Patent Application
20150079040
The present invention relates to probiotic bacteria and particularly, although not exclusively, to medical and cosmetic applications for probiotic bacteria and lysates thereof.
Humans live in constant contact with a multitude of micro-organisms. The gut is by far the most heavily colonised and the gut microbiota has been shown to play multiple roles in normal physiology including nutrient sequestration (3), and development of normal immune responses (22).
Amongst the normal gut microbiota are the so-called probiotic bacteria. Ingestion of these has been shown to prevent or treat gastrointestinal disorders such as antibiotic-associated diarrhoea (47) and inflammatory bowel disease (42). The mechanisms underlying these effects are largely unknown. However, studies have suggested that probiotics can inhibit colonisation of the gut by pathogens. Work in vitro has suggested that probiotics use various mechanisms to inhibit pathogens including direct competition for binding sites on epithelial cells (12) and competition with pathogenic bacteria for nutrients. Probiotic organisms are also able to produce inhibitory substances such as bacteriocins (13) and organic acids (25) that can kill or limit the growth of pathogens. Selected probiotics such as L. plantarum 299v have been shown to upregulate mucin production by epithelial cells thereby preventing pathogen attachment (33). Probiotics may also produce biosurfactants that allow attachment of the probiotic while inhibiting attachment of pathogenic bacteria to cells (43).
In contrast to the gut, very little is known about the normal interactions between the skin microflora and the epidermis. Recent work suggests that skin commensals may also be able to limit the colonisation of the skin by pathogenic bacteria (48). Studies also suggest that certain skin diseases (such as acne vulgaris and atopic dermatitis) may be associated with disruptions to the normal microflora (4, 6, 8). Therefore, the idea that the skin microflora may be modulated using specific skin commensals to promote health or inhibit disease has received some attention (30, 31). However, the skin commensal bacteria can also be pathogenic under certain circumstances (16). By contrast probiotics are generally regarded as safe (GRAS) and therefore, these bacteria could potentially be used topically if they have therapeutic value (17). So far the limited amount of research in this area suggests that conventional probiotic bacteria may be of significant value when used on the skin. For example, topical application of a B. longum reuter lysate has been shown to induce clinical improvement of “reactive skin”. This is skin that is more sensitive to physical changes such as atmospheric temperature, and to chemical changes such as seen with topically applied products (20). Application of the B. longum lysate to the skin of volunteers was shown to decrease sensitivity and decrease transepidermal water loss (TEWL) after tape stripping. Additionally, application of the lysate to ex-vivo skin was shown to decrease signs of inflammation such as vasodilation, oedema and TNF-α release (20). Topical application of L. plantarum has also been demonstrated to improve tissue repair in a burned mouse model and prevent infection in chronic leg ulcers and burns in humans (40, 41, 46). However, in general, the mechanisms underlying these effects are unknown.
Staphylococcus aureus is a transient coloniser of skin predominantly in the moist, warm regions of the body such as the groin, axilla and the anterior nares (28). Up to 60% of people are intermittent carriers while 20% of people may be stably colonised (28). While normal carriage is asymptomatic, S. aureus may invade tissues (e.g. through broken skin) where it causes diseases ranging from the relatively minor impetigo and scalded skin syndrome, to life threatening conditions such as septicaemia (29). Furthermore, S. aureus infection is often a secondary phenomenon in skin with underlying conditions such as atopic dermatitis (27).
Tight junctions (TJs) are multi protein complexes sealing the paracellular space between adjacent epithelial cells and limiting transport through this pathway to small, hydrophilic molecules and ions (55). Transepithelial electrical resistance (TEER) is a measure of TJ function (56, 57). TJ function is often reflected in the expression levels of particular proteins involved in the complexes. The main TJ proteins expressed by keratinocytes include claudin 1, claudin 4, ZO-1 and occludin.
Changes in the expression levels of claudins in particular have been shown many times previously to be linked to changes in barrier function. To date 24 mammalian claudins have been identified and these generally fall into two classes—those which strengthen the barrier and those which form selective pores (58). Several lines of evidence point to a role for claudins 1 and 4 as barrier strengthening claudins. In cell lines, overexpression of claudin 1 increased the TEER and decreased the permeability of cells to paracellular markers. Claudin 4 seals the paracellular space against the passage of ions and in doing so increases the TEER of monolayers [59, 60]. Increases in ZO-1 expression enhance the TEER of A431 cells (61) and the hormone GLP1 also enhances TJ function in caco-2 cells by increasing ZO-1 and occludin expression (62). The existence of TJs in skin has been discovered only relatively recently. Therefore, at present the contribution of particular TJ protein species to skin barrier function is largely unknown. However, genetic loss of claudin 1 is known to be lethal in mice (63).
Keratinocytes sense the presence of bacteria via pattern recognition receptors such as toll like receptors (TLRs). Several lines of evidence in the gut have pointed to a relationship between TLR activation and changes in TJ barrier function. Recent work by Yuki et al. (53) demonstrated TLR-mediated augmentation of TJ function in keratinocytes in response to bacterial ligands such as peptidoglycan.
WO2011/029784 describes the anti-inflammatory action of a strain of Lactobacillus rhamnosus strain LMG P-25211 on the skin, and use in preventing or treating inflammatory or allergic skin conditions.
WO2010/056198 describes preparations useful for the treatment of Staphylococcus induced infections which comprise a combination of one or more viable α-Streptococcus strains and one or more viable Lactobacillus strains chosen from the groups of Lactobacillus rhamnosus strain LB21, Lactobacillus plantarum strain LB3, Lactobacillus plantarum strain LB7.
WO2006/000992 describes cosmetic treatments intended for preventing and/or treating dry or sensitive skin, where a probiotic microorganism, fraction or metabolite thereof is combined with at least one divalent inorganic cation.
Marsella et al (Veterinary Immunology and Immunopathology 146 (2012) 185-189) describe the immunomodulatory effects of oral administration of Lactobacillus rhamnosus GG in a model of allergic/atopic dermatitis.
EP2161329A1 describes the immunomodulatory effects of extracts from Lactobacillus bacteria and describes use in the treatment of inflammatory disorders.
Hoang et al (Inflammation & Allergy—Drug Targets, 2010, 9, 192-196) describes the use of orally administered Lactobacillus rhamnosus cell lysate as a daily immunobiotic supplement in the treatment of atopic eczema.
WO2011/045471 describes the use of Lactobacillus rhamnosus GG in combination with Lactobacillus rhamnosus LC705 for use in the treatment of a respiratory infection, in particular for use against viruses causing respiratory infections, e.g. influenza virus.
The invention provides probiotic bacteria and lysates thereof for use in methods of medical treatment, in methods of cosmetic treatment and for use as an anti-bacterial agent. Use in the treatment of infections, including skin infections and methods involving regeneration or repair of the skin barrier are disclosed.
The invention provides a probiotic bacterium for use in a method of treatment. The probiotic bacterium may be provided in the form of a lysate. In preferred embodiments the probiotic bacterium is a strain of L. rhamnosus, L. reuteri, or Bifidobacterium longum. Preferably, the probiotic bacterium is L. rhamnosus GG. L. rhamnosus GG is deposited with ATCC under accession number 53103. The method of treatment may comprise administering L. rhamnosus GG to the patient. The method may comprise administering L. rhamnosus GG to the skin of the patient. The methods of the invention result in the treatment or alleviation of the condition in the patient undergoing the treatment.
In some embodiments the method of medical treatment is a method of treatment or prevention of infection, for example a bacterial infection such as a Staphylococcus infection. The infection may be a Staphylococcus aureus infection. The infection may be a skin infection. The probiotic bacterium, lysate or composition may be administered separately, sequentially or simultaneously with the infective agent, for example to a wound or other breach in the skin barrier.
In some embodiments (or additionally), the method of medical treatment is a method involving repair or regeneration of the skin barrier. In methods involving the repair or regeneration of the skin barrier the probiotic bacterium may be a strain of L. rhamnosus, L. reuteri or Bifidobacterium longum. The probiotic bacterium may be L. rhamnosus GG. In these methods two different strains of probiotic bacterium may be administered in the same method of treatment, for example L. reuteri and L. rhamnosus GG may be co-administered. The probiotic bacterium may be administered live, inactivated, or as a lysate. Where two or more probiotic bacteria are to be administered, one or more may be administered as a lysate.
Methods of treatment involving repair or regeneration of the skin barrier may involve the repair or regeneration of the skin after injury.
Methods of treatment involving repair or regeneration of the skin barrier may be useful in the treatment of psoriasis, icthyosis, dermatitis, wound healing, acne (including Hiradenitis Suppurativa), Burns, Diaper Rash, Netherton's Syndrome, Actinic Keratosis, Dermatomycoses, Dermatosis or Ectodermal Dysplasia or other disorders associated with damage or breakdown of the skin barrier. Other such disorders will be readily appreciable to the person skilled in the art. Preferably, the methods involve the administration of the probiotic bacterium to the skin.
