US20150366920A1 - Method for preventing and/or treating infections, colonisations, or illnesses related to staphylococcus aureus, pseudomonas aeruginosa, streptococcus pyogenes, enterococcus faecium, enterobacter cloacae, proteus mirabilis, bacteroides fragilis, staphylococcus epidermidis, propionibacterium acnes, candida albicans and/or malassezia furfur - Google Patents

Method for preventing and/or treating infections, colonisations, or illnesses related to staphylococcus aureus, pseudomonas aeruginosa, streptococcus pyogenes, enterococcus faecium, enterobacter cloacae, proteus mirabilis, bacteroides fragilis, staphylococcus epidermidis, propionibacterium acnes, candida albicans and/or malassezia furfur Download PDF

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US20150366920A1
US20150366920A1 US14/648,837 US201314648837A US2015366920A1 US 20150366920 A1 US20150366920 A1 US 20150366920A1 US 201314648837 A US201314648837 A US 201314648837A US 2015366920 A1 US2015366920 A1 US 2015366920A1
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bacterium
bacteria
cncm
lactobacillus
wounds
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Nicolas Desroche
Patrice Arbault
Jean Guzzo
Sandra Rodrigues
Christelle Laurensou
Sylvain Mery
Laurent Apert
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Laboratoires Urgo SAS
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Laboratoires Urgo SAS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/02—Local antiseptics
    • 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
    • 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
    • C12R2001/25—Lactobacillus plantarum
    • 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
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Definitions

  • the present invention relates to bacteria having antagonist activities (hereinafter referred to as “antagonistic bacteria”) with respect to pathogenic bacteria or yeasts belonging to the genera and species Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pyogenes, Enterococcus faecium, Enterobacter cloacae, Proteus mirabilis, Bacteroides fragilis, Staphylococcus epidermidis, Propionibacterium acnes, Candida albicans and/or Malassezia furfur and to the use thereof as an active ingredient or in a medical device, in particular in the treatment and/or prevention of colonization and/or infections related to these pathogenic bacteria or yeasts.
  • antagonist activities hereinafter referred to as “antagonistic bacteria” with respect to pathogenic bacteria or yeasts belonging to the genera and species Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus py
  • the bacteria which are antagonistic with respect to pathogenic bacteria or yeasts have been selected for their abilities to inhibit the adhesion and the development and/or the proliferation of pathogenic bacteria or yeasts and thus to stabilize and/or regulate the ecosystem.
  • the invention relates to care products containing one or more non-pathogenic antagonistic strains intended for the prevention or treatment of infections or colonizations on the skin, wounds, mucosae and superficial body growths.
  • Staphylococcus aureus and Pseudomonas aeruginosa are responsible for a certain number of pathological conditions, such as skin and mucosae infections, wound infections, in particular infections of chronic wounds (ulcers, diabetic wounds), burns and surgical wounds. These bacterial species are in particular known to be responsible for delayed healing. They are also involved in other pathological conditions, such as gastrointestinal, urinary and pulmonary infections, otitis or sinusitis.
  • Pseudomonas aeruginosa can, under certain conditions, be pathogenic.
  • This very resistant bacterium is, with other gram-negative bacteria, increasingly often involved in nosocomial infections.
  • This strain has the ability to become organized as a biofilm, which makes it all the more resistant to antibiotics. It is one of the most difficult bacteria to treat clinically.
  • the mortality rate reaches 50% in vulnerable (immunodepressed) patients. It is a ubiquitous microorganism which is very resistant to numerous antiseptics, which is frequent in the hospital environment, and which leads to the appearance (because of its resistance to antibiotics) of actual hospital strains.
  • Staphylococcus aureus is the most pathogenic species of the Staphylococcus genus. It is responsible for food poisoning, suppurative localized infections and, in certain extreme cases, physical septicemia (graft, cardiac prostheses). The species proves to be an opportunistic pathogen in certain locations or under certain circumstances. It is a ubiquitous microorganism which has good resistance to natural purification mechanisms. S. aureus is also found in the commensal flora (in 15% to 30% of healthy individuals in the nasal fossae and the throat, it can also be found in small amounts in the digestive tract and often at the level of the perineum).
  • S. aureus has pathogenic capacities, in particular an invasive capacity, a capacity to multiply and to spread in the organism, and also a toxic capacity. S. aureus has a great capacity for developing antibiotic-resistant mutants (by way of example, mention may be made of methicillin-resistant Staphylococcus aureus , or MRSA, which is one of the main causes of nosocomial infections). As a result, it shares, with pyocyanic bacillus , the primary role in infections of hospital origin.
