Sessile Legionella pneumophila is able to grow on surfaces and generate structured monospecies biofilms

Currently, models for studying Legionella pneumophila biofilm formation rely on multi-species biofilms with low reproducibility or on growth in rich medium, where planktonic growth is unavoidable. The present study describes a new medium adapted to the growth of L. pneumophila monospecies biofilms in vitro. A microplate model was used to test several media. After incubation for 6 days in a specific biofilm broth not supporting planktonic growth, biofilms consisted of 5.36 ± 0.40 log (cfu cm−2) or 5.34 ± 0.33 log (gu cm−2). The adhered population remained stable for up to 3 weeks after initial inoculation. In situ confocal microscope observations revealed a typical biofilm structure, comprising cell clusters ranging up to 300 μm in height. This model is adapted to growing monospecies L. pneumophila biofilms that are structurally different from biofilms formed in a rich medium. High reproducibility and the absence of other microbial species make this model useful for studying genes involved in biofilm formation

Apport de modèles de biofilms à Pseudomonas aeruginosa et Legionella pneumophila à la maîtrise de la qualité microbiologique des réseaux d'eaux minérales naturelles

Le thermalisme est une médecine ancestrale utilisant les eaux minérales naturelles (EMN) qui concerne chaque année 500000 patients. Les canalisations d'eau des établissements thermaux pouvant être le support de biofilms, à l'origine de la libération de pathogènes potentiels dans l'EMN (dont Pseudomonas aeruginosa et Legionella pneumophila), les désinfections de ces réseaux doivent être fréquentes et efficaces. L'objectif de cette thèse est l'évaluation des traitements à disposition des exploitants, contre P. aeruginosa et L. pneumophila, en prenant en compte la capacité des bactéries à produire un biofilm et la spécificité des réseaux d'EMN. Des techniques in vitro et en conditions réelles de traitements (réseau-pilote) ont été testées. Les résultats montrent une perte de sensibilité de P . aeruginosa vis-à-vis des traitements sous forme biofilm et qui conduit à une efficacité partielle en réseau-pilote. Pour L. pneumophila, la majeure partie des travaux a consisté à mettre au point un modèle de formation de biofilms mono-espèce in vitro qui a permis de démontrer qu'un choc thermique éliminait les bactéries cultivables du biofilm. L'évolution de ce modèle permettra d'intégrer les paramètres conduisant à l'échec des traitements en réseaux d'EMN.Spa treatment is a traditional form of medicine using natural mineral waters, that concerns 500,000 patients each year in France. Water pipes are often colonized by biofilms that harbor potential pathogens like Pseudomonas aeruginosa and Legionella pneumophila. Hence, their disinfection must be frequent and effective. The objective of the thesis is the evaluation of treatments available to spa owners against P. aeruginosa and L. pneumophila, evaluating the capacity of these bacteria to form biofilms and the specific conditions found in spa buildings. In vitro and real conditions assays were used. Results show a loss of sensitivity to treatments of P. aeruginosa in biofilm form that leads to partial effectiveness in real condition assays. For L. pneumophila, most of the work consisted in designing an in vitro biofilm formation model. The use of this model showed that thermal disinfection destroys all cultivable bacteria. The evolution of the model will integrate parameters that explain the failure of disinfection treatments in natural mineral water systems

New insights into Legionella pneumophila biofilm regulation by c-di-GMP signaling

The waterborne pathogen Legionella pneumophila grows as a biofilm, freely or inside amoebae. Cyclic-di-GMP (c-di-GMP), a bacterial second messenger frequently implicated in biofilm formation, is synthesized and degraded by diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), respectively. To characterize the c-di-GMP-metabolizing enzymes involved in L. pneumophila biofilm regulation, the consequences on biofilm formation and the c-di-GMP concentration of each corresponding gene inactivation were assessed in the Lens strain. The results showed that one DGC and two PDEs enhance different aspects of biofilm formation, while two proteins with dual activity (DGC/PDE) inhibit biofilm growth. Surprisingly, only two mutants exhibited a change in global c-di-GMP concentration. This study highlights that specific c-di-GMP pathways control L. pneumophila biofilm formation, most likely via temporary and/or local modulation of c-di-GMP concentration. Furthermore, Lpl1054 DGC is required to enable the formation a dense biofilm in response to nitric oxide, a signal for biofilm dispersion in many other species

Biofilm colonizing the Nam Theun 2 power plant Penstock (Lao PDR) - mechanism and potential evolution

