Roscovitine Worsens Mycobacterium abscessus Infection by Reducing DUOX2-mediated Neutrophil Response.

Persistent neutrophilic inflammation associated with chronic pulmonary infection causes progressive lung injury and, eventually, death in individuals with cystic fibrosis (CF), a genetic disease caused by biallelic mutations in the CF transmembrane conductance regulator (CFTR) gene. Therefore, we examined whether roscovitine, a cyclin-dependent kinase inhibitor that (in other conditions) reduces inflammation while promoting host defense, might provide a beneficial effect in the context of CF. Herein, using CFTR-depleted zebrafish larvae as an innovative vertebrate model of CF immunopathophysiology, combined with murine and human approaches, we sought to determine the effects of roscovitine on innate immune responses to tissue injury and pathogens in the CF condition. We show that roscovitine exerts antiinflammatory and proresolution effects in neutrophilic inflammation induced by infection or tail amputation in zebrafish. Roscovitine reduces overactive epithelial reactive oxygen species (ROS)-mediated neutrophil trafficking by reducing DUOX2/NADPH-oxidase activity and accelerates inflammation resolution by inducing neutrophil apoptosis and reverse migration. It is important to note that, although roscovitine efficiently enhances intracellular bacterial killing of Mycobacterium abscessus in human CF macrophages ex vivo, we found that treatment with roscovitine results in worse infection in mouse and zebrafish models. By interfering with DUOX2/NADPH oxidase-dependent ROS production, roscovitine reduces the number of neutrophils at infection sites and, consequently, compromises granuloma formation and maintenance, favoring extracellular multiplication of M. abscessus and more severe infection. Our findings bring important new understanding of the immune-targeted action of roscovitine and have significant therapeutic implications for safely targeting inflammation in CF.

A piperidinol-containing molecule is active against Mycobacterium tuberculosis by inhibiting the mycolic acid flippase activity of MmpL3.

Mycobacterium tuberculosis, the causative agent of tuberculosis, remains a major human pathogen, and current treatment options to combat this disease are under threat because of the emergence of multidrug-resistant and extensively drug-resistant tuberculosis. High-throughput whole-cell screening of an extensive compound library has recently identified a piperidinol-containing molecule, PIPD1, as a potent lead compound against M. tuberculosis Herein, we show that PIPD1 and related analogs exert in vitro bactericidal activity against the M. tuberculosis strain mc26230 and also against a panel of multidrug-resistant and extensively drug-resistant clinical isolates of M. tuberculosis, suggesting that PIPD1's mode of action differs from those of most first- and second-line anti-tubercular drugs. Selection and DNA sequencing of PIPD1-resistant mycobacterial mutants revealed the presence of single-nucleotide polymorphisms in mmpL3, encoding an inner membrane-associated mycolic acid flippase in M. tuberculosis Results from functional assays with spheroplasts derived from a M. smegmatis strain lacking the endogenous mmpL3 gene but harboring the M. tuberculosis mmpL3 homolog indicated that PIPD1 inhibits the MmpL3-driven translocation of trehalose monomycolate across the inner membrane without altering the proton motive force. Using a predictive structural model of MmpL3 from M. tuberculosis, docking studies revealed a PIPD1-binding cavity recently found to accommodate different inhibitors in M. smegmatis MmpL3. In conclusion, our findings have uncovered bactericidal activity of a new chemical scaffold. Its anti-tubercular activity is mediated by direct inhibition of the flippase activity of MmpL3 rather than by inhibition of the inner membrane proton motive force, significantly advancing our understanding of MmpL3-targeted inhibition in mycobacteria.

Efficacy of Bedaquiline, Alone or in Combination with Imipenem, against Mycobacterium abscessus in C3HeB/FeJ Mice.