The invention further provides the use of a probiotic bacterium or lysate thereof in the manufacture of a medicament for the treatment of skin infection and/or repair or regeneration of the skin barrier. The probiotic bacterium may be an L. rhamnosus, L. reuteri, or Bifidobacterium longum strain. It may be L. rhamnosus GG. The medicament may comprise two or more strains of probiotic bacterium, or may be co-administered with another strain of probiotic bacterium or lysate thereof. The medicament may be useful in the repair or regeneration of the skin after injury, or in the treatment of psoriasis, icthyosis, dermatitis, wound healing, acne (including Hiradenitis Suppurativa), Burns, Diaper Rash, Netherton's Syndrome, Actinic Keratosis, Dermatomycoses, Dermatosis or Ectodermal Dysplasia or other disorders associated with damage or breakdown of the skin barrier. Other such disorders will be readily appreciable to the skilled person.
The invention also provides a pharmaceutical composition comprising a probiotic bacterium. The pharmaceutical composition may comprise an L. rhamnosus probiotic bacterium such as L. rhamnosus GG or L. reuteri, or may comprise a Bifidobacterium longum probiotic bacterium. The pharmaceutical composition may further comprise another probiotic bacterium strain such as one of L. reuteri, L. rhamnosus GG or Bifidobacterium longum. The pharmaceutical composition may comprise lysates of one or more of the probiotic bacterial strains, or a mixture of lysates, live or inactivated probiotic bacteria. The pharmaceutical composition may be formulated for administration to the skin of the patient in need of such treatment.
The invention further provides methods of cosmetic treatment involving the administration of probiotic bacterium or lysate thereof to a subject, or use of such probiotic bacterium or lysate thereof in a method of cosmetic treatment. Such embodiments do not involve the treatment of the human or animal body by therapy or surgery. In such cosmetic methods the probiotic bacterium preferably comprises an L. rhamnosus probiotic bacterium or lysate thereof, for example L. rhamnosus GG, or an L. reuteri probiotic bacterium, or lysate thereof, or a Bifidobacterium longum probiotic bacterium, or lysate thereof. The methods may involve the administration of the probiotic bacterium to the skin of the subject.
The invention also provides cosmetic compositions comprising an L. rhamnosus probiotic such as L. rhamnosus GG, L. reuteri, Bifidobacterium longum or lysates thereof. The cosmetic composition may comprise one or more of an L. rhamnosus probiotic bacterium such as L. rhamnosus GG an L. reuteri, a Bifidobacterium longum or lysates or one, two or each of these probiotic bacteria.
The invention further provides antibacterial compositions comprising a probiotic bacterium or lysate thereof. The probiotic bacterium may be L. rhamnosus GG. Such compositions may be useful for killing or inhibiting the action or growth of bacteria. The compositions may be useful for cleaning or pre-treating a surface which has, or is suspected to have, bacteria on it, or is likely to be exposed to bacteria. In some embodiments the antibacterial composition may be formulated so as not to be suitable for administration to a patient or subject.
The invention also relates to methods of preparing compositions, e.g. suitable for therapeutic and/or cosmetic and/or anti-bacterial use. The method may comprise subjecting a population of probiotic bacteria to lysis and formulating the resulting lysate into a composition. Alternatively, the method may comprise formulating intact bacteria into a composition. The method may comprise the step of treating the bacteria or lysate so as to remove or inactivate intact bacteria, for example, by partitioning intact bacteria from the lysate. The lysate may be subject to purification or filtration steps, e.g. to remove intact bacteria, bacterial growth media or contaminants.
The compositions prepared by such a method may be suitable for use in treatment, for example in treating bacterial infections, or for repairing the skin barrier. Such methods may involve the addition of one or more pharmaceutically or cosmetically acceptable excipients. Other compositions may not be suitable for use in treatment, for example antibacterial compositions such as washes.
The following numbered paragraphs (paras.) contain statements of broad combinations of the inventive technical features herein disclosed:—
1. A probiotic bacterium or a lysate thereof for use in a method of treatment, wherein the probiotic bacterium is Lactobacillus rhamnosus GG.
2. The probiotic bacterium or lysate thereof for use according to paragraph 1 wherein the method of treatment involves administration of the probiotic bacterium or lysate thereof to the skin of the patient being treated.
3. The probiotic bacterium or lysate thereof for use according to paragraph 1 or paragraph 2 for use in a method of treatment or prevention of infection.
4. The probiotic bacterium or lysate thereof for use according to paragraph 3 wherein the infection comprises a bacterial infection.
5. The probiotic bacterium or lysate thereof for use according to paragraph 4 wherein the bacterial infection is a Staphylococcus infection, preferably an S. aureus infection.
6. The probiotic bacterium or lysate thereof for use according to any one of the preceding paragraphs wherein the method of treatment is for a skin infection.
7. A probiotic bacterium or lysate thereof for use in a method of treatment involving repair or regeneration of the skin barrier wherein the probiotic bacterium is an L. rhamnosus or L. reuteri probiotic bacterium.
8. The probiotic bacterium or lysate thereof for use according to paragraph 7 wherein the method involves administration of the probiotic bacterium or lysate thereof to the skin.
9. The probiotic bacterium or lysate thereof for use according to paragraph 7 wherein the probiotic bacterium is L. rhamnosus GG.
10. The probiotic bacterium for use according to any one of paragraphs 7-9 wherein the method is for treatment of psoriasis, icthyosis, dermatitis, wound healing, acne (including Hiradenitis Suppurativa), Burns, Diaper Rash, Netherton's Syndrome, Actinic Keratosis, Dermatomycoses, Dermatosis, or Ectodermal Dysplasia.
11. The probiotic bacterium for use according to any one of the preceding paragraphs wherein the probiotic bacterium is co-administered with a probiotic bacterium of a different strain, or a lysate thereof.
12. The probiotic bacterium or lysate thereof according to any one of the preceding paragraphs wherein the method of treatment involves co-administration with L. reuteri or a lysate thereof.
13. A pharmaceutical composition comprising L. rhamnosus GG or a lysate thereof.
14. The pharmaceutical composition according to paragraph 13 further comprising a probiotic bacterium of a strain other than L. rhamnosus, or a lysate thereof, wherein preferably the probacterium of a strain other than L. rhamnosus is L. reuteri.
15. A cosmetic method comprising administration of one or more of an L. rhamnosus probiotic bacterium such as L. rhamnosus GG, L. reuteri, or lysates thereof to a subject.
16. The cosmetic method according to paragraph 15 wherein the probiotic bacterium or lysate is administered to the skin of the subject.
17. A cosmetic composition comprising an L. rhamnosus probiotic bacterium, or a lysate thereof and/or an L. reuteri probacterium, or a lysate thereof.
18. A method of medical treatment comprising administering L. rhamnosus GG or a lysate thereof to a patient, thereby treating the patient.
19. The method according to paragraph 18 wherein the probiotic bacterium or lysate thereof is administered to the skin of the patient.
20. The method of medical treatment according to paragraph 18 or paragraph 19 further comprising administering a probiotic bacterium other than L. rhamnosus GG, or a lysate thereof, to the patient, wherein the probiotic bacterium other than L. rhamnosus is preferably L. reuteri.
21. The method of medical treatment according to paragraph 18 wherein the medical treatment is the treatment or prevention of infection.
22. The method of medical treatment according to any one of paragraphs 18 to 21 wherein the medical treatment is the treatment or prevention of skin infection, and wherein the skin infection is treated or alleviated following the treatment.
23. A method of medical treatment comprising administering L. rhamnosus, L. rhamnosus GG or L. reuteri to a patient in need thereof, wherein the treatment is for the repair or regeneration of the skin barrier, wherein the skin barrier is repaired or regenerated following the treatment.
24. The method of medical treatment according to paragraph 23 wherein the L. rhamnosus, L. rhamnosus GG or L. reuteri is administered to the skin of the patient.
25. The method according to paragraph 22 or paragraph 23 wherein the method of treatment is for psoriasis, icthyosis, dermatitis, wound healing, acne (including Hiradenitis Suppurativa), Burns, Diaper Rash, Netherton's Syndrome, Actinic Keratosis, Dermatomycoses, Dermatosis, or Ectodermal Dysplasia.
26. Use of a probiotic bacterium or a lysate thereof in the manufacture of a medicament for use in the treatment of infection, wherein the probiotic bacterium is L. rhamnosus GG.
27. Use of a probiotic bacterium or a lysate thereof in the manufacture of a medicament for use in treatment, wherein the treatment involves repair or regeneration of the skin barrier, and wherein the probiotic bacterium is an L. rhamnosus or L. reuteri probiotic bacterium.
28. Use according to paragraph 26 or paragraph 27 wherein the medicament is formulated for administration to skin.
29. Use according to paragraph 28 wherein the method is for treatment of psoriasis, icthyosis, dermatitis, wound healing, acne (including Hiradenitis Suppurativa), Burns, Diaper Rash, Netherton's Syndrome, Actinic Keratosis, Dermatomycoses, Dermatosis, or Ectodermal Dysplasia.