  • Biofilms are defined as complex assemblies of microorganisms (or microbial communities) which adhere to a biotic surface (living tissues, skin, etc.) or an abiotic surface (inert material such as silicone or steel) and are trapped in a matrix of organic polymers (Costerton et al., 1999, Dunne, 2002). These biofilms may thus be monospecific, i.e. compounds of a single bacterial or yeast species, or mixed when they are composed of several bacterial or yeast species.
  • bacterial strains which are antagonistic with respect to pathogenic species.
  • the term bacteriotherapy is then used.
  • These antagonistic strains are capable, by virtue of their metabolism, of producing antimicrobial molecules and/or of interfering with the adhesion of pathogenic bacteria and yeasts and/or of disrupting cell communication between pathogenic bacteria and yeasts and/or of possessing angiogenic activities. They can also control inflammation (immunomodulatory properties).
  • These bacteria form a positive biofilm at the surface of the skin, wounds, mucosae or superficial body growths, which can take hold temporarily and limit the implantation of pathogenic bacteria and yeasts.
  • These antagonistic strains generally belong to the family of lactic acid bacteria. These strains have been isolated from various matrices and mainly from feces. The activities of these strains are widely used for the prevention and treatment of disorders at the level of the intestinal mucosa; the term probiotic bacteria is then used. Other applications at the level of the mucosa of the ear, nose and throat and of the urogenital mucosa have also been described.
  • patent EP 871 503 which relates to a diaper or sanitary napkin comprising microorganisms chosen from the genera Lactobacillus curvatus, Lactobacillus plantarum or Lactococcus lactis having antagonistic properties which allow them to combat strains of undesirable microorganisms present or forming in the absorbent article, or in the urogenital zone.
  • Patent application WO 99/53932 relates to a composition for the treatment of otitis, comprising microorganisms chosen from Streptococcus sanguis, Streptococcus oralis and Streptococcus mitis.
  • the applicant has particularly focused on the strains which are antagonistic with respect to the pathogenic strains S. aureus and P. aeruginosa.
  • One of the objects of the present invention also potentially consists in creating a transient positive biofilm at the surface of the skin, superficial body growths, mucosae and/or wounds, which prevents or limits the implantation of pathogens and the proliferation thereof.
  • the antagonistic bacteria according to the present invention are particularly advantageous for use thereof in pathological conditions involving the pathogenic species S. aureus and P. aeruginosa , such as gastrointestinal, urinary and pulmonary infections, otitis or sinusitis, wounds or pathological conditions involving colonization of wounds, of the skin, of superficial body growths or of mucosae.
  • pathological conditions involving the pathogenic species S. aureus and P. aeruginosa such as gastrointestinal, urinary and pulmonary infections, otitis or sinusitis, wounds or pathological conditions involving colonization of wounds, of the skin, of superficial body growths or of mucosae.
  • the antagonistic bacteria according to the present invention are particularly advantageous for use thereof in the various types of wounds (chronic, acute, burns), but also in skin conditions (such as, for example, folliculitis, impetigo, eczema, boils, anthrax, whitlow, atopies, perleche, superinfections of lesions related to viruses such as the herpes virus or the chickenpox virus).
  • a wound is a lesion subsequent to a trauma, causing a loss of skin or an opening of the skin.
  • the healing process is set up in response to the formation of a wound.
  • the natural healing of a wound occurs mainly according to three successive phases, each of these phases being characterized by specific cell activities which bring about the progression of the repair process according to precise chronological sequences: the inflammatory phase, the granulation phase (or proliferative phase), and the scar formation phase.
  • the rapidity and the quality of the healing of a wound depend on the general condition of the affected organism, on the etiology of the wound, on the condition and the location of the wound, on the possible occurrence of an infection, and also on genetic factors possibly predisposing to healing disorders.
  • Tom or cut skin can no longer act as a barrier against microbes, which can then penetrate into the organism, causing an infection.
  • Colonization is the presence of a certain amount of bacteria and/or of yeasts within the wound without this leading to an inflammatory response. After multiplication of the microorganisms on the wound, and adhesion thereof to epithelial cells, an equilibrium is established between the patient and the microbial flora thereof. The microorganisms remain at the surface of the wound and adhere thereto so as to form a biofilm. From a quantitative point of view, colonization is characterized by an amount of microorganism of less than 10 5 bactreria/mm 3 . If the amount of bacteria and/or of yeasts exceeds this number, some authors cite critical colonization, then strictly local superficial infection in the presence of considerable bacterial and/or yeast colonization. This considerable presence of bacteria and/or of yeasts is detrimental to the correct operating of the healing process and induces delayed healing.
  • infection will be used when the presence of the bacterial and/or yeast microorganisms leads to a locoregional inflammatory response and the appearance of general signs with clinical signs which reflect tissue invasion by the microorganisms present (large amount of the microorganisms present, bacterial virulence, decrease in immune defense mechanisms of the patient).
