Acinetobacter baumannii is a bacterium classified as 'Critical' by the World Health Organization due to the rapid development of antibiotic resistance leading to many therapeutic failures. However, the existence of persister bacterial subpopulations may also partly explain these therapeutic failures. Persister cells are a bacterial subpopulation that may survive to higher concentrations of antibiotics compared to isogenic individuals. This temporary and reversible phenotype may eventually lead to the development of new antibiotic resistance or to the recurrence of infection making it important to study. The mechanisms involved in the selection of persister cells are multiple and complex involving toxin/antitoxin systems, second messengers, the SOS response, the phenyl acetic catabolic pathway or membranes modifications. Some of these mechanisms, like toxin/antitoxin systems presenting a great diversity, appear to be species- or strain-specific. This review focuses on current advances in knowledge concerning the molecular mechanisms involved in the physiology of persister cells in A. baumannii, and the eradication strategies developed to combat this dormant subpopulation.

Persister cells constitute a bacterial subpopulation able to survive to high concentrations of antibiotics. This phenotype is temporary and reversible, and thus could be involved in the recurrence of infections and emergence of antibiotic resistance. To better understand how persister cells survive to such high antibiotic concentration, we examined changes in their lipid composition. We thus compared the lipidome of Acinetobacter baumannii ATCC 19606T persister cells formed under ciprofloxacin treatment with the lipidome of control cells grown without antibiotic. Using matrix assisted laser desorption ionisation-Fourier transform ion cyclotron resonance mass spectrometry, we observed a higher abundance of short chains and secondary chains without hydroxylation for lipid A in persister cells. Using liquid chromatography-tandem mass spectrometry, we found that persister cells produced particular phosphatidylglycerols, as LPAGPE and PAGPE, but also lipids with particular acyl chains containing additional hydroxyl group or uncommon di-unsaturation on C18 and C16 acyl chains. In order to determine the impact of these multiple lipidome modifications on membrane fluidity, fluorescence anisotropy assays were performed. They showed an increase of rigidity for the membrane of persister cells, inducing likely a decrease membrane permeability to protect cells during dormancy. Finally, we highlighted that A. baumannii persister cells also produced particular wax esters, composed of two fatty acids and a fatty diol. These uncommon storage lipids are key metabolites allowing a rapid bacterial regrow when antibiotic pressure disappears. These overall changes in persister lipidome may constitute new therapeutic targets to combat these particular dormant cells.

Acinetobacter baumannii, classified as priority number one by the World Health Organization (WHO), is an opportunistic pathogen responsible for infection and is able to develop antibiotic resistance easily. Membranes are bacteria's first line of defense against external aggression, such as antibiotics. A chemical modification of a lipid family or a change in lipid composition can lead to resistance to antibiotics. In this work, we analyzed different A. baumannii strains from various environments with different antibiotic resistance profiles, using matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR MS). This study shows that it is possible to describe the main lipidome (phospholipids and lipid A) from the simple preparation of lysed cells, and that despite the complexity of the mixture. This ultra-high resolution mass spectrometry technique enables the separation of isobaric ion, to report a new class of lipids. Given its performance, this technique can be used to quickly and reliably characterize the lipidome of clinical strains from different environments. (c) 2024 Elsevier B.V. and Soci & eacute;t & eacute; Fran & ccedil;aise de Biochimie et Biologie Mol & eacute;culaire (SFBBM). All rights are reserved, including those for text and data mining, AI training, and similar technologies.

