Composition containing probiotics with antibacterial and re-epithelialization effects

Abstract

The present invention relates to Lactobacillus plantarum strain NCIMB 43029; Lactobacillus acidophilus strain NCIMB 43030; a composition having antibacterial and re-epithelialization effects, the composition comprising Lactobacillus plantarum and Lactobacillus acidophilus and optionally comprising Streptococcus thermophilus and / or Bacillus amyloliquefaciens, for use as a medicament for re-epithelialization of an injury or wound; a bandage comprising the composition and a kit comprising such a composition.

Description

Field of the Invention

[0001] The present invention relates to Lactobacillus plantarum strain NCIMB 43029; Lactobacillus acidophilus strain NCIMB 43030; a composition having antibacterial and re-epithelialization effects, the composition comprising Lactobacillus plantarum and Lactobacillus acidophilus and optionally comprising Streptococcus thermophilus and / or Bacillus amyloliquefaciens, the composition being used as a pharmaceutical product, especially for the re-epithelialization process of an injury or wound; and a bandage or kit comprising the composition. Background Art

[0002] The success of skin regeneration is a major challenge since injury or wound healing is a highly dynamic and complex process which involves many stages starting from the hemostasis and coagulation stages, formation of a temporary wound matrix, followed by the stages of inflammation, re-epithelialization and tissue remodelling and restoration.

[0003] When an injury or wound becomes chronic, the difficulty of healing the injury or wound increases.

[0004] One of the representative problems most commonly associated with the wound healing process is that the wound may become infected. Open wounds, especially chronic wounds, are at high risk of being colonized by pathogens. In particular, the wound bed provides an ideal environment for bacterial proliferation. Due to the presence of permissive conditions such as reduced blood circulation and insufficient oxygen supply, bacteria can colonize the wound bed to form multicellular aggregates which adhere to the surface through a biofilm having a specific structure. The biofilm is composed of an extracellular polysaccharide matrix which allows the formation of microbial aggregates embedded in the matrix. Such structures are present in 60% of chronic and 6% of acute injuries or wounds.

[0005] The biofilm is a microbial colonization factor which not only allows strong adhesion of microorganisms to a large number of biological and non-biological surfaces, but also, by encapsulating the microbial aggregates, allows the latter to be protected from antimicrobial agents such as antibiotics and fungicides. In addition, it also promotes the spread of infection. In these cases, bacterial infections are difficult to eradicate and often require removal of the infected tissue or administration of high doses of antimicrobial agents such as antibiotics and disinfectants.

[0006] Even though the administration of antibiotics for oral and topical use is an effective treatment strategy, it is also one of the main causes of the emergence of the phenomenon of drug resistance, which poses serious problems for the treatment of infected injuries / wounds, thus preventing the healing of said injuries / wounds. Regarding the use of topical disinfectants, it should be noted that they are usually highly aggressive in many cases, and this high aggressiveness is associated with damage to the epithelial areas where these substances are applied.

[0007] In order to be able to act on wound healing, it is necessary to understand the biochemical aspects of this process.

[0008] It is well known that nitric oxide (NO) plays a key role in the healing process of injuries or wounds, and particularly in its re-epithelialization and scar formation. Nitric oxide is produced by inducible nitric oxide synthase (iNOS). In particular, nitric oxide is a biological mediator that affects all stages of the wound healing process and acts on all cell types involved in said process.

[0009] Although the use of probiotics for the treatment of skin changes has been studied, the results obtained do not seem to be clear and / or entirely satisfactory.

[0010] In particular, the identification of probiotics or probiotic-containing compositions that play a relevant antibacterial role and an effective re-epithelialization role (especially mediated by iNOS) in tissues with injuries or wounds is still an objective to be achieved.

[0011] Therefore, there is a need for probiotics that are particularly suitable for the treatment of injuries or wounds and have a significant antibacterial effect and an effective re-epithelialization effect, as well as compositions that are suitable for the treatment of injuries or wounds, are free of side effects at the same time, are mild to the application areas that need re-epithelialization, and are easy to set up and inexpensive. Summary of the Invention

[0012] The inventors have surprisingly found that Lactobacillus plantarum NCIMB 43029 strain containing the hypervariable regions V1-V3 of the 16S rRNA-encoding gene of SEQ ID NO:4, the groEL gene of SEQ ID NO:5, and the pheS gene of SEQ ID NO:6; Lactobacillus acidophilus NCIMB 43030 strain containing the hypervariable regions V1-V3 of the 16S rRNA-encoding gene of SEQ ID NO 1, the groEL gene of SEQ ID NO:2, and the pheS gene of SEQ ID NO:3; and Streptococcus thermophilus significantly increase the expression and activation of iNOS, and thus increase the level of nitric oxide (NO), have a highly positive effect on the re-epithelialization of injuries and wounds, and also exert important antibacterial activity.

[0013] Furthermore, the inventors of the present invention have discovered a composition containing probiotics that exhibit antibacterial and re-epithelialization effects, which allows overcoming all the drawbacks of the compositions in the prior art, and which in particular exhibits not only effective antibacterial and re-epithelialization effects, but also no side effects, is gentle on the applied area, easy to apply and inexpensive.

[0014] The composition is particularly effective in treating difficult-to-heal injuries or wounds, especially wounds with a biofilm of pathogenic bacteria (which may be resistant to antibiotics).

[0015] A first object of the present invention is a Lactobacillus plantarum strain deposited on April 20, 2018 at the NCIMB Limited Storage Centre under accession number 43029, which Lactobacillus plantarum strain contains the hypervariable regions V1-V3 of the 16S rRNA-encoding gene of SEQ ID NO:4, the groEL gene of SEQ ID NO:5 and the pheS gene of SEQ ID NO:6 (hereinafter referred to as Lactobacillus plantarum NCIMB 43029).

[0016] A second subject of the present invention is a Lactobacillus acidophilus strain deposited on April 20, 2018 at the NCIMB Limited Storage Centre under accession number 43030, which Lactobacillus acidophilus strain contains the hypervariable regions V1-V3 of the 16S rRNA-encoding gene of SEQ ID NO:1, the groEL gene of SEQ ID NO:2 and the pheS gene of SEQ ID NO:3 (hereinafter referred to as Lactobacillus acidophilus NCIMB 43030).

[0017] As previously indicated, the probiotics activate or induce the expression of iNOS. Accordingly, another object of the present invention is the use of the Lactobacillus plantarum strain NCIMB 43029 and / or the Lactobacillus acidophilus strain NCIMB 43030 as defined previously as a medicament.