  • the infection is characterized by the locoregional and general clinical signs.
  • Said infection can result in wound extension with exposure of underlying anatomical structures such as ligaments or bones.
  • Bacterial colonization does not require particular therapeutic approaches, whereas an infection requires the setting up of local and general antibacterial treatments.
  • the infection is generally the determining factor in the non-healing or delayed healing of wounds, directly or indirectly. Any bacterial and/or yeast contamination of a wound increases the inflammation. This may be beneficial in the case of moderate contamination (inevitable in the case of an open wound), but becomes deleterious in the case of infection of the wound which results in delayed healing.
  • the immediate complication of healing is first and foremost the infection which prevents the initiation of the healing process from being set up; a wound is considered to be infected when the amount of bacteria and/or yeasts present in the wound hinders the healing process or worsens the wound.
  • the healing usually requires the use of dermatological medicaments and of surgical tools (surgical knife, curette, etc.).
  • surgical tools surgical knife, curette, etc.
  • antiseptics such as chlorhexidine or hexamidine
  • proteolytic medicaments i.e. medicaments of which the purpose is to destroy the strips of dead skin which contaminate the wound.
  • active agents commonly used for treating wound infections mention may also be made of silver, copper, octenidine, iodine, PHMB to (polyhexamethylene biguanide) and honey.
  • antibacterial agents and/or antiseptics are not recommended in wounds.
  • the effectiveness of these active agents is short lived, they are inactivated by organic substances, they may be cytotoxic with respect to cell components, they have a broad spectrum, and, consequently, they will not only attack the pathogenic bacteria, but will also destroy the commensal flora.
  • a certain number of bacterial strains have developed a resistance to antiseptics/antibacterial agents.
  • bacterial strains which are antagonistic with respect to pathogenic strains. These strains are capable, by virtue of their metabolism, of producing antimicrobial molecules and/or interfering with the adhesion of pathogenic bacteria and/or yeasts. These bacteria can also form a positive biofilm at the surface of the skin, the wound, the mucosae or the superficial body growths, which can be established temporarily and limit the implantation of pathogenic bacteria and/or yeasts.
  • These antagonistic strains generally belong to the family of lactic acid bacteria and/or have been isolated from the commensal flora of the mucosae of human beings.
  • the activities of these strains are widely used for the prevention and treatment of disorders at the level of the intestinal mucosa, of the ENT mucosa and of the vaginal mucosa.
  • Such strains are described in WO 96/38159, WO 00/61201, EP 1 140 226, EP 1 461 012 and WO 2006/013441.
  • the antagonistic effect of these strains is due to various mechanisms, such as:
  • the applications using probiotic strains on the skin and wounds also generally make use of one of the activities previously described.
  • Probiotic strains of Bifidobacterium longum reuter, of Lactobacillus paracasei CNCM I-2116, of Lactobacillus johnsonii La1 and of Vitreoscilla filiformis , in topical forms, have also been used to regulate inflammation phenomena and dysregulations at the level of the skin (Gueniche et al., 2006, Gueniche et al., 2008a, Gueniche et al., 2008b, Gueniche et al., 2008c, Gueniche et al., 2008d, Gueniche et al., 2009, Gueniche et al., 2010).
  • the authors emphasize the immunomodulatory capacity of the strains.
  • Such strains are described in patent applications EP 1 593 382 and WO 2006/037922.
  • aeruginosa Peral et al., 2010, Ramos et al., 2010, Ramos et al., 2012.
  • a formulation of this strain in an alginate film has been described by Brachkova et al. (Brachkova et al., 2011) for the prevention of burn infections by P. aeruginosa.
  • a strain of Lactobacillus fermentum RC-14 has demonstrated notable capacities for inhibiting Staphylococcus aureus adhesion and limiting infections related to this microorganism at the level of surgical implants (Gan et al., 2002). This antimicrobial effect has been attributed to the release of a biosurfacant.
  • the objective of the present invention is to provide novel active agents based on bacteria which are antagonistic with respect to the pathogenic bacteria Staphylococcus aureus and/or Pseudomonas aeruginosa , and which are effective in the treatment and/or prevention of colonization and/or infections related to these pathogenic bacteria, in particular at the level of the skin, wounds, mucosae and superficial body growths.
  • these active agents can be administered directly in contact with the skin, wounds, mucosae and superficial body growths, or can easily be incorporated into compositions, in particular compositions suitable for topical application, and are non-invasive.
  • the antagonistic bacteria have been preselected on the basis of several criteria: (i) their capacities to inhibit the growth of pathogenic organisms, namely S. aureus and P. aeruginosa , (ii) their capacities to adhere and to form a positive biofilm on collagen and epidermis, (iii) their capacities to limit the adhesion of pathogenic microorganisms on matrices containing collagen and (iv) their capacities to limit the inflammatory reaction (TNF- ⁇ production by macrophages).
