53 research outputs found
Heterogeneous hydrolytic features for OXA-48-like Ī²-lactamases
Objectives Carbapenem-hydrolysing class D Ī²-lactamases of the OXA-48 type are increasingly reported from Enterobacteriaceae. Ī²-Lactamase OXA-48 hydrolyses penicillins very efficiently, but carbapenems only weakly and spares broad-spectrum cephalosporins. Recently, diverse OXA-48-like Ī²-lactamases have been identified worldwide (OXA-162, OXA-181, OXA-163, OXA-204 and OXA-232). They differ by few amino acid substitutions or by amino acid deletions. Methods blaOXA-48, blaOXA-162, blaOXA-163, blaOXA-181, blaOXA-204 and blaOXA-232 were cloned into the same expression vector and expressed in the same Escherichia coli background. Kinetic studies were performed with enzymes purified by ion-exchange chromatography. Determination of hydrolytic activities was performed by UV spectrophotometry. MICs were determined for all recombinant strains, using as background either the WT E. coli TOP10 strain or a porin-deficient E. coli strain. Results Kinetic studies showed that OXA-162 and OXA-204 shared the same hydrolytic properties as OXA-48. On the other hand, OXA-181 possessed a higher ability to hydrolyse carbapenems, while OXA-232 hydrolysed those substrates less efficiently. In contrast to the other OXA-48-like Ī²-lactamases, OXA-163 hydrolysed broad-spectrum cephalosporins very efficiently, but did not possess significant carbapenemase activity. Although several of these OXA-48-like enzymes possess low activity against carbapenems, MICs of carbapenems were significantly elevated when determined for strains possessing permeability defects. Conclusions A detailed comparative analysis of the kinetic properties of the OXA-48-like Ī²-lactamases is provided here. It clarifies the respective features of each OXA-48-like variant and their respective impacts in terms of carbapenem resistanc
Heterogeneous hydrolytic features for OXA-48-like Ī²-lactamases
Objectives: Carbapenem-hydrolysing class D Ī²-lactamases of the OXA-48 type are increasingly reported from Enterobacteriaceae. Ī²-Lactamase OXA-48 hydrolyses penicillins very efficiently, but carbapenems only weakly and spares broad-spectrum cephalosporins. Recently, diverse OXA-48-like Ī²-lactamases have been identified worldwide (OXA-162, OXA-181, OXA-163, OXA-204 and OXA-232). They differ by few amino acid substitutions or by amino acid deletions.Methods: blaOXA-48, blaOXA-162, blaOXA-163, blaOXA-181, blaOXA-204 and blaOXA-232 were cloned into the same expression vector and expressed in the same Escherichia coli background. Kinetic studies were performed with enzymes purified by ion-exchange chromatography. Determination of hydrolytic activities was performed by UV spectrophotometry. MICs were determined for all recombinant strains, using as background either the WT E. coli TOP10 strain or a porin-deficient E. coli strain.Results: Kinetic studies showed that OXA-162 and OXA-204 shared the same hydrolytic properties as OXA-48. On the other hand, OXA-181 possessed a higher ability to hydrolyse carbapenems, while OXA-232 hydrolysed those substrates less efficiently. In contrast to the other OXA-48-like Ī²-lactamases, OXA-163 hydrolysed broad-spectrum cephalosporins very efficiently, but did not possess significant carbapenemase activity. Although several of these OXA-48-like enzymes possess low activity against carbapenems, MICs of carbapenems were significantly elevated when determined for strains possessing permeability defects.Conclusions: A detailed comparative analysis of the kinetic properties of the OXA-48-like Ī²-lactamases is provided here. It clarifies the respective features of each OXA-48-like variant and their respective impacts in terms of carbapenem resistance
Genetic and biochemical characterization of FRI-1, a carbapenem-hydrolyzing class A Ī²-Lactamase from Enterobacter cloacae