30. Use of a probiotic bacterium or lysate according to any one of paragraphs 26-29 wherein the treatment involves co-administration of more than one probiotic bacterium or lysate thereof.
31. A lysate of L. rhamnosus GG.
32. A composition formulated for administration to the skin and comprising L. rhamnosus GG, L. rhamnosus and/or L. reuteri, or a lysate of one or more of said bacteria.
33. An antibacterial composition comprising L. rhamnosus GG or a lysate thereof.
The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
Aspects and embodiments of the present invention will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.
Skin is the primary barrier between the organism and the outside world. It must both prevent invasion by pathogens but at the same time, prevent water and electrolyte loss from the body. To date, the barrier function of skin has thought to be solely the role of the stratum corneum. However, complexes known as tight junctions are also critical to the skins' barrier function. Occasionally, the barrier of skin is breached due to wounds or abrasions. In these instances, the skin must repair itself. However, occasionally, repair does not occur quickly enough and this leaves the skin open to infection. We have screened a range of probiotic bacteria for their ability to:
Agents that increase and repair the barrier function of keratinocytes, have great potential not only as therapeutic agents but also as cosmetic skin improving agents. Augmentation of the barrier function increases the skins hydration level and is known to improve the appearance of the skin and lead to a healthier cosmetic appearance.
Our observations broaden the utility and application of probiotics. Our initial observations indicate that probiotics could be applied to a number of applications in skin care that include: wound healing, coating technology for wound dressings, improved skin barrier creams for cosmetic and therapeutic uses (e.g. atopic dermatitis); antibacterial products such as hand washes and soaps, and cosmetic preparations aimed at maintenance/repair of the skin's barrier function and treatments to restore a damaged barrier due to sun exposure and ageing.
With the potential to prevent the detrimental effects of harmful bacteria and simultaneously actively repair a damaged skin barrier the commercial applications for probiotics in skin care are significant. Increasingly people are demanding natural, safe and effective treatments for common skin alignments and our technology represents a significant advance in meeting these market needs
Our technology could not only provide a new therapeutic agent for establishment/repair of the skin barrier, but could potentially circumvent the need for existing aggressive therapies e.g. antibiotics. Current mainstay prevention methods include antibacterial agents that although effective have a number of detrimental effects. Treatment relies on the use of antibiotic to kill harmful bacterial infections. Our approach not only inhibits harmful skin pathogens, but actively promotes repair of the barrier at a cellular level. Since a decline in barrier function/repair goes hand in hand with reduced ability to repel infection, our lysate represents a technology that enhances both the barrier and its ability to guard against pathogens.
There are currently very few products on the market with live bacteria as components (e.g. Nude live skin probiotic cosmetic range). Our purified lysate approach has a number of functional and commercial benefits over these approaches by offering a controllable and definable activity at lower concentrations; increased stability and ease of production; and an improved shelf life. Taken together these make our technology more amenable to therapeutic and cosmetic uses, unlike difficult to manufacture and distribute live bacterial creams.
Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:
a. L. reuteri does not inhibit the growth of S. aureus in co-culture. Chart showing results of competition assay revealing no significant difference between groups over time (P=0.146).
b (Table 1). L. reuteri does not affect staphylococcal viability in cell culture. S. aureus grown in the absence of cells had a viable count of 8.0 (log cfu). S. aureus grown in the presence of keratinocytes had a viable count of 8.6±0.2. S. aureus co-incubated with L. reuteri in the absence of cells had a viable count of 8.4±0.4 while S. aureus co-incubated with L. reuteri in the presence of cells had a viable count of 8.0±0.2. There was no significant difference found between groups (P=0.34). Results are expressed as the mean±SEM.
The details of one or more embodiments of the invention are set forth in the accompanying description below including specific details of the best mode contemplated by the inventors for carrying out the invention, by way of example. It will be apparent to one skilled in the art that the present invention may be practiced without limitation to these specific details.
Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.
The invention relates to the use of probiotic bacteria. Probiotics are commonly defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. Studies in the gut have demonstrated the ability of probiotic bacteria to inhibit colonisation by pathogens through mechanisms including exclusion, competition and displacement of pathogen attachment to the host tissues. As used herein, the term “probiotic bacterium” may also refer to such bacteria when they are no longer alive, for example following inactivation by heat or radiation.
Lactobacillus rhamnosus
The invention particularly relates to probiotic bacteria of the species Lactobacillus rhamnosus. Such bacteria were originally considered a subspecies of Lactobacillus casei, but later genetic research found it to be a species of its own. A number of L. rhamnosus strains are known. For example strains I-1720 (Pasteur Collection Nationale de Cultures de Microorganismes), AC413, GR-1 (Karlsson et al., BMC microbiology 2012, 12:15), JB-1 (Bravo et al., PNAS 2011, 108(38) 16050-16055), GG and Lc705 (Savijok et al., J. Proteome Res. 2011 10(8): 3460-3473). Other strains of L. rhamnosus may be readily isolated. Optionally, some embodiments of the present invention do not include the use of Lactobacillus rhamnosus strain LB21.
In particular, the invention relates to L. rhamnosus GG. L. rhamnosus GG (also referred to herein as LGG) is deposited at ATCC (American Tissue Culture Collection) under accession number ATCC 53103. L. rhamnosus GG was isolated in 1983 from the intestinal tract of a healthy human being by Gorbach and Goldin,
Lactobacillus reuteri
The invention also relates to Lactobacillus reuteri strains. L. reuteri is a gram-positive bacterium that naturally inhabits the gut of mammals and birds. A number of L. reuteri strains are known. For example, DSM 17938 and ATCC deposits 23272, 53608, 53609 and 55148 and 55739. L. reuteri strains are described in, for example, U.S. Pat. No. 6,872,565, and U.S. Pat. No. 7,517,681. Other strains of L. reuteri may be readily isolated.
A particularly preferred L. reuteri strain according to the invention is deposited at ATCC under accession number ATCC 55730, described in U.S. Pat. No. 5,837,238.
Bifidobacterium longum
The invention also relates to Bifidobacterium longum strains. Bifidobacterium longum is a species of gram-positive bacterium found in the intestines of infant humans. A number of Bifidobacterium longum strains are known. For example, ATCC deposits 15708, 55816, 55818, 15707, 35183, and 51870. Other strains of Bifidobacterium longum may be readily isolated.
A particularly preferred Bifidobacterium longum strain according to the invention is Bifidobacterium longum reuter. In some embodiments the Bifidobacterium longum reuter is deposited at ATCC under accession number ATCC 51870.
Probiotic bacteria or their lysates according to the invention may be formulated as pharmaceutical compositions for clinical use and may comprise a pharmaceutically acceptable carrier, diluent or adjuvant. They may be formulated for topical administration.
Administration is preferably in a “therapeutically effective amount”, this being an amount sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins. It will be appreciated by one of skill in the art that appropriate dosages of the active compounds, and compositions comprising the active compounds, can vary from patient to patient.
The compounds and compositions of the present invention are useful in the treatment of a wide range of diseases and conditions. In particular, the compounds and compositions of the present invention are useful in the treatment of disorders, diseases and conditions of the skin and find application in the treatment of fibrosis (including post-surgical fibrosis), tissue adhesion formation, and scar formation. The compounds and compositions of the invention are useful in the treatment of acne, eczema, ichthyosis and psoriasis. The probiotic bacteria and their lysates according to the invention are useful in wound healing.
Probiotic bacteria and lysates thereof for use in therapeutic applications may be formulated as medicaments, that is to say formulated as a medicine. The medicament may include other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents. The formulation may further comprise other active agents, for example, other therapeutic or prophylactic agents.
The invention relates to the treatment of infections. The probiotic bacteria and lysates of the invention exhibit anti-infective agent activity. For example, anti-bacterial activity. Thus, they are useful for the treatment of infections including bacterial infections. Thus, they are useful in the treatment of multi-drug resistant bacterial infections, hospital acquired bacterial infections, antibiotic resistant bacterial infections, infections by gram-negative and/or gram-positive bacterial infections.
The probiotic bacteria and lysates of the invention are also useful for the treatment of infections by Staphylococcus spp., Pseudomonas spp., Staphyloccus saprophyticus, Staphyloccocus xylosus, Staphyloccocus lugdunensis, Staphyloccocus schleiferi, Stapylococcus caprae staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus warneri, Staphylococcus aureus, Staphylococcus hominis, Methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, Vancomycin-resistant Enterococcus (VRE), Proprionibacterium acnes, Bacillus cereus, Bacillus subtilis, Listeria monocytogenes, Streptococcus pyrogenes, Streptococcus salivariu, Streptococcus mutans and Streptococcus pneumonia.
In particular, the probiotic bacteria and lysates of the invention exhibit anti-Staphylococcus activity, and are thus useful for the treatment or prevention of Staphylococcus infection. For example, the probiotic bacteria and lysates of the invention exhibit anti-Staphylococcus aureus activity, and are thus useful in the treatment or prevention or Staphylococcus aureus infections.