  • These bacteria are capable of developing a barrier effect at the level of the skin, wounds, mucosae and superficial body growths and thus of preventing and limiting the risks of colonizations and infections.
  • the inventors have been particularly interested in selecting strains which are antagonistic mainly with respect to the S. aureus and P. aeruginosa , which are the pathogenic species predominantly involved in infections, in particular nosocomial infections.
  • the inventors have selected bacterial strains which, in addition to inhibiting strains of S. aureus and P. aeruginosa , inhibit the development of other pathogenic species, such as Streptococcus pyogenes, Enterococcus faecium, Enterobacter cloacae, Proteus mirabilis, Bacteroides fragilis, Staphylococcus epidermidis, Propionibacterium acnes, Candida albicans and Malassezia furfur . These microorganisms are more particularly involved in wound and skin infections.
  • Propionibacterium acnes is an anaerobic gram-positive bacillus which grows relatively slowly and is normally present in the skin, the hair and the mucosae. It is responsible for acne.
  • the bacterium is widely present in the skin flora of most adults in good health, but it is also responsible for post-operative infections, which, in particular in the case of the presence of an implant, are potentially severe: central nervous system infections, endophthalmitis, endocarditis, infections of the ENT and pulmonary sphere, spondylodiscitis, peritonitis and osteoarticular infections.
  • Staphylococcus epidermidis is a commensal bacterium of the skin in virtually 100% of human beings; its lipolytic properties allow it to prosper in sebum. It is normally harmless, but it causes authentic infections in patients whose immune system is compromised or patients who have catheters or prostheses. These may be dermatological infections and nasal infections, such as sinusitis or else urinary infections in women and more rarely in men. These bacteria have the capacity to produce biofilms which allow them to adhere to the surfaces of medical prostheses.
  • red plaques covered with fatty and yellowish squamae, which are more or less prurigineous, predominant in the zones rich in sebaceous glands, the seborrheic zones.
  • the topography of the lesions is suggestive: grooves between the nose and the lips, root of the eyebrows, scalp, wings of the nose, folds of the pinnae, concha of the ears, external auditory canals.
  • the scalp frequent involvement results in a more or less seborrheic dandruff condition.
  • two common zones are noted in humans: the sternum and the region between the two shoulder blades.
  • Pityriasis versicolor is a superficial mycosis, related to the proliferation on the skin of Malassezia furfur .
  • This yeast normally lives at the surface of the skin, but, in certain situations, it multiplies at great speed and causes these small brown or discolored marks.
  • This fungus has a liking particularly for oily skin, lying on the thorax, but also on the neck and the shoulders, on the upper back and on the upper limbs, rarely on the lower limbs.
  • a subject of the present invention is therefore a bacterium chosen from the strains of Lactobacillus saniviri also denoted in the present application by F3C5p (registered on Jul. 12, 2012, under No. CNCM I-4650 at the Collection Nationale de Cultures de Microorganismes [National Collection of Microorganism Cultures], Institut Pasteur, 25 rue du Dondel Roux, 75724 Paris Cedex 15, France, hereinafter denoted “CNCM”), Lactobacillus salivarius also denoted in the present application by F50C2p, F52C3p and F41C3p (registered respectively under Nos.
  • a subject of the present invention is also a bacterium according to the invention for use as an immunomodulator.
  • the subject of the present invention is also the use of such a bacterium as an active ingredient or as a medical device or as a cosmetic or else as a food supplement.
  • the bacterium according to the invention may also be used as an active ingredient in a medicament, in a medical device, in a cosmetic or in a food supplement.
  • the present invention is also directed toward a bacterium according to the invention for use in the treatment and/or prevention of colonization and/or infections related to at least one bacterium or one yeast chosen from P. aeruginosa, S. aureus, S. pyogenes, E. faecium, E. cloacae, P. mirabilis, B. fragilis, Staphylococcus epidermidis, Propionibacterium acnes, Candida albicans and Malassezia furfur.
  • bacterium or one yeast chosen from P. aeruginosa, S. aureus, S. pyogenes, E. faecium, E. cloacae, P. mirabilis, B. fragilis, Staphylococcus epidermidis, Propionibacterium acnes, Candida albicans and Malassezia furfur.
  • the present invention is directed toward a bacterium according to the invention for use in the treatment and/or prevention of colonization and/or infections related to P. aeruginosa and/or S. aureus.
  • the present invention relates more particularly to the treatment and/or prevention of wound and skin colonization and/or infections by pathogenic bacteria or yeasts.
  • treatment means reducing and/or inhibiting the development of this infection or colonization.