Mycobacterium abscessus lung infections remain difficult to treat. Recent studies have recognized the power of new combinations of antibiotics, such as bedaquiline and imipenem, although in vitro data have questioned this combination. We report that the efficacy of bedaquiline-imipenem combination treatment relies essentially on the activity of bedaquiline in a C3HeB/FeJ mice model of infection with a rough variant of M. abscessus The addition of imipenem contributed to clearing the infection in the spleen.

Insights into the smooth-to-rough transitioning in Mycobacterium bolletii unravels a functional Tyr residue conserved in all mycobacterial MmpL family members.

In mycobacteria, MmpL proteins represent key components that participate in the biosynthesis of the complex cell envelope. Whole genome analysis of a spontaneous rough morphotype variant of Mycobacterium abscessus subsp. bolletii identified a conserved tyrosine that is crucial for the function of MmpL family proteins. Isogenic smooth (S) and rough (R) variants differed by a single mutation linked to a Y842H substitution in MmpL4a. This mutation caused a deficiency in glycopeptidolipid production/transport in the R variant and a gain in the capacity to produce cords in vitro. In zebrafish, increased virulence of the M. bolletii R variant over the parental S strain was found, involving massive production of serpentine cords, abscess formation and rapid larval death. Importantly, this finding allowed us to demonstrate an essential role of Tyr842 in several different MmpL proteins, including Mycobacterium tuberculosis MmpL3. Structural homology models of MmpL4a and MmpL3 identified two additional critical residues located in the transmembrane regions TM10 and TM4 that are facing each other. We propose that these central residues are part of the proton-motive force that supplies the energy for substrate transport. Hence, we provide important insights into mechanistic/structural aspects of MmpL proteins as lipid transporters and virulence determinants in mycobacteria.

A new piperidinol derivative targeting mycolic acid transport in Mycobacterium abscessus.

The natural resistance of Mycobacterium abscessus to most commonly available antibiotics seriously limits chemotherapeutic treatment options, which is particularly challenging for cystic fibrosis patients infected with this rapid-growing mycobacterium. New drugs with novel molecular targets are urgently needed against this emerging pathogen. However, the discovery of such new chemotypes has not been appropriately performed. Here, we demonstrate the utility of a phenotypic screen for bactericidal compounds against M. abscessus using a library of compounds previously validated for activity against M. tuberculosis. We identified a new piperidinol-based molecule, PIPD1, exhibiting potent activity against clinical M. abscessus strains in vitro and in infected macrophages. Treatment of infected zebrafish with PIPD1 correlated with increased embryo survival and decreased bacterial burden. Whole genome analysis of M. abscessus strains resistant to PIPD1 identified several mutations in MAB_4508, encoding a protein homologous to MmpL3. Biochemical analyses demonstrated that while de novo mycolic acid synthesis was unaffected, PIPD1 strongly inhibited the transport of trehalose monomycolate, thereby abrogating mycolylation of arabinogalactan. Mapping the mutations conferring resistance to PIPD1 on a MAB_4508 tridimensional homology model defined a potential PIPD1-binding pocket. Our data emphasize a yet unexploited chemical structure class against M. abscessus infections with promising translational development possibilities.

Bedaquiline Inhibits the ATP Synthase in Mycobacterium abscessus and Is Effective in Infected Zebrafish.

