EP4263800A1 - Use of extracellular membrane vesicles for anti-biofilm purposes - Google Patents

Use of extracellular membrane vesicles for anti-biofilm purposes

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Publication number
EP4263800A1
EP4263800A1 EP21851620.1A EP21851620A EP4263800A1 EP 4263800 A1 EP4263800 A1 EP 4263800A1 EP 21851620 A EP21851620 A EP 21851620A EP 4263800 A1 EP4263800 A1 EP 4263800A1
Authority
EP
European Patent Office
Prior art keywords
biofilm
probiotic
membrane vesicles
genus
extracellular membrane
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21851620.1A
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German (de)
French (fr)
Inventor
Aurélie RIEU
David DA SILVA BARREIRA
Jean Guzzo
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universite de Bourgogne
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Universite de Bourgogne
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Filing date
Publication date
Application filed by Universite de Bourgogne filed Critical Universite de Bourgogne
Publication of EP4263800A1 publication Critical patent/EP4263800A1/en
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the use of biological tools to prevent or reduce the formation of a biofilm on the surface of a material.
  • the present invention finds applications in many fields of industry, such as for example in the food industry, the piping or surface treatment industry or even in the medical sector.
  • Biofilms are in the form of a viscous film made up of microorganisms, often bacteria, yeasts, fungi or algae.
  • planktonic bacteria isolated bacteria, in suspension
  • EPS Extracellular Polymeric Substances
  • Biofilms form on the surfaces of industrial equipment and colonize all industrial surfaces, such as pipes or membrane filters.
  • biofilms can form if manufacturers are not particularly vigilant. They are thus regularly responsible for serious contamination of finished products and numerous food poisonings.
  • Biofilms are for example a major concern in the dairy processing industry. Bacterial biofilms can also develop on implants or during chronic infections; they constitute reservoirs of pathogens and can be the source of nosocomial infections.
  • biofilms The adhesion capacity of bacteria from biofilms is very high (often greater than that of planktonic cells) whether on natural or artificial surfaces. Bacteria within biofilms are resistant to all kinds of stress which makes them difficult to eliminate. Biofilms are very resistant to chemical (antibiotics for example) and mechanical (fluid for example) stresses. Thus, biofilms are resistant to most conventional cleaning methods, and tend to grow more in water or in aqueous media. However, many factors favor the formation of biofilms such as temperature or access and the nature of available metabolic resources.
  • nanotechnologies for the prevention of the formation of biofilms is currently an important strategy. It involves in particular the incorporation of anti-bacterial agents in the inert supports. The release of nanoparticles is nevertheless to be taken into account depending on the applications.
  • enzymes are used to disperse the biofilm. These are mainly hydrolases ( ⁇ -amylases, proteases, ribonucleases for example), oxidoreductases (such as glucose-oxidases or haloperoxidases), transferases (such as transaminase) or lyases (such as alginate lyase). Since these enzymes do not kill bacteria, they are usually combined with bactericidal methods.
  • hydrolases ⁇ -amylases, proteases, ribonucleases for example
  • oxidoreductases such as glucose-oxidases or haloperoxidases
  • transferases such as transaminase
  • lyases such as alginate lyase
  • the vesicles are derived from probiotics, which confers an advantage for their use, in particular for health.
  • This origin guarantees a safe, natural, eco-responsible, non-polluting product, with a broad spectrum of action, which can even constitute a protective film, with a preventive action.
  • a first object of the invention relates to the use of extracellular membrane vesicles of at least one probiotic to prevent or reduce the formation of a biofilm on the surface of a material.
  • extracellular membrane vesicles within the meaning of the present invention, means any vesicle of a lipid nature, released spontaneously or induced (by culture conditions or by physicochemical treatments) in the medium by the probiotic, and containing at least one active principle belonging to this producing bacterium.
  • active principles can be lipids, proteins, nucleic acids or exopolysaccharides.
  • probiotic within the meaning of the present invention, means any living microorganism which, when ingested in sufficient quantity, has a beneficial effect on the health of the host.
  • probiotic bacteria or yeasts in particular a bacterium such as a lactobacillus, a bifidobacterium, an enterococcus, a propionibacterium, a streptococcus and a bacterium of the genus Bacillus, or a yeast such as Saccharomyces cerevisiae and Saccharomyces boulardi or a mixture thereof.
  • the probiotic bacteria can be chosen from: L. acidophilus, L. crispatus, L. gasseri, L.
  • the probiotic bacteria are L. casei, L. paracasei and L. plantarum or a mixture thereof.
  • the probiotic yeasts suitable for the present invention can be chosen from: Saccharomyces cerevisiae and Saccharomyces boulardii or one of their mixtures.
  • vesicles originating for example from different types of probiotic bacteria. It is for example possible to use vesicles originating from one or more different bacterial species, the number of different species not being limited. It may for example be a mixture of L. casei and L. paracasei.
  • the vesicles can be used in combination with at least one antimicrobial with curative or preventive properties, which the person skilled in the art can choose from known antimicrobials depending on the intended application.
  • biofilm within the meaning of the present invention, a multicellular community of microorganisms adhering to each other and to a surface, and secreting an adhesive and protective EPS matrix.
  • the biofilm can be a bacterial biofilm, a yeast biofilm or a mixed biofilm.
  • bacterial biofilm within the meaning of the present invention, means a biofilm whose multicellular community of microorganisms consists essentially of bacteria.
  • yeast biofilm within the meaning of the present invention, means a biofilm whose multicellular community of microorganisms consists essentially of yeasts.
  • the bacterial biofilm can be formed by at least one bacterial species chosen from the Enterobacteriaceae family, in particular Salmonella enterica Enteritidis, Hafnia alvei and/or Citrobacter freundii, the Staphylococcus genus, in particular Staphylococcus aureus or Staphylococcus epidermidis, the Bacillus genus, in particular Bacillus cereus or Bacillus subtilis, the genus Pseudomonas, in particular Pseudomonas aeruginosa and the genus Enterococcus, in particular Enterococcus faecalis.
  • yeast biofilm can be formed by the yeast species Candida albicans.
  • mixed biofilm within the meaning of the present invention, means a biofilm composed of a community of different types of microorganisms, which may include in particular bacteria, yeasts and/or phages.
  • the mixed biofilm may comprise a mixture of at least one bacterium chosen from the enterobacteriaceae family, in particular Salmonella enterica Enteritidis, Hafnia alvei and/or Citrobacter freundii, the genus Staphylococcus, in particular Staphylococcus aureus or Staphylococcus epidermidis, the genus Bacillus , in particular Bacillus cereus or Bacillus subtilis, the genus Pseudomonas, in particular Pseudomonas aeruginosa and the genus Enterococcus, in particular Enterococcus faecalis, and the yeast species Candida albicans.
  • enterobacteriaceae family in particular Salmonella enterica Enteritidis,
  • preventing the formation of a biofilm within the meaning of the present invention, the action of preventing on a surface devoid of biofilm, the formation thereof.
  • the vesicles prevent the adhesion of bacteria to the treated surface.
  • the preventive effect of the extracellular membrane vesicles can take place for a period of up to several weeks to several months after the treatment of the surface, in particular if the surface is conditioned with the vesicles and the material is stabilized, for example by drying.
  • the reduction can be a reduction of at least 20% in the formation of a biofilm, compared to an identical surface and stored under the same conditions, in the absence of treatment.
  • the biofilm-reducing effect of extracellular membrane vesicles may occur for up to several weeks to several months after surface treatment, especially if the surface is conditioned with the vesicles and stabilized. the material, for example by drying.
  • the antibiofilm effect of the extracellular vesicles is not accompanied, or not necessarily, by an antimicrobial effect.
  • the antimicrobial activity is to be distinguished from the antibiofilm activity.
  • the extracellular vesicles prevent the adhesion of bacteria to the treated surface, this not being due to an anti-microbial effect. This characteristic is all the more advantageous in that in certain cases, it is desirable to have an antibiofilm effect without having an antimicrobial effect.
  • the extracellular membrane vesicles can be produced according to any process known to those skilled in the art.
  • the production process may for example comprise the following steps:
  • step (b) separating the at least one probiotic and the extracellular membrane vesicles produced in step (a) and,
  • Culture step (a) can be carried out under standard conditions known to those skilled in the art, depending on the nature of the probiotic.
  • the culture in in the case of lactobacilli, the culture can be carried out in the MRS medium, at 37° C., for 24 h.
  • the separation step (b) can also be carried out under standard conditions known to those skilled in the art, depending on the nature of the probiotic. This may be, for example, a filtration step. For example, in the case of lactobacilli, centrifugation can be carried out at 4000 g for 20 min and filtration can be carried out with a filter having a pore size of approximately 0.22 ⁇ m.
  • Step (c) of purification and concentration can be carried out under standard conditions known to those skilled in the art, depending on the nature of the probiotic.
  • the purification and concentration step may comprise at least one technique known to those skilled in the art, such as for example differential centrifugation, illustrated by Zaborowska et al. ([4]), the density gradient, illustrated by Kim et al. ([5]) or Dean et al. ([6]), exclusion chromatography, illustrated by Kuhn et al. ([7]), ultrafiltration, illustrated by Mata Forsberg et al. ([8]), Dominguez Rubio et al. ([9]), Choi et al., 2020 ([10]) or Kim et al.