  • prevention of or “preventing” an infection or colonization means reducing and/or avoiding the appearance of the symptoms of the infection or colonization.
  • infection or colonization of wounds is intended to mean an infection or a colonization chosen from diabetic foot ulcers, leg ulcers of arterial origin, leg ulcers of venous origin, bedsores, whitlow, infections related to acute wounds, infections related to traumatic wounds, such as wounds caused by severing, penetrating wounds, wounds caused by thermal or caustic agents and burns; and infections related to post-operative wounds, such as simple sutured wounds of a surgical incision, complex sutured wounds after skin excision, surgical dermabrasions and non-suturable wounds requiring second-intention healing.
  • the bacterium according to the invention can be incorporated into a composition, for instance a medical device.
  • a medical device is intended to mean prostheses, catheters, dressings, ostomy bags, surgical drapes, urinary catheters, endotracheal tubes, tympanostomy tubes, bandages, support bandages, orthopedic and breast implants, contact lenses, intrauterine devices, or else materials for sutures.
  • a subject of the present invention is also a composition comprising at least one bacterium according to the invention.
  • Said composition comprises, in an acceptable medium, at least one bacterium according to the invention.
  • acceptable medium is intended to mean a medium that is compatible with the skin, wounds, mucosae and superficial body growths.
  • composition according to the invention may be administered topically, orally or parenterally.
  • such a composition or such a bacterium is suitable for topical application to the skin, wounds, superficial body growths or mucosae, such as the nose and throat, urogenital, digestive or respiratory mucosa.
  • the composition may thus be directly applied to the skin, wounds or mucosae.
  • topical application is intended to mean application to the skin, wounds, mucosae and/or superficial body growths.
  • composition according to the invention preferably comprises 10 3 to 10 12 bacteria, preferably 10 6 to 10 11 .
  • the topical composition may be in liquid, paste or solid form, and more particularly in the form of a salve, a cream, a milk, an ointment, a powder, an impregnated pad, a syndet, a wipe, a solution, a gel, a spray, a foam, a suspension, a lotion, a stick, a shampoo or washing base. It may also be in the form of a suspension of microspheres or nanospheres or of lipid or polymeric vesicles or of microcapsules or of a polymeric patch and a hydrogel for controlled release.
  • This composition for topical application may be in anhydrous form, in aqueous form or in emulsion form.
  • the parenteral composition may be in the form of a solution for subcutaneous injection (injectable solution).
  • the oral composition may be in the form of a solution, a powder, a gel capsule or a tablet, or may be integrated into a food product, such as a dairy product.
  • the bacterium according to the invention may be incorporated into the composition in the form of an inactivated cell, in particular inactivated by heat, by UV radiation or by any other process. It may also be incorporated in the form of a living cell which may be encapsulated or nonencapsulated, immobilized or nonimmobilized, and lyophilized or nonlyophilized, or else in the form of a cell extract. It may also be a cell lysate or any other type of presentation known to those skilled in the art.
  • the antagonistic bacteria used in the context of the present invention or a galenic formulation containing them, will be applied directly to the wound, either in the form of a topical composition, or integrated into a synthetic or nonsynthetic matrix which is part of the composition of a dressing.
  • the bacteria are formulated in a dressing, or else formulated in a composition which is itself included in a dressing or may be included at the time of application of the dressing.
  • the dressing according to the invention therefore comprises at least one bacterium according to the invention, or at least one composition according to the invention.
  • the antagonistic bacteria according to the invention or the compositions according to the invention containing them may be incorporated into any component of the structure of a dressing, with the proviso that said bacteria can directly or indirectly come into contact with the surface of the wound.
  • the bacterium (or a composition according to the invention containing it), will be incorporated into the layer of the dressing which comes into contact with the wound, or will be deposited on the surface of the dressing which comes into contact with the wound.
  • the bacterium (or a composition according to the invention containing it) may thus be deposited, in a continuous or discontinuous manner, on the surface intended to come into contact with the wound:
  • a dressing is intended to denote all types of dressings used for the treatment of wounds.
  • a dressing comprises at least one adhesive or nonadhesive layer or matrix.
  • the layer or surface which comes into contact with the wound may consist of any material or combination of materials normally used for this purpose in the dressings field.
  • materials mention may be made of absorbent foams, in particular polyurethane-based hydrophilic foams; textile materials, in particular nonwovens based on absorbent or superabsorbent fibers; hydrogels; or a combination of these materials; an absorbent or nonabsorbent adhesive material; an adherent or nonadherent interface structure.
  • the galenic or the structure of the dressing may be adjusted in order to obtain a specific release profile for the bacterium, which is rapid or delayed, as required.