Pulmonary infections caused by Mycobacterium abscessus are emerging as a global threat, especially in cystic fibrosis patients. Further intensifying the concern of M. abscessus infection is the recent evidence of human-to-human transmission of the infection. M. abscessus is a naturally multidrug-resistant fast-growing pathogen for which pharmacological options are limited. Repurposing antitubercular drugs represents an attractive option for the development of chemotherapeutic alternatives against M. abscessus infections. Bedaquiline (BDQ), an ATP synthase inhibitor, has recently been approved for the treatment of multidrug-resistant tuberculosis. Herein, we show that BDQ has a very low MIC against a vast panel of clinical isolates. Despite being bacteriostatic in vitro, BDQ was highly efficacious in a zebrafish model of M. abscessus infection. Remarkably, a very short period of treatment was sufficient to protect the infected larvae from M. abscessus-induced killing. This was corroborated with reduced numbers of abscesses and cords, considered to be major pathophysiological signs in infected zebrafish. Mode-of-action studies revealed that BDQ triggered a rapid depletion of ATP in M. abscessusin vitro, consistent with the drug targeting the FoF1 ATP synthase. Importantly, despite a failure to select in vitro for spontaneous mutants that are highly resistant to BDQ, the transfer of single nucleotide polymorphisms leading to D29V or A64P substitutions in atpE conferred high resistance, thus resolving the target of BDQ in M. abscessus Overall, this study indicates that BDQ is active against M. abscessusin vitro and in vivo and should be considered for clinical use against the difficult-to-manage M. abscessus pulmonary infections.

Vibrating-mesh nebulizer maintenance by CF patients: Results from a French survey.

PURPOSE OF THE STUDY: Vibrating-mesh nebulizers are widely used at home for cystic fibrosis (CF) treatment, with a therapeutic efficiency closely linked to the mesh performance. This national study looks at the maintenance at home by CF patients and their families of the mesh of the eFlow®rapid nebulizer. Ninety-two patients from 34 French CF centers, treated at home with inhaled drugs delivered with a vibrating-mesh nebulizer, answered to a phone standardized questionnaire specifying the different techniques of maintenance of the nebulizer. PRINCIPAL RESULTS: Patients were aged from 2 to 68 years (58.7% of children). They inhaled a mean of 1.8 nebulizations per day. Maintenance was assumed by patients in 36% of the cases. All steps of the theoretical maintenance of a nebulizer were respected in 66% of the cases. The mesh was not cleaned in 45.6%, not disinfected or not thermically disinfected in 32.5%, and not rinsed after chemical disinfection in 45.5% of the case. Only 49% of the patients knew the role of the MeshCare® system in the mesh maintenance, and only 39% had already used it when the nebulization duration reached 10 min. CONCLUSIONS: Efforts about education, particularly for the maintenance of the mesh, are needed for CF patients using a vibrating-mesh nebulizer at home.

European recommendations on the proper indication and use of peripheral venous access devices (the ERPIUP consensus): A WoCoVA project.

Since several innovations have recently changed the criteria of choice and management of peripheral venous access (new devices, new techniques of insertion, new recommendations for maintenance), the WoCoVA Foundation (WoCoVA = World Conference on Vascular Access) has developed an international Consensus with the following objectives: to propose a clear and useful classification of the currently available peripheral venous access devices; to clarify the proper indication of central versus peripheral venous access; discuss the indications of the different peripheral venous access devices (short peripheral cannulas vs long peripheral cannulas vs midline catheters); to define the proper techniques of insertion and maintenance that should be recommended today. To achieve these purposes, WoCoVA have decided to adopt a European point of view, considering some relevant differences of terminology between North America and Europe in this area of venous access and the need for a common basis of understanding among the experts recruited for this project. The ERPIUP Consensus (ERPIUP = European Recommendations for Proper Indication and Use of Peripheral venous access) was designed to offer systematic recommendations for clinical practice, covering every aspect of management of peripheral venous access devices in the adult patient: indication, insertion, maintenance, prevention and treatment of complications, removal. Also, our purpose was to improve the standardization of the terminology, bringing clarity of definition, and classification.

WoCoVA consensus on the clinical use of in-line filtration during intravenous infusions: Current evidence and recommendations for future research.

The need for filtering intravenous infusions has long been recognized in the field of venous access, though hard scientific evidence about the actual indications for in-line filters has been scarce. In the last few years, several papers and a few clinical studies have raised again this issue, suggesting that the time has come for a proper definition of the type of filtration, of its potential benefit, and of its proper indications in clinical practice. The WoCoVA Foundation, whose goal is to increase the global awareness on the risk of intravenous access and on patients' safety, developed the project of a consensus on intravenous filtration. A panel of experts in different aspects of intravenous infusion was chosen to express the current state of knowledge about filtration and to indicate the direction of future research in this field. The present document reports the final conclusions of the panel.