Infections occur where disease causing microorganisms invade the tissues of the body. Multiplication of those microorganisms and the toxins that they produce react with the tissues of the body, often causing immune reactions by the infected host. Infections may be caused by bacteria, viruses, viroids, fungi and other parasites. Infections may occur via any of the tissues of the body, such as the skin, gut or membranes. In some embodiments of the invention the probiotic bacteria or lysates of the invention are used to treat infection of tissues other than the gut, for example in some embodiments the probiotic bacterium or lysate according to the invention is not used for the treatment of infection of the alimentary canal, esophagus, stomach, intestines, rectum or anus. In particular aspects the invention relates to the treatment or prevention of infection of the external surface of the body, and particularly of the skin.
The probiotic bacterium, lysates and compositions according to the invention may be used in the treatment or prevention of skin infections. The infection may be due to a bacterium, such as Staphylococcus bacteria. The infective agent may be Staphylococcus aureus. The probiotic bacterium, lysate or composition may be applied separately, sequentially or simultaneously with exposure to the infective agent. In some cases the probiotic bacterium, lysate or composition is applied after exposure to the infective agent.
The probiotic bacteria and lysates of the present invention have barrier repair, reinforcement and/or regeneration activity. Skin is composed of two compartments, the deep compartment (the dermis) and the surface compartment (the epidermis). This constitutes a barrier against external attacks, in particular chemical, mechanical or infectious attacks, and, therefore, a number of defensive reactions against environmental factors (climate, ultraviolet rays, tobacco, etc) and/or xenobiotic, such as, for example, microorganisms, occur therein. This property is referred to as barrier function and is mainly provided by the most superficial layer of the epidermis, namely the horny layer, referred to as the stratum corneum. Detrimental changes in the barrier can be reflected by cutaneous discomfort, sensory phenomena and in particular unpleasant phenomena, or cutaneous dryness measured by imperceptible water loss. Compositions according to the invention are useful for preventing a reduction in the barrier and/or to repair or regenerate barrier function. Reduction of barrier function is associated with a number of disorders, such as those set out below. If the barrier is breached, for example by wounds or abrasions, the skin must repair itself. However, occasionally, repair does not occur quickly enough and this leaves the skin open to infection.
Disorders associated with disruption of the skin barrier include Psoriasis, Acne (including Hiradenitis Suppurativa), Burns, Diaper Rash, Netherton's Syndrome, Actinic Keratosis, Dermatomycoses, Dermatosis or Ectodermal Dysplasia, Atopic Dermatitis, Contact Dermatitis, Dermatitis, Seborrehic Dermatitis, Icthyosis Vulgaris or other disorders associated with damage or breakdown of the skin barrier
Eczema (atopic dermatitis) is a particular type of inflammatory reaction of the skin in which there are typically vesicles (tiny blister-like raised areas) in the first stage followed by erythema (reddening), edema (swelling), papules (bumps), and crusting of the skin followed, finally, by lichenification (thickening) and scaling of the skin. Eczema characteristically causes itching and burning of the skin.
Eczema is very common and can first occur at any age. It is frequently chronic and difficult to treat, and it tends to disappear and recur. Itching can be extreme and severe. There are a number of types of eczema. The prevalence of eczema is high with an estimated 33 million people in the USA alone affected.
Ichthyosis vulgaris is a genetic skin disease that is characterised by the presence of excessive amounts of dry surface scales on the skin caused by abnormal epidermal differentiation or metabolism. It is usually most severe over the legs but may also involve the arms, hands, and trunk in some cases. It may also be associated with atopic dermatitis, keratosis pilaris (small bumps on the back of the arms), or other skin disorders. It usually disappears during adulthood, but may recur when elderly.
Other types of ichthyosis include X-linked ichthyosis, ichthyosis lamellaris, epidermolytic hyperkeratosis, harlequin type ichthyosis, Netherton's syndrome and Sjogren-Larsson syndrome, although ichthyosis vulgaris, accounts for 95% of all ichthyosis cases. Hereditary (congenital) ichthyosis vulgaris accounts for more than 95% of cases of ichthyosis vulgaris It first appears in early childhood. The gene responsible for ichthyosis vulargaris has been mapped to chromosome band 1q21. The product of this gene is a substance called filaggrin (FLG) which may act as the “keratin matrix protein” in cells of the stratum corneum. The inheritance pattern is autosomal dominant. Acquired ichthyosis vulgaris, typically develops in adulthood and results from an internal disease or the use of certain medications. Hereditary ichthyosis vulgaris is a common disease in United States, with a prevalence of approximately 1 case in 300 persons. Because symptoms improve with age, the true prevalence is probably higher. Acquired ichthyosis is extremely rare. Its prevalence in United States is unknown. In the United Kingdom, the incidence of ichthyosis vulgaris was reported to be 1 in 250. In China, ichthyosis vulgaris has a prevalence of 2.29%.
Psoriasis is a chronic, genetic, non-contagious skin disorder characterised by itchy or sore patches of thick, red skin with silvery scales. The scaly patches caused by psoriasis, called psoriatic plaques, are areas of inflammation and excessive skin production. The disorder is a chronic recurring condition which varies in severity from minor localised patches to complete body coverage. Psoriasis can also cause inflammation of the joints, which is known as psoriatic arthritis. There are a number of forms of the disease. According to the National Institutes of Health (NIH), as many as 7.5 million Americans (approximately 2.2 percent of the population) have psoriasis, with an estimated 125 million being affected globally. Between 150,000 and 260,000 new cases of psoriasis occurring each year. Incidence of psoriasis tends to be affected by the climate and genetic heritage of the population. It is less common in the tropics and in dark-skinned persons, and most common in Caucasians. Total direct and indirect health care costs of psoriasis for patients are calculated at $11.25 billion annually, with work loss accounting for 40% of the cost burden16. Approximately 60% of psoriasis patients missed an average of 26 days of work a year due to their illness17.
As used herein wound healing refers to the process by which the skin repairs itself after injury. In normal skin, the epidermis (outermost layer, predominantly made up of keratinocytes) and dermis (inner or deeper layer) exists in a steady-state equilibrium, forming a protective barrier against the external environment. Once the protective barrier is broken, i.e. by wounding, the normal (physiologic) process of wound healing is immediately set in motion. As used herein the term “wound” refers to a type of injury in which the skin is torn, cut or punctured, or where the skin is damaged following a blunt force trauma or medical condition, resulting in a breach in the skin barrier. The probiotic bacteria or lysates thereof according to the invention may be useful in wound healing in vivo, including tissue repair, regeneration and/or replacement. For example, they may be used in healing scar tissue, acceleration of wound healing, or where tissue grafts such as skin grafts are used.
In some embodiments repair or regeneration of the skin barrier includes repair or regeneration of a mucous membrane. Mucous membranes include mucosa of the mouth (including mucosa of the cheek, the soft palate, the tongue, including the under surface of the tongue and the floor of the mouth), the nose, the throat (including mucosa of the pharynx, the larynx, the trachea and the esophagus), the bronchi, the lungs, the eye, the ear, the vagina, the penis, the urethra, the bladder, and the anus.
The patient to be treated may be any animal or human. The patient is preferably a non-human mammal, or a human patient. The patient may be male or female.
In some aspects the invention relates to a cosmetic treatment comprising the administration of a probiotic bacterium or lysate according to the invention. “Cosmetic” as used herein is non-therapeutic. Such methods do not involve the treatment of the human or animal body by therapy. The cosmetic treatment may be used to improve the appearance and/or texture of the skin.
The invention also contemplates the use of probiotic bacteria or lysates thereof in a method of cosmetic treatment, and methods of cosmetic treatment using the probiotic bacteria or their lysates. For example, in a method of improving the hydration level, or appearance of the skin. As used herein the term “cosmetic method” does not refer to a method for treatment of the human or animal body by surgery or therapy, or diagnostic methods practised on the human or animal body according to Article 53(c)EPC.
The invention also provides a cosmetic composition comprising a probiotic bacterium. The composition may be used to improve the appearance of the skin. For example, the composition may enhance the hydration level of the skin. Increased hydration level is known to improve the appearance of the skin and lead to a healthier cosmetic appearance. Cosmetic compositions may be formulated similarly to pharmaceutical compositions, as described below.
The subject to be treated may be any animal or human. The subject may be a non-human mammal, but is more preferably human. The subject may be male or female. The subject does not require repair of their skin barrier. In some cases the subject does not require treatment for an infection, such as a bacterial infection. In some cases the subject does not require treatment at the site at which the cosmetic treatment is to be applied.
The cosmetic methods according to the invention preferably involve the administration of a “cosmetically effective amount”. This pertains to the administration of compounds, ingredients, materials, compositions, dosage forms, etc. in an amount effective to induce a cosmetic benefit. This is within the scope of sound judgement of a relevant practitioner. It will be appreciated by one of skill in the art that appropriate dosages of the active compounds, and compositions comprising the active compounds, can vary from subject to subject.