  • the bacterium (or a composition according to the invention containing it) may be integrated into any type of existing dressing, such as, without these examples being limiting:
  • the amount of bacterium or bacteria used in the galenic formulation or in the dressing will be adjusted according to the desired kinetics and also the specific constraints related to its nature, solubility, heat resistance, etc.
  • the bacterium or bacteria according to the invention will be incorporated in an amount such that the amount of bacteria released to into the wound exudates is between 0.001 g/l and 50 g/l, and preferably between 0.01 and 10 g/l.
  • the use of the dressing may require prior impregnation or hydration of the dressing with a solution, for instance physiological saline, in order to activate the antagonistic bacteria.
  • a solution for instance physiological saline
  • composition according to the invention may comprise one or more antagonistic strains, optionally combined with at least one compound chosen from probiotics, prebiotics and yeasts.
  • prebiotics mention may be made, by way of example, of fructans such as inulin, fructooligosaccharides or trans-galactooligosaccharides, or else long-chain or branched-chain sugars.
  • the bacterium according to the invention may also be combined with active agents in particular chosen from antifungal agents, painkillers, anti-inflammatories, agents which promote healing, moisturizing agents, keratolytic agents, restructuring active agents, and anesthetics.
  • active agents in particular chosen from antifungal agents, painkillers, anti-inflammatories, agents which promote healing, moisturizing agents, keratolytic agents, restructuring active agents, and anesthetics.
  • the active agents which can be introduced into the composition according to the invention may be chosen from:
  • FIG. 1 represents the inhibition halos observed after contact of the bacteria according to the invention with pathogenic bacteria on agar medium after 48 hours of incubation.
  • FIG. 2 represents an inhibition halo formed after contact with the bacteria according to the invention, incorporated into a dressing, with pathogenic bacteria on agar medium after 48 hours of incubation.
  • FIG. 3 represents the capacity of the bacteria according to the invention to adhere on reconstituted epidermises (as amount of bacteria adhered in CFU/cm 2 ).
  • FIG. 4 represents the results of tests for competition of adhesion on collagen surfaces of the bacteria according to the invention with respect to S. aureus (amount of S. aureus adhered after 24 h of incubation (CFU/cm 2 )).
  • FIG. 5 represents the results of the tests for exclusion on collagen surfaces of the bacteria according to the invention with respect to S. aureus (amount of S. aureus after 24 h of incubation (CFU/cm 2 )).
  • FIG. 6 represents the results of the tests for competition of adhesion on collagen surfaces of the bacteria according to the invention with respect to P. aeruginosa (amount of P. aeruginosa adhered after 24 h of incubation (CFU/cm 2 )).
  • FIG. 7 represents the results of the tests for exclusion on collagen surfaces of the bacteria according to the invention with respect to P. aeruginosa (amount of P. aeruginosa after 24 h of incubation (CFU/cm 2 )).
  • FIGS. 8 a and 8 b represent the amount of TNF- ⁇ released (pg/ml) by macrophages after 3.5 hours of contact with the bacteria according to the invention, without or with stimulation by LPS from E. coli O127:B8.
  • Lactobacillus saniviri F3C5p (CNCM I-4650), Lactobacillus salivarius F50C2p (CNCM I-4651), Lactobacillus salivarius F52C3p (CNCM I-4652), Lactobacillus salivarius F41C3p (CNCM I-4653). Streptococcus mitis F3C2v (CNCM I-4654) and Lactobacillus pentosus or plantarum L1C1 (CNCM I-4655)
  • the Lactobacillus saniviri F3C5p (CNCM I-4650), Lactobacillus salivarius F50C2p, F52C3p and F41C3p (respectively CNCM I-4651, CNCM I-4652 and CNCM I-4653), Streptococcus mitis F3C2v (CNCM I-4654) and Lactobacillus pentosus or plantarum L1C1 (CNCM I-4655) bacteria were cultured in Man Rogosa Sharpe (MRS) medium for 16 hours, at 37° C. and under anaerobic conditions.
  • MRS Man Rogosa Sharpe
  • TSA Tryptic Soy Agar
  • MRSA methicillin-resistant Staphylococcus aureus
  • MRSA methicillin-resistant Staphylococcus aureus
  • a negative control was carried out by depositing 10 ⁇ l of sterile MRS medium.
  • the plates were incubated under anaerobic conditions at 37° C. for 24 and 48 hours. After incubation, the diameter of the inhibition halos formed around the drops (in mm) was measured.
