Thierry Jouenne

Antibacterial surfaces developed from bio-inspired approaches.

Prevention of bacterial adhesion and biofilm formation on the surfaces of materials is a topic of major medical and societal importance. Various synthetic approaches based on immobilization or release of bactericidal substances such as metal derivatives, polyammonium salts and antibiotics were extensively explored to produce antibacterial coatings. Although providing encouraging results, these approaches suffer from the use of active agents which may be associated with side-effects such as cytotoxicity, hypersensibility, inflammatory responses or the progressive alarming phenomenon of antibiotic resistance. In addition to these synthetic approaches, living organisms, e.g. animals and plants, have developed fascinating strategies over millions of years to prevent efficiently the colonization of their surfaces by pathogens. These strategies have been recently mimicked to create a new generation of bio-inspired biofilm-resistant surfaces. In this review, we discuss some of these bio-inspired methods devoted to the development of antibiofilm surfaces. We describe the elaboration of antibacterial coatings based on natural bactericidal substances produced by living organisms such as antimicrobial peptides, bacteriolytic enzymes and essential oils. We discuss also the development of layers mimicking algae surfaces and based on anti-quorum-sensing molecules which affect cell-to-cell communication. Finally, we report on very recent strategies directly inspired from marine animal life and based on surface microstructuring.

Antibacterial and Antifouling Polymer Brushes Incorporating Antimicrobial Peptide

Surface-initiated atom transfer radical polymerization (ATRP) has been used to prepare antifouling copolymer brushes based on 2-(2-methoxyethoxy)ethyl methacrylate (MEO(2)MA) and hydroxyl-terminated oligo(ethylene glycol) methacrylate (HOEGMA). The amount of hydroxyl reactive groups incorporated into the brushes was varied by changing the composition of the monomer mixture. These coatings were subsequently functionalized by a natural antibacterial peptide, magainin I, via an oriented chemical grafting on hydroxyl groups, which maintains the activity of the peptide. The antibacterial activity of the functionalized brushes was successfully tested against two different strains of gram-positive bacteria.

Temperature-Responsive Polymer Brushes Switching from Bactericidal to Cell-Repellent

Materials exhibiting antibacterial properties at room temperature and turning biocompatible and non-adhesive for in vivo conditions, are extremely attractive for devices that have to be ultimately introduced in living beings. Indeed, infections related to the use of invasive biomedical and medical items are still one of the main medical complications that cause high rates of mortality. [ 1 ] Despite sanitation protocols, a well-identifi ed route for patient bacterial infection is transmission through contaminated instruments such as intubation tubes, catheters, surgical drains or endoscopes that bypass the natural protective barriers of the body. [ 1 ]

Isolation, characterization, and distribution of a novel neuropeptide, Rana RFamide (R-RFa), in the Brain of the european green frog Rana esculenta

A novel neuropeptide of the RFamide peptide family was isolated in pure form from a frog (Rana esculenta) brain extract by using reversed‐phase high performance liquid chromatography in combination with a radioimmunoassay for mammalian neuropeptide FF (NPFF). The primary structure of the peptide was established as Ser‐Leu‐Lys‐ Pro‐Ala‐Ala‐Asn‐Leu‐Pro‐Leu‐ Arg‐Phe‐NH2. The sequence of this neuropeptide, designated Rana RFamide (R‐RFa), exhibits substantial similarities with those of avian LPLRFamide, gonadotropin‐inhibitory hormone, and human RFRP‐1. The distribution of R‐RFa was investigated in the frog central nervous system by using an antiserum directed against bovine NPFF. In the brain, immunoreactive cell bodies were primarily located in the hypothalamus, i.e., the anterior preoptic area, the suprachiasmatic nucleus, and the dorsal and ventral hypothalamic nuclei. The most abundant population of R‐RFa–containing neurons was found in the periependymal region of the suprachiasmatic nucleus. R‐RFa– containing fibers were widely distributed throughout the brain from the olfactory bulb to the brainstem, and were particularly abundant in the external layer of the median eminence. In the spinal cord, scattered immunoreactive neurons were found in the gray matter. R‐RFa–positive processes were found in all regions of the spinal cord, but they were more abundant in the dorsal horn. This study provides the first characterization of a member of the RFamide peptide family in amphibians. The occurrence of this novel neuropeptide in the hypothalamus and median eminence and in the dorsal region of the spinal cord suggests that, in frog, R‐RFa may exert neuroendocrine activities and/or may be involved in the transmission of nociceptive stimuli. J. Comp. Neurol. 448:111–127, 2002.