An Enterobacter cloacae isolate was recovered from a rectal swab from a patient hospitalized in France with previous travel to Switzerland. It was resistant to penicillins, narrow- and broad-spectrum cephalosporins, aztreonam, and carbapenems but remained susceptible to expanded-spectrum cephalosporins. Whereas PCR-based identification of the most common carbapenemase genes failed, the biochemical Carba NP test II identified an Ambler class A carbapenemase. Cloning experiments followed by sequencing identified a gene encoding a totally novel class A carbapenemase, FRI- 1, sharing 51 to 55% amino acid sequence identity with the closest carbapenemase sequences. However, it shared conserved residues as a source of carbapenemase activity. Purified Ī²-lactamase FRI-1 hydrolyzed penicillins, aztreonam, and carbapenems but spared expanded-spectrum cephalosporins. The 50% inhibitory concentrations (ICā
ās) of clavulanic acid and tazobactam were 10-fold higher than those found for Klebsiella pneumoniae carbapenemase (KPC), IMI, and SME, leading to lower sensitivity of FRI-1 activity to Ī²-lactamase inhibitors. The bla FRI-1 gene was located on a ca. 110-kb untypeable, transferable, and non-self-conjugative plasmid. A putative LysR family regulator-encoding gene at the 5ā² end of the Ī²-lactamase gene was identified, leading to inducible expression of the blaFRI-1 gene
Genetic and biochemical characterization of OXA-405, an OXA-48-Type extended-spectrum Ī²-lactamase without significant carbapenemase activity
The epidemiology of carbapenemases worldwide is showing that OXA-48 variants are becoming the predominant carbapenemase type in Enterobacteriaceae in many countries. However, not all OXA-48 variants possess significant activity toward carbapenems (e.g., OXA-163). Two Serratia marcescens isolates with resistance either to carbapenems or to extended-spectrum cephalosporins were successively recovered from the same patient. A genomic comparison using pulsed-field gel electrophoresis and automated Rep-PCR typing identified a 97.8% similarity between the two isolates. Both strains were resistant to penicillins and first-generation cephalosporins. The first isolate was susceptible to expanded-spectrum cephalosporins, was resistant to carbapenems, and had a significant carbapenemase activity (positive Carba NP test) related to the expression of OXA-48. The second isolate was resistant to expanded-spectrum cephalosporins, was susceptible to carbapenems, and did not express a significant imipenemase activity, (negative for the Carba NP test) despite possessing a blaOXA-48-type gene. Sequencing identified a novel OXA-48-type Ī²-lactamase, OXA-405, with a four-amino-acid deletion compared to OXA-48. The blaOXA-405 gene was located on a ca. 46-kb plasmid identical to the prototype IncL/M blaOXA-48-carrying plasmid except for a ca. 16.4-kb deletion in the tra operon, leading to the suppression of self-conjugation properties. Biochemical analysis showed that OXA-405 has clavulanic acid-inhibited activity toward expanded-spectrum activity without significant imipenemase activity. This is the first identification of a successive switch of catalytic activity in OXA-48-like Ī²-lactamases, suggesting their plasticity. Therefore, this report suggests that the first-line screening of carbapenemase producers in Enterobacteriaceae may be based on the biochemical detection of carbapenemase activity in clinical settings
Biochemical and Structural Characterization of OXA-405, an OXA-48 Variant with Extended-Spectrum Ī²-Lactamase Activity