[0018] An object of the present invention is also the use of the Lactobacillus plantarum strain NCIMB 43029 and / or the Lactobacillus acidophilus strain NCIMB 43030 as defined previously for activating or inducing the expression of iNOS.

[0019] A further object of the present invention is the use of the Lactobacillus plantarum strain NCIMB 43029 and / or the Lactobacillus acidophilus strain NCIMB 43030 as defined previously for the re-epithelialization of injuries or wounds and / or as an antibacterial agent.

[0020] It has also been unexpectedly discovered that a composition comprising Lactobacillus plantarum and Lactobacillus acidophilus exerts a combined strong antibacterial and re-epithelialization effect and can be used for treating injuries or wounds.

[0021] Accordingly, another object of the present invention is a composition having antibacterial and re-epithelializing effects, which comprises Lactobacillus plantarum and Lactobacillus acidophilus.

[0022] In one aspect of the present invention, the composition comprises the Lactobacillus plantarum strain NCIMB 43029 as defined previously and / or the Lactobacillus acidophilus strain NCIMB 43030 as defined previously.

[0023] In another aspect of the present invention, the composition further comprises at least one strain of the Streptococcus thermophilus species, which can highly significantly increase the expression and activation of iNOS, and thus increase the level of nitric oxide (NO); and / or at least one strain of the Bacillus amyloliquefaciens species, which can exert antifungal activity.

[0024] In a preferred embodiment, Streptococcus thermophilus comprises the hypervariable regions V1-V2 of the 16S rRNA-encoding gene of SEQ ID NO:7, the recA gene of SEQ ID NO:8, and the secA gene of SEQ ID NO:9.

[0025] In another preferred embodiment, the Bacillus amyloliquefaciens strain is a known strain characterized by SEQ ID NO:10 encoding 16S rRNA.

[0026] Another object of the present invention is a composition according to the present invention, which is used as a drug, especially in the process of re-epithelialization of injuries or wounds.

[0027] A preferred aspect of the present invention is a composition according to the present invention, which is used in the process of re-epithelialization of injuries or wounds by activating or inducing iNOS expression.

[0028] Another object of the present invention is a bandage, which comprises the composition according to the present invention.

[0029] Another object of the present invention is a bandage comprising the composition according to the present invention, and the bandage is used as a drug, especially in the re-epithelialization of injuries or wounds.

[0030] Another object of the present invention is a kit comprising a sterile compressible container or a single-dose or multi-dose pump, which contains the composition according to the present invention and corresponding package instructions. Description of the Drawings

[0031] Figure 1 : Effect of bacterial extracts on the re-epithelialization of scratched monolayers of the human keratinocyte cell line HaCaT at different time points.

[0032] (A) Effect of bacterial extract on wound closure rate (%, relative to corresponding T0) of scratched monolayers at 50 μg / ml concentration at 20 and 28 h. Data are expressed as mean ± SEM of three independent experiments performed in duplicate. Two-way ANOVA followed by Dunnett's post-hoc test was used for comparative analysis of data sets. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, relative to control (untreated).

[0033] (B) Images represent re-epithelialization (magnification 10×) of untreated (control) or bacterial extract (50 μg / ml)-treated HaCaT cell monolayers at 20 and 28 h after wound generation.

[0034] Figure 2 : Effect of probiotic extract on iNOS levels in scratched keratinocyte monolayers.

[0035] Immunoblot analysis of iNOS was performed on scratched monolayers treated with 50 μg / ml probiotic extract for 28 h. Values generated by densitometric analysis of the bands were normalized relative to those for β-actin and compared to untreated controls.

[0036] (A) Densitometric results are expressed as iNOS / β-actin (fold relative to control).

[0037] Data were derived from three independent experiments performed in duplicate, and values are expressed as mean ± SEM. One-way ANOVA followed by Dunnett's post-hoc test was used to assess the presence of statistically significant differences between data sets. *P < 0.05, ***P < 0.01, ****P < 0.0001, relative to control (untreated).

[0038] (B) Image showing immunoblot representing iNOS.

[0039] Figure 3 : Nitrite levels in the medium of scratched monolayers of HaCaT cells in the presence or absence of aminoguanidine, AG, an iNOS inhibitor.

[0040] Before treatment with the bacterial extracts, the scratched monolayers of HaCaT cells were incubated for 15 minutes in the presence or absence of 20 μM concentration of AG. The nitrite levels in the medium were analyzed after 28 hours by Griess reagent. The data shown represent the mean ± SEM of three independent experiments performed in duplicate. Two-way ANOVA followed by Bonferroni's post-hoc test was used to evaluate the presence of statistically significant differences between data groups: *P < 0.05, **P < 0.01, ****P < 0.0001, relative to control (untreated). #P < 0.05, ##P < 0.01, P < 0.001, #P < 0.0001, relative to the respective cultures in the absence of AG.

[0041] Figure 4 : Effect of the iNOS inhibitor AG on the re-epithelialization of scratched monolayers of HaCaT cells induced by Streptococcus thermophilus, Lactobacillus plantarum, and Lactobacillus acidophilus.

[0042] (A) Effect on the relative rate of closure of scratched monolayers pretreated with 20 μM concentration of AG for 15 minutes and treated with 50 μg / ml concentration of probiotic extracts for 20 hours. The data represent the mean ± SEM of three independent experiments performed in duplicate. Two-way ANOVA was followed by Bonferroni's post-hoc test to evaluate statistically significant differences. #P < 0.05, ##P < 0.01, relative to the respective cultures in the absence of AG.

[0043] (B) Images represent scratched monolayers (magnification 10×) of HaCaT cells subsequently exposed to 50 μg / ml concentration of Streptococcus thermophilus, Lactobacillus plantarum, or Lactobacillus acidophilus extracts for 20 hours in the presence and absence of pretreatment with 20 μM concentration of AG.

[0044] Figure 5 : Effect of the iNOS inhibitor AG on the re-epithelialization of scratched monolayers of HaCaT cells induced by addition of 20 μg / ml concentration of Bacillus amyloliquefaciens extracts for 20 hours.

[0045] (A) Effect of pretreatment with 20 μM AG for 15 minutes on the relative rate of closure of scratched monolayers treated with 20 μg / ml Bacillus amyloliquefaciens extract for 20 hours. Data are represented as mean ± SD of experiments performed in duplicate, representing 3 independent experiments performed in duplicate. Two-way ANOVA followed by Bonferroni post-test was used to evaluate statistically significant differences between groups of data. Relative to each corresponding culture in the absence of AG, (#P < 0.05, ##P < 0.01). Relative to the control, **P < 0.01.