Channel Formation by CarO, the Carbapenem Resistance-Associated Outer Membrane Protein of Acinetobacter baumannii

ABSTRACT It has been recently shown that resistance to both imipenem and meropenem in multidrug-resistant clinical strains of Acinetobacter baumannii is associated with the loss of a heat-modifiable 25/29-kDa outer membrane protein, called CarO. This study aimed to investigate the channel-forming properties of CarO. Mass spectrometry analyses of this protein band detected another 25-kDa protein (called Omp25), together with CarO. Both proteins presented similar physicochemical parameters (Mw and pI). We overproduced and purified the two polypeptides as His-tagged recombinant proteins. Circular dichroism analyses demonstrated that the secondary structure of these proteins was mainly a β-strand conformation with spectra typical of porins. We studied the channel-forming properties of proteins by reconstitution into artificial lipid bilayers. In these conditions, CarO induced ion channels with a conductance value of 110 pS in 1 M KCl, whereas the Omp25 protein did not form any channels, despite its suggested porin function. The pores formed by CarO showed a slight cationic selectivity and no voltage closure. No specific imipenem binding site was found in CarO, and this protein would rather form unspecific monomeric channels.

Characterization of bacterial biofilms formed on urinary catheters

BACKGROUND The formation of bacterial biofilms on urinary catheters is a leading cause of urinary tract infections in intensive care units. Cytobacteriological examination of urine from patients is often misleading, due to the formation of these biofilms. Therefore, characterizing these biofilms and identifying the bacterial species residing on the surface of catheters are of major importance. METHODS We studied the formation of biofilms on the inner surface of urinary catheters using microbiological culture techniques, with the direct contact of catheter pieces with blood agar. The bacterial species on the surface were characterized by scanning electron microscopy, and the kinetic profile of biofilm formation on a silicone substrate for an imipenem-resistant Acinetobacter baumannii bacterium was evaluated with a crystal violet staining assay. RESULTS The bacterial species that constituted these biofilms were identified as a variety of gram-negative bacilli, with a predominance of strains belonging to Pseudomonas aeruginosa. The other isolated strains belonged to A baumannii and Klebsiella ornithinolytica. Kinetic profiling of biofilm formation identified the transient behavior of A baumannii between its biofilm and planktonic state. This strain was highly resistant to all of the antibiotics tested except colistin. Scanning electron microscopy images showed that the identified isolated species formed a dense and interconnected network of cellular multilayers formed from either a single cell or from different species that were surrounded and enveloped by a protective matrix. CONCLUSIONS Microbiological analysis of the intraluminal surface of the catheter is required for true identification of the causative agents of catheter-associated urinary tract infections. This approach, combined with a routine cytobacteriological examination of urine, allows for the complete characterization of biofilm-associated species, and also may help prevent biofilm formation in such devices and help guide optimum antibiotic treatment.