  • step (c) can make it possible to obtain a solution of vesicles with a concentration of approximately 10 11 particles/mL, this number being given as an indication and which can vary according to the conditions of implementation of the different steps. of protocol c) and according to the bacteria producing the vesicles.
  • the stabilization and preservation of the extracellular membrane vesicles can be carried out under standard conditions known to those skilled in the art.
  • the stabilization and preservation step may include a drying and/or freezing step.
  • the material on which the extracellular membrane vesicles are used can be any material on which a biofilm is likely to form. form. It may in particular be a material chosen from among metals, metal alloys, polymers, glass, ceramics, and foodstuffs.
  • the extracellular membrane vesicles can be incorporated into products making it possible to treat these materials. It may be, for example, a spray, or a covering product of the paint, lacquer or varnish type.
  • the use means an ex vivo, non-therapeutic use.
  • Another object of the invention relates to a process for treating a surface of a material to prevent or reduce the formation of a biofilm on said surface, said process comprising a step of bringing vesicles into contact extracellular membranes from at least one probiotic with said surface.
  • treatment means the application of a layer of extracellular membrane vesicles to the surface of the material.
  • the application takes place in particular under normal conditions of use of the material, for example at ambient temperature and atmospheric pressure.
  • the amount of vesicles applied to the surface can be determined by those skilled in the art, depending on the material and the type of vesicle.
  • Another object of the invention relates to a material comprising at its surface extracellular membrane vesicles of at least one probiotic.
  • the vesicles cover at least partly, and preferably totally, the surface of the material to be protected. They can thus form a layer, with a thickness that can be between 10 and 500 nm. The thickness will be determined by those skilled in the art depending on the applications.
  • the material may be a packaging material, in particular food packaging material, a water pipe, a heat exchanger, a pipeline, or a catheter.
  • the material can also be a material used in the medical sector, because in addition to the anti-biofilm activity, the vesicles can provide anti-inflammatory activity (Mata Forsberg et al. ([8]), Kim et al. ( [13]), Nahui Palomino et al. ([14]), Yamasaki-Yashiki et al. ([12]), (Bâuerl et al. ([12]), Choi et al. ([10]), Kuhn et al. ([7]).
  • the vesicles can be incorporated into dressings, creams or cover certain medical devices.
  • Figure 1 shows the diagram illustrating the steps of the protocol carried out in Examples 1 and 2.
  • FIG. 2 represents part of the action spectrum of the antibiofilm effect of the extracellular vesicles of L. casei BL23 (dotted lines) and L. paracasei ATCC334 (closer dotted lines).
  • subtilis ( Figure 2D) is determined either without the addition of extracellular vesicles (close hatching) or with the addition of 0.04 ⁇ g/ ⁇ l of vesicles.
  • a control is carried out with the vesicles purified from the MRS culture medium (hatched).
  • the formation of the biofilm by the pathogens after 24 h at 37° C. is quantified by crystal violet staining.
  • the condition without the addition of extracellular vesicles (close hatching) corresponds to the NT condition (untreated).
  • Figure 3 shows an antibiofilm effect of L. casei BL23 and L. paracasei ATCC334 vesicles against S aureus, S. epidermidis, H. alvei, S. enterica, E. faecalis, P. aeruginosa and B. subtilis.
  • FIG. 3 represents: A) the quantification (in percentage relative to the NT) of the formation of a biofilm of S. enterica, either without addition of extracellular vesicles (close hatching), or with addition of 0.04 pg / ⁇ l of extracellular vesicles of L. casei BL23 (dotted lines), or with addition of 0.04 ⁇ g/ ⁇ l of vesicles purified from the MRS culture medium (hatched).
  • the formation of the biofilm by S. enterica after 24 h at 37° C. is quantified by crystal violet staining; the condition without the addition of extracellular vesicles (close hatching) corresponds to the NT condition (untreated); B) the growth curve of S.
  • FIG. 4 represents the quantification (in percentage relative to NT) of the formation of the biofilm by S. enterica at 0 h, 4 h, 8 h and 15 h at 37° C. by crystal violet staining.
  • S. enterica was either untreated with vesicles (close hatching), treated with 0.04 pg/pl of L. casei BL23 extracellular vesicles (dashed lines) or treated with 0.04 pg/pl of vesicles purified from the MRS culture medium (control, hatched).
  • the condition without the addition of extracellular vesicles (close hatching) corresponds to the NT condition (untreated).
  • Figure 4 shows that the antibiofilm effect of L. casei BL23 vesicles against S. enterica relates to the early stages of biofilm formation.
  • Example 1 Isolation of membrane vesicles
  • Step 2 Concentration and isolation of the vesicles by filtration and ultrafiltration
  • the biological material is maintained at 4°C under aseptic conditions during step 2
  • Example 2 Antibiofilm activity of the vesicular fraction of lactobacilli
  • Step 1 Formation of a biofilm on polystyrene microplates and treatment with the concentrated vesicle fraction
  • Step 2 Quantification of biofilm formation by crystal violet staining -Remove the bacteria in suspension present in each of the wells of the polystyrene plate

Abstract

The present invention relates to a use of extracellular membrane vesicles of at least one probiotic for preventing or reducing the formation of a biofilm on the surface of a material. The present invention also relates to a method for treating a surface of a material in order to prevent or reduce the formation of a biofilm on the surface, the method comprising a step of bringing extracellular membrane vesicles derived from at least one probiotic into contact with the surface. The present invention further relates to a material comprising extracellular membrane vesicles of at least one probiotic on its surface, or into which extracellular membrane vesicles of at least one probiotic are incorporated.

Description

UTILISATION ANTI-BIOFILM DE VESICULES MEMBRANAIRES EXTRACELLULAIRES ANTI-BIOFILM USE OF EXTRACELLULAR MEMBRANE VESICLES
[0001] Domaine technique [0001] Technical area
[0002] La présente invention se rapporte à l’utilisation d’outils biologiques pour prévenir ou réduire la formation d’un biofilm à la surface d’un matériau. The present invention relates to the use of biological tools to prevent or reduce the formation of a biofilm on the surface of a material.
[0003] La présente invention trouve des applications dans de nombreux domaines de l’industrie, comme par exemple dans l’industrie agroalimentaire, l’industrie de la tuyauterie ou du traitement de surfaces ou encore dans le secteur médical. The present invention finds applications in many fields of industry, such as for example in the food industry, the piping or surface treatment industry or even in the medical sector.
[0004] Dans la description ci-dessous, les références entre crochets ([ ]) renvoient à la liste des références présentées à la fin du texte. In the description below, the references in square brackets ([]) refer to the list of references presented at the end of the text.
[0005] Etat de la technique [0005] State of the art
[0006] Les biofilms se présentent sous forme d’une pellicule visqueuse constituée de micro-organismes, souvent des bactéries, des levures, des champignons ou des algues. [0006] Biofilms are in the form of a viscous film made up of microorganisms, often bacteria, yeasts, fungi or algae.
[0007] La formation d’un biofilm est un processus dynamique et non-figé qui se déroule en plusieurs étapes détaillées ci-dessous : [0007] The formation of a biofilm is a dynamic and non-fixed process which takes place in several stages detailed below:
[0008] - adhésion réversible : les bactéries planctoniques (bactéries isolées, en suspension) arrivent à proximité du support par gravité, par diffusion ou grâce à des flux dynamiques et adhèrent de manière réversible par le biais de phénomènes physicochimiques tels que des forces électrostatiques, des forces de Van der Waals, ainsi que des interactions hydrophobes ; [0008] - reversible adhesion: the planktonic bacteria (isolated bacteria, in suspension) arrive close to the support by gravity, by diffusion or thanks to dynamic flows and adhere reversibly by means of physicochemical phenomena such as electrostatic forces, Van der Waals forces, as well as hydrophobic interactions;
[0009] - adhésion irréversible : la production de polymères extracellulaires et la mise en place de liaisons entre les appendices bactériens (pilis, flagelles, protéines d’adhésion) et la surface permettent l’adhésion irréversible des bactéries entre- elles et à la surface. L’élimination des cellules irréversiblement adhérées est difficile ; [0009] - irreversible adhesion: the production of extracellular polymers and the establishment of bonds between the bacterial appendages (pili, flagella, adhesion proteins) and the surface allow the irreversible adhesion of the bacteria to each other and to the surface . The elimination of irreversibly adhered cells is difficult;
[0010] - formation de microcolonies : les bactéries s’agrègent et se multiplient pour former des microcolonies. Les cellules bactériennes au sein des microcolonies sont liées dans une matrice de substances exopolymériques abrégés EPS (Extracellular Polymeric Substances). Les EPS sont produits en réponse à l’adhésion et à des stimuli environnementaux comme la pression, le pH, la température ou l’appauvrissement du milieu en nutriments ; [0010] - formation of microcolonies: the bacteria aggregate and multiply to form microcolonies. Bacterial cells within microcolonies are bound in a matrix of exopolymeric substances abbreviated as EPS (Extracellular Polymeric Substances). EPS are produced in response to adhesion and environmental stimuli such as pressure, pH, temperature or nutrient depletion of the medium;
[0011] - maturation : les microcolonies se développent et le biofilm se structure de manière tridimensionnelle. Plusieurs conditions affectent la structure de ce biofilm mature comme la surface, la composition de la communauté microbienne, la disponibilité en nutriments et les conditions hydrodynamiques. [0011] - maturation: the microcolonies develop and the biofilm is structured in a three-dimensional manner. Several conditions affect the structure of this mature biofilm such as surface area, microbial community composition, nutrient availability and hydrodynamic conditions.