International audienceOXA-48-producing Enterobacterales have now widely disseminated globally. A sign of their extensive spread is the identification of an increasing number of OXA-48 variants. Among them, three are particularly interesting, OXA-163, OXA-247 and OXA-405, since they have lost carbapenem activities and gained expanded-spectrum cephalosporin hydrolytic activity subsequent to a four amino-acid (AA) deletion in the Ī²5-Ī²6 loop. We investigated the mechanisms responsible for substrate specificity of OXA-405. Kinetic parameters confirmed that OXA-405 has a hydrolytic profile compatible with an ESBL (hydrolysis of expanded spectrum cephalosporins and susceptibility to class A inhibitors). Molecular modeling techniques and 3D structure determination show that the overall dimeric structure of OXA-405 is very similar to that of OXA-48, except for the Ī²5-Ī²6 loop, which is shorter for OXA-405, suggesting that the length of the Ī²5-Ī²6 loop is critical for substrate specificity. Covalent docking with selected substrates and molecular dynamics simulations evidenced the structural changes induced by substrate binding, as well as the distribution of water molecules in the active site and their role in substrate hydrolysis. All this data may represent the structural basis for the design of new and efficient class D inhibitors
Genomic characterization of an NDM-9-producing Acinetobacter baumannii clinical isolate and role of Glu152Lys substitution in the enhanced cefiderocol hydrolysis of NDM-9
Here, we characterized the first French NDM-9-producing Acinetobacter baumannii isolate. A. baumannii 13A297, which belonged to the STPas25 (international clone IC7), was highly resistant to Ī²-lactams including cefiderocol (MIC >32āmg/L). Whole genome sequencing (WGS) using both Illumina and Oxford Nanopore technologies revealed a 166-kb non-conjugative plasmid harboring a blaNDM-9 gene embedded in a Tn125 composite transposon. Complementation of E. coli DH5Ī± and A. baumannii CIP70.10 strains with the pABEC plasmid carrying the blaNDM-1 or blaNDM-9 gene, respectively, resulted in a significant increase in cefiderocol MIC values (16 to >256-fold), particularly in the NDM-9 transformants. Interestingly, steady-state kinetic parameters, measured using purified NDM-1 and NDM-9 (Glu152Lys) enzymes, revealed that the affinity for cefiderocol was 3-fold higher for NDM-9 (Kmā=ā53āĪ¼M) than for NDM-1 (Kmā=ā161āĪ¼M), leading to a 2-fold increase in catalytic efficiency for NDM-9 (0.13 and 0.069āĪ¼Mā1.sā1, for NDM-9 and NDM-1, respectively). Finally, we showed by molecular docking experiments that the residue 152 of NDM-like enzymes plays a key role in cefiderocol binding and resistance, by allowing a strong ionic interaction between the Lys152 residue of NDM-9 with both the Asp223 residue of NDM-9 and the carboxylate group of the R1 substituent of cefiderocol
CaractĆ©risation molĆ©culaire et biochimique des carbapĆ©nĆØmases les plus rĆ©pandues chez les EntĆ©robactĆ©ries associĆ©es Ć des infections sĆ©vĆØres en vue de dĆ©velopper de nouveaux inhibiteurs
Antibiotics, and particularly Ć- lactams, have long been considered the ultimate weapon for the treatment of bacterial infections, because of their effectiveness, good tolerance and low cost. However, bacteria have developed mechanisms of resistance, including against Ī²- lactams with the broadest spectrum of activity, carbapenems. The latter are considered as last resort antibiotics for the treatment of severe infections due to Gram negative bacilli in hospital. This emerging resistance to carbapenems in Enterobacteriaceae is mainly due to the expression of enzymes called carbapenemases capable of inactivating carbapenems. The worldwide exponential spread of these carbapenemase-producing enterobacteria (CPE) represents a major public health issue. Currently, the most common carbapenemases in the world are KPC (Klebsiella pneumoniae carbapenemase), NDM-1 (New Delhi metallo-Ī²-lactamase) and OXA-48 (Oxacillinase). The first KPC was identified in 1996 in the United States, NDM-1 in 2009 in Sweden in a patient from India and OXA-48 in 2003 in Turkey. In France, OXA-48- type are the most abundant carbapenemases with nearly 70% of the EPCs. Thus, the repercussion of their dissemination and the development of new inhibitors capable of inactivating carbapenemases is becoming more and more