Experimental study of the POP technique: focus on the physical basis of the process.

INTRODUCTION: Central or peripheral vascular access devices have been in use for many decades. However, despite adequate care and maintenance, complete occlusion may occur, and its impact cannot be overlooked. A new procedure using a percussion technique has been published and referred as 'the POP technique'. METHODS: A hydrodynamic bench was used permitting both the recording of the movement of the piston with a fast camera and the pressure variations in the polyurethane and silicone catheters while connected to 2- and 3-piece syringes. RESULTS: The results are twofold. First the upward movement of the piston leads to the installation of a saturation vapour pressure in the body of the syringe. During this sequence, the clot is submitted to a force of aspiration. Then the release of the plunger leads to a pulse pressure whose dynamics and intensity are dependent of the types of syringes and catheters. CONCLUSIONS: The experiments bring to light the importance of practical features such as the orientation of the syringe and the nature of the polyurethane or silicone catheters. Then the analysis enables the definition of practical rules for safe practice of the POP technique. This study will impact clinicians as many may be tempted to use the technique in hope to resolve the occlusion safely, in a timely manner.

Structure-Based Design and Synthesis of Piperidinol-Containing Molecules as New Mycobacterium abscessus Inhibitors.

Non-tuberculous mycobacterium (NTM) infections, such as those caused by Mycobacterium abscessus, are increasing globally. Due to their intrinsic drug resistance, M. abscessus pulmonary infections are often difficult to cure using standard chemotherapy. We previously demonstrated that a piperidinol derivative, named PIPD1, is an efficient molecule both against M. abscessus and Mycobacterium tuberculosis, the agent of tuberculosis, by targeting the mycolic acid transporter MmpL3. These results prompted us to design and synthesize a series of piperidinol derivatives and to determine the biological activity against M. abscessus. Structure-activity relationship (SAR) studies pointed toward specific sites on the scaffold that can tolerate slight modifications. Overall, these results identified FMD-88 as a new promising active analogue against M. abscessus. Also, we determined the pharmacokinetics properties of PIPD1 and showed that intraperitoneal administration of this compound resulted in promising serum concentration and an elimination half-life of 3.2 hours.

Cystic fibrosis in the modern therapeutic era: Give the shower a thought!

Fifty-two meshes of e-flow rapid® were characterized for tobramycin delivery with a laser diffractometer after 6 months of home use by cystic fibrosis patients treated with various nebulized drugs. Three meshes were out of order and 30 considered to be defective for tobramycin delivery. The use of the specific mesh cleaning shower system permitted 14 defective meshes to be in the expected range of nebulized volume.

CFTR Protects against Mycobacterium abscessus Infection by Fine-Tuning Host Oxidative Defenses.

Infection by rapidly growing Mycobacterium abscessus is increasingly prevalent in cystic fibrosis (CF), a genetic disease caused by a defective CF transmembrane conductance regulator (CFTR). However, the potential link between a dysfunctional CFTR and vulnerability to M. abscessus infection remains unknown. Herein, we exploit a CFTR-depleted zebrafish model, recapitulating CF immuno-pathogenesis, to study the contribution of CFTR in innate immunity against M. abscessus infection. Loss of CFTR increases susceptibility to infection through impaired NADPH oxidase-dependent restriction of intracellular growth and reduced neutrophil chemotaxis, which together compromise granuloma formation and integrity. As a consequence, extracellular multiplication of M. abscessus expands rapidly, inducing abscess formation and causing lethal infections. Because these phenotypes are not observed with other mycobacteria, our findings highlight the crucial and specific role of CFTR in the immune control of M. abscessus by mounting effective oxidative responses.