Bio-based multilayer films were prepared by using the innovative nanolayer coextrusion process to produce films with a number of alternating layers varying from 3 to 2049. For the first time, a semicrystalline polymer was confined by another semicrystalline polymer by nanolayering in order to develop high barrier polyamide (PA11)/polylactic acid (PLA) films without compromising thermal stability and mechanical behavior. This process allows the preparation of nanostratified films with thin layers (down to nanometric thicknesses) in which a confinement effect can be induced. The stratified structure has been investigated, and the layer thicknesses have been measured. Barrier properties were successfully correlated to the microstructure, as well as the thermal behavior, and mechanical properties. The layer continuity was fully achieved for most of the films, but some layer breakups have been observed on the film with the thinnest PLA layer (2049-layers film). Coextruding PLA with PA11 has induced an increase in PLA crystallinity (from 4 to 16%) along with an increase in thermal stability of the multilayer films without impacting PA11 properties. Gas barrier properties were driven by the PLA confined layers due to the microstructural rearrangement by increasing crystallinity, whereas water barrier properties were governed by the PA11 confining layers due to its lower water affinity. As a consequence, a decrease of water permeability (up to 11 times less permeable for the 6M film) but an increase of gas barrier properties (barrier improvement factor (BIF) of 66% for the 0M film for N2 and BIF of 36% for the 6M film for CO2 for instance) were evidenced as the layer number was increased. This study paves the way for the development of ecofriendly materials with outstanding barrier performances and highlights the importance of nonmiscible polymers adhesion at melt state and additives presence.

Identification of the molecular mechanisms governing neuroendocrine secretion and resulting intercellular communication is one of the great challenges of cell biology to better understand organism physiology and neurosecretion disruption-related pathologies such as hypertension, neurodegenerative, or metabolic diseases. To visualize molecule distribution and dynamics at the nanoscale, many imaging approaches have been developed and are still emerging. In this review, we provide an overview of the pioneering studies using transmission electron microscopy, atomic force microscopy, total internal reflection microscopy, and super-resolution microscopy in neuroendocrine cells to visualize molecular mechanisms driving neurosecretion processes, including exocytosis and associated fusion pores, endocytosis and associated recycling vesicles, and protein-protein or protein-lipid interactions. Furthermore, the potential and the challenges of these different advanced imaging approaches for application in the study of neuroendocrine cell biology are discussed, aiming to guide researchers to select the best approach for their specific purpose around the crucial but not yet fully understood neurosecretion process.

Pseudomonas aeruginosa is an opportunistic pathogen highly resistant to a wide range of antimicrobial agents, making its infections very difficult to treat. Since microorganisms need to perpetually adapt to their surrounding environment, understanding the effect of carbon sources on P. aeruginosa physiology is therefore essential to avoid increasing drug-resistance and better fight this pathogen. By a global proteomic approach and phenotypic assays, we investigated the impact of various carbon source supplementations (glucose, glutamate, succinate, and citrate) on the physiology of the P. aeruginosa PA14 strain. A total of 581 proteins were identified as differentially expressed in the 4 conditions. Most of them were more abundant in citrate supplementation and were involved in virulence, motility, biofilm development, and antibiotic resistance. Phenotypic assays were performed to check these hypotheses. By coupling all this data, we highlight the importance of the environment in which the bacterium evolves on its metabolism, and thus the necessity to better understand the metabolic pathways implied in its adaptative response according to the nutrient availability.

N-[2-(acryloyloxy)ethyl]-N,N-dimethyl-N-butylammonium iodide was successfully photo-polymerized from native and thiolated PDMS surface, in the presence of benzophenone. The directly and indirectly grafted surfaces exhibited quaternary ammonium densities of about 1015 and 1017 N+.cm−2, respectively, and very high hydrophilicity compared to non-grafted surfaces. The live and dead tests performed by fluorescence microscopy revealed an effective contact bactericidal effect of this surface against Escherichia coli and Staphylococcus epidermidis.

Vinylbenzyl dimethylbutylammonium chloride was successfully grafted and photo-polymerized from a PDMS surface, in presence of benzophenone. The obtained surface exhibited quaternary ammonium density above 10(17) charge/cm(2) and very high hydmphilicity compared to ungrafted surfaces. Bacterial enumeration and fluorescence microscopy revealed an efficient contact killing of this surface against Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis.