urgent.The aims of this thesis were to better understand the mode of action of the 3 main carbapenemases from a molecular and biochemical point of view, in order to identify the structural elements necessary for their functioning, but also to lay the basis for the development of "pan-inhibitors" and noveldiagnostic tools. For this purpose, the biochemical characterization of carbapenemases of the KPC, NDM and OXA-48 type, their natural variants and mutants generated in vitro has been undertaken.We could highlight the involvement of specific residues and structural elements necessary for the hydrolysis of carbapenems. The crystallographic and NMR study as well as the in silico (modeling) studies of these enzymes and their respective mutants, allowed us to better understand the enzyme-substrate interaction mode. We could thus understand the basis of the impressive capacity of these carbapenemases to adapt and therefore to evolve according to the selection pressures exerted.In collaboration with several chemists we participated in the development of different series of "pan-inhibitors" that were able to inhibit the 3 classes of carbapenemases. Thus we could show the inhibitory properties of some compounds of the family of flavonoids including myricetin, the most active molecule. We have also been able to identify a series of compounds, imidazolines, possessing a pan- inhibitory effect with sub-micromolar IC50 values and therefore compatible with in vivo use. Finally, we participated in the development (in collaboration with the CEA) of a rapid diagnostic tool based on theimmunochromatographic, allowing the detection of EPCs in less than 15 minutes.Les antibiotiques, et plus particulieĢrement les Ī²-lactamines, furent pendant longtemps consideĢreĢs comme lāarme absolue pour le traitement des infections bacteĢriennes, du fait de leur efficaciteĢ, leur bonne toleĢrance et de leur faible couĢt. Cependant, les bacteĢries ont deĢveloppeĢ des meĢcanismes de reĢsistance, y compris vis-aĢ-vis des Ī²-lactamines posseĢdant le spectre dāactiviteĢ le plus large, les carbapeĢneĢmes. Ces derniers sont consideĢreĢs comme les antibiotiques de derniers recours pour le traitement des infections seĢveĢres aĢ bacilles aĢ Gram neĢgatif en milieu hospitalier. Chez les enteĢrobacteĢries, cette reĢsistance eĢmergente aux carbapeĢneĢmes est principalement due aĢ lāexpression dāenzymes appeleĢes les carbapeĢneĢmases capables dāinactiver les carbapeĢneĢmes. LāeĢmergence exponentielle aĢ lāeĢchelle mondiale de ces enteĢrobacteĢries productrices de carbapeĢneĢmases (EPC) constitue un probleĢme majeur de santeĢ publique. Actuellement, les carbapeĢneĢmases les plus reĢpandues dans le monde sont les KPC (Klebsiella pneumoniae carbapenemase), NDM-1 (New Delhi metallo- Ī²- lactamase) et OXA-48 (Oxacillinase). La premieĢre KPC a eĢteĢ identifieĢe en 1996 aux Etats- Unis, NDM-1 en 2009 en SueĢde chez une patiente en provenance dāInde et OXA-48 en 2003 en Turquie. En France, les carbapeĢneĢmases de type OXA-48 sont largement majoritaires et repreĢsentent preĢs de 70% des EPC. Ainsi, le retentissement de leur disseĢmination et le besoin de deĢvelopper de nouveaux inhibiteurs capables dāinactiver les carbapeĢneĢmases se fait de plus en plus pressante.Cette theĢse a pour objectif de mieux comprendre le mode dā action de ces 3 carbapeĢneĢmases dāun point de vue moleĢculaire et biochimique, afin dā identifier les eĢleĢments structuraux neĢcessaires aĢ leur fonctionnement, mais aussi de poser les bases du deĢveloppement de Ā« pan-inhibiteurs Ā» et de nouveaux outils de diagnostics. Pour cela, la caracteĢrisation biochimique des carbapeĢneĢmases de type KPC, NDM et OXA-48, de leurs variants naturels et de mutants geĢneĢreĢs in vitro a eĢteĢ entreprise. Il a pu eĢtre mis en exergue lāimplication de reĢsidus speĢcifiques et dāeĢleĢments structuraux neĢcessaires aĢ lā hydrolyse des carbapeĢneĢmes. L ā eĢtude cristallographique et