Characterization of BRP_MBL the bleomycin resistance protein associated with the carbapenemase NDM

The metallo-β-lactamase NDM-1 is among the most worrisome resistance determinants and is spreading worldwide among Gram-negative bacilli. A bleomycin resistance gene, bleMBL, downstream of the blaNDM-1 gene has been associated with resistance almost systematically. Here, we characterized the corresponding protein, BRPMBL, conferring resistance to bleomycin, an antitumoral glycopeptide molecule. We have determined whether the expression of the blaNDM-1-bleMBL operon is inducible in the presence of carbapenems and/or bleomycin-like molecules using quantitative reverse transcription-PCR (qRT-PCR), determination of imipenem and zeocin MICs, and carbapenemase-specific activity assays. We showed that the blaNDM- 1-bleMBL operon is constitutively expressed. Using electrophoretic mobility shift and DNA protection assays performed with purified glutathione S- transferase (GST)-BRPMBL, we demonstrated that BRPMBL is able to bind and sequester bleomycin-like molecules, thus preventing bleomycin-dependent DNA degradation. In silico modeling confirmed that the mechanism of action required the dimerization of the BRPMBL protein in order to sequester bleomycin and prevent DNA damage. BRPMBL acts specifically on bleomycin-like molecules since cloning and expression of bleMBL in Staphyloccoccus aureus did not confer cross-resistance to any other antimicrobial glycopeptides such as vancomycin and teicoplanin

The role of Bosniak classification in the assessment of renal cystic masses and in the therapeutical protocol

One of the most frequent kidney pathologies encountered in daily practice is represented by the presence of renal cysts. Most of them are asymptomatic and are found accidentally during periodical check-ups because they don’t have clinical signs until they grow and compress the surrounding organs. We have reviewed the current data regarding this pathology, in order to underline the risk of malignant transformation and its impact on the patient’s life. It is estimated that the prevalence rate of renal cysts in the general population is approximately 10% and it increases with age.Imaging investigations, such as contrast tomography or magnetic resonance imaging, are essential for establishing the cysts characteristics, especially when ultrasonography raises the suspicion of a modified renal cyst, as well as in guiding the therapeutical protocol. The Bosniak classification is based on contrast tomography scans and has allowed the standardization of the kidney cysts, considering their characteristics. More attention should be given to Bosniak IIF and III cystic renal masses, which contain thickened walls and more septa, but no enhanced nodules/soft tissue components, because more than half of these cysts can have a malignant component

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 β-lactams including cefiderocol (MIC >32 mg/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 DH5α 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 (Km = 53 μM) than for NDM-1 (Km = 161 μM), leading to a 2-fold increase in catalytic efficiency for NDM-9 (0.13 and 0.069 μM−1.s−1, 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

Azetidinimines as carbapenemases inhibitors

The present application relates to novel azetidinimine of formula (I). Wherein R1-R6 are as defined in claim 1. The azetidinimine of the invention are useful as antibiotics and as inhibitors of a carbapenemases. The present invention thus further relates totheir use in antibiotic therapies and their methods of synthesis

The antimalarial compound ELQ-400 is an unusual inhibitor of the \textitbc _\textrm1 complex, targeting both \textitQ _\textrmo and \textitQ _\textrmi sites

International audienc

Evaluation of Clostridium botulinum A5 neurotoxin actions in vivo and ex vivo at the mouse skeletal neuromuscular junction

International audienceClostridium botulinum neurotoxins (BoNTs), the most potent toxins known, are the cause of a worldwide lifethreatening disease in humans and animals known as botulism. This disease usually manifests as descending symmetrical flaccid paralysis of skeletal muscles together with autonomic dysfunction. BoNTs include a large family of zinc metalloproteases that can be immunologically distinguished by homologous antitoxins into seven primary serotypes, designated A to G. Additionally, for BoNT/A, at least 8 subtypes (/A1-/A8) have been identified from gene sequence analysis. In France, several cases of human botulism due to BoNT/A5, /A6 or /A7 have been reported, though BoNT/A1 and /A2 are the prevalent forms of type-A botulism. The characterization of the C. botulinum strains involved in two cases of BoNT/A5-poisoning revealed that they possess the gene encoding for BoNT/A5 identical to the one previously reported. In the present study, we produced the BoNT/A5 crude complex (3 x 107 LD50/mg) from the C. botulinum A5 strain 126.07 [1], and studied in vivo and ex vivo, in murine models, the skeletal neuromuscular block caused by BoNT/A5, at different times after a single local toxin injection into the hind-limbs. The results show that the duration and degree of paralysis depended on the dose of BoNT/A5 and on the mouse strain studied. The transgenic Thy-1-YFP-16 black C57BL6 mice [2] were more sensitive to the action of the toxin than Swiss mice, as revealed by the digit abduction score (DAS) assay and by compound muscle action potential (CMAP) recordings from the same mouse in vivo, at different times after toxin injection. functional and morphological ex vivo studies on muscles locally injected with BoNT/A5 in vivo reveal (a) the presence of axonal outgrowths (nodal and nerve terminal sprouts), (b) an extension of nicotinic acetylcholine receptor clusters, (c) a reduction of both muscle weight and muscle fiber cross sectional area, and (d) the prolonged atrophy of muscle fibers. Pre- and post-synaptic remodeling was completely abolished by an immune-purified rabbit polyclonal antibody directed against the BoNT/A1-Heavy chain (HcA1), when injected together with BoNT/A5. In conclusion, our results show that the actions of BoNT/A5 have many similarities to those of BoNT/A1 previously reported, although no direct comparison was performed in the present study. BoNT/A5 differed from BoNT/A1 by 5 identified amino acids in the Light chain domain (Lc) and by 32 amino acids in the Heavy chain domain (Hc). The fact that the antibody directed to the HcA1 completely prevented the lethal effect of BoNT/A5, as well as the block of the CMAP in vivo, and the morphological changes induced by the neurotoxin suggest that the antibody blocks the entry of BoNT/A5 into motor nerve terminals of the neuromuscular junction