A combined 15N tracing/proteomics study in Brassica napus reveals the chronology of proteomics events associated with N remobilisation during leaf senescence induced by nitrate limitation or starvation

Our goal was to identify the leaf proteomic changes which appeared during N remobilisation that were associated or not associated with senescence of oilseed rape in response to contrasting nitrate availability. Remobilisation of N and leaf senescence status were followed using 15N tracing, patterns of chlorophyll level, total protein content and a molecular indicator based on expression of senescence‐associated gene 12/Cab genes. Three phases associated with N remobilisation were distinguished. Proteomics revealed that 55 proteins involved in metabolism, energy, detoxification, stress response, proteolysis and protein folding, were significantly induced during N remobilisation. Four proteases were specifically identified. FtsH, a chloroplastic protease, was induced transiently during the early stages of N remobilisation. Considering the dynamics of N remobilisation, chlorophyll and protein content, the pattern of FtsH expression indicated that this protease could be involved in the degradation of chloroplastic proteins. Aspartic protease increased at the beginning of senescence and was maintained at a high level, implicating this protease in proteolysis during the course of leaf senescence. Two proteases, proteasome beta subunit A1 and senescence‐associated gene 12, were induced and continued to increase during the later phase of senescence, suggesting that these proteases are more specifically involved in the proteolysis processes occurring at the final stages of leaf senescence.

Immobilized-cell physiology: current data and the potentialities of proteomics

Despite the sustained development of immobilized-cell (IC) technology over the last 20 years, our knowledge of IC physiology has remained for a long time in the shade of the technological advances involving IC systems. This review surveys the main physiological responses of artificially and naturally immobilized microorganisms and presents recent data on protein expression in ICs that support the existence of a specific metabolic behaviour of microbial cells in the immobilized state.

Adhesion of Yersinia ruckeri to fish farm materials: influence of cell and material surface properties

Abstract Two strains (an environmental strain and a reference one coming from a national culture collection) of Yersinia ruckeri , a fish pathogenic bacterium, are characterised according to the ability to adhere on wood, concrete, polyvinylchloride (PVC) and fibreglass, four materials commonly found in fish farms. The relationships between adherence, bacterial and support hydrophobicities and surface roughness are investigated. The results show that: (i) Y. ruckeri is strongly hydrophilic; (ii) the environmental strain exhibits a higher ability to adhere than the reference one; and (iii) for the two strains a strong correlation is observed between roughness amplitude (RA) of the support material and adhesion ability.

Growth of Acinetobacter baumannii in Pellicle Enhanced the Expression of Potential Virulence Factors

Background Interestingly, Acinetobacter baumannii presents an enhanced capacity to form biofilms (also named pellicles) at the air-liquid interface as compared to the other Acinetobacter species. This characteristic questions the contribution of this phenotype to an increased risk of clinical infections by this pathogen. Methodology/Principal Findings By a proteomic approach using 2-D gel electrophoresis-LC-MS/MS mass spectrometry, we compared the membrane protein patterns of A. baumannii 77, a pellicle-forming clinical isolate, grown in planktonic and in sessile modes. We identified 52 proteins with a differential expression, including 32 up-regulated and 20 down-regulated in the pellicle state. Several proteins, differentially expressed during pellicle development, were of particular interest. We determined the over-expression of four siderophore iron uptake systems including the acinetobactin and enterobactin receptors and confirmed that the development of this type of biofilm is promoted by ferric ions. Two over-expressed proteins, CarO and an OprD-homologue, putative carbapenem-resistance associated porins, would be involved in the transport of specific compounds, like ornithine, a biosynthesis precursor of a siderophore from the hydroxamate family. We evidenced the overexpression of a lipase and a transporter of LCFA that may be involved in the recycling of lipids inside the pellicle matrix. Finally, we demonstrated both by proteomic and by AFM studies that this particular type of biofilm required multiple pili systems to maintain this cohesive structure at the air-liquid interface; two of these systems have never been described in A. baumannii. Conclusions/Significance Our study demonstrated that several proteins, overexpressed at a late state of pellicle development, could be potentially involved in virulence processes. Therefore, regarding the number of potential virulence factors that are over-expressed in this growth mode, the pellicle-forming clinical isolates should be kept under survey.