Probiotic bacteria, lysates thereof, and compositions according to the invention have barrier maintenance and repair activity. As such, they are useful for preventing a reduction in and/or reinforcing barrier function. This may be useful for improving hydration of the skin and/or improving the appearance of the skin.
The following represent independent optional and separable embodiments which in some cases may not form part of the present invention.
Optionally, some embodiments of the present invention do not include the combination of a probiotic bacterium or lysate thereof with a Streptococcus strain, e.g. an α-Streptococcus strain.
Optionally, some embodiments of the present invention do not include the combination of a Lactobacillus rhamnosus GG (or a lysate thereof) with Lactobacillus rhamnosus LC705 (or a lysate thereof).
Optionally, some embodiments of the present invention do not include cosmetic or dermatological treatment of dry and/or sensitive skin.
Optionally, in some embodiments methods of treatment involving repair or regeneration of the skin barrier are not for the treatment of an allergic disease/condition or inflammatory disease/condition. For example, such embodiments optionally do not include treatment of one or more of atopic dermatitis, seborrhoeic dermatitis, eczema, atopic eczema, ichthyosis, psoriasis, acne or allergic disease, or inflammatory conditions.
Optionally, some embodiments of the present invention do not include anti-inflammatory or anti-viral use of the probiotic bacterium, or lysate thereof.
Optionally, some embodiments of the present invention do not include the treatment of respiratory infections.
The probiotic bacteria according to the invention may be administered as live bacteria, an inactivated or killed bacterium, or as lysates thereof. As used herein “lysates” refers to a sample of the probiotic bacterium which has been subject to lysis. A lysate may contain one or more soluble metabolites of the probiotic bacterium useful in the methods described herein. Lysis may occur by chemical or physical disruption, for example by addition of an osmotic agent or enzyme to the bacteria, or by the application of physical pressure, for example through sonic disruption. Gueniche et al., for example, prepared a lysate by ultrasound (20).
The bacteria may be washed prior to lysis. The lysate may be filtered and/or sterilized after lysis to remove any whole bacteria remaining. The lysate may comprise a cell free supernatant. The lysate may be a mixture comprising the cellular contents of the probiotic bacterium. For example, one or more of fragments of cell wall or cell membrane, proteins, nucleic acids, carbohydrates, and organelles (disrupted or intact). The lysate may be suspended, for example in aqueous medium.
Lysed cells may allow the release of many substances, e.g. soluble metabolites, from inside the bacterial cell membrane that may contribute to bioactivity. Therefore in some cases use of a cell-lysate may be advantageous over the use of whole bacteria.
In some embodiments a cell free supernatant is the supernatant of a cell culture from which the cells have been removed. Cells may be removed by centrifugation, and the supernatant may be filtered. As such, the supernatant (as a form of lysate) may be used directly in the formulations of the present invention. A cell free supernatant may contain one or more soluble metabolites.
A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
The probiotic bacterium or lysate thereof may be dissolved in, suspended in, or admixed with one or more other pharmaceutically acceptable ingredients. The probiotic bacterium or lysate thereof may be presented in a liposome or other microparticulate.
In some embodiments, probiotic bacterium may be provided as a suspension in a pharmaceutically acceptable excipient, diluent or carrier. In some embodiments probiotic bacterium may be provided as a lyophilisate.
In particularly preferred embodiments the probiotic bacteria or lysate thereof according to the invention is formulated for topical administration, particularly for use or application to, or on, the skin.
Formulations suitable for transdermal administration include gels, pastes, ointments, creams, lotions, and oils, as well as patches, adhesive plasters, bandages, dressings, depots, cements, glues, and reservoirs.
Ointments are typically prepared from the probiotic bacteria or lysate thereof and a paraffinic or a water-miscible ointment base.
Creams are typically prepared from the probiotic bacterium or lysate and an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the active compound through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.
Emulsions are typically prepared from the probiotic bacterium or lysate and an oily phase, which may optionally comprise merely an emulsifier (otherwise known as an emulgent), or it may comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabiliser. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabiliser(s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
Suitable emulgents and emulsion stabilisers include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulphate. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations may be very low. Thus the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
Other formulations include dental sprays, mouthwashes, toothpastes, lozenges, antibacterial washes, drinks (e.g. milk, yoghurt), food items (such as yoghurt, ice cream, candy bars), or powdered foods (such as powdered milk).
Formulations may suitably be provided as a patch, adhesive plaster, bandage, dressing, or the like which is impregnated with, or coated with, one or more probiotic bacteria or lysate thereof according to the invention and optionally one or more other pharmaceutically acceptable ingredients, including, for example, penetration, permeation, and absorption enhancers. The probiotic bacteria or lysates may also be provided in the form of coatings for medical devices such as implants, prosthetics, surgical instruments, gloves, catheters, valves, pacemakers and the like.
A formulation may contain a single (unit) dose of probiotic bacteria, or lysate thereof. Suitable doses of probiotic bacteria (intact or lysed) may be in the range 104 to 1012 cfu, e.g. one of 104 to 1010, 104 to 108, 106 to 1012, 106 to 1010, or 106 to 108 cfu. In some embodiments doses may be administered once or twice daily.
In some embodiments a formulation for use according to the present invention may comprise at least about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.5%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, about 10.0%, about 11.0%, about 12.0%, about 13.0%, about 14.0%, about 15.0%, about 16.0%, about 17.0%, about 18.0%, about 19.0%, about 20.0%, about 25.0%, about 30.0%, about 35.0%, about 40.0%, about 45.0%, about 50.0% by weight of probiotic bacteria or lysate thereof.
In some embodiments the formulation may comprise, one of at least about 0.01% to about 30%, about 0.01% to about 20%, about 0.01% to about 5%, about 0.1% to about 30%, about 0.1% to about 20%, about 0.1% to about 15%, about 0.1% to about 10%, about 0.1% to about 5%, about 0.2% to about 5%, about 0.3% to about 5%, about 0.4% to about 5%, about 0.5% to about 5%, about 1% to 10 about 5%, by weight of probiotic bacteria or lysate thereof.
The invention also provides antibacterial compositions in the form of cleaning products, washes, surface coatings or other compositions which are not for medical treatment of the human or animal body.
Such agents may be useful for removing, killing, or preventing the accumulation of bacteria on a surface, or inhibiting the action or growth of the bacteria.
Anti-bacterial compositions according to the invention may be useful for treating biomaterials, implants and prosthesis (including stents, valves, eyes, hearing aids, gastric bands, dentures, artificial joint replacements etc), surgical instruments or other medical devices prior to administration to, or treatment of, or use with, a patient or subject. The antibacterial compositions may be useful for treating surfaces prone to colonisation or exposure to bacterial, such as handrails, food preparation surfaces, kitchen surfaces or equipment, tables, sinks, toilets or other bathroom hardware,
Antibacterial compositions may comprise agents in addition to the lysate, such as cleaning agents, stabilisers, anionic surfactants, perfumes, chelating agents, acids, alkalis, buffers or detergents. Such agents may facilitate or enhance the antibacterial properties of the agent, such as killing or inhibiting bacteria, or preventing the recolonisation of the cleaned surface.
The invention also relates to methods of preparing compositions. The method may comprise subjecting a population of probiotic bacteria to lysis and formulating the resulting lysate into a composition. The method may comprise the step of treating the lysate so as to remove or inactivate intact bacteria in the lysate, for example, by partitioning intact bacteria from the lysate. The lysate may be subject to purification of filtration steps to remove other components, such as bacterial growth media or contaminants.
In alternative compositions intact bacteria are formulated as a composition. The bacteria may be inactivated prior to, or after, formulation into a composition. The bacteria may be subject to a purification or filtration step so as to remove other components, such as bacterial growth media or contaminants.
The compositions prepared by such a method may be suitable for use in treatment, for example in treating bacterial infections, or for repairing the skin barrier. Such methods may involve the addition of one or more pharmaceutically or cosmetically acceptable excipients.
Other compositions may be not suitable for use in treatment, for example antibacterial compositions such as washes. Methods for the preparation of antibacterial compositions may involve the addition of one or more agents to the lysate or bacteria.
Probiotic bacteria were grown routinely to stationary phase in Wilkins-Chalgren Broth (WCB) (Oxoid) at 37° C. in a Mark 3 Anaerobic Work Station (Don Whitley Scientific, Shipley, UK). S. aureus was grown aerobically with at 37° C. in Nutrient Broth (Oxoid). Culture densities were adjusted spectrophotometrically with medium to contain the number of bacteria required. For experiments utilising keratinocytes, bacteria were washed twice in 0.85% NaCl solution, centrifuged and reconstituted in keratinocyte medium. Selective agar was used in some experiments. This was Mannitol Salt Agar—MSA (Oxoid) or Man-Rogosa Sharpe agar (MRS) for S. aureus or Lactobacilli respectively. For experiments utilising heat killed bacteria, L. reuteri was centrifuged and resuspended in 0.4% glucose and heat inactivated by placing in a water bath at 85° C. for 45 minutes. Samples were gram stained to ensure lysis of bacterial cells had not occurred, and plated onto growth media to ensure they had been killed. For experiments using probiotic lysates, 10 ml of 108 cfu/ml L. reuteri was centrifuged, washed, concentrated in 1 ml keratinocyte media and lysed using a bead beater (FastPrep™ FP120, Thermo Electron Corporation). Samples were filter sterilised to remove any whole bacteria remaining. Approximately 100 μl of this lysate was used to treat keratinocytes.