RMN ainsi que lāeĢtude in silico (modeĢlisation) de ces enzymes et de leurs mutants respectifs, nous a permis de mieux comprendre le mode dā interaction enzyme- substrat. Nous avons ainsi pu comprendre les bases de la capaciteĢ impressionnante de ces carbapeĢneĢmases aĢ sāadapter et aĢ eĢvoluer en fonction des pressions de seĢlections exerceĢes.En collaborations avec diffeĢrentes eĢquipes de chimistes nous avons deĢveloppeĢs diffeĢrentes seĢries de moleĢcules Ā« pan-inhibiteurs Ā» capables dāinhiber les 3 classes de carbapeĢneĢmases. Ainsi nous avons montreĢ un effet inhibiteur de certains composeĢs de la famille des flavonoiĢdes dont la myreĢceĢtine, moleĢcule la plus active. Nous avons eĢgalement identifieĢ une seĢrie de composeĢs, les imidazolines, posseĢdant un effet pan-inhibiteur avec des valeurs de CI50 sub-micromolaires et donc compatible avec une utilisation in vivo.Enfin, nous avons participeĢ au deĢveloppement (en collaboration avec le CEA) dā un outil de diagnostic rapide baseĢ sur lāimmunochromatographie, permettant deĢtection des EPC en moins de 15 minutes
MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF THE MOST POPULAR CARBAPENEMASES IN ENTEROBACTERIES ASSOCIATED WITH SEVERE INFECTIONS IN ORDER TO DEVELOP NEW INHIBITORS
Les antibiotiques, et plus particulieĢrement les Ī²-lactamines, furent pendant longtemps consideĢreĢs comme lāarme absolue pour le traitement des infections bacteĢriennes, du fait de leur efficaciteĢ, leur bonne toleĢrance et de leur faible couĢt. Cependant, les bacteĢries ont deĢveloppeĢ des meĢcanismes de reĢsistance, y compris vis-aĢ-vis des Ī²-lactamines posseĢdant le spectre dāactiviteĢ le plus large, les carbapeĢneĢmes. Ces derniers sont consideĢreĢs comme les antibiotiques de derniers recours pour le traitement des infections seĢveĢres aĢ bacilles aĢ Gram neĢgatif en milieu hospitalier. Chez les enteĢrobacteĢries, cette reĢsistance eĢmergente aux carbapeĢneĢmes est principalement due aĢ lāexpression dāenzymes appeleĢes les carbapeĢneĢmases capables dāinactiver les carbapeĢneĢmes. LāeĢmergence exponentielle aĢ lāeĢchelle mondiale de ces enteĢrobacteĢries productrices de carbapeĢneĢmases (EPC) constitue un probleĢme majeur de santeĢ publique. Actuellement, les carbapeĢneĢmases les plus reĢpandues dans le monde sont les KPC (Klebsiella pneumoniae carbapenemase), NDM-1 (New Delhi metallo- Ī²- lactamase) et OXA-48 (Oxacillinase). La premieĢre KPC a eĢteĢ identifieĢe en 1996 aux Etats- Unis, NDM-1 en 2009 en SueĢde chez une patiente en provenance dāInde et OXA-48 en 2003 en Turquie. En France, les carbapeĢneĢmases de type OXA-48 sont largement majoritaires et repreĢsentent preĢs de 70% des EPC. Ainsi, le retentissement de leur disseĢmination et le besoin de deĢvelopper de nouveaux inhibiteurs capables dāinactiver les carbapeĢneĢmases se fait de plus en plus pressante.Cette theĢse a pour objectif de mieux comprendre le mode dā action de ces 3 carbapeĢneĢmases dāun point de vue moleĢculaire et biochimique, afin dā identifier les eĢleĢments structuraux neĢcessaires aĢ leur fonctionnement, mais aussi de poser les bases du deĢveloppement de Ā« pan-inhibiteurs Ā» et de nouveaux outils de diagnostics. Pour cela, la caracteĢrisation biochimique des carbapeĢneĢmases de type KPC, NDM et OXA-48, de leurs variants naturels et de mutants geĢneĢreĢs in vitro a eĢteĢ entreprise. Il a pu eĢtre mis en exergue lāimplication de reĢsidus speĢcifiques et dāeĢleĢments structuraux neĢcessaires aĢ lā hydrolyse des carbapeĢneĢmes. L ā eĢtude cristallographique et RMN ainsi que lāeĢtude in silico (modeĢlisation) de ces enzymes et de leurs mutants respectifs, nous a permis de mieux comprendre le mode dā interaction enzyme- substrat. Nous avons ainsi pu comprendre les bases de la capaciteĢ impressionnante de ces carbapeĢneĢmases aĢ sāadapter et aĢ eĢvoluer en fonction des pressions de seĢlections exerceĢes.En collaborations avec diffeĢrentes eĢquipes de chimistes