[0046] (B) Images represent scratched monolayers (magnification 10x) of HaCaT cells pretreated with 20 μM AG for 15 minutes or without pretreatment and then treated with 20 μg / ml Bacillus amyloliquefaciens extract for 20 hours.

[0047] Figure 6 : Effect of different combinations of bacterial extracts at the indicated concentrations on the re-epithelialization of scratched monolayers of HaCaT cells 20 hours after scratching. Data are represented as the mean increase rate (%) of re-epithelialization relative to untreated controls.

[0048] Figure 7 :

[0049] Figure A: Photograph of the right foot of a patient with a diabetic ulcer and a postoperative wound after toe amputation (day 36).

[0050] Figure B: Figure 7 Magnified photograph of the foot of A.

[0051] Figure 8 :

[0052] Figure A: Figure 7 Photograph of the foot of A in lateral view on day 48.

[0053] Figure B: Figure 8 Magnified photograph of the foot of A in front view.

[0054] Figure 9 :

[0055] Figure A: Figure 7 Photograph of the foot of A in front view on day 57.

[0056] Figure B: Figure 9 Magnified photograph of the foot of A in lateral view.

[0057] Figure 10 :

[0058] Figure A: Figure 7 Photograph of the foot of A in front view on day 170.

[0059] Figure B: Figure 10 Enlarged photograph of the foot of A in side view. Detailed implementation mode

[0060] The Lactobacillus plantarum strain NCIMB 43029 proposed above according to the present invention was deposited on April 20, 2018 at the NCIMB Ltd. collection centre, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, Scotland, UK, and is a Lactobacillus plantarum strain with the NCIMB accession number 43029.

[0061] The Lactobacillus plantarum NCIMB 43029 has been deposited with NCIMB Ltd. by Professor Claudio De Simone (51 Route des Chenolettes 1660, Chateau-d'OEx, Switzerland).

[0062] Professor Claudio De Simone has authorized the owner of this patent application to mention Lactobacillus plantarum NCIMB 43029 in the present invention document, and has agreed to make Lactobacillus plantarum NCIMB 43029 available to the public in accordance with R. 33 EPC.

[0063] The main characteristic demonstrated by Lactobacillus plantarum NCIMB 43029 as previously defined is the ability to highly activate and induce iNOS expression to significantly increase the nitric oxide level, as demonstrated by the nitrite dose, and thus significantly act on the re-epithelialization of injuries or wounds.

[0064] In vitro and in vivo evidence also shows that Lactobacillus plantarum NCIMB 43029 exerts a highly antibacterial effect.

[0065] The Lactobacillus acidophilus NCIMB 43030 according to the present invention was deposited on April 20, 2018 at the NCIMB Ltd. collection centre, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, Scotland, UK, and is a Lactobacillus acidophilus strain with the accession number NCIMB 43030.

[0066] The Lactobacillus acidophilus NCIMB 43030 has been deposited with NCIMB Ltd. by Professor Claudio De Simone (51 Route des Chenolettes 1660, Chateau-d'OEx, Switzerland).

[0067] Professor Claudio De Simone has authorized the owner of this patent application to mention Lactobacillus acidophilus NCIMB 43030 in this invention document and has given consent for Lactobacillus acidophilus NCIMB 43030 to be made available to the public according to R.33EPC.

[0068] The main characteristics demonstrated by Lactobacillus acidophilus NCIMB 43030, as previously defined, are the ability to highly activate and induce the expression of iNOS to significantly increase the nitric oxide level, as demonstrated by the nitrite dose, and thus significantly act on the re-epithelialization of injuries or wounds.

[0069] In vitro and in vivo evidence also shows that Lactobacillus acidophilus NCIMB 43030 exerts a highly antibacterial effect.

[0070] The probiotic strain is novel and the subject of this invention, as previously mentioned.

[0071] It has also been unexpectedly found that Lactobacillus plantarum and Lactobacillus acidophilus in combination show enhanced antibacterial and re-epithelialization effects, which have never been observed in compositions containing probiotics currently known in the art.

[0072] Therefore, an object of the present invention is a composition comprising Lactobacillus plantarum and Lactobacillus acidophilus, said composition having antibacterial and re-epithelialization effects.

[0073] According to a preferred aspect of the present invention, the composition of the present invention further comprises Streptococcus thermophilus and / or Bacillus amyloliquefaciens.

[0074] According to a particularly preferred aspect of the present invention, the composition having antibacterial and re-epithelialization effects of the present invention comprises Lactobacillus plantarum, Lactobacillus acidophilus and Streptococcus thermophilus.

[0075] According to another particularly preferred aspect of the present invention, the composition having antibacterial, re-epithelialization and antifungal effects comprises Lactobacillus plantarum, Lactobacillus acidophilus, Streptococcus thermophilus and Bacillus amyloliquefaciens.

[0076] The composition according to the present invention is generally a composition preferably for local use in the form of powder, granules, gingival tablets or vaginal tablets or preferably for oral use in the form of capsules or gels.

[0077] The composition according to the present invention exerts an enhanced antibacterial effect because Lactobacillus plantarum and Lactobacillus acidophilus in combination are able to synergistically act on molecules of bacterial origin.

[0078] The composition according to the invention exerts a re-epithelialization effect because Lactobacillus plantarum, Lactobacillus acidophilus and Streptococcus thermophilus are able to activate and induce the expression of iNOS and thus cause the production of nitric oxide.

[0079] Preferably, the Lactobacillus plantarum in the composition according to the invention is the strain NCIMB 43029 as previously identified.

[0080] Preferably, the Lactobacillus acidophilus in the composition according to the invention is the strain NCIMB 43030 as previously identified.

[0081] Preferably, the Streptococcus thermophilus in the composition according to the invention is a strain comprising the hypervariable regions V1-V2 of the 16S rRNA-encoding gene of SEQ ID NO:7, the recA gene of SEQ ID NO:8 and the secA gene of SEQ ID NO:9.

[0082] Preferably, the Bacillus amyloliquefaciens in the composition according to the invention is a known strain characterized by SEQ ID NO:10 encoding 16S rRNA.

[0083] Based on the total weight of the composition, the weight percentage of Lactobacillus plantarum in the composition ranges from 1% to 40% by weight.