Lsr2 Is an Important Determinant of Intracellular Growth and Virulence in Mycobacterium abscessus.

Mycobacterium abscessus, a pathogen responsible for severe lung infections in cystic fibrosis patients, exhibits either smooth (S) or rough (R) morphotypes. The S-to-R transition correlates with inhibition of the synthesis and/or transport of glycopeptidolipids (GPLs) and is associated with an increase of pathogenicity in animal and human hosts. Lsr2 is a small nucleoid-associated protein highly conserved in mycobacteria, including M. abscessus, and is a functional homolog of the heat-stable nucleoid-structuring protein (H-NS). It is essential in Mycobacterium tuberculosis but not in the non-pathogenic model organism Mycobacterium smegmatis. It acts as a master transcriptional regulator of multiple genes involved in virulence and immunogenicity through binding to AT-rich genomic regions. Previous transcriptomic studies, confirmed here by quantitative PCR, showed increased expression of lsr2 (MAB_0545) in R morphotypes when compared to their S counterparts, suggesting a possible role of this protein in the virulence of the R form. This was addressed by generating lsr2 knock-out mutants in both S (Δlsr2-S) and R (Δlsr2-R) variants, demonstrating that this gene is dispensable for M. abscessus growth. We show that the wild-type S variant, Δlsr2-S and Δlsr2-R strains were more sensitive to H2O2 as compared to the wild-type R variant of M. abscessus. Importantly, virulence of the Lsr2 mutants was considerably diminished in cellular models (macrophage and amoeba) as well as in infected animals (mouse and zebrafish). Collectively, these results emphasize the importance of Lsr2 in M. abscessus virulence.

A Simple and Rapid Gene Disruption Strategy in Mycobacterium abscessus: On the Design and Application of Glycopeptidolipid Mutants.

Little is known about the disease-causing genetic determinants that are used by Mycobacterium abscessus, increasingly acknowledged as an important emerging pathogen, notably in cystic fibrosis. The presence or absence of surface exposed glycopeptidolipids (GPL) conditions the smooth (S) or rough (R) M. abscessus subsp. abscessus (M. abscessus) variants, respectively, which are characterized by distinct infective programs. However, only a handful of successful gene knock-out and conditional mutants have been reported in M. abscessus, testifying that genetic manipulation of this mycobacterium is difficult. To facilitate gene disruption and generation of conditional mutants in M. abscessus, we have designed a one-step single cross-over system that allows the rapid and simple generation of such mutants. Cloning of as small as 300 bp of the target gene allows for efficient homologous recombination to occur without additional exogenous recombination-promoting factors. The presence of tdTomato on the plasmids allows easily sifting out the large background of mutants spontaneously resistant to antibiotics. Using this strategy in the S genetic background and the target gene mmpL4a, necessary for GPL synthesis and transport, nearly 100% of red fluorescent clones exhibited a rough morphotype and lost GPL on the surface, suggesting that most red fluorescent colonies obtained after transformation incorporated the plasmid through homologous recombination into the chromosome. This system was further exploited to generate another strain with reduced GPL levels to explore how the presence of these cell wall-associated glycolipids influences M. abscessus hydrophobicity as well as virulence in the zebrafish model of infection. This mutant exhibited a more pronounced killing phenotype in zebrafish embryos compared to its S progenitor and this effect correlated with the production of abscesses in the central nervous system. Overall, these results suggest that the near-complete absence of GPL on the bacterial surface is a necessary condition for optimal pathogenesis of this mycobacterium. They also suggest that GPL content affects hydrophobicity of M. abscessus, potentially altering the aerosol transmission, which is of particular importance from an epidemiological and clinical perspective.

Does repeated disinfection of the e-Flow rapid nebulizer affect in vitro performance?