Normal human epidermal keratinocytes (NHEK) (Promocell, Heidelberg, Germany) were maintained in keratinocyte basal medium (Promocell, Heidelberg, Germany) containing a supplement mix (bovine pituitary extract 0.004 mg/ml, epidermal growth factor (recombinant human) 0.125 ng/ml, insulin (recombinant human) 5 pg/ml, hydrocortisone 0.33 pg/ml, epinephrine 0.39 pg/ml and transferrin, holo (human) 10 pg/ml) and 0.06 mM CaCl2 (Promocell Heidelberg, Germany). Medium was substituted twice weekly and cells were cultured at 37° C. in a humid atmosphere of 5% CO2. Cells were detached once grown to 80% confluency and re-seeded at approximately 5×103 cells/cm2. For experiments using differentiated cells, calcium chloride in the medium was increased to 1.8 mM and the cells were grown in this for 24 hours prior to experimentation.
Aliquots (100 μl) of overnight cultures of L. reuteri and S. aureus were inoculated into fresh 10 ml broths (both as axenic cultures and as co-cultures). The pH and optical density of cultures was measured at 0 and 48 hours. At regular intervals (3, 6, 24, 30, and 48 hours), bacteria were counted by serial dilution plate counts using selective and non-selective agars. At 48 hours, BacLite™ Live/Dead stains were performed according to the manufacturer's instructions (Invitrogen).
Production of Inhibitory Substances by L. reuteri
Inhibition of the growth of S. aureus by L. reuteri was determined using well diffusion assays, as described by Holder and Boyce (23). For inhibition studies, L. reuteri was grown in either Man-Rogosa-Sharpe (MRS) media (Oxoid) or Wilkins-Chalgren Broth (WCB) (Oxoid) anaerobically at 37° C. to stationary phase. Whole cell (WC) cultures of L. reuteri grown in WCB (1 g/l glucose) or MRS (20 g/l glucose) were used. In other experiments cells were sedimented in a microfuge at 15,000×g for 5 minutes and the cell-free supernatant (CFS) extracted for use. The plates were then incubated at 37° C. for 48 h and zones of inhibition calculated. To measure the production of organic acids by L. reuteri, the pH of WC and CFS was measured. In a separate experiment to determine whether L. reuteri produced any other inhibitory substances, cell free supernatants were neutralised using 1M NaOH prior to use in the well diffusion assay. To determine if L. reuteri produced organic acids in cell culture, the pH of infected cell cultures was measured.
NHEK cells were grown in 24 well tissue culture plates to confluency. Cell culture medium was replaced with medium containing the indicated concentration of bacteria. For pre-exposure and post-exposure experiments, NHEK were treated with L. reuteri for periods of between 1 and 4 hours prior to the addition/after the addition of S. aureus. In all experiments, cells were incubated at 37° C. in a humidified atmosphere of 5% CO2 for 24 hours. The medium was then removed and cells were washed with sterile PBS. Cells were detached using trypsin (0.4%)/EDTA (0.3%) (Promocell) and cell viability determined by trypan blue exclusion assay (45).
Measurement of S. aureus Viability
To determine if L. reuteri or NHEK were able to inhibit the growth of S. aureus in cell culture, cells were grown to confluency in a 24 well plate. These were infected with either S. aureus or S. aureus and L. reuteri together. In parallel, wells without any cells also had these combinations added to determine the effect of the keratinocytes themselves on staphylococcal viability. After 24 hours exposure, the medium was removed and centrifuged to pellet extracellular bacteria. The cells were then trypsinised and 500 μl of 0.25% Triton-X-100 (Sigma-Aldrich) in PBS was added for approximately 30 minutes to lyse the cells. The well contents were then combined with the cell pellet and serial dilution plate counts using Mannitol Salt Agar (MSA) performed to determine the total number of viable staphylococci.
Adhesion Assays for L. reuteri, L. salivarius and S. aureus
Confluent differentiating NHEK were exposed to either the probiotic or S. aureus for 1 hour. After incubation, cells were washed three times in PBS to remove non adherent bacteria. The cells were trypsinised and serial dilution plate counts performed to assess the number of adherent bacteria. Selective agar was used for growth of staphylococci. In separate experiments, cells were exposed to L. reuteri 1 hour before the addition of S. aureus (exclusion), at the same time (competition) or 30 minutes after S. aureus infection had begun (displacement) to determine whether L. reuteri could inhibit S. aureus adhesion to keratinocytes. To determine whether L. reuteri could inhibit invasion of keratinocytes by staphylococci, cells were either exposed to S. aureus or S. aureus and L. reuteri for 1 hour. Cells were washed three times in sterile PBS to remove non-adherent bacteria, and the growth medium replaced with medium containing 100 μg/ml Gentamicin (Sigma-Aldrich) for 2 hours. Cells were then washed three times in sterile PBS and trypsinised and lysed in 0.25% Triton-X-100 for 30 minutes to release internalised bacteria. Bacteria in lysates were counted using serial dilutions. In experiments utilising conditioned media (CM), NHEK were exposed to L. reuteri and incubated for 1 hour at 37° C. in 5% CO2. The CM from the exposed cells was removed and centrifuged at 15000×g for 3 minutes to pellet the bacterial cells and filtered through a 0.22 μm pore size filter (Millipore, USA). In experiments exploring the role of the α5β1 integrin in S. aureus adhesion, NHEK were pre-exposed to 60 pg/ml of an anti-α5β1 integrin antibody (Millipore, USA) for 1 hour prior to infection of cells. An alternative method for assessing the number of adherent bacteria was utilised for dead preparations of L. reuteri. Cells were grown on LabTek chamber slides (Thermo-Scientific). Adhesion assays were performed as usual, using adjusted amounts of bacteria. After the cells were exposed to bacteria, cells were washed twice in PBS and fixed in methanol for 20 minutes before performing a gram stain. The number of adherent bacteria per 100 cells was then assessed using a Keyence All in one Type Fluorescence microscope.
All experiments were performed a minimum of three times, with three replicates each time. For experiments comparing two treatments, a students T-test was used. For experiments comparing two or more treatments, a one way ANOVA with blocking by experiment and post hoc Tukey test was utilised. Dose response and competition assays were analyzed using linear regression and 2-way ANOVA respectively. Results were considered significant if P<0.05.
We initially characterised the effects of S. aureus on keratinocyte viability by performing trypan blue exclusion assays on NHEK incubated with S. aureus at different doses. At a concentration of 105 cfu/ml S. aureus, only 49.4% of keratinocytes were viable after 24 hours infection (
L. reuteri Protects Keratinocytes from S. aureus Induced Cell Death
The ability of three strains of lactobacilli (L. reuteri ATCC 55730, L. rhamnosus AC413 and L. salivarius UCC118) to protect keratinocytes from the effects of S. aureus was investigated (
We next investigated whether the timing of L. reuteri application relative to S. aureus infection was critical to the protective effect of the probiotic. We assessed the viability of keratinocytes pre- or post-exposed to L. reuteri for 1, 2 or 4 hours prior to infection with S. aureus for 24 hours. Pre-exposed keratinocytes had similar viabilities to co-infected cells (P>0.05) (
L. reuteri does not Inhibit Growth of S. aureus
We considered that the mechanism(s) underlying the protective effects of S. aureus might be due to direct effects of the probiotic on the pathogen. However, competition assays showed no significant reduction in either L. reuteri or S. aureus viability during co-culture for 48 hours (
L. reuteri Inhibits Adhesion and Invasion of Staphylococci to Keratinocytes
One of the mechanisms by which probiotics are known to protect gut epithelia is by inhibition of pathogenic adhesion to epithelial cells. Therefore, we explored whether L. reuteri could inhibit adhesion of S. aureus to NHEK.
The graph in
L. salivarius UCC118 does not Prevent S. aureus from Adhering to Keratinocytes
We considered that the protective effects of L. reuteri might be connected to its ability to inhibit pathogen adhesion to NHEK. To further explore this, we asked whether inhibition of S. aureus binding to NHEK resulted in a decrease in its toxicity. S. aureus is known to adhere to cells by means of a fibronectin binding protein which interacts with extracellular fibronectin (26, 36). Fibronectin itself binds to the keratinocyte receptor, the α5β1 integrin. As shown in previous studies (27) pre-treatment of cells with a blocking antibody to α5β1 integrin resulted in decreased S. aureus adhesion. Therefore, we treated NHEK with the α5β1 blocking antibody and investigated its ability to inhibit adherence and toxicity to NHEK (
In this study, we have made a preliminary step towards characterizing the utility of probiotics for topical use. We have investigated the ability of three strains of lactobacilli with known probiotic potential in the gut and mouth (9, 38, 44) to protect keratinocytes from the effects of the common skin pathogen, S. aureus.