[0084] Based on the total weight of the composition, the weight percentage of Lactobacillus acidophilus in the composition ranges from 1% to 40% by weight.

[0085] Based on the total weight of the composition, the weight percentage of Streptococcus thermophilus in the composition ranges from 0.5% to 20% by weight.

[0086] Based on the total weight of the composition, the weight percentage of Bacillus amyloliquefaciens in the composition ranges from 0.1% to 10% by weight.

[0087] A preferred aspect of the invention is a composition having antibacterial and re-epithelialization effects, comprising:

[0088] 10% to 90% by weight of Lactobacillus plantarum, and

[0089] 90% to 10% by weight of Lactobacillus acidophilus,

[0090] both based on the total weight of the composition.

[0091] A preferred aspect of the invention is also a composition having antibacterial and re-epithelialization effects, comprising:

[0092] 20% to 80% by weight of Lactobacillus plantarum,

[0093] 40% to 10% by weight of Lactobacillus acidophilus, and

[0094] from 40% to 10% by weight of Streptococcus thermophilus,

[0095] both calculated on the total weight of the composition.

[0096] A preferred aspect of the present invention is also a composition having antibacterial, re-epithelializing and antifungal effects, which comprises:

[0097] from 10% to 80% by weight of Lactobacillus plantarum,

[0098] from 40% to 10% by weight of Lactobacillus acidophilus,

[0099] from 40% to 9% by weight of Streptococcus thermophilus, and

[0100] from 10% to 1% by weight of Bacillus amyloliquefaciens,

[0101] both calculated on the total weight of the composition.

[0102] In a preferred aspect of the present invention, the bacteria Lactobacillus plantarum, Lactobacillus acidophilus, Streptococcus thermophilus and / or Bacillus amyloliquefaciens in the composition are live bacteria.

[0103] Alternatively, the bacteria Lactobacillus plantarum, Lactobacillus acidophilus, Streptococcus thermophilus and / or Bacillus amyloliquefaciens are non-viable bacteria, usually made non-viable by irradiation or by alternative techniques.

[0104] Preferably, the composition according to the present invention comprises at least one pharmaceutically acceptable excipient selected from the group consisting of: seaweed, bee pollen, honey, aerated clay and zeolite.

[0105] Another object of the present invention is a composition according to the present invention for use as a medicament, in particular for the re-epithelialization of injuries or wounds. Preferably, the re-epithelialization process is carried out by activating or inducing iNOS.

[0106] According to the present invention, the term "wound" means an open wound, a chronic wound, an acute wound, a burn wound, a post-surgical wound, a traumatic wound, dermabrasion, an ulcer such as a diabetic ulcer, a pressure ulcer or a gastrointestinal ulcer formation.

[0107] A preferred aspect is a composition according to the present invention for use in the process of re-epithelializing an injury or wound selected from the group consisting of an open wound, a chronic wound, an acute wound, a burn wound, a post-surgical wound, a traumatic wound, dermabrasion, an ulcer such as a diabetic ulcer, a pressure ulcer.

[0108] Said injury or wound may generally affect the skin or mucosa, in particular the oral, vaginal or rectal mucosa, but may also be a gastrointestinal injury.

[0109] Accordingly, a preferred aspect of the present invention is the composition for use in the process of re-epithelialization of an injury or wound of the skin, oral cavity, vaginal or rectal mucosa, or gastrointestinal mucosa.

[0110] Preferably, when the composition is used in the process of re-epithelialization of an injury or wound of the skin, oral cavity, vaginal or rectal mucosa, ulcers such as diabetic ulcers or pressure sores, the composition is for topical use in the form of granules or powders, tablets for the gums or tablets for the vagina.

[0111] Preferably, when the composition is used in the process of re-epithelialization of an injury or wound of the gastrointestinal mucosa, the composition is for oral use in the form of capsules or gels.

[0112] According to the present invention, the injury or wound may be infected by a biofilm composed of the following: Enterococcus faecalis, Candida Albicans, and Gram-negative bacteria such as Klebsiella pneumoniae and Proteus mirabilis.

[0113] Another object of the present invention is a bandage comprising the composition according to the present invention as previously defined.

[0114] A preferred object of the present invention is a bandage comprising the composition according to the present invention, which is used as a medicament, especially in the process of re-epithelialization of an injury or wound.

[0115] Another object of the present invention is a kit comprising a sterile compressible container or a single-dose or multi-dose pump, which contains the composition according to the present invention and corresponding package instructions.

[0116] Experimental Section

[0117] Effect of bacterial strain extracts on wound healing on a scratch monolayer generated from HaCaT cells (human keratinocyte cell line) Figure 1

[0118] The present inventors have used an in vitro artificial wound model to evaluate the ability of extracts obtained from selected bacterial strains to affect wound re-epithelialization. The closure rate of the scratched monolayer in untreated cells and in cells treated with each bacterial extract at a concentration of 50 μg / ml was shown and evaluated by observing the repopulation of the area between the wound edges at different time points after wound generation. To quantitatively analyze the effect of the bacterial extracts on the closure of the wound area, images obtained by using an inverted phase contrast microscope at 0, 20, 28, and 45 hours after scratching were acquired and expressed as a closure percentage by using an automated calculation system to evaluate the percentage of the area occupied by the cells relative to the total surface area analyzed. In all experiments, the scratched monolayer of control cells (untreated) healed completely after 36 - 42 hours. In the presence or absence of the bacterial extracts, the percentage of re-epithelialization at 20 and 28 hours was compared with the wound of the corresponding monolayer at T0. The results are expressed as the percentage of relative re-epithelialization (mean ± SEM from three independent experiments performed in duplicate) and the images representing the microscopic observations of the scratched monolayer are shown respectively in iNOS expression and nitrite levels Figures A and 1B. Treatment with extracts from Streptococcus thermophilus or Lactobacillus plantarum significantly accelerated the repair rate relative to the untreated control 20 hours and 28 hours after wound generation. In contrast, treatment with extracts from Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium breve significantly delayed the repair process of the monolayer relative to the untreated control. The extract from Lactobacillus bulgaricus did not seem to significantly affect the wound closure rate relative to the control at the two observation time points.