Repeated disinfection of a nebulizer may modify its performance. During 60 cycles of disinfection with the Nuk steam sterilizer (120 nebulizations), the median volume diameter of tobramycin or colistin, nebulization time and various parameters correlated with the membrane weight of the e-Flow rapid are not modified. Results are similar when tap or purified water is used for washing and disinfection.

1H-1,2,3-triazole-tethered uracil-ferrocene and uracil-ferrocenylchalcone conjugates: Synthesis and antitubercular evaluation.

Copper-catalyzed azide-alkyne [3 + 2] cycloaddition has been utilized for preparing a series of 1H-1,2,3-triazoles with the purpose of probing structure-activity relationships among a uracil-ferrocene-triazole conjugate family. The antitubercular evaluation studies revealed an improvement in activity with the introduction of a ferrocene nucleus among N-alkylazido-uracil precursors, with a preference for a bromo-substituent along with moderate chain lengths of n = 2-6. The reported protocol is a successful approach for integrating uracil-ferrocene-chalcone functionalities tethered via 1H-1,2,3-triazole rings with apparent physicochemical stability.

Targeting Mycolic Acid Transport by Indole-2-carboxamides for the Treatment of Mycobacterium abscessus Infections.

Mycobacterium abscessus is a fast-growing, multidrug-resistant organism that has emerged as a clinically significant pathogen in cystic fibrosis (CF) patients. The intrinsic resistance of M. abscessus to most commonly available antibiotics seriously restricts chemotherapeutic options. Herein, we report the potent activity of a series of indolecarboxamides against M. abscessus. The lead compounds, 6 and 12, exhibited strong activity in vitro against a wide panel of M. abscessus isolates and in infected macrophages. High resistance levels to the indolecarboxamides appear to be associated with an A309P mutation in the mycolic acid transporter MmpL3. Biochemical analyses demonstrated that while de novo mycolic acid synthesis remained unaffected, the indolecarboxamides strongly inhibited the transport of trehalose monomycolate, resulting in the loss of trehalose dimycolate production and abrogating mycolylation of arabinogalactan. Our data introduce a hereto unexploited chemical structure class active against M. abscessus infections with promising translational development possibilities for the treatment of CF patients.

Impact of the shape of the needle tip on the efficacy of flushing implantable port chambers.

INTRODUCTION: To follow international guidelines and protect healthcare workers from needle stick injuries, manufacturers now provide safety non-coring needles. METHODS: Numerical simulations show the potential efficacy of infusion and flushing flow in straight and curved side wall port chambers from a beveled non-coring needle (BNCN) or a bevel-free cannula (BFC). RESULTS: In straight and curved side wall port chambers, the stream of flow with a BNCN is determined by the direction of the bevel, whereas the outlet stream from a BFC leads to a cylindrical symmetric flow. This, along with the position of the BFC impacts the wall shear rate of the port chamber, and the hydraulic resistance within the chamber. DISCUSSION: This last factor has never been the subject of extensive hydrodynamic study. Caregiver safety and patient comfort are paramount but physics show that the shape of the needle tip affects the hydrodynamics of the injected flow. CONCLUSIONS: The presence of a bevel at the needle tip is a key factor for ensuring efficient flushing of the port. The absence of a bevel may increase hydraulic resistance and deposits in the port chamber. Implantable port flushing protocols must take this notion into account if the patency of port chambers is to be maintained. The clinical impact of this study has yet to be determined.

[Long-term intravenous devices, continuity of care from the specialist doctor to the nurse]. / Gestion des risques des dispositifs intraveineux de longue durée.

Long-term intravenous devices have become an essential tool in the treatment of patients requiring the administration of medication over more than one month. The choice of device must be established through consultation between the nursing team and the patient. Several types of complications can be observed immediately after the insertion of the device and during treatment. Protocols in the community and at hospital are therefore necessary in order that doctors, nurses, specialists and experts work together. Clinical and translational research should enable the incidence of certain complications, notably infections and thrombosis, to be further reduced.