s) for activity.
The current knowledge on β-lactamase structure-function relationships will be reviewed, with a particular focus on carbapenemases, such as KPC-2, VIM-2 and NDM-1 metallo-β-lactamases and OXA-type carbapenem-hydrolyzing enzymes, which currently represent among the most worrisome resistance determinants currently emerging in Pseudomonas aeruginosa, Acinetobacter baumannii and Enterobacteriaceae.
The identification and development of new β-lactamase inhibitors, whose clinical need is stronger than ever, is an active field of research. The properties of novel β-lactamase inhibitors, some of which are under clinical development (e. g. NXL104), will be discussed
Structure-Function Relationships of Class D Carbapenemases
No full textClass D carbapenemases, also known as Carbapenem-Hydrolyzing class D β-Lactamases (CHDLs) are of increasingly high clinical relevance, as they have been found in various important human pathogens, such as Acinetobacter baumannii and Klebsiella pneumoniae and contribute to the evolution of these pathogens towards extensively or totally-drug resistance (XDR/TDR) phenotypes. Essentially two main groups of phylogenetically-related enzymes have been described: one including the acquired OXA-23, OXA-24/40, OXA-51 and OXA-58 enzymes mostly in Acinetobacter baumannii, and the other including the OXA-48-related variants, i.e. OXA-54, OXA-162, OXA-163 and OXA- 181. In this article, the biochemical and structural features of class D carbapenemases will be discussed. Furthermore, the mechanistic hypothesis based on recently obtained crystal structures of the native forms of class D carbapenemases and mutants thereof, in complex with relevant substrates or inhibitors, will be critically reviewed. Finally, the mechanism of inhibition by available inhibitors, some of which are currently in clinical development, will be discussed
Interaction of Ceftaroline with Clinically-Relevant β-Lactamases
No full textBackground: Ceftaroline fosamil is a cephalosporin approved for community-acquired pneumonia and acute bacterial skin and skin structure infections. Ceftaroline, the active metabolite of ceftaroline fosamil, has broad-spectrum in vitro activity against Gram-positive pathogens (including MRSA) and some Gram-negative organisms. To our knowledge, the hydrolysis of ceftaroline by clinically-relevant β-lactamases (BLs) has not been systematically studied. We thus investigated the hydrolytic activity of various BLs, including ESBLs, AmpC-type chromosomal and plasmid-mediated enzymes and serine- and metallo-carbapenemases, on ceftaroline and their potential contribution to ceftaroline resistance.
Methods: Ceftaroline MICs were determined according to CLSI with isogenic laboratory E. coli strains producing representative BLs of class A (TEM-1, TEM-72, TEM-76, SHV-5, SHV-12, CTX-M-2, CTX-M-15, BEL-1, BEL-2, KPC-2), class B (IMP-1, IMP-18, VIM-2, NDM-1), class C (P. aeruginosa AmpC, E. cloacae AmpC, CMY-2, CMY-4) and class D (OXA-10, OXA-23, OXA-40, OXA-46, OXA-48) and compared to those of cephalothin, cefotaxime and ceftazidime. The kinetic parameters for the hydrolysis of these antibiotics by the 23 aforementioned purified BLs were determined by spectrophotometry.
Results: All BLs efficiently hydrolyzed ceftaroline (kcat/Km ≥105 M-1.s-1), except TEM-76, BEL-2 and OXA-48. When compared to other tested cephalosporins, ceftaroline was hydrolyzed with similar (cephalothin and ceftriaxone) or higher (ceftazidime) efficiencies. Large differences in the individual kinetic parameters were observed, with turnover rates ranging 0.5 to 650 s-1. Class C enzymes and NDM-1 exhibited the lowest turnover rates for ceftaroline hydrolysis. Ceftaroline MICs for isogenic BL-producing E. coli strains ranged 0.12 to 256 μg/ml, most being resistant. Interestingly, the strains susceptible or intermediate to ceftaroline (according to CLSI criteria) were those producing AmpC, CMY-2, NDM-1 and OXA-48, characterized by low turnover rates (kcat ≤ 2 s-1).
Conclusions: Although ceftaroline was readily hydrolyzed by most tested BLs, remarkable differences in turnover rates (3 orders of magnitude) were observed. The ceftaroline MIC data correlated with the kinetic parameters and showed that enzymes with low turnover rates were unable to confer ceftaroline resistance in an E. coli laboratory strain
Structure-fucntion relationship of the NDM-1 metallo-beta-lactamase: Importance of Trp-87 in enzyme structure, function and stability.
No full textBackground. NDM-1, an acquired subclass B1 metallo-beta-lactamase (MBL), represents a new worrisome global health issue, due to its rapid dissemination among clinical isolates in several countries and further aggravated by the unavailability of clinically-useful MBL inhibitors. NDM-1 thus represents an extremely relevant target for MBL inhibitors, and its structure-function relationship deserves investigation. In a previous study performed on VIM-2, the highly conserved Trp-87 residue was identified as an essential determinant for the enzyme stability and folding. In this work, we probed the impact of Trp-87 substitutions in NDM-1 on the enzyme properties and bacterial resistance.
Methods. The role of Trp-87 of NDM-1 was investigated by saturation mutagenesis. A library of blaNDM-1 mutants was obtained by means of a mutagenic PCR, using plasmid pLBII-NDM-1 as the template, and the recombinant plasmids transformed in E. coli. The clones carrying the various blaNDM-1 mutants genes were subjected to sequence analysis and their antimicrobial susceptibility profile determined using the broth microdulition technique as recommended by CLSI. Various NDM-1 variants were also subjected by biochemical characterization using kinetic assays.
Results. Antimicrobial susceptibility data obtained with E. coli strains producing the various Trp-87 NDM-1 variants consistently showed a significant reduction of the MIC values with several β-lactam antibiotics (e. g., the ceftazidime MIC values were decreased up to 128-fold, as compared to that shown by the strain producing the wild-type NDM-1). However, the substitution of Trp-87 did not compromise per se the activity of the purified enzyme, as revealed by kinetic assays. These observations are very similar to those previously obtained with the VIM-2 MBL, indicating that residue Trp-87, although not directly involved in catalysis, is a crucial determinant for conferring β-lactam resistance, likely due to its relevance in enzyme stability and proper in vivo folding.
Conclusions. These data overall support and validate the hypothesis that the conserved Trp-87 of MBLs is an essential determinant for enzyme stability and in vivo folding and thus would represent an important structural determinant to consider for the development of rationally-designed MBL inhibitors
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