[0119] Figure 2

[0120] To investigate the potential involvement of iNOS in the effect of the above bacterial extracts on the repair process of HaCaT cell monolayers, the present inventors initially have analyzed the iNOS protein level in the monolayer 28 hours after scratch generation in the absence or presence of the bacterial extracts at a concentration of 50 μg / ml by Western blot analysis. The values obtained by densitometric analysis of the iNOS bands were normalized relative to the values for β-actin. The data expressed as mean ± SEM from three independent experiments performed in duplicate and the images representing the iNOS immunoblots are shown respectively in Figure 3In A and 2B. The results indicate that treatment with extracts of Streptococcus thermophilus, Lactobacillus plantarum, and Lactobacillus acidophilus is consistent with their ability to accelerate the re-epithelialization of monolayers, resulting in a significant overregulation of iNOS protein expression relative to that observed in control cells. The highest degree of related effects was observed in the case of Lactobacillus acidophilus and Lactobacillus plantarum. Although to a lesser extent, the extract of Streptococcus thermophilus was also able to significantly increase the expression level of iNOS protein. The extract of Lactobacillus bulgaricus, which had no significant effect on the healing of wounds generated on the monolayer relative to that observed for the cell monolayer used as a control, was also unable to regulate iNOS expression. On the other hand, HaCaT cell monolayers exposed to extracts of Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium breve (all strains were able to inhibit the wound closure rate) showed significantly lower levels of iNOS protein relative to the untreated situation.

[0121] The ability of bacterial extracts to regulate iNOS was further analyzed by measuring the nitrite levels in the supernatants of scratched monolayers of HaCaT cells pretreated with AG (a selective iNOS inhibitor) or not treated with such an inhibitor (known from T.P. Misko et al., Eur. J. Pharmacol, 1993, 233: 119 - 125).

[0122] As Figure 4 shown, AG pretreatment significantly prevented the increase in nitrite production due to its ability to inhibit iNOS activity induced by scratching the control monolayer. Treatment with extracts of Streptococcus thermophilus, Lactobacillus acidophilus, and Lactobacillus plantarum, which were able to regulate iNOS expression, induced a significant increase in the nitrite level in the culture medium relative to the control monolayer, even though to different extents. Notably, the stimulatory effect of extracts of Streptococcus thermophilus, Lactobacillus acidophilus, and Lactobacillus plantarum on nitrite production was completely or partially blocked by pretreatment with AG, further supporting the evidence of the ability of these probiotics to induce iNOS expression and activity.

[0123] Conversely, extracts of Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium breve induced a significant decrease in nitrite levels relative to the control, confirming their inhibitory effect on the degree of iNOS expression. According to the results of the experiments on iNOS expression, pretreatment with AG did not significantly affect the nitrite levels in cell cultures treated with Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium breve relative to monolayers treated only with bacterial extracts. Treatment with the extract of Lactobacillus bulgaricus did not significantly affect the nitrite levels relative to the untreated cultures, while AG pretreatment significantly decreased the nitrite levels relative to the corresponding samples not treated with the inhibitor.

[0124] The effect of AG (iNOS inhibitor) on the wound closure rate was also evaluated based on the ability of extracts from Lactobacillus plantarum, Streptococcus thermophilus, and Lactobacillus acidophilus to accelerate the wound closure process in vitro. The results, expressed as the mean ± SEM of three experiments repeated twice, relate to the re-epithelialization % at 20 h in cell monolayers in the presence or absence of bacterial extracts at a concentration of 50 μg / ml, relative to the scratched area generated at T0 ( Figure 4 A). Images representing the microscopic observations are also shown ( Figure 5 B). As expected, in accordance with the role of iNOS activity in wound healing, pretreatment with AG had a strong effect on the physiological repair of the control monolayers and significantly blocked the stimulatory effect of all these bacterial extracts in terms of the monolayer repair rate expressed as re-epithelialization % relative to the scratched area generated at the start of the experiment.

[0125] Taken together, these results strongly suggest that an increase in the expression and activity of iNOS plays a key role in the re-epithelialization process promoted by the selected probiotic extracts.

[0126] The ability to accelerate the re-epithelialization process was also confirmed after treatment with Bacillus amyloliquefaciens extract at a concentration of 20 μg / ml ( Figure 5 ). Moreover, in this case, the involvement of iNOS was confirmed by the ability of its specific inhibitor aminoguanidine (20 μM) to block the stimulatory effect induced by treatment with Bacillus amyloliquefaciens. Conclusion A shows the re-epithelialization % after 20 h of treatment with Bacillus amyloliquefaciens extract, relative to the scratched area generated at T0, expressed as the mean ± SD. The results represent 3 independent experiments repeated twice. #P < 0.05 and ##P < 0.01 relative to the corresponding samples in the absence of AG; **P < 0.01 relative to the untreated control.

[0127] To evaluate the effect of treatments with different combinations of probiotics on the re-epithelialization process of scratched HaCaT cell monolayers, the use concentration of individual strains (Lactobacillus plantarum, Lactobacillus acidophilus, Streptococcus thermophilus, and Bacillus amyloliquefaciens) showed no significant effect on the repair of injury when used alone. In fact, 20 hours after injury, treatment of the scratched monolayer with individual bacterial extracts at a concentration of 10 μg / ml did not significantly affect the wound closure % (re-epithelialization < 10%) compared to the untreated control. On the other hand, treatment with a combination of Lactobacillus plantarum and Lactobacillus acidophilus, both at a concentration of 10 μg / ml, increased the repair rate of the injured monolayer 20 hours after injury (about 35%). A combination of Lactobacillus plantarum (10 μg / ml), Lactobacillus acidophilus (10 μg / ml), and Streptococcus thermophilus (10 μg / ml) caused a significant increase in the repair rate (about 60%) in the injured monolayer observed 20 hours after treatment. Similarly, combination treatment with Lactobacillus plantarum (10 μg / ml), Lactobacillus acidophilus (10 μg / ml), and Bacillus amyloliquefaciens (10 μg / ml) caused an increase in the repair rate (more than 70%) 20 hours after treatment. A combination of Lactobacillus plantarum (10 μg / ml), Lactobacillus acidophilus (10 μg / ml), Streptococcus thermophilus (10 μg / ml), and Bacillus amyloliquefaciens (10 μg / ml) was even more effective in accelerating the re-epithelialization of the damaged monolayer, causing an increase in the wound closure rate (97% - 98%) 20 hours later.

[0128] Materials and Methods

[0129] Considering the important barrier function exerted by the epidermis at the time of wound appearance, it is necessary to restore tissue integrity as quickly and effectively as possible by promoting the re-epithelialization process.

[0130] Proliferation and migration of keratinocytes are fundamental steps in the re-epithelialization process during wound healing.