nous avons deĢveloppeĢs diffeĢrentes seĢries de moleĢcules Ā« pan-inhibiteurs Ā» capables dāinhiber les 3 classes de carbapeĢneĢmases. Ainsi nous avons montreĢ un effet inhibiteur de certains composeĢs de la famille des flavonoiĢdes dont la myreĢceĢtine, moleĢcule la plus active. Nous avons eĢgalement identifieĢ une seĢrie de composeĢs, les imidazolines, posseĢdant un effet pan-inhibiteur avec des valeurs de CI50 sub-micromolaires et donc compatible avec une utilisation in vivo.Enfin, nous avons participeĢ au deĢveloppement (en collaboration avec le CEA) dā un outil de diagnostic rapide baseĢ sur lāimmunochromatographie, permettant deĢtection des EPC en moins de 15 minutes.Antibiotics, and particularly Ć- lactams, have long been considered the ultimate weapon for the treatment of bacterial infections, because of their effectiveness, good tolerance and low cost. However, bacteria have developed mechanisms of resistance, including against Ī²- lactams with the broadest spectrum of activity, carbapenems. The latter are considered as last resort antibiotics for the treatment of severe infections due to Gram negative bacilli in hospital. This emerging resistance to carbapenems in Enterobacteriaceae is mainly due to the expression of enzymes called carbapenemases capable of inactivating carbapenems. The worldwide exponential spread of these carbapenemase-producing enterobacteria (CPE) represents a major public health issue. Currently, the most common carbapenemases in the world are KPC (Klebsiella pneumoniae carbapenemase), NDM-1 (New Delhi metallo-Ī²-lactamase) and OXA-48 (Oxacillinase). The first KPC was identified in 1996 in the United States, NDM-1 in 2009 in Sweden in a patient from India and OXA-48 in 2003 in Turkey. In France, OXA-48- type are the most abundant carbapenemases with nearly 70% of the EPCs. Thus, the repercussion of their dissemination and the development of new inhibitors capable of inactivating carbapenemases is becoming more and more urgent.The aims of this thesis were to better understand the mode of action of the 3 main carbapenemases from a molecular and biochemical point of view, in order to identify the structural elements necessary for their functioning, but also to lay the basis for the development of "pan-inhibitors" and noveldiagnostic tools. For this purpose, the biochemical characterization of carbapenemases of the KPC, NDM and OXA-48 type, their natural variants and mutants generated in vitro has been undertaken.We could highlight the involvement of specific residues and structural elements necessary for the hydrolysis of carbapenems. The crystallographic and NMR study as well as the in silico (modeling) studies of these enzymes and their respective mutants, allowed us to better understand the enzyme-substrate interaction mode. We could thus understand the basis of the impressive capacity of these carbapenemases to adapt and therefore to evolve according to the selection pressures exerted.In collaboration with several chemists we participated in the development of different series of "pan-inhibitors" that were able to inhibit the 3 classes of carbapenemases. Thus we could show the inhibitory properties of some compounds of the family of flavonoids including myricetin, the most active molecule. We have also been able to identify a series of compounds, imidazolines, possessing a pan- inhibitory effect with sub-micromolar IC50 values and therefore compatible with in vivo use. Finally, we participated in the development (in collaboration with the CEA) of a rapid diagnostic tool based on theimmunochromatographic, allowing the detection of EPCs in less than 15 minutes
In silico prediction of Ī²-lactamase hydrolysis efficiency: Finding the right balance between kinetic and thermodynamic terms