Biofilms are the most common bacterial life mode on Earth. These tri-dimension bacterial structures occur at a substratum-liquid interface. Due to their intrinsic properties(niche for pathogens, resistance to biocide treatments, etc.), they cause major problems in various industries. In water systems, the physical and chemical characteristics of biofilms (viscoelastic behavior, roughness) may lead to the lowering of flow velocity. A rough biofilm has developed in the Penstock of the Nam Theun 2 hydropower plant (Khammouane Province, Lao PDR). This biofilm is thought to lead to additional head losses and to slightly affect the power production. The mineral, chemical and microbiological compositions of the biofilm were investigated in order to propose solutions to reduce its effect. Samples were taken during two water drainages in 2011 and 2012. In order to complete the knowledge from the water quality monitoring, major elements, trace elements and rare earth element (REE) contents in samples were measured using ICP-AES and ICP-MS. Crystalline phases were identified and quantified by X-ray diffraction (XRD). The microbial composition of the biofilm was first assessed by culture (2011) and then monitored according to the location and the time after water drainage by molecular biology methods (2012). Results show that the chemical composition of the biofilm is dominated by ferric iron Fe3+ and its mineralogy is mostly constituted of lepidocrocite and magnesioferrite. The bacterial population was dominated by beta-Proteobacteria but population profiles varied strongly according to the layer of the biofilm, the nature of the substratum and the time during which the biofilm was subjected to the conditions of the water drainage. These observations are concordant with the modification of the biofilm properties and the reduction in head losses when returning to functioning regimes in the Penstoc

Dynamic Mechanisms of the Bactericidal Action of an Al2O3-TiO2-Ag Granular Material on an Escherichia coli Strain.

The bactericidal activity of an Al2O3-TiO2-Ag granular material against an Escherichia coli strain was confirmed by a culture-based method. In particular, 100% of microorganisms were permanently inactivated in 30 to 45 min. The present work aimed to investigate the mechanisms of the bactericidal action of this material and their dynamics on Escherichia coli using different techniques. Observations by transmission electron microscopy (TEM) at different times of disinfection revealed morphological changes in the bacteria as soon as they were put in contact with the material. Notably highlighted were cell membrane damage; cytoplasm detachment; formation of vacuoles, possibly due to DNA condensation, in association with regions exhibiting different levels of electron density; and membrane lysis. PCR and flow cytometry analyses were used to confirm and quantify the observations of cell integrity. The direct exposure of cells to silver, combined with the oxidative stress induced by the reactive oxygen species (ROS) generated, was identified to be responsible for these morphological alterations. From the first 5 min of treatment with the Al2O3-TiO2-Ag material, 98% of E. coli isolates were lysed. From 30 min, cell viability decreased to reach total inactivation, although approximately 1% of permeable E. coli cells and 1% of intact cells (10(5) genomic units·ml(-1)) were evidenced. This study demonstrates that the bactericidal effect of the material results from a synergic action of desorbed and supported silver. Supported silver was shown to generate the ROS evidenced

High refractive index in low metal content nanoplasmonic surfaces from self-assembled block copolymer thin films

Materials with a high and tunable refractive index are attractive for nanophotonic applications. In this contribution, we propose a straightforward fabrication technique of high-refractive index surfaces based on self-assembled nanostructured block copolymer thin films. The selective and customizable metal incorporation within out-of-plane polymer lamellae produces azimuthally isotropic metallic nanostructures of defined geometries, which were analysed using microscopy and small-angle X-ray scattering techniques. Variable-angle spectroscopic ellipsometry was used to relate the geometrical parameters of the metallic features and the resulting refractive index of the patterned surfaces. In particular, nanostructured gold patterns with a high degree of homogeneity and a gold content as low as 16 vol% reach a refractive index value of more than 3 in the visible domain. Our study thus demonstrates a new route for the preparation of high refractive index surfaces with a low metal content for optical applications

Protons accumulation during anodic phase turned to advantage for oxygen reduction during cathodic phase in reversible bioelectrodes

Reversible bioelectrodes were designed by alternating acetate and oxygen supply. It was demonstrated that the protons produced and accumulated inside the biofilm during the anodic phase greatly favored the oxygen reduction reaction when the electrode was switched to become the biocathode. Protons accumulation, which hindered the bioanode operation, thus became an advantage for the biocathode. The bioanodes, formed from garden compost leachate under constant polarization at −0.2 V vs. SCE, were able to support long exposure to forced aeration, with only a slight alteration of their anodic efficiency. They produced a current density of 16 ± 1.7 A/m2 for acetate oxidation and up to −0.4 A/m2 for oxygen reduction. Analysis of the microbial communities by 16S rRNA pyrosequencing revealed strong selection of Chloroflexi (49 ± 1%), which was not observed for conventional bioanodes not exposed to oxygen. Chloroflexi were found as the dominant phylum of electroactive biofilms for the first time

Cutibacterium acnes (Propionibacterium acnes) and acne vulgaris: a brief look at the latest updates