[0131] In this study, the present inventors compared the effects exerted by seven different probiotic strains on an in vitro wound healing model under the same experimental conditions. The results obtained demonstrated that extracts of Lactobacillus plantarum, Lactobacillus acidophilus, Streptococcus thermophilus, and Bacillus amyloliquefaciens promoted the re-epithelialization of scratched monolayers of HaCaT cells. In contrast, extracts of Bifidobacterium infantis, Bifidobacterium breve, and Bifidobacterium longum inhibited the re-epithelialization process, while the bacterial extract of Lactobacillus delbrueckii subsp. bulgaricus had no effect on in vitro wound repair.

[0132] The underlying mechanisms of the increased re-epithelialization induced by Lactobacillus plantarum, Lactobacillus acidophilus, Streptococcus thermophilus, and Bacillus amyloliquefaciens involve increased expression and activity of iNOS, as demonstrated by immunoblot data, nitrite level analysis, and the effect of pretreatment with aminoguanidine (a specific inhibitor of iNOS). On the other hand, Bifidobacterium infantis, Bifidobacterium breve, and Bifidobacterium longum significantly decreased the expression and activity of iNOS in scratched HaCaT cell monolayers. In this context, it was also shown that the bacterial extract of Lactobacillus bulgaricus had no effect.

[0133] Taken together, these results strongly suggest that, as also supported by experiments conducted in the presence of AG, pro-healing or anti-healing properties strictly depend on the ability of probiotics to upregulate or downregulate the expression and activity of iNOS. The data obtained by the present inventors extend the scope of the underlying mechanisms of the effect of probiotics on the epithelialization process and can demonstrate their use in the treatment of chronic wounds. The evidence provided should be the subject of further detailed studies to investigate the potential therapeutic use of probiotics, especially with regard to dose standardization and detailed characterization of beneficial effects. In addition, changes in the production process should be closely monitored in terms of safety and / or efficacy, which may cause harmful differences in probiotic products.

[0134] From the above, it follows that the selection of probiotic strains is also very important, as the effects of these bacteria can be highly strain-specific.

[0135] Preparation of bacterial samples for cell treatment

[0136] Cell lines and culture conditions

[0137] Lactobacillus plantarum, Lactobacillus acidophilus, and Streptococcus thermophilus were obtained from DuPont Danisco, Wilmington, Delaware, USA. Lactobacillus bulgaricus and Bifidobacterium longum were obtained from Bioprox, Noyant, France. Bifidobacterium breve and Bifidobacterium infantis were obtained from Centro Sperimentale del Latte S.r.l., Zelo Buon Persico LO, Italy. Bacillus amyloliquefaciens was obtained from Sanzyme Biologics Private Limited, Hyderabad, Telangana, India.

[0138] For the preparation of bacterial extracts, a stock solution of 1 g of each freeze-dried bacterium resuspended in phosphate buffered saline (PBS, Euro Clone, West York, UK) was centrifuged at 8,600×g, washed twice, resuspended in 10 ml of PBS and sonicated (30 min, alternating 10 s sonication and 10 s pause) using a Vibracell sonicator (Sonic and Materials, Danbury, CT).

[0139] Bacterial cell lysis was verified by measuring the absorbance of the samples at 590 nm (Eppendorf Hamburg, Germany) before and after each sonication step. The samples were then centrifuged at 17,949×g and the supernatant was filtered using a 0.22 mm pore filter (Corning Incorporated, Corning, NY, USA) to remove any remaining whole bacteria. The total protein content was determined by BioRad DC protein assay (BioRad, Hercules, CA) using bovine serum albumin (BSA, Sigma Aldrich, Saint Louis, MO, USA) as a standard. For in vitro experiments, the bacterial preparations were added to cell cultures at different time intervals, as indicated below, using different amounts to obtain different concentrations, expressed as μg protein / ml as the final concentration.

[0140] In vitro wound healing model

[0141] The HaCaT cell line of spontaneously immortalized human keratinocytes was purchased from Cell LinesService GmbH (Eppelheim, Germany). HaCaT cells were cultured in DMEM supplemented with 10% (v / v) fetal calf serum (FCS), 2 mM L-glutamine, 100 U / ml penicillin and 100 μg / ml streptomycin (Euro Clone, West York, UK). The culture conditions were kept constant at 37 °C in a humid atmosphere with 5% CO2. After reaching 80% confluence, the cells were seeded at 18,000 cells / cm 2Cells were seeded at the indicated concentrations into 6- or 12-well plates (Becton Dickinson, San José, CA) for sterile cell culture as specified below. Non-adherent cells were removed by gentle washing in phosphate buffered saline (PBS, pH = 7.4). Cells grown in 12-well plates were harvested at 20, 28, and 45 hours and viable cells were counted using a trypan blue dye exclusion test (Euro Clone, West Yorkshire, UK). Cells were incubated with various concentrations of bacterial extracts for different time intervals (20 - 48 hours), washed with PBS thereafter, centrifuged at 400×g for 10 minutes, and the pellets were incubated with a 0.04% trypan blue solution for 5 minutes to analyze the total cell number and their viability. Untreated cells were also analyzed and used as a negative control. Cells were transferred to a Bürker counting chamber and counted under a microscope Eclipse 50i (Nikon Corporation, Japan). To select a suitable concentration of bacterial extract expressed as μg protein / ml, the viability of HaCaT keratinocytes was analyzed using the trypan blue exclusion test. After incubation with various concentrations of bacterial extracts for 20 - 48 hours, no significant effect on cell viability or proliferation levels was detected relative to untreated control cells (data not shown). On the other hand, treatment with Triton X detergent (0.1%) caused a significant decrease in cell viability (positive control) (P ≤ 0.01). For most experiments, a concentration of bacterial lysate in the range of 10 - 50 μg / ml was used.

[0142] Western blot analysis of iNOS expression

[0143] As previously described for in vitro wound healing assays, HaCaT cells were grown in 6-well microplates using the culture conditions described above and were then allowed to proliferate until they reached approximately 90% confluence; subsequently, DMEM was removed from the wells and the cell monolayer was scratched with the tip of a 200 μl pipette to create a uniform cell-free area (wound) of reproducible size. Debris was removed from the culture by gently washing with sterile PBS. The cells were then incubated in fresh medium at 37 °C in a humidified atmosphere with 5% CO2 in the presence or absence of bacterial extracts at the indicated final concentrations (in the range of 10 - 50 μg protein / ml). Where indicated, the cells were pretreated with 20 μM aminoguanidine (AG) (selective iNOS inhibitor) (Sigma Aldrich, St. Louis, MO, USA) for 15 minutes. Cell migration was monitored using a phase contrast inverted microscope Eclipse TS100 (Nikon) and images were taken at the start of the experiment (T0) and at different time points up to 45 hours post-injury. The experiment was repeated twice with at least three - six regions being evaluated for each condition. To calculate the wound closure %, the acquired images were quantitatively analyzed using TScratch software. The relative re-epithelialization was quantified using the following equation:

[0144]

[0145] where T is a specific time point after scratching.