International audienceDuring recent decades, an alarming worldwide spread and diversification of Ī²-lactamases has been obsd. in Gram-neg. species, conferring resistance to Ī²-lactam antibiotics. In this context, the prediction of future Ī²-lactamase mutants becomes an essential issue and we have developed a protocol that allows the evaluation of the energetic cost (thermodn.) assocd. with a mutation in the active site of a Ī²-lactamase, in the presence of the Ī²-lactam substrate. The catalytic efficiency for the Ī²-lactamase-mediated hydrolysis of Ī²-lactam antibiotics is represented by the ratio of a kinetic term (kcat) and a thermodn. term (Km). Here we present an approach based on quantum calcns. and mol. dynamics simulations allowing to est. the kcat term by evaluating Ī²-lactamases from different classes, in the presence of several Ī²-lactam substrates. Therefore, we are now able to predict in silico the overall catalytic efficiency for a large panel of Ī²-lactamases. The precision of this prediction, which depends in turn on the precision for predicting the individual terms kcat and Km, will be discussed and compared with the precision of exptl. values. These results will ultimately provide essential information for the fight against resistance to Ī²-lactam antibiotics
A greater than expected variability among OXA-48-like carbapenemases
International audienceBackground: OXA-48-like carbapenemases represent a major health concern given their difficult detection, their epidemic behavior and their propensity to modify their spectrum of hydrolysis through point mutations. Objective: To get an extensive view on the current variability among OXA-48-like enzymes, we haveretrieved all the sequences available from NCBI (National Center for Biotechnology Information).Method: We carried out several BLAST (Basic Local Alignment Search Tool) searches in the NCBIās ānrā and ānr_envā databases (downloaded on December 20th, 2016) using known members of OXA-48-likesubfamily as query.Results: While 23 variants have assigned OXA-numbers, 62 novel alleles have been identified. Theycorrespond to novel enzymes with mutations located in some cases within the conserved active site motives. The important number of novel variants identified by this study is of great interest, since it provides a more realistic assessment of OXA-48-like variants.Conclusion: A large variety of OXA-48-like enzymes has been unraveled through our bioinformatic search for variants. The finding of OXA-48-like enzymes in environmental isolates may reflect the contamination by Enterobactericeae producing OXA-48-like enzymes and/or the presence of Shewanella spp. isolates.Introducere: Carbapenemazele OXA-48-like reprezintÄ o problemÄ majorÄ pentru sÄnÄtate, avĆ¢nd Ć®nvedere detectarea lor dificilÄ, comportamentul epidemic Či tendinČa lor de a-Či modifica spectrul de hidrolizÄprin mutaČii punctiforme.Obiectiv: Pentru a obČine o imagine amplÄ asupra variabilitÄČii actuale a enzimelor OXA-48-like, amrecuperat toate secvenČele disponibile de la NCBI (National Center for Biotechnology Information).MetodÄ: Am efectuat mai multe cÄutÄri BLAST (Basic Local Alignment Search Tool) Ć®n bazele de dateānrā Či ānr_envā ale NCBI (descÄrcate pe 20 decembrie 2016), utilizĆ¢nd ca interogare membrii cunoscuČi aisub-familiei OXA-48.Rezultate: Ćn timp ce unui numÄr de 23 de variante li s-au atribuit numere OXA, au fost identificate 62 dealele noi. Acestea corespund noilor enzime cu mutaČii localizate Ć®n unele cazuri Ć®n cadrul motivelor conservateale site-ului activ. NumÄrul important de variante noi identificate Ć®n acest studiu este de mare interes, deoareceoferÄ o evaluare mai realistÄ a variantelor de tip OXA-48-like.Concluzie: O mare varietate de enzime OXA-48-like a fost descoperitÄ Ć®n urma cÄutÄrii bioinformaticea variantelor. Detectarea enzimelor OXA-48-like Ć®n izolate din mediu poate reflecta contaminarea cuEnterobactericeae producÄtoare de enzime OXA-48-like Či/sau prezenČa de izolate Shewanella spp.Cuvinte-cheie: OXA-48, variabilitate, variante
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