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 NCBI’s “nr” and “nr_env” 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 reprezintă o problemă majoră pentru sănătate, având învedere detectarea lor dificilă, comportamentul epidemic și tendința lor de a-și modifica spectrul de hidrolizăprin mutații punctiforme.Obiectiv: Pentru a obține o imagine amplă asupra variabilității actuale a enzimelor OXA-48-like, amrecuperat toate secvențele disponibile de la NCBI (National Center for Biotechnology Information).Metodă: Am efectuat mai multe căutări BLAST (Basic Local Alignment Search Tool) în bazele de date„nr” și „nr_env” ale NCBI (descărcate pe 20 decembrie 2016), utilizând ca interogare membrii cunoscuți aisub-familiei OXA-48.Rezultate: În timp ce unui număr de 23 de variante li s-au atribuit numere OXA, au fost identificate 62 dealele noi. Acestea corespund noilor enzime cu mutații localizate în unele cazuri în cadrul motivelor conservateale site-ului activ. Numărul important de variante noi identificate în acest studiu este de mare interes, deoareceoferă o evaluare mai realistă a variantelor de tip OXA-48-like.Concluzie: O mare varietate de enzime OXA-48-like a fost descoperită în urma căutării bioinformaticea variantelor. Detectarea enzimelor OXA-48-like în izolate din mediu poate reflecta contaminarea cuEnterobactericeae producătoare de enzime OXA-48-like și/sau prezența de izolate Shewanella spp.Cuvinte-cheie: OXA-48, variabilitate, variante

Poly(γ-benzyl-l-glutamate)-PEG-alendronate multivalent nanoparticles for bone targeting.

International audienceHydroxyapatite (HAP), a highly specific component of bone tissue, is the main target in order to impart osteotropicity. Bone targeted nanoparticles can increase the strength of the interaction with HAP through multivalency and thus constitute a valuable strategy in the therapeutics of skeletal diseases. PBLG10k-b-PEG6k-alendronate nanoparticles (∼75nm) were prepared by a simple nanoprecipitation method. The calcium affinity (KCa(+2)=1.8×10(4)M(-1)) of these nanoparticles was evaluated using isothermal titration calorimetry. The multivalent interaction with HAP surfaces (KHAP) was studied by fluorescence and was estimated to be 1.1×10(10)M(-1), which is more than 4000 times stronger than the reported monovalent interaction between alendronate and HAP surfaces. Molecular modeling suggests that the number of binding sites available at the HAP surface is in large excess than what is required for the whole surface coverage by alendronate decorated nanoparticles. The lower calcium affinity of these nanoparticles than for HAP allows calcium bound nanoparticles to interact with HAP, which yields a deeper understanding of bone targeted carriers and could potentially improve their bone targeting properties

Conformational study of glycal-type neuraminidase inhibitors

International audienceConformational study of glycal-type neuraminidase inhibitors The conformational flexibility of two glycal-type neuraminidase inhibitors has been studied, using several molecular modeling techniques. In agreement with the experimental data available, an intramolecular hydrogen bond, representing a key structural feature that controls the conformer distribution in solution, has been identified. The contribution of each substituent on the overall equilibrium was evaluated using simplified derivatives. Additionally, four methods for estimating NMR coupling constants from dihedral angles were evaluated and the Haasnoot method was found to be appropriate for this class of sugars. These results should allow a better understanding of the structural parameters governing physico-chemical properties of glycal-like compounds