[0146] Nitrite level analysis

[0147] For Western blot analysis, scratched monolayers of untreated cells and cells treated with bacterial extract for 28 h were collected, washed in PBS and lysed in RIPA buffer (RIPA lysis buffer, Merck KGaA, Darmstadt, Germany), which contained protease inhibitor mixture (carboxypeptidase inhibitor, 5 μg / ml trypsin inhibitor, 1 mM PMSF, 10 μg / ml leupeptin, 10 μg / ml aprotinin, 10 μg / ml pepstatin) (Sigma Aldrich, St. Louis, MO, USA). Protein content of the samples was tested using BioRad DC protein assay (Bio-Rad, Hercules, CA) with BSA as a standard. 25 μg of protein was mixed with sample buffer, boiled at 100 °C for 5 min, and separated by polyacrylamide gel electrophoresis, 10% SDS. Proteins were transferred onto 0.45 mm nitrocellulose membrane sheets (Bio-Rad, Hercules, CA) at 70 V for 1 h at 4 °C using a Mini Trans-Blot Cell (Bio-Rad, Hercules, CA) device. The membranes were blocked with 5% fatty acid-free milk for 1 h at room temperature and then incubated overnight at 4 °C with 1:500 polyclonal rabbit anti-iNOS antibody (Cell Signaling Technology, CA) or 1:1000 anti-β-actin antibody (Santa Cruz Biotechnology, CA). For anti-iNOS antibody, goat anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase (HRP) at a recommended dilution of 1:5000 was used, and for anti-β-actin antibody (Millipore EMD, Darmstadt, Germany), rabbit anti-goat IgG secondary antibody conjugated to horseradish peroxidase (HRP) at a recommended dilution of 1:5000 was used. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech) according to the manufacturer's instructions. Relative band densities were determined using an ALLIANCE chemiluminescence detection system (UVITEC, Cambridge, UK) and the values were expressed as relative units. Immunoblot data were normalized to the corresponding protein levels of β-actin.

[0148] Statistical analysis

[0149] The Griess method was used to indirectly evaluate NO production by measuring nitrite levels using nitrate reductase and the Griess reaction based on colorimetric analysis. Briefly, the supernatant (20 μl) of the scratched monolayer of untreated cells and cells treated with bacterial extract for 28 h was added to a 96-well microtiter plate together with 50 mM Hepes, 5 μM FAD, 0.1 mM NADPH, 0.2 U / ml nitrate reductase, 1,500 U / ml lactate dehydrogenase, and 100 mM pyruvate, and finally Griess reagent. Absorbance was measured by spectrophotometric reading at 550 nm using a microplate reader (Bio-Rad, Hercules, California, USA). The values were interpolated with a standard curve of known concentrations of KNO3.

[0150] Example 1 - Application of the composition of the present invention in powder form to the foot of a female patient infected with ulcers caused by diabetes

[0151] Data were analyzed using Prism 6.0 software (GraphPad, San Diego, CA). Results are expressed as mean ± SEM of three experiments performed in duplicate. Results were considered statistically significant if P < 0.05. For comparison of groups, ANOVA test was used followed by Tukey or Dunnett post hoc tests. For statistical analysis of data, *, # for P < 0.05, **, ## for P < 0.01, ***, for P < 0.001, and ****, # for P < 0.0001 were used herein.

[0152] Regarding in vivo evaluation, the inventors have tested the probiotics of the present invention, and in particular, the compositions according to the present invention for treating injuries or wounds, in particular postoperative wounds and diabetic ulcers, in particular biofilms resistant to antibiotic therapy, in order to better study the antibacterial effect and re-epithelialization effect.

[0153] Examples of applying the compositions according to the present invention to in vivo wounds and the corresponding results are provided below.

[0154] and with a toe amputation wound Figure 7

[0155] The efficacy of compositions comprising Lactobacillus plantarum NCIMB 43029 as previously identified, Lactobacillus acidophilus NCIMB 43030 as previously identified, and Streptococcus thermophilus known as previously identified on diabetic ulcers and surgical wounds has been studied.

[0156] In particular, an 83-year-old female with severe critical limb ischemia (CLI) caught our attention, presenting with ulcerative skin lesions on her right leg that extended to the second and third toes. Her medical history showed type II diabetes, systemic arterial hypertension, ischemic heart disease (with previous coronary artery bypass surgery), and atrial fibrillation (AF). She was also an active smoker (20 cigarettes per day for 20 years). According to clinical observations, bilateral femoral artery pulsations were present, but the remaining peripheral artery (periferic) pulsations (popliteal artery, posterior tibial artery, and pedal arteries (pepidia)) were absent. The ulcer on the right leg extended to the toes, with necrotic epidermis (excara) on the anterior and posterior surfaces of the leg, and parchment-like necrosis of the second and third toes; there was pain on pressure, and motor function was preserved. The ankle-brachial index (ABI) of both legs could not be monitored.

[0157] Upon admission, blood tests yielded the following results: C-reactive protein (CRP), 101,000 μg / l; erythrocyte sedimentation rate (ESR), 100 mm / h; hemoglobin (HGB), 8.0 g / dl; platelets, 454,000 μg / l; white blood cells (WBC), 12,800 / μl; international normalized ratio (INR), 1.87; partial thromboplastin time (PTT), ratio 2.3. Surgical treatment consisted of: recanalization of the superficial femoral artery (SFA) and the right popliteal artery and percutaneous transluminal angioplasty (PTA) using a drug-eluting balloon (DEB) Ranger (5×100 mm), followed by surgical debridement of the necrotic lesions on the forefoot and amputation of the second toe of the right foot. Before the surgical intervention, the infectious disease specialist reported: “…inflammatory parameters are significantly increased. It is recommended to initiate antibiotic therapy with (piperacillin + tazobactam), 4.5 g every 8 hours.”.