[0012] Les biofilms se forment sur les surfaces des équipements industriels et colonisent toutes les surfaces industrielles, comme les canalisations, ou les filtres à membrane. Plusieurs études ont mis en évidence que lors de certaines opérations, notamment dans les échangeurs à plaques, des biofilms peuvent se former si les industriels ne se montrent pas particulièrement vigilants. Ils sont ainsi responsables, régulièrement, de graves contaminations de produits finis et de nombreuses toxi-infections alimentaires. Les biofilms sont par exemple une préoccupation majeure dans l’industrie de la transformation laitière. Les biofilms bactériens peuvent également se développer sur des implants ou lors d’infections chroniques ; ils constituent des réservoirs de pathogènes et peuvent être à l’origine d’infections nosocomiales. [0012] Biofilms form on the surfaces of industrial equipment and colonize all industrial surfaces, such as pipes or membrane filters. Several studies have shown that during certain operations, particularly in plate heat exchangers, biofilms can form if manufacturers are not particularly vigilant. They are thus regularly responsible for serious contamination of finished products and numerous food poisonings. Biofilms are for example a major concern in the dairy processing industry. Bacterial biofilms can also develop on implants or during chronic infections; they constitute reservoirs of pathogens and can be the source of nosocomial infections.
[0013] La capacité d’adhésion de bactéries issues de biofilms est très grande (souvent supérieure à celle des cellules planctoniques) que ce soit sur des surfaces naturelles ou artificielles. Les bactéries au sein des biofilms sont résistantes à toute sorte de stress ce qui les rend difficile à éliminer. Les biofilms résistent en effet très bien aux stress chimiques (antibiotiques par exemple) et mécaniques (fluide par exemple). Ainsi, les biofilms résistent à la plupart des méthodes classiques de nettoyage, et ont tendance à se développer davantage dans l’eau ou dans les milieux aqueux. Toutefois, de nombreux facteurs favorisent la formation des biofilms tels que la température ou l’accès et la nature des ressources métaboliques disponibles. [0013] The adhesion capacity of bacteria from biofilms is very high (often greater than that of planktonic cells) whether on natural or artificial surfaces. Bacteria within biofilms are resistant to all kinds of stress which makes them difficult to eliminate. Biofilms are very resistant to chemical (antibiotics for example) and mechanical (fluid for example) stresses. Thus, biofilms are resistant to most conventional cleaning methods, and tend to grow more in water or in aqueous media. However, many factors favor the formation of biofilms such as temperature or access and the nature of available metabolic resources.
[0014] Les stratégies actuelles permettant de limiter la formation d’un biofilm sont diverses (Rendueles O & Ghigo JM ([1]) ; Venkatesan N, Perumal G & Doble M ([2]) ; Rao PK & Sreenivasa MY ([3])). Certaines de ces stratégies sont préventives et visent à empêcher l’adhésion et la formation du biofilm. Une de ces stratégies est l’utilisation de molécules de signalisation synthétiques, qui brouillent le système de communication de cellules à cellules, essentiel à la formation du biofilm. Cette dernière application en est encore au stade de l’exploration. [0014] Current strategies for limiting the formation of a biofilm are diverse (Rendueles O & Ghigo JM ([1]); Venkatesan N, Perumal G & Doble M ([2]); Rao PK & Sreenivasa MY ([ 3])). Some of these strategies are preventive and aim to prevent the adhesion and formation of biofilm. One such strategy is the use of synthetic signaling molecules, which scramble the cell-to-cell communication system essential for biofilm formation. This latter application is still in the exploration stage.
[0015] Le recours aux nanotechnologies pour la prévention de la formation de biofilms est à ce jour une stratégie importante. Elle met en jeu notamment l’incorporation d’agents anti-bactériens dans les supports inertes. Le relargage de nanoparticules est néanmoins à prendre en compte selon les applications. [0015] The use of nanotechnologies for the prevention of the formation of biofilms is currently an important strategy. It involves in particular the incorporation of anti-bacterial agents in the inert supports. The release of nanoparticles is nevertheless to be taken into account depending on the applications.
[0016] D’autres stratégies sont curatives et visent à éradiquer les biofilms. Il s’agit de méthodes bactéricides, ou de méthodes de dispersion et de désagrégation d’un biofilm déjà formé. Parmi les méthodes bactéricides, on peut distinguer les approches physiques, chimiques ou biologiques. [0016] Other strategies are curative and aim to eradicate biofilms. These are bactericidal methods, or methods of dispersion and disintegration of an already formed biofilm. Among bactericidal methods, physical, chemical or biological approaches can be distinguished.
[0017] Des traitements physiques et mécaniques, comme les radiations ionisantes, les radiations UV et les ultrasons, ont été expérimentés par le passé. Leur efficacité est partielle, mais il est possible de cumuler ces méthodes pour en potentialiser l’effet anti-biofilm. Dans le cas des traitements par ultrasons, de nombreux effets délétères ont été rapportés sur la qualité de l’aliment, sa composition physique et sa flaveur. [0017] Physical and mechanical treatments, such as ionizing radiation, UV radiation and ultrasound, have been tried out in the past. Their effectiveness is partial, but it is possible to combine these methods to potentiate their anti-biofilm effect. In the case of ultrasonic treatments, many deleterious effects have been reported on food quality, physical composition and flavor.
[0018] L’utilisation de désinfectants, notamment pour les produits frais, est fréquente. Ceux-ci sont cependant beaucoup plus actifs sur les cellules planctoniques. Les bactéries présentes dans les biofilms ont des capacités de résistance accrues aux désinfectants. En effet, l’organisation au sein du réseau matriciel que constitue le biofilm assure aux bactéries une protection efficace contre les agents chimiques. D’une part le biofilm constitue une barrière de perméabilité permettant de réduire l’exposition des bactéries aux agents chimiques et d’autre part les bactéries présentes dans les biofilms ont des activités métaboliques réduites permettant ainsi de réduire davantage l’action des agents chimiques. Dans les industries alimentaires, les surfaces sont nettoyées avec des dérivés chlorés, du peroxide d’hydrogène, de l’iode, des isothiazolinones, l’ozone, l’acide peracétique, les composes acides, les biocides à base d’aldéhyde, les phénols, les biguanides, les surfactants, les halogens et les ammoniums quaternaires. En général, ces agents n’éradiquent pas totalement le biofilm, ne sont pas éco-responsables et sont à l’origine dans de nombreux cas de la corrosion des surfaces. L’effet des huiles essentielles sur la destruction des biofilms est également à l’étude. [0018] The use of disinfectants, in particular for fresh products, is frequent. These are however much more active on planktonic cells. Bacteria present in biofilms have increased resistance to disinfectants. Indeed, the organization within the matrix network that constitutes the biofilm provides bacteria with effective protection against chemical agents. On the one hand, the biofilm constitutes a permeability barrier making it possible to reduce the exposure of bacteria to chemical agents and, on the other hand, the bacteria present in the biofilms have reduced metabolic activities, thus making it possible to further reduce the action of chemical agents. In food industries, surfaces are cleaned with chlorine derivatives, hydrogen peroxide, iodine, isothiazolinones, ozone, peracetic acid, acid compounds, aldehyde-based biocides, phenols, biguanides, surfactants, halogens and quaternary ammoniums. In general, these agents do not totally eradicate the biofilm, are not environmentally responsible and are the cause in many cases of surface corrosion. The effect of essential oils on the destruction of biofilms is also being studied.
[0019] L’utilisation de bactériophages est actuellement développée pour leur propriété bactéricide. [0019] The use of bacteriophages is currently being developed for their bactericidal properties.
[0020] Récemment, il a été proposé l’incorporation de biosurfactants dans des liposomes avec une activité antibiofilm. Il s’agit dans ce cas de vésicules artificielles reconstituées. Des effets ont été mis en évidence contre un biofilm de S. aureus, pour des applications potentielles relatives à des maladies de peau. [0020] Recently, the incorporation of biosurfactants into liposomes with antibiofilm activity has been proposed. In this case, these are reconstituted artificial vesicles. Effects have been demonstrated against a biofilm of S. aureus, for potential applications relating to skin diseases.
[0021] Par ailleurs, des enzymes sont utilisées pour disperser le biofilm. Il s’agit majoritairement d’hydrolases (a-amylases, protéases, ribonucléases par exemple), d’oxydo-réductases (comme les glucose-oxydases ou les haloperoxidases), de transférases (comme la transaminase) ou de lyases (comme l’alginate lyase). Ces enzymes ne tuant pas les bactéries, elles sont généralement combinées avec des méthodes bactéricides. [0021] Furthermore, enzymes are used to disperse the biofilm. These are mainly hydrolases (α-amylases, proteases, ribonucleases for example), oxidoreductases (such as glucose-oxidases or haloperoxidases), transferases (such as transaminase) or lyases (such as alginate lyase). Since these enzymes do not kill bacteria, they are usually combined with bactericidal methods.