While the commensal bacterium Propionibacterium acnes (P. acnes) is involved in the maintenance of a healthy skin, it can also act as an opportunistic pathogen in acne vulgaris. The latest findings on P. acnes shed light on the critical role of a tight equilibrium between members of its phylotypes and within the skin microbiota in the development of this skin disease. Indeed, contrary to what was previously thought, proliferation of P. acnes is not the trigger of acne as patients with acne do not harbour more P. acnes in follicles than normal individuals. Instead, the loss of the skin microbial diversity together with the activation of the innate immunity might lead to this chronic inflammatory condition. This review provides results of the most recent biochemical and genomic investigations that led to the new taxonomic classification of P. acnes renamed Cutibacterium acnes (C. acnes), and to the better characterisation of its phylogenetic cluster groups. Moreover, the latest data on the role of C. acnes and its different phylotypes in acne are presented, providing an overview of the factors that could participate in the virulence and in the antimicrobial resistance of acne-associated strains. Overall, this emerging key information offers new perspectives in the treatment of acne, with future innovative strategies focusing on C. acnes biofilms and/or on its acne-associated phylotypes

Characterisation of Cutibacterium acnes phylotypes in acne and in vivo exploratory evaluation of Myrtacine®

OBJECTIVE : Our main objective was to compare Cutibacterium acnes (C. acnes) skin colonisation in patients with mild to moderate acne versus healthy controls and secondly, to evaluate a Myrtacine® -based cream on C. acnes total population and antibioresistant Cutibacteria in patients with acne. METHODS:In 60 acne patients (Global Acne Severity Scale, GEA grades 2-3), of mean age 20 [15-30] years and in 24 age- and sex- matched healthy controls, forehead strips samplings were performed for microbiological analysis of comedones by colony forming unit (CFU) counts of global C. acnes and erythromycin (EryR) or clindamycin-resistant (ClnR) populations of Cutibacterium and determination of phylotypes by MALTI-TOF. Clinical evaluations of acne patients (GEA, lesion count, porphyrin fluorescence) were performed at baseline and after 56 days of twice-daily application of a Myrtacine® -based cream. RESULTS : We first showed (i) high and similar levels of C. acnes colonisation in superficial pilosebaceous follicles and detection of EryR and ClnR strains in both acne and control groups; (ii) different repartition of phylotypes in acne patients versus healthy control, with a predominance of phylotype IA in acne patients and a link between phylotype IA and erythromycin resistance. Besides, after treatment with the Myrtacine® -based cream in acne patients, there was no change in C. acnes total load, but a significant decrease of EryR Cutibacteria, reduced porphyrin production by C. acnes, a decrease in acne severity (GEA), associated with reduced retentional and inflammatory lesions. CONCLUSION : Cutibacterium acnes colonisation was not significantly different in acne versus control groups. Phylotype IA was predominant in acne patient and in EryR C. acnes. A Myrtacine® -based cream significantly reduced the level of EryR Cutibacteria in vivo and improved acne lesions

A Myrtus communis extract enriched in myrtucummulones and ursolic acid reduces resistance of Propionibacterium acnes biofilms to antibiotics used in acne vulgaris

Recent works present evidence of Propionibacterium acnes growing as a biofilm in cutaneous follicles. This formation of clusters is now considered as an explanation for the in vivo resistance of P. acnes to the main antimicrobials prescribed in acne vulgaris. Purpose : Our objective was to explore this hypothesis and propose a new therapeutic approach focusing on anti-biofilm activity of Myrtacine® New Generation (Mediterranean Myrtle extract–Botanical Expertise P. Fabre) alone or combined with antibiotics. Methods/Results : Using in vitro models able to promote the growth of adhered bacteria, the loss of sensitivity of P. acnes biofilms (48 h) towards erythromycin and clindamycin was checked considering either sensitive or resistant strains. In the same time, the activity of Myrtacine® New Generation against biofilm formation and mature biofilm (48 h) was evaluated. Using a dynamic model of biofilm formation, we noted an inhibition of biofilm formation (addition of Myrtacine® New Generation at T 0) and a significant effect on mature biofilm (48 h) for 5 min of contact. This effect was also checked using the static model of biofilm formation for Myrtacine® New Generation concentrations ranging from 0.03% to 0.0001%. A significant, dose-dependent anti-biofilm effect was observed and notable even at a concentration lower than the active concentration on planktonic cells, i.e. 0.001%. Finally, the interest of the combination of Myrtacine® New Generation with antibiotics was explored. An enhanced efficacy was noted when erythromycin (1000 mg/l) or clindamycin (500 mg/l) was added to 0.001% Myrtacine®, leading to significant differences in comparison to each compound used alone