  • the sign “ ⁇ ” indicates an absence of inhibition halo
  • a sign “+” indicates an inhibition halo with a diameter of less than 1 mm
  • a sign “++” indicates an inhibition halo with a diameter of between 1 mm and 3 mm
  • a sign “+++” indicates an inhibition halo with a diameter of greater than 3 mm
  • Lactobacillus saniviri F3C5p (CNCM I-4650), Lactobacillus salivarius F50C2p (CNCM I-4651), Lactobacillus salivarius F52C3p (CNCM I-4652), Lactobacillus salivarius F41C3p (CNCM I-4653), Streptococcus mitis F3C2v (CNCM I-4654) and Lactobacillus pentosus or plantarum L1C1 (CNCM I-4655) after Incorporation into a Dressing
  • the F3C5p, F50C2p, F52C3p, F41C3p, F3C2v and L1C1 bacteria were cultured in Man Rogosa Sharpe (MRS) medium for 16 hours, at 37° C. and under anaerobic conditions.
  • MRS Man Rogosa Sharpe
  • Pieces of dressings (1 cm ⁇ 1 cm) composed of polyurethane foam and of a lipido-colloid mesh were impregnated with 500 ⁇ l of the culture of bacteria according to the invention (concentration of between 10 9 and 10 10 CFU.ml ⁇ 1 ) and deposited at the surface of Tryptic Soy Agar (TSA) media preinoculated in the body of the agar media with 10 6 to 10 7 cells of Staphylococcus aureus MRSA ATCC 43300 or Pseudomonas aeruginosa ATCC 9027.
  • TSA Tryptic Soy Agar
  • a negative control was carried out by depositing 500 ⁇ l of sterile MRS medium.
  • the plates were incubated under anaerobic conditions at 37° C. for 24 and 48 hours. After incubation, the antimicrobial activity is demonstrated by the presence of an inhibition halo. After incubation, the inhibition halos are visible around the dressings impregnated with the bacteria Lactobacillus saniviri F3C5p, Lactobacillus salivarius F50C2p, Lactobacillus salivarius F52C3p, Lactobacillus salivarius F41C3p, Streptococcus mitis F3C2v and Lactobacillus pentosus or plantarum L1C1 (cf. FIG. 2 ). No inhibition halo is visible around the dressings impregnated with the MRS medium.
  • Tests for adhesion of the Lactobacillus saniviri F3C5p, Lactobacillus salivarius F50C2p, Lactobacillus salivarius F52C3p, Lactobacillus salivarius F41C3p, Streptococcus mitis F3C2v and Lactobacillus pentosus or plantarum L1C1 bacteria were carried out on reconstituted epidermises of EpiSkin (SkinEthic) type (1.07 cm 2 ) aged 13 days.
  • the inserts containing the epidermises were placed in a 12-well plate with 2 ml of maintenance medium and incubated for 24 hours at 35° C. ⁇ 2° C. in order to regenerate the epidermises.
  • the Lactobacillus saniviri F3C5p, Lactobacillus salivarius F50C2p, Lactobacillus salivarius F52C3p, Lactobacillus salivarius F41C3p, Streptococcus mitis F3C2v and Lactobacillus pentosus or plantarum L1C1 bacteria are capable of adhering to the epidermis and of persisting for 24 hours.
  • the Lactobacillus saniviri F3C5p strain exhibits the greatest adhesion capacity (cf. FIG. 3 ).
  • Lactobacillus saniviri F3C5p The capacity of the Lactobacillus saniviri F3C5p, Lactobacillus salivarius F50C2p, Lactobacillus salivarius F52C3p, Lactobacillus salivarius F41C3p, Streptococcus mitis F3C2v and Lactobacillus pentosus or plantarum L1C1 bacterial strains to limit and/or inhibit the adhesion of pathogenic species ( S. aureus MRSA ATCC 43300 or P. aeruginosa ATCC 9027) on matrices containing collagen was evaluated. The tests were carried out in 24-well microplates coated with type I collagen (BD BiocoatTM Collagen I).
  • the pathogenic bacteria were cultured for 16 hours at 37° C. in Tryptic Soy Broth (TSB) medium. After incubation, the cultures were diluted in TSB medium to concentrations of approximately 2.5 ⁇ 10 7 CFU.ml ⁇ 1 , 2.5 ⁇ 10 5 CFU.ml ⁇ 1 and 2.5 ⁇ 10 3 CFU.ml ⁇ 1 .
  • Lactobacillus saniviri F3C5p Lactobacillus salivarius F50C2p, Lactobacillus salivarius F52C3p, Lactobacillus salivarius F41C3p, Streptococcus mitis F3C2v and Lactobacillus pentosus or plantarum L1C1 bacteria were cultured in Man Rogosa Sharpe (MRS) medium for 16 hours, at 37° C. and under anaerobic conditions.