[0022] Il existe donc un réel besoin d’outils palliant les défauts, inconvénients et obstacles de l’art antérieur, en particulier d’un outil permettant de prévenir ou réduire la formation d’un biofilm à la surface d’un matériau. [0022]There is therefore a real need for tools that overcome the defects, drawbacks and obstacles of the prior art, in particular for a tool that makes it possible to prevent or reduce the formation of a biofilm on the surface of a material.
[0023] Description de l’invention [0023] Description of the invention
[0024] Aux termes d’importantes recherches, la Demanderesse a réussi à démontrer que l’utilisation de vésicules membranaires, naturellement produites par des probiotiques, empêche la formation de biofilm par des micro-organismes pathogènes ou indésirables sur des surfaces biotiques et abiotiques. [0024] Following significant research, the Applicant has succeeded in demonstrating that the use of membrane vesicles, naturally produced by probiotics, prevents the formation of biofilm by pathogenic or undesirable microorganisms on biotic and abiotic surfaces.
[0025] De manière très avantageuse, les vésicules sont issues de probiotiques, ce qui confère un avantage pour leur utilisation, notamment pour la santé. Cette origine garantit un produit sûr, naturel, éco-responsable, non polluant, ayant un spectre d’action large, pouvant même constituer un film protecteur, avec une action préventive. [0025] Very advantageously, the vesicles are derived from probiotics, which confers an advantage for their use, in particular for health. This origin guarantees a safe, natural, eco-responsible, non-polluting product, with a broad spectrum of action, which can even constitute a protective film, with a preventive action.
[0026] Avantageusement, plusieurs effets sont envisageables, dont l’effet antibiofilm et immunomodulateur, selon la souche probiotique utilisée pour isoler les vésicules. [0027] Ainsi, un premier objet de l’invention se rapporte à l’utilisation de vésicules membranaires extracellulaires d’au moins un probiotique pour prévenir ou réduire la formation d’un biofilm à la surface d’un matériau. [0026] Advantageously, several effects can be envisaged, including the antibiofilm and immunomodulatory effect, depending on the probiotic strain used to isolate the vesicles. Thus, a first object of the invention relates to the use of extracellular membrane vesicles of at least one probiotic to prevent or reduce the formation of a biofilm on the surface of a material.
[0028] On entend par « vésicules membranaires extracellulaires », au sens de la présente invention, toute vésicule de nature lipidique, libérée spontanément ou de manière induite (par les conditions de culture ou par des traitements physicochimiques) dans le milieu par le probiotique, et renfermant au moins un principe actif appartenant à cette bactérie productrice. Avantageusement, il est envisageable de produire des vésicules chargées en principes actifs pouvant être des lipides, des protéines, des acides nucléiques ou des exopolysaccharides. The term “extracellular membrane vesicles”, within the meaning of the present invention, means any vesicle of a lipid nature, released spontaneously or induced (by culture conditions or by physicochemical treatments) in the medium by the probiotic, and containing at least one active principle belonging to this producing bacterium. Advantageously, it is possible to envisage producing vesicles loaded with active principles which can be lipids, proteins, nucleic acids or exopolysaccharides.
[0029] On entend par « probiotique », au sens de la présente invention, tout microorganisme vivant qui, lorsqu’il est ingéré en quantité suffisante, a un effet bénéfique sur la santé de l’hôte. Il peut s’agir notamment de bactéries ou de levures probiotiques, notamment une bactérie telle qu’un lactobacille, une bifidobactérie, un entérocoque, une propionibactérie, un streptocoque et une bactérie du genre Bacillus, ou une levure telle que Saccharomyces cerevisiae et Saccharomyces boulardi ou un de leurs mélanges. Les bactéries probiotiques peuvent être choisies parmi : L. acidophilus, L. crispatus, L. gasseri, L. delbrueckii, L. salivarius, L. casei, L. paracasei, L. plantarum, L. rhamnosus, L. reuteri, L. brevis, L. buchneri, L. fermentum, B. adolescentis, B. angulation, B. bifidum, B. breve, B. catenulatum, B. infantis, B. lactis, B. longum, B. pseudocatenulatum, S. thermophiles, ou un de leurs mélanges, de préférence les bactéries probiotiques sont L. casei, L. paracasei et L. plantarum ou un de leurs mélanges. Les levures probiotiques convenant pour la présente invention peuvent être choisies parmi : Saccharomyces cerevisiae et Saccharomyces boulardii ou un de leurs mélanges. The term "probiotic", within the meaning of the present invention, means any living microorganism which, when ingested in sufficient quantity, has a beneficial effect on the health of the host. These may be in particular probiotic bacteria or yeasts, in particular a bacterium such as a lactobacillus, a bifidobacterium, an enterococcus, a propionibacterium, a streptococcus and a bacterium of the genus Bacillus, or a yeast such as Saccharomyces cerevisiae and Saccharomyces boulardi or a mixture thereof. The probiotic bacteria can be chosen from: L. acidophilus, L. crispatus, L. gasseri, L. delbrueckii, L. salivarius, L. casei, L. paracasei, L. plantarum, L. rhamnosus, L. reuteri, L. brevis, L. buchneri, L. fermentum, B. adolescentis, B. angulation, B. bifidum, B. breve, B. catenulatum, B. infantis, B. lactis, B. longum, B. pseudocatenulatum, S. thermophiles, or a mixture thereof, preferably the probiotic bacteria are L. casei, L. paracasei and L. plantarum or a mixture thereof. The probiotic yeasts suitable for the present invention can be chosen from: Saccharomyces cerevisiae and Saccharomyces boulardii or one of their mixtures.
[0030] Selon l’invention, des combinaisons de différents types de vésicules, provenant par exemple de différents types de bactéries probiotiques, peuvent être réalisées. On peut par exemple utiliser des vésicules provenant d’une ou plusieurs espèces bactériennes différentes, le nombre d’espèces différentes n’étant pas limité. Il peut s’agir par exemple d’un mélange de L. casei et L. paracasei. Eventuellement, les vésicules peuvent être utilisées en association avec au moins un antimicrobien aux propriétés curatives ou préventives, que l’homme du métier pourra choisir parmi les antimicrobiens connus en fonction de l’application visée. [0030] According to the invention, combinations of different types of vesicles, originating for example from different types of probiotic bacteria, can be produced. It is for example possible to use vesicles originating from one or more different bacterial species, the number of different species not being limited. It may for example be a mixture of L. casei and L. paracasei. Optionally, the vesicles can be used in combination with at least one antimicrobial with curative or preventive properties, which the person skilled in the art can choose from known antimicrobials depending on the intended application.
[0031] On entend par « biofilm », au sens de la présente invention, une communauté multicellulaire de micro-organismes adhérant entre eux et à une surface, et sécrétant une matrice EPS adhésive et protectrice. The term "biofilm", within the meaning of the present invention, a multicellular community of microorganisms adhering to each other and to a surface, and secreting an adhesive and protective EPS matrix.
[0032] Selon l’invention, le biofilm peut être un biofilm bactérien, un biofilm levurien ou un biofilm mixte. On entend par « biofilm bactérien », au sens de la présente invention, un biofilm dont la communauté multicellulaire de micro-organismes est constituée essentiellement de bactéries. On entend par « biofilm levurien », au sens de la présente invention, un biofilm dont la communauté multicellulaire de micro-organismes est constituée essentiellement de levures. Par exemple, le biofilm bactérien peut être formé par au moins une espèce bactérienne choisie parmi la famille des entérobactéries, notamment Salmonella enterica Enteritidis, Hafnia alvei et/ou Citrobacter freundii, le genre Staphylococcus, notamment Staphylococcus aureus ou Staphylococcus epidermidis, le genre Bacillus, notamment Bacillus cereus ou Bacillus subtilis, le genre Pseudomonas, notamment Pseudomonas aeruginosa et le genre Enterococcus, notamment Enterococcus faecalis. Par exemple, le biofilm levurien peut être formé par l’espèce levurienne Candida albicans. On entend par « biofilm mixte », au sens de la présente invention, un biofilm composé d’une communauté de différents types de micro-organismes, pouvant comprendre notamment des bactéries, des levures et/ou des phages. Par exemple, le biofilm mixte peut comprendre un mélange d’au moins une bactérie choisie parmi la famille des entérobactéries, notamment Salmonella enterica Enteritidis, Hafnia alvei et/ou Citrobacter freundii, le genre Staphylococcus, notamment Staphylococcus aureus ou Staphylococcus epidermidis, le genre Bacillus, notamment Bacillus cereus ou Bacillus subtilis, le genre Pseudomonas, notamment Pseudomonas aeruginosa et le genre Enterococcus, notamment Enterococcus faecalis, et de l’espèce levurienne Candida albicans. According to the invention, the biofilm can be a bacterial biofilm, a yeast biofilm or a mixed biofilm. The term “bacterial biofilm”, within the meaning of the present invention, means a biofilm whose multicellular community of microorganisms consists essentially of bacteria. The term “yeast biofilm”, within the meaning of the present invention, means a biofilm whose multicellular community of microorganisms consists essentially of yeasts. For example, the bacterial biofilm can be formed by at least one bacterial species chosen from the Enterobacteriaceae family, in particular Salmonella enterica Enteritidis, Hafnia alvei and/or Citrobacter freundii, the Staphylococcus genus, in particular Staphylococcus aureus or Staphylococcus epidermidis, the Bacillus genus, in particular Bacillus cereus or Bacillus subtilis, the genus Pseudomonas, in particular Pseudomonas aeruginosa and the genus Enterococcus, in particular Enterococcus faecalis. For example, yeast biofilm can be formed by the yeast species Candida albicans. The term "mixed biofilm", within the meaning of the present invention, means a biofilm composed of a community of different types of microorganisms, which may include in particular bacteria, yeasts and/or phages. For example, the mixed biofilm may comprise a mixture of at least one bacterium chosen from the enterobacteriaceae family, in particular Salmonella enterica Enteritidis, Hafnia alvei and/or Citrobacter freundii, the genus Staphylococcus, in particular Staphylococcus aureus or Staphylococcus epidermidis, the genus Bacillus , in particular Bacillus cereus or Bacillus subtilis, the genus Pseudomonas, in particular Pseudomonas aeruginosa and the genus Enterococcus, in particular Enterococcus faecalis, and the yeast species Candida albicans.