Poly(dimethyl siloxane) (PDMS) is one of the most widely used materials in the biomedical field, but due to its hydrophobic character it is prone to bacterial adhesion and biofilm formation. Prevention of bacterial adhesion by contact-killing surface is one of the promising strategies, although bacterial resistance to conventional active molecules complicates antibacterial control. A prepared PDMS surface with alkyne groups was covalently grafted with an azidated claramine-derivative, through the simple and orthogonal CuI-catalyzed Huisgen 1,3-dipolar click cycloaddition. The azidated claramine derivative was prepared in a four-step synthesis pathway from deoxycholic acid. The alkyne groups were introduced onto the PDMS surface via a silanization reaction. X-Ray Photolectron Spectroscopy (XPS) analysis has demonstrated the covalent grafting of the claramine with a total conversion of the azide group into a triazole ring. Fluorescent microscopy has showed an antibacterial activity of the modified surface against both Gram – (Escherichia coli) and Gram + (Staphyloccocus epidermidis) bacteria, which could be derived from electrostatic attraction followed by a membrane destabilization. These results confirmed the importance of using a chemioselective reaction to control the orientation of covalently immobilized antibacterial molecules in order to keep their activity. PDMS surfaces based on claramine-derivative can be potentially useful for the elaboration of biomaterials preventing biofilm formation and addressing the issue of antibacterial resistance.

The skin constitutes with its microbiota the first line of body defense against exogenous stress including air pollution. Especially in urban or sub-urban areas, it is continuously exposed to many environmental pollutants including gaseous nitrogen dioxide (gNO2). Nowadays, it is well established that air pollution has major effects on the human skin, inducing various diseases often associated with microbial dysbiosis. However, very few is known about the impact of pollutants on skin microbiota. In this study, a new approach was adopted, by considering the alteration of the cutaneous microbiota by air pollutants as an indirect action of the harmful molecules on the skin. The effects of gNO2 on this bacterial skin microbiota was investigated using a device developed to mimic the real-life contact of the gNO2 with bacteria on the surface of the skin. Five strains of human skin commensal bacteria were considered, namely Staphylococcus aureus MFP03, Staphylococcus epidermidis MFP04, Staphylococcus capitis MFP08, Pseudomonas fluorescens MFP05, and Corynebacterium tuberculostearicum CIP102622. Bacteria were exposed to high concentration of gNO2 (10 or 80 ppm) over a short period of 2 h inside the gas exposure device. The physiological, morphological, and molecular responses of the bacteria after the gas exposure were assessed and compared between the different strains and the two gNO2 concentrations. A highly significant deleterious effect of gNO2 was highlighted, particularly for S. capitis MFP08 and C. tuberculostearicum CIP102622, while S. aureus MFP03 seems to be the less sensitive strain. It appeared that the impact of this nitrosative stress differs according to the bacterial species and the gNO2 concentration. Thus the exposition to gNO2 as an air pollutant could contribute to dysbiosis, which would affect skin homeostasis. The response of the microbiota to the nitrosative stress could be involved in some pathologies such as atopic dermatitis.

Chromogranin A (CgA) is a key luminal actor of secretory granule biogenesis at the trans-Golgi network (TGN) level but the molecular mechanisms involved remain obscure. Here, we investigated the possibility that CgA acts synergistically with specific membrane lipids to trigger secretory granule formation. We show that CgA preferentially interacts with the anionic glycerophospholipid phosphatidic acid (PA). In accordance, bioinformatic analysis predicted a PA-binding domain (PABD) in CgA sequence that effectively bound PA (36:1) or PA (40:6) in membrane models. We identified PA (36:1) and PA (40:6) as predominant species in Golgi and granule membranes of secretory cells, and we found that CgA interaction with these PA species promotes artificial membrane deformation and remodeling. Furthermore, we demonstrated that disruption of either CgA PABD or phospholipase D (PLD) activity significantly alters secretory granule formation in secretory cells. Our findings show for the first time the ability of CgA to interact with PLD-generated PA, which allows membrane remodeling and curvature, key processes necessary to initiate secretory granule budding.

BACKGROUND:Pseudomonas aeruginosa is a bacterium able to induce serious pulmonary infections in cystic fibrosis (CF) patients. This bacterium is very often antibiotic resistant, partly because of its membrane impermeability, which is linked to the membrane lipid composition. This work aims to study the membrane phospholipids of P. aeruginosa grown in CF sputum-like media.METHODS:Three media were used: Mueller Hilton broth (MHB), synthetic cystic fibrosis medium (SCFM) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) complemented SCFM (SCFM-PC). Lipids were extracted and LC-MS/MS analyses were performed. Growth curves, atomic force microscopy images and minimal inhibitory concentration determination were performed in order to compare the growth and potentially link lipid modifications to antibiotic resistance.RESULTS:Semi-quantification showed phospholipid quantity variation depending on the growth medium. Phosphatidylcholines were detected in traces in SCFM. MS/MS experiments showed an increase of phospholipids derived from DOPC in SCFM-PC. We observed no influence of the medium on the bacterial growth and a minor influence on the bacterial shape. MIC values were generally higher in SCFM and SCFM-PC than in MHB.CONCLUSIONS:We defined a CF sputum-like media which can be used for the membrane lipid extraction of P. aeruginosa. We also showed that the growth medium does have an influence on its membrane lipid composition and antibiotic resistance, especially for SCFM-PC in which P. aeruginosa uses DOPC, in order to make its own membrane.GENERAL SIGNIFICANCE:Our results show that considerable caution must be taken when choosing a medium for lipid identification and antibiotic testing -especially for phospholipids-enriched media.