  • MRS Man Rogosa Sharpe
  • the F3C5p, F50C2p and F52C3p bacteria and the pathogenic bacterium were simultaneously added at a respective concentration of approximately 2.5 ⁇ 10 7 CFU.ml ⁇ 1 and 2.5 ⁇ 10 7 CFU.ml ⁇ 1 or 2.5 ⁇ 10 5 CFU.ml ⁇ 1 or 2.5 ⁇ 10 3 CFU.ml ⁇ 1 for the pathogenic bacterium.
  • the F41C3p, F3C2v and L1C1 bacteria and the pathogenic bacterium ( S. aureus or P. aeruginosa ) were simultaneously added at a respective concentration of approximately 2.5 ⁇ 10 7 CFU.ml ⁇ 1 and 2.5 ⁇ 10 7 CFU.ml ⁇ 1 .
  • the F3C5p, F50C2p and F52C3p bacteria were introduced into the wells at a concentration of approximately 2.5 ⁇ 10 7 CFU.ml ⁇ 1 and incubated at 37° C. for 24 hours. After adhesion, the wells were washed with physiological saline and the pathogenic strain ( S. aureus or P. aeruginosa ) was introduced into the wells at concentrations of approximately 2.5 ⁇ 10 7 CFU.ml ⁇ 1 or 2.5 ⁇ 10 5 CFU.ml ⁇ 1 or 2.5 ⁇ 10 3 CFU.ml ⁇ 1 .
  • the F41C3p, F3C2v and L1C1 bacteria were tested at a concentration of approximately 2.5 ⁇ 10 7 CFU.ml ⁇ 1 with the pathogenic strain ( S. aureus or P. aeruginosa ) at a concentration of approximately 2.5 ⁇ 10 7 CFU.ml ⁇ 1 .
  • the pathogenic bacteria were specifically counted on Baird-Parker medium supplemented with an emulsion of egg yolk containing potassium tellurite for S. aureus or cephalosporin/fucidin/cetrimide (CFC) medium for P. aeruginosa . After 24 h of incubation at 35° C. ⁇ 2° C., the characteristic colonies were counted. A nontreated control with the bacteria according to the invention was carried out for each of the tests.
  • the F3C5p, F50C2p, F52C3p, F41C3p, F3C2v and L1C1 bacteria make it possible to strongly limit the adhesion of S. aureus and the colonization of the collagen-coated substrate compared with the nontreated control for the three concentrations tested (cf. FIGS. 4 and 5 ).
  • the F3C5p, F50C2p, F52C3p, F41C3p, F3C2v and L1C1 bacteria make it possible to limit the adhesion of P. aeruginosa and the colonization of the collagen-coated substrate compared with the nontreated control when the initial contamination is less than 10 5 CFU/cm 2 (cf. FIGS. 6 and 7 ).
  • Lactobacillus saniviri F3C5p, Lactobacillus salivarius F50C2p, Lactobacillus salivarius F52C3, Lactobacillus pentosus/plantarum L1C1, Lactobacillus salivarius F41C3p and Streptococcus mitis F3C2v strains were cultured in Man Rogosa Sharpe (MRS) medium for 16 hours, at 37° C. and under anaerobic conditions. The bacteria were recovered by centrifugation and then ultrasound-inactivated or heat-inactivated (95° C.) in order to obtain bacterial extracts.
  • MRS Man Rogosa Sharpe
  • THP1 monocyte cells (ATCC TIB-202) were cultured in Roswell Park Memorial Institute (RPMI) medium and were differentiated to macrophages by addition of phorbol-12-myristate-13-acetate (PMA). After 24 hours of culture, the macrophages obtained were exposed (i) either to the bacterial extracts alone, (ii) or to LPS from E. coli O127:B8, (iii) or to LPS from E. coli O127:B8 in the presence of cell extract. After 3.5 hours of contact, the concentration of tumor necrosis factor (TNF- ⁇ ) was measured in the culture supernatant using an ELISA method.
  • RPMI Roswell Park Memorial Institute
  • the stimulation of the macrophages with the LPS from E. coli brings about a considerable release of TNF- ⁇ in the absence of bacterial extracts (concentration about 500 pg/ml).
  • the simultaneous addition of the cell extracts prepared from the various bacterial strains (F3C5p, F41C3p, F50C2p, F52C3p, L1C1 or F3C2v) with the LPS makes it possible to significantly reduce (by a factor of 2 to 5) the release of TNF- ⁇ by the macrophages.

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US20180036356A1 (en) 2018-02-08
CA2892478A1 (fr) 2014-06-26
JP6309967B2 (ja) 2018-04-11
CN105189732B (zh) 2018-11-09
BR112015014118A2 (pt) 2017-07-11
CN105189732A (zh) 2015-12-23
ES2645099T3 (es) 2017-12-04
EP2931924B1 (fr) 2017-10-04
FR2999601A1 (fr) 2014-06-20
US10449222B2 (en) 2019-10-22

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