The application of S. aureus at physiologically relevant concentrations killed 69.5% of NHEK within 24 h. By contrast, none of the strains of lactobacilli significantly affected the viability of NHEK under the same conditions, suggesting that probiotics were well tolerated by keratinocytes. When NHEK were exposed to both pathogen and L. reuteri, the probiotic protected the cells from the effects of the pathogen, as indicated by the significantly higher percentage viability of S. aureus infected NHEK in the presence vs. absence of L. reuteri (
While, to our knowledge, this is the first evidence showing the ability of a probiotic to protect keratinocytes from the pathogenic effects of S. aureus in vitro, L. fermentum has been demonstrated to inhibit S. aureus infection of surgical implants and abscess formation in rats (18). Probiotics have previously been shown to exert protective effects against pathogen-induced cell death in other epithelia. For example, L. rhamnosus, L. reuteri and L. oris were shown to inhibit S. pyogenes induced cell death of pharyngeal epithelial cells (34). Probiotic cell free supernatants have also been shown to be protective. L. delbruekii supernatants protect enterocytes from C. difficile pathogenicity (5).
The protective effect of L. reuteri against S. aureus-induced cell death of keratinocytes could result from several different mechanisms. Lactobacilli are known to produce organic acids and bacteriocins that can have direct antimicrobial effects on pathogens. Although we could demonstrate in diffusion assays that L. reuteri could produce acid, this does not appear to be responsible for the protective effects observed because the pH of the keratinocyte culture medium did not change in the presence of lactobacilli. Similarly, we did not detect any other antimicrobial activity since in a competition assay, the growth rate and productivity of S. aureus in the presence of L. reuteri was identical to that of axenic cultures (
At least part of the mechanism by which L. reuteri protects keratinocytes appears to be linked to the ability of L. reuteri to prevent adhesion of S. aureus. Several lines of evidence point to this: Firstly, S. aureus was less toxic to the cells when its adhesion was inhibited using an integrin blocking antibody (
The timing of L. reuteri addition to the cells appears to be critical to its protective effects. When L. reuteri was added before, or at the same time as S. aureus, NHEK were protected from the effects of the pathogen (
How S. aureus induces cell death in keratinocytes is a complex process. In general, the first step in S. aureus pathogenesis is considered to be adherence of S. aureus to keratinocytes prior to invasion (27). S. aureus utilises a number of adhesins to do this, collectively called the Microbial Surface Components Recognising Adhesive Matrix Molecules (MSCRAMMS) (11). Staphylococcal adhesins known to be involved in adhesion to keratinocytes include Fibronectin binding proteins (FnBPs) (27, 36), protein A, clumping factors and coagulase (35). Teichoic acid mediates binding of S. aureus to nasal epithelial cells, though whether this is true for keratinocytes is not known (1). However, integrin blocking did not wholly inhibit binding of S. aureus to NHEK, suggesting that more than one adhesin is used (
Our study and others (27) have shown that blocking fibronectin receptors on the keratinocytes inhibits adhesion of S. aureus (
To date, there are no published reports on the ability of probiotics to adhere to epidermal keratinocytes. Adhesion of probiotics to epithelia elsewhere has been suggested to occur via collagen binding proteins (21), mucus binding proteins (37), lipoteichoic acids (19) and s-layer proteins (10, 14). The relevant adhesins may be proteinacious, carbohydrate or a mixture of the two, and more than one adhesin may be in use. L. reuteri was able to inhibit staphylococcal adhesion to keratinocytes when cells were pre-exposed to the probiotic (exclusion of binding sites) (
It seems likely that L. reuteri employs multiply mechanisms to attach to host epithelia and may use several strategies to prevent S. aureus from adhering. It is possible that L. reuteri may influence the expression of adhesive structures on the surfaces of keratinocytes, an area which has yet to be explored. Previous work has shown that probiotic L. rhamnosus GG and L. plantarum 299v can induce upregulation of genes involved in mucin expression in the intestine, which in turn inhibits adhesion of enteropathogenic E. coli (33). In a study in 1999, application of the lactic acid bacterium S. thermophilus to keratinocytes in vitro and skin in vivo resulted in increased ceramide production (15). Another study in 2006 used a probiotic fermented whey product on NHEK and found increased mRNA expression of the differentiation markers keratin-10 and involucrin (2) suggesting that “postbiotics” or metabolites of probiotics may also be of use for skin maladies. Another example of the positive effects of metabolites of probiotics was demonstrated in a recent paper where a probiotic patch for skin was developed. Lyophilised L. fermentum suspended in alginate with glucose and nitrite salts was able to produce nitric oxide gas, a substance able to inhibit the growth of pathogenic bacteria (24).
We have provided evidence that a probiotic has a protective effect against the pathogenic effects of S. aureus on keratinocyte viability in vitro. These results suggest that a topical probiotic for skin could exclude pathogenic organisms and therefore prevent infections, and agrees with the work of others. The topical administration of L. plantarum to burns in mice has been shown to inhibit P. aeruginosa colonisation of burns, promote healing and modify the inflammatory response (46). This organism has also been compared to and found to be as effective as traditional silver suphadiazine treatments of burn wounds for limiting the bacterial load of wounds and healing in vivo (41). In addition, a probiotic may also be of use in the treatment of healthy skin for increased moisturisation and barrier function. In 2010 a Bifidobacterium lysate was applied topically to the skin of volunteers and found to increase barrier function of skin through decreased trans-epidermal water loss measurements (20). While further investigations are required to determine the full effect probiotics have on the skin, our findings show that L. reuteri can protect keratinocytes from the pathogenic effects of S. aureus through the exclusion and competition for binding sites. We suggest that the prophylactic use of probiotics or their lysates in barrier creams and soaps may aid in the fight against staphylococcal skin colonisation and subsequent infections.
As set out above, we demonstrated that Lactobacillus reuteri can partially protect primary human keratinocytes from the effects of the skin pathogen, Staphylococcus aureus. In this study, to further our understanding of the potential of probiotics, we have evaluated the role of a second probiotic, L. rhamnosus GG as well as combinations of lactobacilli, and bacterial lysates in the inhibition of S. aureus infection of keratinocytes. L. rhamnosus GG was isolated from human faeces (see U.S. Pat. No. 4,839,281 and U.S. Pat. No. 5,032,399), and is deposited under accession number ATCC 53103.
L. reuteri Protects Keratinocytes from S. aureus Induced Cell Death
Normal human primary keratinocytes (KCs) were exposed to S. aureus (106/ml) in the presence or absence of L. rhamnosus GG, L reuteri either alone or in combination (all at 108/ml). The viability of the KCs was measured after 24 hours using a trypan blue exclusion assay.
When KCs were treated with S. aureus alone, only 39% of the KCs remained viable at 24 hours post infection (p<0.001). However, in the presence of either L. reuteri or L. rhamnosus GG, the viability of pathogen infected KCs increased to 60% and 59% respectively (p=0.008 and 0.009 respectively). Furthermore, when we used both strains in combination on S. aureus infected cells, the viability of the KCs increased to 75% (p=0.0025).
Interestingly, a lysate of L rhamnosus GG also provided significant protection to KCs with 70% of cells being viable following a 24 hour incubation with S. aureus in the presence of the probiotic lysate.
L. rhamnosus Inhibits Growth of S. aureus
S. aureus was grown in the presence of L. rhamnosus GG or L. rhamnosus GG lysate. As shown in
This inhibitory effect was not observed in L. rhamnosus AC413. As shown in
L. rhamnosus Kills S. aureus
To further elucidate the effect of L. rhamnosus GG on S. aureus we investigated the effect of L. rhamnosus or its lysate on S. aureus viability. As shown in
Thus, we have identified a strain of probiotic bacterium with skin cell protecting and regenerative properties. Significantly, lysates of this strain improve skin keratinocytes ability to resist harmful toxicity induced by S. aureus (SA) (
L. rhamnosus GG and L. reuteri Speed Up Recovery of a Scratched Keratinocyte Monolayer and Increase Keratinocyte Migration
Agents that increase and repair the barrier function of keratinocytes, have great potential not only as therapeutic agents but also as cosmetic skin improving agents. Augmentation of the barrier function increases the skins hydration level and is known to improve the appearance of the skin and lead to a healthier cosmetic appearance.
We have also demonstrated that when keratinocyte monolayers are scratched to remove cells (i.e. a model wound), the presence of our propriety probiotic lysates significantly and surprisingly speeds up the recovery of the monolayer and re-establishment of the barrier (
LGG and L. reuteri lysates were also able to increase the rate of keratinocyte migration. As shown in
These data demonstrate that different strains of probiotic Lactobacilli either alone or in combination can protect human keratinocytes from the effects of the skin pathogen, S aureus. Our data suggest that probiotics may potentially have applications, for example, in topical formulations to protect the skin from adventitious pathogens.