Collision cross section (CCS) values are descriptors of the 3D structure of ions which can be determined by ion mobility spectrometry (IMS). Currently, most lipidomic studies involving CCS value determination concern eukaryote samples (e.g. human, bovine) and to a lower extent prokaryote samples (e.g. bacteria). Here, we report CCS values obtained from traveling wave ion mobility spectrometry (TWCCSN2) measurements from the bacterial membrane of Pseudomonas aeruginosa-a bacterium ranked as priority 1 for the R&D of new antibiotics by the World Health Organization. In order to cover the lack of reference compounds which could cover the m/z and CCS ranges of the membrane lipids of P. aeruginosa, three calibrants (polyalanine, dextran and phospholipids) were used for the TWCCSN2 calibration. A shift from the published lipid CCS values was systematically observed (ΔCCS% up to 9%); thus, we proposed a CCS correction strategy. This correction strategy allowed a reduction in the shift (ΔCCS%) between our measurements and published values to less than 2%. This correction was then applied to determine the CCS values of Pseudomonas aeruginosa lipids which have not been published yet. As a result, 32 TWCCSN2 values for [M+H]+ ions and 24 TWCCSN2 values for [M-H]- ions were obtained for four classes of phospholipids (phosphatidylethanolamines (PE), phosphatidylcholines (PC), phosphatidylglycerols (PG) and diphosphatidylglycerols-known as cardiolipins (CL)). Graphical abstract.

The increasing threat of Acinetobacter baumannii as a nosocomial pathogen is mainly due to the occurrence of multidrug-resistant strains that are associated with the real problem of its eradication from hospital wards. The particular ability of this pathogen to form biofilms contributes to its persistence, increases antibiotic resistance, and promotes persistent/device-related infections. We previously demonstrated that virstatin, which is a small organic compound known to decrease virulence of Vibrio cholera via an inhibition of T4-pili expression, displayed very promising activity to prevent A. baumannii biofilm development. Here, we examined the antibiofilm activity of mono-unsaturated chain fatty acids, palmitoleic (PoA), and myristoleic (MoA) acids, presenting similar action on V. cholerae virulence. We demonstrated that PoA and MoA (at 0.02 mg/mL) were able to decrease A. baumannii ATCC 17978 biofilm formation up to 38% and 24%, respectively, presented a biofilm dispersing effect and drastically reduced motility. We highlighted that these fatty acids decreased the expression of the regulator abaR from the LuxIR-type quorum sensing (QS) communication system AbaIR and consequently reduced the N-acyl-homoserine lactone production (AHL). This effect can be countered by addition of exogenous AHLs. Besides, fatty acids may have additional non-targeted effects, independent from QS. Atomic force microscopy experiments probed indeed that PoA and MoA could also act on the initial adhesion process in modifying the material interface properties. Evaluation of fatty acids effect on 22 clinical isolates showed a strain-dependent antibiofilm activity, which was not correlated to hydrophobicity or pellicle formation ability of the tested strains, and suggested a real diversity in cell-to-cell communication systems involved in A. baumannii biofilm formation.