[0033] On entend par « prévenir la formation d’un biofilm », au sens de la présente invention, l’action d’empêcher sur une surface dépourvue de biofilm, la formation de celui-ci. En particulier, les vésicules empêchent l’adhésion des bactéries sur la surface traitée. L’effet préventif des vésicules membranaires extracellulaires peut avoir lieu pendant une durée pouvant atteindre plusieurs semaines à plusieurs mois après le traitement de la surface, notamment si on conditionne la surface avec les vésicules et qu’on stabilise le matériau, par séchage par exemple. The term "preventing the formation of a biofilm", within the meaning of the present invention, the action of preventing on a surface devoid of biofilm, the formation thereof. In particular, the vesicles prevent the adhesion of bacteria to the treated surface. The preventive effect of the extracellular membrane vesicles can take place for a period of up to several weeks to several months after the treatment of the surface, in particular if the surface is conditioned with the vesicles and the material is stabilized, for example by drying.
[0034] On entend par « réduire la formation d’un biofilm », au sens de la présente invention, l’action d’empêcher en partie, sur une surface dépourvue de biofilm, la formation de celui-ci. La réduction peut être une réduction d’au moins 20% de la formation d’un biofilm, par rapport à une surface identique et conservée dans les mêmes conditions, en l’absence de traitement. L’effet de réduction de la formation d’un biofilm des vésicules membranaires extracellulaires peut avoir lieu pendant une durée pouvant atteindre plusieurs semaines à plusieurs mois après le traitement de la surface, notamment si on conditionne la surface avec les vésicules et qu’on stabilise le matériau, par séchage par exemple. The term "reducing the formation of a biofilm", within the meaning of the present invention, the action of preventing in part, on a surface devoid of biofilm, the formation of the latter. The reduction can be a reduction of at least 20% in the formation of a biofilm, compared to an identical surface and stored under the same conditions, in the absence of treatment. The biofilm-reducing effect of extracellular membrane vesicles may occur for up to several weeks to several months after surface treatment, especially if the surface is conditioned with the vesicles and stabilized. the material, for example by drying.
[0035] Avantageusement, l’effet antibiofilm des vésicules extracellulaires ne s’accompagne pas, ou pas nécessairement, d’un effet antimicrobien. En effet, l’activité antimicrobienne est à distinguer de l’activité antibiofilm. Avantageusement, les vésicules extracellulaires empêchent l’adhésion des bactéries sur la surface traitée, ceci n'étant pas dû à un effet anti-microbien. Cette caractéristique est d’autant plus avantageuse que dans certains cas, il est souhaitable d’avoir un effet antibiofilm sans avoir un effet anti-microbien. [0035] Advantageously, the antibiofilm effect of the extracellular vesicles is not accompanied, or not necessarily, by an antimicrobial effect. Indeed, the antimicrobial activity is to be distinguished from the antibiofilm activity. Advantageously, the extracellular vesicles prevent the adhesion of bacteria to the treated surface, this not being due to an anti-microbial effect. This characteristic is all the more advantageous in that in certain cases, it is desirable to have an antibiofilm effect without having an antimicrobial effect.
[0036] Les vésicules membranaires extracellulaires peuvent être produites selon tout procédé connu de l’homme du métier. Le procédé de production peut par exemple comprendre les étapes suivantes : The extracellular membrane vesicles can be produced according to any process known to those skilled in the art. The production process may for example comprise the following steps:
(a) culture d’au moins un probiotique dans des conditions adaptées à la production de vésicules membranaires extracellulaires, (a) cultivation of at least one probiotic under conditions suitable for the production of extracellular membrane vesicles,
(b) séparation de l’au moins un probiotique et des vésicules membranaires extracellulaires produites à l’étape (a) et, (b) separating the at least one probiotic and the extracellular membrane vesicles produced in step (a) and,
(c) purification et concentration des vésicules membranaires extracellulaires. (c) purification and concentration of extracellular membrane vesicles.
[0037] L’étape (a) de culture peut être réalisée dans des conditions standard connues de l’homme du métier, en fonction de la nature du probiotique. Par exemple, dans le cas des lactobacilles, la culture peut être réalisée dans le milieu MRS, à 37°C, pendant 24 h. Culture step (a) can be carried out under standard conditions known to those skilled in the art, depending on the nature of the probiotic. For example, in in the case of lactobacilli, the culture can be carried out in the MRS medium, at 37° C., for 24 h.
[0038] L’étape (b) de séparation peut être elle aussi réalisée dans des conditions standard connues de l’homme du métier, en fonction de la nature du probiotique. Il peut s’agir par exemple d’une étape de filtration. Par exemple, dans le cas des lactobacilles, la centrifugation peut être réalisée à 4000 g pendant 20 min et la filtration peut être réalisée avec un filtre ayant une taille de pores d’environ 0,22 pm. The separation step (b) can also be carried out under standard conditions known to those skilled in the art, depending on the nature of the probiotic. This may be, for example, a filtration step. For example, in the case of lactobacilli, centrifugation can be carried out at 4000 g for 20 min and filtration can be carried out with a filter having a pore size of approximately 0.22 µm.
[0039] L’étape (c) de purification et de concentration peut être réalisée dans des conditions standard connues de l’homme du métier, en fonction de la nature du probiotique. L’étape de purification et de concentration peut comprendre au moins une technique connue de l’homme du métier, comme par exemple la centrifugation différentielle, illustrée par Zaborowska ét al. ([4]), le gradient de densité, illustré par Kim et al. ([5]) ou Dean et al. ([6]), la chromatographie d’exclusion, illustré par Kuhn et al. ([7]), l’ultrafiltration, illustrée par Mata Forsberg et al. ([8]), Dominguez Rubio et al. ([9]), Choi et al., 2020 ([10]) ou Kim et al. ([5]), l’immunocapture (IC), illustrée par Wubbolts et al. ([11]), comme par exemple l’IC sur colonne, par billes magnétiques couplées à des anticorps ou toute autre surface couplée à un anticorps spécifique, ou encore la précipitation, comme illustrée par Bâuerl et al. ([12]) cette liste n’étant pas limitative. Dans le cas des lactobacilles, l’ultrafiltration peut être une filtration d’exclusion à environ 100 kDa. Avantageusement, l’étape (c) peut permettre d’obtenir une solution de vésicules d’une concentration d’environ 1011 particules / mL, ce nombre étant donné à titre indicatif et pouvant varier selon les conditions de mise en œuvre des différentes étapes du protocole c) et selon les bactéries productrices des vésicules. [0039] Step (c) of purification and concentration can be carried out under standard conditions known to those skilled in the art, depending on the nature of the probiotic. The purification and concentration step may comprise at least one technique known to those skilled in the art, such as for example differential centrifugation, illustrated by Zaborowska et al. ([4]), the density gradient, illustrated by Kim et al. ([5]) or Dean et al. ([6]), exclusion chromatography, illustrated by Kuhn et al. ([7]), ultrafiltration, illustrated by Mata Forsberg et al. ([8]), Dominguez Rubio et al. ([9]), Choi et al., 2020 ([10]) or Kim et al. ([5]), immunocapture (IC), illustrated by Wubbolts et al. ([11]), such as for example IC on a column, by magnetic beads coupled to antibodies or any other surface coupled to a specific antibody, or even precipitation, as illustrated by Bâuerl et al. ([12]) this list not being exhaustive. In the case of lactobacilli, the ultrafiltration can be an exclusion filtration at around 100 kDa. Advantageously, step (c) can make it possible to obtain a solution of vesicles with a concentration of approximately 10 11 particles/mL, this number being given as an indication and which can vary according to the conditions of implementation of the different steps. of protocol c) and according to the bacteria producing the vesicles.
[0040] La stabilisation et la conservation des vésicules membranaires extracellulaires peuvent être réalisées dans des conditions standard connues de l’homme du métier. Par exemple, l’étape de stabilisation et de conservation peut comprendre une étape de séchage et/ou de congélation. The stabilization and preservation of the extracellular membrane vesicles can be carried out under standard conditions known to those skilled in the art. For example, the stabilization and preservation step may include a drying and/or freezing step.
[0041] Le matériau sur lequel sont utilisées les vésicules membranaires extracellulaires peut être tout matériau sur lequel un biofilm est susceptible de se former. Il peut s’agir notamment d’un matériau choisi parmi les métaux, les alliages de métaux, les polymères, le verre, la céramique, et les aliments. [0041] The material on which the extracellular membrane vesicles are used can be any material on which a biofilm is likely to form. form. It may in particular be a material chosen from among metals, metal alloys, polymers, glass, ceramics, and foodstuffs.