All probiotic strains (B. longum reuter ATCC-51870, L. plantarum, ATCC-10241, L. reuteri, ATCC-55730, L. rhamnosus Goldwin and Gorbach, ATCC-53103, L. fermentum, ATCC-14932) were purchased from LGC Itd (UK) and were routinely grown to stationary phase in Wilkins-Chalgren Broth or on Wilkins-Chalgran agar at 37° C. in a Mark 3 anaerobic work station (Don Whitley scientific, UK). Cultures were adjusted spectrophotometrically to contain 108 cfu/ml. 10 ml of these cultures was centrifuged, washed and then concentrated in 1 ml of keratinocyte basal medium (Promocell, Heidelberg, Germany). The sample was then lysed using a bead beater (FastPrep FP120, Thermo Electron Corporation, UK) and then filter sterilised to remove any remaining whole bacteria. 100 μl of this lysate was used to treat keratinocyte cultures.
Normal human epidermal keratinocytes (NHEK) were obtained and cultured as described in Prince et al (50). For experiments measuring TJ function, cells were plated on 12-well, permeable polycarbonate Thincert™ cell culture inserts with 0.4 um pore size (Greiner Bio-one Ltd, UK). NHEK were grown in keratinocyte basal medium (Promocell, Heidelberg, Germany) until confluent. At this point, the medium was replaced with CNT-02-3DP5 high calcium medium (CELLnTEC Advanced cell systems, Switzerland) which induces TJ formation. TJ function was measured using an epithelial voltmeter fitted with chopstick electrodes (World Precision Instruments Ltd, UK). All experiments were repeated at least three times with triplicate wells within individual experiments. In some experiments, lysates of probiotic bacteria were added to the apical side of the inserts and the TEER was measured at the times indicated post treatment.
3-{4,5-dimethylthiazol-2-yl}diphenyltetrazolium bromide (MTT, purchased from the Sigma-Aldrich Ltd., Poole, UK) was prepared as a stock solution of 5 mg/ml in phosphate buffered saline. NHEK were grown to confluence in 96 well plates and then treated with bacterial lysates for 24 hours. Medium was then replaced with fresh medium containing 10% MTT. The plates were incubated for 4 hours at 37° C. and the medium was replaced with dimethyl sulphoxide. The absorbance of each well was then read at 570 nm in a plate reader.
Extraction of Protein from NHEK and Immunoblotting.
Protein was extracted from NHEK cells according to the method described in Mclaughlin et al (51). Briefly, cells from a single Thincert™ were scraped into 100 μl extraction buffer (NaCl (120 mM), HEPES, pH 7.5 (25 mM), Triton X-100 (1%), EDTA (2 mM), NaF (25 mM), NaVO4 (1 mM), SDS (0.2%), Aprotinin (10 μg/ml), Leupeptin, (10 μg/ml) and pepstatin A (10 μg/ml) and then incubated on ice for 30 minutes. Following centrifugation in a microfuge, the supernatant was recovered to a fresh tube and used for analysis of TJ protein expression. SDS-PAGE was performed according to the method of Laemmli et al (52) and proteins were electrophoretically transferred onto PVDF membranes. These were subsequently washed, blocked in 5% (w/v) skimmed milk and incubated with the primary antibodies overnight. These were rabbit anti-claudin 1, 4, mouse anti occludin, mouse anti-ZO-1 or mouse anti-β-actin (all purchased from InVitrogen). The membranes were subsequently washed and incubated with horse radish peroxidise conjugated secondary antibodies. The immunoblots were developed using enhanced chemiluminescence (Amersham, Bucks, UK) and densitometry performed as described in Mclaughlin et al, 2004 (51).
For inhibition of TLR2, keratinocytes were pre-treated with recombinant human anti-TLR2 antibody at 10 μg/ml (Abcam, UK) for 1 hour before stimulation with probiotic lysates.
All experiments were performed a minimum of three times and analysed using SPSS software version 20. For experiments comparing two treatments, an Independent Samples T-test was used to statistically analyse all data. For experiments comparing two or more treatments, a one-way ANOVA test was used to statistically analyse all data. Results were considered statistically significant at p<0.05. Data are expressed as means±standard errors of the means (SEM) for at least n=3 for each experiment.
To begin with, we investigated whether any of the probiotic lysates under investigated affected the viability of NHEK. To this end, we performed an MTT assay on NHEK that had been incubated with bacterial lysates for 24 hours. The data in
Normal human epidermal keratinocytes develop TJs when they are transferred from medium containing low calcium concentrations (˜0.2 mM) into medium containing high calcium (˜1.8 mM). This ‘calcium switch’ induces assembly of TJs which can be detected as a rise in the TEER of the cells with time. TEER reaches a peak 48 hours post calcium switch and then drops slightly to reach a steady state at 72 hours (
B longum Reuter and L Rhamnosus GG Produce Dose Dependent Effects on TEER in Keratinocytes.
To further define the effects of B. longum reuter and L. rhamnosus GG on TJ function in keratinocytes, we measured TEER in the presence of 100 μl lysate made from bacteria at 106, 104 and 102 CFU/ml. At 106 CFU/ml, lysates from both strains were still able to produces TEERs that were significantly higher than in untreated cells (
Evidence suggests that TJ function is often reflected in the expression levels of particular proteins involved in the complexes. The main TJ proteins expressed by keratinocytes include claudin 1, claudin 4, ZO-1 and occludin. Therefore, we used immunoblotting to investigate the expression of these proteins in untreated NHEK vs NHEK treated with probiotic lysates for 72 hours.
Treatment of NHEK with a lysate of B. longum reuter, produced significant increases in all four TJ proteins (
Bifidobacterium longum Reuter-Induced Modulation of TJ Function is Mediated Via TLR2.
Keratinocytes sense the presence of bacteria via pattern recognition receptors such as toll like receptors (TLRs). Several lines of evidence in the gut have pointed to a relationship between TLR activation and changes in TJ barrier function. Additionally, recent work by Yuki et al, (53) demonstrated TLR-mediated augmentation of TJ function in keratinocytes in response to bacterial ligands such as peptidoglycan. Therefore, we naturally wondered whether the probiotic induced increases in TJ function were mediated by TLRs. Of particular interest was TLR2 because this is the major receptor for gram positive organisms (54).
To investigate this we incubated NHEK in the presence of a TLR2 neutralising antibody for 1 hour prior to the addition of probiotic lysates. The data in
Five different strains of lactobacilli were tested for their effect on TJ function. Of these, lysates of all but one of the strains, L. fermentum, were able to increase the TEER of keratinocytes. L. fermentum actually reduced the TEER which is probably related to our observation that this strain also reduced the viability of keratinocytes. The other 4 strains of lactobacilli all enhanced TJ function but with different efficacies. In this regard, L. rhamnosus GG and B. longum reuter produced greater, and more sustained increases in TEER than L. plantarum or L. reuteri.
These strain dependent effects are probably due to the expression of different proteins and carbohydrates by individual strains of lactobacilli. Indeed, the increase in TEER induced by L. rhamonsus GG and B. longum reuter exhibited dose dependent effects suggesting that a particular molecule(s) and receptive mechanism(s) is involved.
Both L. rhamnosus GG and B. longum reuter increased the expression of TJ proteins in keratinocytes. However, the particular subset of molecules was different in each case. The modulation of protein expression is almost certainly the mechanism by which these lysates increase the TJ barrier function of keratinocytes. Changes in the expression levels of claudins in particular have been shown many times previously to be linked to changes in barrier function.
The mechanism by which B. longum reuter, but not L. rhamnosus GG increases TJ protein expression and TEER is almost certainly associated with signalling through TLR2.
This is demonstrated by two lines of evidence: 1) neutralisation of TLR2 abolishes B. longum reuter-induced increases in TEER and 2) the increase in TJ protein expression is also abolished by blocking TLR2. This is perhaps not surprising given that TLR2 is the major pattern recognition receptor for gram positive bacteria.
The link between the innate immune system and barrier regulation has only recently been established in the gut. Here, activation of TLRs can either increase or decrease epithelial tight junction function depending on the particular TLR activated.
The L. rhamnosus GG lysate appeared to elicit its effects via a mechanism independent of TLR2 because neutralisation of this receptor did not abolish the L. rhamnosus GG-induced increase in TJ function or protein expression. TLR2 is the major receptor for gram positive species and is activated by several different ligands from these bacteria such as lipteichoic acid. The observation that L. rhamnosus GG induced increases in TJ function does not appear to be mediated by TLR 2 may suggest that this lactobacillus possesses molecules that signal via alternative receptors.
Our data suggest that specific strains of Lactobacilli enhance TJ function in human primary keratinocytes. The barrier function of the epidermis is critical for life as terrestrial organisms and recent data has highlighted the essential role of TJs in the epidermal permeability barrier to water. Our data suggest that the augmentation of TJ barrier induced by probiotic bacterial lysates, could have a role in enhancing overall skin barrier function.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1206599.1 | Apr 2012 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2013/050646 | 3/15/2013 | WO | 00 |