RATIONALE:Pseudomonas aeruginosa is an opportunistic pathogen bacterium widely considered to be an excellent research model in several areas of molecular studies, namely genomics and proteomics. However, its lipid metabolism is still not totally decrypted. While it is known that this bacterium has the particularity to produce phosphatidylcholine, a lipid mainly found in eukaryotes, other singularities are still to be discovered.METHODS:P. aeruginosa was grown as planktonic cultures to the stationary state. Membrane pellets were collected and lipids were extracted using the Bligh and Dyer protocol. Lipid extracts were analyzed by Electrospray Ionization Mass Spectrometry (ESI-MS) using high-resolution mass spectrometer (LTQ Orbitrap Elite, Thermo Scientific) in the negative mode. MSn spectra were recorded both in the Orbitrap and in the ion trap analyzer (collision-induced dissociation (CID) or higher energy collision-induced dissociation (HCD) mode).RESULTS:We observed by mass spectrometry and thin layer chromatography that P. aeruginosa produced an unreferenced lipid in classical growth conditions. MS2 analysis of the unknown ion indicates that it is a phosphatidylglycerol derivative. The exact mass shift corresponds to glucosamine which is largely found in the metabolism of this bacterium. MS3 analysis of secondary ions allowed us to conclude that this lipid is a glucosaminylphosphatidylglycerol, a phosphatidylglycerol derivative containing a glucosamine substituted at C4.CONCLUSIONS:We show here that P. aeruginosa is able to produce glucosaminylphosphatidylglycerols via a probable esterification of phosphatidylglycerols by glucosamine.

For optimal growth of a microorganism, the pH of the culture medium should be set at an optimum value. For that reason, growth media require buffering agents. We show in this study that, when grown in a medium supplemented with tris(hydroxymethyl)aminomethane (Tris), Pseudomonas aeruginosa is able to use this organic compound to produce new phospholipids. We thus pointed out that phosphatidyltris(hydroxymethyl)aminomethane as well as diphosphatidyltris(hydroxymethyl)aminomethane was detected in membrane lipid extracts of bacteria grown in Tris-buffered medium. Moreover, the amounts of lysoglycerophospholipids in the lipidome of P. aeruginosa grown in Tris-buffered medium increased leading to the presence of lysophosphatidylglycerol and lysophosphatidyltris(hydroxymethyl)aminomethane as well as other lysophospholipid derivatives. Finally, we investigated the effect of the presence of these exogenous phospholipids on the susceptibility of P. aeruginosa to some antibiotics. We observed a decrease of the minimal inhibitory concentrations of different antibiotic families, i.e., fluoroquinolones, aminoglycosides, ß-lactams and polymyxins, proving the importance of the buffer choice for growth medium and its impact on the lipidome.

Acinetobacter baumannii has emerged as an opportunistic nosocomial pathogen causing infections worldwide. One reason for this emergence is due to its natural ability to survive in the hospital environment, which may be explained by its capacity to form biofilms. Cell surface appendages are important determinants of the A. baumannii biofilm formation and as such constitute interesting targets to prevent the development of biofilm-related infections. A chemical agent called virstatin was recently described to impair the virulence of Vibrio cholerae by preventing the expression of its virulence factor, the toxin coregulated pilus (type IV pilus). The objective of this work was to investigate the potential effect of virstatin on A. baumannii biofilms.

The clinical importance of Acinetobacter baumannii is partly due to its natural ability to survive in the hospital environment. This persistence may be explained by its capacity to form biofilms and, interestingly, A. baumannii can form pellicles at the air-liquid interface more readily than other less pathogenic Acinetobacter species. Pellicles from twenty-six strains were morphologically classified into three groups: I) egg-shaped (27%); II) ball-shaped (50%); and III) irregular pellicles (23%). One strain representative of each group was further analysed by Brewster's Angle Microscopy to follow pellicle development, demonstrating that their formation did not require anchoring to a solid surface. Total carbohydrate analysis of the matrix showed three main components: Glucose, GlcNAc and Kdo. Dispersin B, an enzyme that hydrolyzes poly-N-acetylglucosamine (PNAG) polysaccharide, inhibited A. baumannii pellicle formation, suggesting that this exopolysaccharide contributes to pellicle formation. Also associated with the pellicle matrix were three subunits of pili assembled by chaperon-usher systems: the major CsuA/B, A1S_1510 (presented 45% of identity with the main pilin F17-A from enterotoxigenic Escherichia coli pili) and A1S_2091. The presence of both PNAG polysaccharide and pili systems in matrix of pellicles might contribute to the virulence of this emerging pathogen.