[0042] Les vésicules membranaires extracellulaires peuvent être incorporées dans des produits permettant de traiter ces matériaux. Il peut s’agir par exemple d’un spray, ou d’un produit de couverture du type peinture, laque ou vernis. [0042] The extracellular membrane vesicles can be incorporated into products making it possible to treat these materials. It may be, for example, a spray, or a covering product of the paint, lacquer or varnish type.
[0043] Dans le cadre du traitement d’une surface selon l’invention, l’utilisation s’entend d’une utilisation ex vivo, non thérapeutique. In the context of the treatment of a surface according to the invention, the use means an ex vivo, non-therapeutic use.
[0044] Un autre objet de l’invention se rapporte à un procédé de traitement d’une surface d’un matériau pour prévenir ou réduire la formation d’un biofilm sur ladite surface, ledit procédé comprenant une étape de mise en contact de vésicules membranaires extracellulaires provenant d’au moins un probiotique avec ladite surface. Another object of the invention relates to a process for treating a surface of a material to prevent or reduce the formation of a biofilm on said surface, said process comprising a step of bringing vesicles into contact extracellular membranes from at least one probiotic with said surface.
[0045] On entend par « traitement », au sens de la présente invention, l’application d’une couche de vésicules membranaires extracellulaires à la surface du matériau. L’application a notamment lieu dans des conditions normales d’utilisation du matériau, par exemple à température ambiante et à pression atmosphérique. La quantité de vésicules appliquée sur la surface peut être déterminée par l’homme du métier, en fonction du matériau et du type de vésicule. The term "treatment", within the meaning of the present invention, means the application of a layer of extracellular membrane vesicles to the surface of the material. The application takes place in particular under normal conditions of use of the material, for example at ambient temperature and atmospheric pressure. The amount of vesicles applied to the surface can be determined by those skilled in the art, depending on the material and the type of vesicle.
[0046] Un autre objet de l’invention se rapporte à un matériau comprenant à sa surface des vésicules membranaires extracellulaires d’au moins un probiotique. Another object of the invention relates to a material comprising at its surface extracellular membrane vesicles of at least one probiotic.
[0047] Selon l’invention, les vésicules recouvrent au moins en partie, et de préférence totalement, la surface du matériau à protéger. Elles peuvent ainsi former une couche, d’une épaisseur pouvant être comprise entre 10 et 500 nm. L’épaisseur sera déterminée par l’homme du métier en fonctions des applications. According to the invention, the vesicles cover at least partly, and preferably totally, the surface of the material to be protected. They can thus form a layer, with a thickness that can be between 10 and 500 nm. The thickness will be determined by those skilled in the art depending on the applications.
[0048] Le matériau peut être un matériau d’emballage, notamment d’emballage alimentaire, une conduite d’eau, un échangeur thermique, un pipeline, ou un cathéter. Le matériau peut également être un matériau utilisé dans le secteur médical, car en complément de l’activité anti-biofilm, les vésicules peuvent apporter une activité anti-inflammatoire (Mata Forsberg et al. ([8]), Kim et al. ([13]), Nahui Palomino et al. ([14]), Yamasaki-Yashiki et al. ([12]), (Bâuerl et al. ([12]), Choi et al. ([10]), Kuhn et al. ([7]). Ainsi, les vésicules peuvent être incorporées dans des pansements, des crèmes ou recouvrir certains dispositifs médicaux. The material may be a packaging material, in particular food packaging material, a water pipe, a heat exchanger, a pipeline, or a catheter. The material can also be a material used in the medical sector, because in addition to the anti-biofilm activity, the vesicles can provide anti-inflammatory activity (Mata Forsberg et al. ([8]), Kim et al. ( [13]), Nahui Palomino et al. ([14]), Yamasaki-Yashiki et al. ([12]), (Bâuerl et al. ([12]), Choi et al. ([10]), Kuhn et al. ([7]). Thus, the vesicles can be incorporated into dressings, creams or cover certain medical devices.
[0049] D’autres avantages pourront encore apparaître à l’homme du métier à la lecture des exemples ci-dessous, illustrés par les figures annexées, donnés à titre illustratif. Other advantages may also appear to those skilled in the art on reading the examples below, illustrated by the appended figures, given for illustrative purposes.
[0050] Brève description des figures [0050] Brief description of the figures
[0051] La figure 1 représente le schéma illustrant les étapes du protocole réalisé dans les exemples 1 et 2. Figure 1 shows the diagram illustrating the steps of the protocol carried out in Examples 1 and 2.
[0052] La figure 2 représente une partie du spectre d’action de l’effet antibiofilm des vésicules extracellulaires de L. casei BL23 (pointillés) et L. paracasei ATCC334 (pointillés rapprochés). La quantification (en pourcentage rapporté au NT) de la formation d’un biofilm des pathogènes S aureus (figure 2A), S. epidermidis (figure 2A), H. alvei (figure 2B), S. enterica (figure 2B), E. faecalis (figure 2C), P. aeruginosa (figure 2C) et B. subtilis (figure 2D) est déterminée soit sans ajout de vésicules extracellulaires (hachures rapprochées), soit avec ajout de 0,04 pg/pl de vésicules. Un contrôle est réalisé avec les vésicules purifiées à partir du milieu de culture MRS (hachures). La formation du biofilm par les pathogènes après 24 h à 37°C est quantifiée par coloration au Cristal violet. La condition sans ajout de vésicules extracellulaires (hachures rapprochées) correspond à la condition NT (non traité). La figure 3 montre un effet antibiofilm des vésicules de L. casei BL23 et L. paracasei ATCC334 contre S aureus, S. epidermidis, H. alvei, S. enterica, E. faecalis, P. aeruginosa et B. subtilis. FIG. 2 represents part of the action spectrum of the antibiofilm effect of the extracellular vesicles of L. casei BL23 (dotted lines) and L. paracasei ATCC334 (closer dotted lines). The quantification (in percentage relative to the NT) of the formation of a biofilm of the pathogens S aureus (figure 2A), S. epidermidis (figure 2A), H. alvei (figure 2B), S. enterica (figure 2B), E . faecalis (Figure 2C), P. aeruginosa (Figure 2C) and B. subtilis (Figure 2D) is determined either without the addition of extracellular vesicles (close hatching) or with the addition of 0.04 µg/µl of vesicles. A control is carried out with the vesicles purified from the MRS culture medium (hatched). The formation of the biofilm by the pathogens after 24 h at 37° C. is quantified by crystal violet staining. The condition without the addition of extracellular vesicles (close hatching) corresponds to the NT condition (untreated). Figure 3 shows an antibiofilm effect of L. casei BL23 and L. paracasei ATCC334 vesicles against S aureus, S. epidermidis, H. alvei, S. enterica, E. faecalis, P. aeruginosa and B. subtilis.
[0053] La figure 3 représente : A) la quantification (en pourcentage rapporté au NT) de la formation d’un biofilm de S. enterica, soit sans ajout de vésicules extracellulaires (hachures rapprochées), soit avec ajout de 0,04 pg/pl de vésicules extracellulaires de L. casei BL23 (pointillés), soit avec ajout de 0,04 pg/pl de vésicules purifiées à partir du milieu de culture MRS (hachures). La formation du biofilm par S. enterica après 24 h à 37°C est quantifiée par coloration au Cristal violet ; la condition sans ajout de vésicules extracellulaires (hachures rapprochées) correspond à la condition NT (non traité) ; B) la courbe de croissance de S. enterica suivie par lecture de la DO à 600 nm pendant 24 h à 37°C pour les conditions suivantes : sans ajout de vésicules extracellulaires de L. casei BL23 (rond), ou avec ajout de 0,04 pg/pl de vésicules extracellulaires L. casei BL23 (triangle), ou avec ajout de 0,04 pg/pl de vésicules purifiées à partir du milieu de culture MRS (carré). La figure 3 montre que l’effet antibiofilm des vésicules de L. casei BL23 contre S. enterica ne s’accompagne pas d’un effet antimicrobien. FIG. 3 represents: A) the quantification (in percentage relative to the NT) of the formation of a biofilm of S. enterica, either without addition of extracellular vesicles (close hatching), or with addition of 0.04 pg / μl of extracellular vesicles of L. casei BL23 (dotted lines), or with addition of 0.04 μg/μl of vesicles purified from the MRS culture medium (hatched). The formation of the biofilm by S. enterica after 24 h at 37° C. is quantified by crystal violet staining; the condition without the addition of extracellular vesicles (close hatching) corresponds to the NT condition (untreated); B) the growth curve of S. enterica followed by reading the OD at 600 nm for 24 h at 37°C for the conditions following: without addition of L. casei BL23 extracellular vesicles (circle), or with addition of 0.04 pg/pl of L. casei BL23 extracellular vesicles (triangle), or with addition of 0.04 pg/pl of purified vesicles from MRS culture medium (square). Figure 3 shows that the antibiofilm effect of L. casei BL23 vesicles against S. enterica is not accompanied by an antimicrobial effect.
[0054] La figure 4 représente la quantification (en pourcentage rapporté au NT) de la formation du biofilm par S. enterica à 0 h, 4 h, 8 h et 15 h à 37°C par coloration au Cristal violet. S. enterica a soit été non traitée par des vésicules (hachures rapprochées), soit traitée par 0,04 pg/pl de vésicules extracellulaires L. casei BL23 (pointillés) soit traitée par 0,04 pg/pl de vésicules purifiées à partir du milieu de culture MRS (contrôle, hachures). La condition sans ajout de vésicules extracellulaires (hachures rapprochées) correspond à la condition NT (non traité). La figure 4 montre que l’effet antibiofilm des vésicules de L. casei BL23 contre S. enterica concerne les étapes précoces de la formation du biofilm. FIG. 4 represents the quantification (in percentage relative to NT) of the formation of the biofilm by S. enterica at 0 h, 4 h, 8 h and 15 h at 37° C. by crystal violet staining. S. enterica was either untreated with vesicles (close hatching), treated with 0.04 pg/pl of L. casei BL23 extracellular vesicles (dashed lines) or treated with 0.04 pg/pl of vesicles purified from the MRS culture medium (control, hatched). The condition without the addition of extracellular vesicles (close hatching) corresponds to the NT condition (untreated). Figure 4 shows that the antibiofilm effect of L. casei BL23 vesicles against S. enterica relates to the early stages of biofilm formation.
[0055] EXEMPLES [0055] EXAMPLES
[0056] Exemple 1 : Isolement des vésicules membranaires Example 1: Isolation of membrane vesicles
[0057] Matériel: [0057] Material:
-Milieu de culture MRS (Man, Rogosa, Sharpe) - MRS culture medium (Man, Rogosa, Sharpe)
-Filtres 0,22 pm PES (Polyethersulfone) -0.22µm PES (Polyethersulfone) filters
-Seringues 10 ml - 10ml syringes
-Bouteilles pyrex 250 ml (stérile) - 250 ml pyrex bottles (sterile)
-Tube à centrifuger de 15 ml et de 50 ml (stérile) - 15 ml and 50 ml centrifuge tube (sterile)
-Système d’ultrafiltration (100-kDa-exclusion filter) : -Ultrafiltration system (100-kDa-exclusion filter):
Amicon® Ultra (ref. UFC510008) Amicon® Ultra (ref. UFC510008)
Centricon Plus-70 (ref. UFC710008) Centricon Plus-70 (ref. UFC710008)
-Centrifugeuse Heraeus Multifuge X3R -Heraeus Multifuge X3R Centrifuge
-Adaptateur Rotor swing-out BIOLiner (11646190) -BIOLiner swing-out rotor adapter (11646190)
Adaptateur pour tubes à centrifuger de 50 ml Adaptateur pour flacons de 250 ml Adapter for 50 ml centrifuge tubes Adapter for 250 ml bottles
-Ultracentrifugeuse OPTIMA L séries 90K (NS COL 96J32) -OPTIMA L 90K series ultracentrifuge (NS COL 96J32)
-Beckman SW41 Ti Swinging-Bucket Rotor (ref. 331362) -Beckman SW41 Ti Swinging-Bucket Rotor (ref. 331362)
-Thin-wall polypropylene tube 13.2 mL, 14 x 89 mm (ref. 33137) -Thin-wall polypropylene tube 13.2 mL, 14 x 89 mm (ref. 33137)
-Phosphate Buffered Saline (stérile) -Phosphate Buffered Saline (sterile)
[0058] Protocole [0058] Protocol
[0059] Etape 1 : Mise en culture des lactobacilles Step 1: Cultivation of the lactobacilli
[0060] Un protocole de mise en culture des lactobacilles avec des paramètres standards est proposé ci-dessous. A protocol for culturing lactobacilli with standard parameters is proposed below.
-Inoculer 15 ml de MRS avec environ 50 pl d’un lactobacille conservé dans 20% glycérol à 80°C (Sortie de cryotube) - Inoculate 15 ml of MRS with approximately 50 µl of a lactobacillus stored in 20% glycerol at 80°C (Exit from cryotube)
-Incuber à 37°C, 24 h -Incubate at 37°C, 24 hours
-Inoculer 15 ml de MRS avec la sortie de cryotube. Diluer pour obtenir une densité optique à 600 nm de 0,05 (D0600nm=0,05) (Pré-culture) - Inoculate 15 ml of MRS with the cryotube outlet. Dilute to obtain an optical density at 600 nm of 0.05 (D0600nm=0.05) (Pre-culture)
-Incuber à 37°C, 24 h -Incubate at 37°C, 24 hours
-Inoculer 250 ml de MRS à D0600nm=0,05 avec la pré-culture (Culture de travail)- Inoculate 250 ml of MRS at D0600nm=0.05 with the pre-culture (working culture)
-Incuber à 37°C pendant 24 h -Incubate at 37°C for 24 hours
[0061] Etape 2 : Concentration et isolement des vésicules par filtration et ultrafiltrationStep 2: Concentration and isolation of the vesicles by filtration and ultrafiltration
[0062] Le matériel biologique est maintenu à 4°C dans des conditions d’asepsie au cours de l’étape 2 The biological material is maintained at 4°C under aseptic conditions during step 2
-Centrifuger les 250 ml à 4 000 g pendant 20 min pour précipiter les cellules-Centrifuge the 250 ml at 4000 g for 20 min to precipitate the cells
-Recueillir le surnageant clarifié dans une bouteille stérile de 250 ml -Collect the clarified supernatant in a sterile 250ml bottle
-Filtrer le surnageant clarifié à travers un filtre de 0,22 pm -Filter the clarified supernatant through a 0.22 µm filter
-Concentrer le surnageant en utilisant un système d'ultrafiltration (100-kDa- exclusion filter) (ref. UFC710008) -Concentrate the supernatant using an ultrafiltration system (100-kDa- exclusion filter) (ref. UFC710008)
-Concentrer les 250 ml jusqu’à obtenir un volume de 10-15 ml de liquide-Concentrate the 250 ml until you obtain a volume of 10-15 ml of liquid
-Filtrer à travers un filtre de 0,22 pm (afin d'éliminer les matières agrégées) -Ultracentrifuger le surnageant concentré à 110 000g pendant 2 h à 4°C-Filter through a 0.22 µm filter (to remove aggregated material) -Ultracentrifuge the concentrated supernatant at 110,000g for 2 hours at 4°C
-Eliminer le surnageant et resuspendre le culot avec 500 pl de PBS à 4°C (fraction de vésicules concentrée) - Remove the supernatant and resuspend the pellet with 500 µl of PBS at 4°C (concentrated vesicle fraction)
-Réaliser un dosage protéique (dosage de Bradford) de la fraction vésiculaire concentrée ainsi obtenue -Carry out a protein assay (Bradford assay) of the concentrated vesicular fraction thus obtained
-Conserver la fraction vésiculaire au froid. -Keep the vesicular fraction cold.
[0063] Exemple 2 : Activité antibiofilm de la faction vésiculaire des lactobacilles[0063] Example 2: Antibiofilm activity of the vesicular fraction of lactobacilli
[0064] Matériel [0064] Material
-Milieu de culture TSB -TSB culture medium
-Plaque de culture 96 puits fond plat avec couvercle 1 GREINER 2515432-Flat bottom 96-well culture plate with lid 1 GREINER 2515432
-Pro-Lab Diagnostics™ Solution cristal violet (ref. 12926287) -Pro-Lab Diagnostics™ Crystal Violet Solution (ref. 12926287)
[0065] Protocole [0065] Protocol
[0066] Etape 1 : Formation d'un biofilm sur des microplaques de polystyrène et traitement avec la fraction de vésicules concentrée Step 1: Formation of a biofilm on polystyrene microplates and treatment with the concentrated vesicle fraction
-Inoculer 15 ml de TSB avec environ 50 pl de bactéries conservés dans 20% glycérol à 80°C (Sortie de cryotube) - Inoculate 15 ml of TSB with approximately 50 pl of bacteria preserved in 20% glycerol at 80°C (Exit from cryotube)
-Incuber à 37°C, 24 h (± agitation, ± aérobie selon la bactérie étudiée)- Incubate at 37°C, 24 h (± agitation, ± aerobic depending on the bacterium studied)
-Inoculer 15 ml de TSB avec la sortie de cryotube. Diluer pour obtenir une densité optique à 600nm de 0,05 (D0600nm=0,05) (Pré-culture) -Inoculate 15 ml of TSB with the cryotube outlet. Dilute to obtain an optical density at 600nm of 0.05 (D0600nm=0.05) (Pre-culture)
-Incuber à 37°C, 24 h (± agitation, ± aérobie selon la bactérie étudiée)- Incubate at 37°C, 24 h (± agitation, ± aerobic depending on the bacterium studied)
-Inoculer 20 ml de TSB à D0600nm=0,05 avec la pré-culture (Culture de travail)- Inoculate 20 ml of TSB at D0600nm=0.05 with the pre-culture (working culture)
-Répartir la culture de travail dans les puits d’une plaque de polystyrène et ajouter la fraction vésiculaire à une concentration finale de 0,04 pg/pl dans 100 pl de volume final. - Distribute the working culture in the wells of a polystyrene plate and add the vesicle fraction to a final concentration of 0.04 µg/µl in 100 µl of final volume.
-Incuber à 37°C, 24 h (± agitation, ± aérobie selon la bactérie étudiée) - Incubate at 37°C, 24 h (± agitation, ± aerobic depending on the bacterium studied)
[0067] Etape 2 : Quantification de la formation du biofilm par coloration au cristal violet -Retirer les bactéries en suspension présentes dans chacun des puits de la plaque de polystyrène Step 2: Quantification of biofilm formation by crystal violet staining -Remove the bacteria in suspension present in each of the wells of the polystyrene plate
-Laver 2 fois avec 200 pl d’eau distillée chaque puit de la plaque de polystyrène-Wash twice with 200 µl of distilled water each well of the polystyrene plate
-Ajouter 150 pl de cristal violet à 0.5% (dilution dans de l’eau distillée) dans chaque puit -Add 150 µl of 0.5% crystal violet (dilution in distilled water) to each well
-Incuber 1 h sans agitation -Incubate 1 hour without shaking
-Laver 2 fois avec 200 pl d’eau distillée chaque puit de la plaque de polystyrène-Wash twice with 200 µl of distilled water each well of the polystyrene plate
-Ajouter 150 pl d’éthanol 95% (dilution dans de l’eau distillée) dans chaque puit-Add 150 µl of 95% ethanol (dilution in distilled water) to each well
-Mesurer l’absorbance à 595 nm -Measure absorbance at 595 nm
[0068] LISTE DES REFERENCES [0068] LIST OF REFERENCES
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2. Venkatesan N, Perumal G & Doble M, Bacterial resistance in biofilm-associated Bacteria Future Microbiol 2015;10(11 ):1743-50. DOI: 10.2217/fmb.15.69 2. Venkatesan N, Perumal G & Doble M, Bacterial resistance in biofilm-associated Bacteria Future Microbiol 2015;10(11):1743-50. DOI: 10.2217/fmb.15.69
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Claims

Revendications Claims
[Revendication 1 ] Utilisation de vésicules membranaires extracellulaires d’au moins un probiotique pour prévenir ou réduire la formation d’un biofilm à la surface d’un matériau. [Claim 1] Use of extracellular membrane vesicles of at least one probiotic for preventing or reducing the formation of a biofilm on the surface of a material.
[Revendication 2] Utilisation selon la revendication 1 , dans laquelle l’au moins un probiotique est choisi parmi une bactérie probiotique telle qu’un lactobacille, une bifidobactérie, un entérocoque, une propionibactérie, un streptocoque et une bactérie du genre Bacillus, ou une levure telle que Saccharomyces cerevisiae et Saccharomyces boulardi ou un de leurs mélanges. [Claim 2] Use according to claim 1, in which the at least one probiotic is chosen from a probiotic bacterium such as a lactobacillus, a bifidobacterium, an enterococcus, a propionibacterium, a streptococcus and a bacterium of the genus Bacillus, or a yeast such as Saccharomyces cerevisiae and Saccharomyces boulardi or a mixture thereof.
[Revendication 3] Utilisation selon la revendication 1 ou 2, dans laquelle le biofilm est un biofilm bactérien, un biofilm levurien ou un biofilm mixte. [Claim 3] Use according to claim 1 or 2, wherein the biofilm is bacterial biofilm, yeast biofilm or mixed biofilm.
[Revendication 4] Utilisation selon la revendication 3, dans laquelle : [Claim 4] Use according to claim 3, wherein:
- ledit biofilm bactérien comprend au moins une bactérie choisie parmi la famille des entérobactéries, notamment Salmonella enterica Enteritidis, Hafnia alvei et/ou Citrobacter freundii, le genre Staphylococcus, notamment Staphylococcus aureus ou Staphylococcus epidermidis, le genre Bacillus, notamment Bacillus cereus ou Bacillus subtilis, le genre Pseudomonas, notamment Pseudomonas aeruginosa et le genre Enterococcus, notamment Enterococcus faecalis - said bacterial biofilm comprises at least one bacterium chosen from the Enterobacteriaceae family, in particular Salmonella enterica Enteritidis, Hafnia alvei and/or Citrobacter freundii, the Staphylococcus genus, in particular Staphylococcus aureus or Staphylococcus epidermidis, the Bacillus genus, in particular Bacillus cereus or Bacillus subtilis , the genus Pseudomonas, in particular Pseudomonas aeruginosa and the genus Enterococcus, in particular Enterococcus faecalis
- ledit biofilm levurien comprend l’espèce levurienne Candida albicans- said yeast biofilm comprises the yeast species Candida albicans
- ledit biofilm mixte comprend un mélange d’au moins une bactérie choisie parmi la famille des entérobactéries, notamment Salmonella enterica Enteritidis, Hafnia alvei et/ou Citrobacter freundii, le genre Staphylococcus, notamment Staphylococcus aureus ou Staphylococcus epidermidis, le genre Bacillus, notamment Bacillus cereus ou Bacillus subtilis, le genre Pseudomonas, notamment Pseudomonas aeruginosa et le genre Enterococcus, notamment Enterococcus faecalis, et de l’espèce levurienne Candida albicans. - said mixed biofilm comprises a mixture of at least one bacterium chosen from the Enterobacteriaceae family, in particular Salmonella enterica Enteritidis, Hafnia alvei and/or Citrobacter freundii, the Staphylococcus genus, in particular Staphylococcus aureus or Staphylococcus epidermidis, the Bacillus genus, in particular Bacillus cereus or Bacillus subtilis, the genus Pseudomonas, in particular Pseudomonas aeruginosa and the genus Enterococcus, in particular Enterococcus faecalis, and the yeast species Candida albicans.
[Revendication 5] Utilisation selon l’une quelconque des revendications précédentes, dans laquelle les vésicules membranaires extracellulaires sont produites par un procédé comprenant les étapes suivantes: (a) culture d’au moins un probiotique dans des conditions adaptées à la production de vésicules membranaires extracellulaires, [Claim 5] Use according to any preceding claim, wherein the extracellular membrane vesicles are produced by a process comprising the following steps: (a) culture of at least one probiotic under conditions suitable for the production of extracellular membrane vesicles,
(b) séparation de l’au moins un probiotique et des vésicules membranaires extracellulaires produites à l’étape (a) et, (b) separating the at least one probiotic and the extracellular membrane vesicles produced in step (a) and,
(c) purification et concentration des vésicules membranaires extracellulaires. (c) purification and concentration of extracellular membrane vesicles.
[Revendication 6] Utilisation selon la revendication 5, dans laquelle ladite étape de purification et de concentration comprend au moins une étape choisie parmi la centrifugation différentielle, le gradient de densité, la chromatographie d’exclusion, l’ultrafiltration, l’immunocapture et la précipitation. [Claim 6] Use according to claim 5, wherein said purification and concentration step comprises at least one step selected from differential centrifugation, density gradient, exclusion chromatography, ultrafiltration, immunocapture and precipitation.
[Revendication 7] Utilisation selon l’une quelconque des revendications précédentes, dans laquelle ledit matériau est choisi parmi les métaux, les alliages de métaux, les polymères, le verre, la céramique, et les aliments.[Claim 7] Use according to any preceding claim, wherein said material is selected from metals, metal alloys, polymers, glass, ceramics, and foods.
[Revendication 8] Procédé de traitement d’une surface d’un matériau pour prévenir ou réduire la formation d’un biofilm sur ladite surface, ledit procédé comprenant une étape de mise en contact de vésicules membranaires extracellulaires provenant d’au moins un probiotique avec ladite surface. [Claim 8] A method of treating a surface of a material to prevent or reduce the formation of a biofilm on said surface, said method comprising a step of bringing extracellular membrane vesicles from at least one probiotic into contact with said surface.
[Revendication 9] Matériau comprenant à sa surface des vésicules membranaires extracellulaires d’au moins un probiotique, ou dans lequel sont incorporées des vésicules membranaires extracellulaires d’au moins un probiotique. [Claim 9] Material comprising on its surface extracellular membrane vesicles of at least one probiotic, or in which are incorporated extracellular membrane vesicles of at least one probiotic.
[Revendication 10] Matériau selon la revendication 9, dans lequel lesdites vésicules forment une couche d’une épaisseur comprise entre 10 et 500 nm sur ladite surface. [Claim 10] Material according to claim 9, wherein said vesicles form a layer of thickness between 10 and 500 nm on said surface.
[Revendication 11] Matériau selon la revendication 9, choisi parmi un matériau d’emballage, notamment d’emballage alimentaire, une conduite d’eau, un échangeur thermique, un pipeline, un cathéter, un pansement, une crème et un dispositif médical. [Claim 11] Material according to claim 9, selected from a packaging material, in particular food packaging, a water pipe, a heat exchanger, a pipeline, a catheter, a dressing, a cream and a medical device.
EP21851620.1A 2020-12-18 2021-12-17 Use of extracellular membrane vesicles for anti-biofilm purposes Pending EP4263800A1 (en)

Applications Claiming Priority (2)

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FR2013717A FR3118060A1 (en) 2020-12-18 2020-12-18 ANTI-BIOFILM USE OF EXTRACELLULAR MEMBRANE VESICLES
PCT/FR2021/052374 WO2022129808A1 (en) 2020-12-18 2021-12-17 Use of extracellular membrane vesicles for anti-biofilm purposes

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MA50086A (en) * 2017-09-08 2020-07-15 Evelo Biosciences Inc BACTERIAL EXTRACELLULAR (EV) VESICLES

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FR3118060A1 (en) 2022-06-24
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