[0158] After 8 days, with the improvement of the inflammatory markers, the infectious disease physician changed the antibiotic therapy to (minocycline) 100 mg, 1 tablet × 2 for 15 days, and the patient was discharged after a total of 21 days of hospitalization. At home, the patient attended outpatient clinics twice a week, and dressings with an antibacterial solution were applied locally to the edges of the wound and the resection site. First, the treatment area was cleaned and a polymer film ( Ferries Mfg.) was applied routinely.

[0159] Thirty-six days after discharge and home treatment, due to the worsening clinical picture with a significant increase in wound exudate, the patient presented to our institution despite being empirically prescribed amoxicillin (500 mg, orally every 12 hours) for a four-week antibiotic therapy by the attending physician. The wound was moist, had a large amount of discharge, and was covered with fibrin. A wound swab was immediately taken, and Klebsiella pneumoniae, Enterococcus faecalis, and Proteus mirabilis were confirmed positive. Although the patient was afebrile, three blood cultures were also taken, with negative results.

[0160] Given the diversity of the microorganisms present in the wound and the overall condition of the patient, who was severely ill and bedridden, it was decided to use a dressing with 10% povidone iodine skin solution ( 10% skin solution) for local use. After 30 days, no improvement was observed, and systemic antibiotic treatment was discontinued. Out of compassion, the patient was informed and gave consent to apply a probiotic mixture in dry powder form to the wound. The probiotic preparation was a composition comprising Lactobacillus plantarum NCIMB43029, Lactobacillus acidophilus NCIMB 43030, and the known Streptococcus thermophilus (SEQ ID NOs: 7-9). 0.5 g of the composition was applied once daily.

[0161] One week after probiotic treatment, the condition of the wound stabilized. Two weeks later, the microbial analysis improved, and in subsequent time periods, slow but progressive wound healing was clearly observed. The wound swab was negative for Enterococcus faecalis on day 48 (12 days after starting probiotic application), and negative for Klebsiella pneumoniae and Proteus mirabilis on day 57 (21 days after local probiotic application). The probiotic preparation was discontinued on day 60, 24 days after treatment.

[0162] Within the subsequent 90 days, the wound healed, albeit very slowly. Treatment with PolyMem at home improved the comfort of the patient, who was able to walk again and perform his daily tasks independently.

[0163] Figure 8 A / 7B shows a photograph of the patient's right foot on the 36th day after discharge.

[0164] Figure 9 A / 8B shows a photograph of the patient's right foot on the 48th day after discharge.

[0165] Figure 10 A / 9B shows a photograph of the patient's right foot on the 57th day after discharge.

[0166] ​ A / 10B shows a photograph of the patient's right foot on the 170th day after discharge.

[0167] It should be noted that before treatment with the composition of the present invention, the patient had been treated with antibiotics in order to eradicate the bacterial biofilm present on the foot ulcer, but the results were almost nil.

[0168] Conversely, the composition of the present invention was well tolerated by the patient, who was also extremely satisfied with the healing results obtained on the ulcers and wounds affecting his foot.

[0169] Therefore, it has been found that the composition of the present invention is safe and extremely effective against bacterial biofilms or wounds that are also resistant to antibiotics, in terms of antibacterial and re-epithelialization effects. Sequence Listing <110> EOS2021 S.p.A. <120> Composition with antibacterial and re-epithelialization action containing probiotics <140> IT102019000009951 <141> 2019-06-24 <160> 10 <170> BiSSAP 1.3.6 <210> 1 <211> 551 <212> DNA <213> Lactobacillus acidophilus <220> <223> Hypervariable regions V1-V3 of the 16S rRNA-encoding gene of Lactobacillus acidophilus NCIMB43030 <400> 1 agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60 gagctgaacc aacagattca cttcggtgat gacgttggga acgcgagcgg cggatgggtg 120 agtaacacgt ggggaacctg ccccatagtc tgggatacca cttggaaaca ggtgctaata 180 ccggataaga aagcagatcg catgatcagc ttataaaagg cggcgtaagc tgtcgctatg 240 ggatggcccc gcggtgcatt agctagttgg tagggtaacg gcctaccaag gcaatgatgc 300 atagccgagt tgagagactg atcggccaca ttgggactga gacacggccc aaactcctac 360 gggaggcagc agtagggaat cttccacaat ggacgaaagt ctgatggagc aacgccgcgt 420 gagtgaagaa ggttttcgga tcgtaaagct ctgttgttgg tgaagaagga tagaggtagt 480 aactggcctt tatttgacgg taatcaacca gaaagtcacg gctaactacg tgccagcagc 540 cgcggtaata c 551 <210> 2 <211> 1629 <212> DNA <213> Lactobacillus acidophilus <220> <223> Lactobacillus acidophilus NCIMB43030 - groEL gene <400> 2 atggcaaaag atattaaatt cgcagaaaac gcaagacgtt ctcttttaaa gggtgttgac 60 aagttagctg ataccgttaa gactactatt ggtcctaagg gtagaaacgt tgttttggaa 120 caaagttacg gcaacccaga cattactaat gatggtgtta ctattgctaa gtcaattgaa 180 ttaaaagacc attacgaaaa catgggtgct aagcttgttg ctgaagctgc acaaaagact 240 aacgacattg ccggtgacgg tactactact gcaactgttt tgactcaagc aattgctcgt 300 gaaggtatga agaacgttac tgctggtgct aaccctgtag gcattcgtcg cggtattgaa 360 aaggcaacta aggctgctgt tgatgaatta cacaagatta gccacaaggt tgaatcaaag 420 gaacaaattg ctaacgtagc tgctgtttct tcagcatcta aagaagttgg tgaattgatc 480 gctgacgcta tggaaaaggt tggtcacgat ggtgttatta ctatcgaaga ttcacgtggt 540 atcaatactg aactttcagt agttgaaggt atgcaattcg atcgtggtta cttgtcacaa 600 tacatggtaa ctgacaacga caagatggaa gcagaccttg ataacccata catcttgatt 660 actgacaaga agatttcaaa tattcaagac atcttgccac ttcttcaaga aatcgttcaa 720 caaggtaagt cattattgat cattgctgac gatgttactg gtgaagctct tccaactctt 780 gttttgaaca agattcgtgg tactttcaac gttgtagctg ttaaggcacc tggttttggt 840 gaccgtcgta aagctcaact tgaagacatt gcagccctta ctggtggtac agtaattacc 900 gatgacttag gctttgaatt aaaggacact aagattgatc aattaggtca agcacgtcgt 960 gtaactgtaa ctaaggactc aactactatt gttgacggtg ctggttcaaa ggacgctatt 1020 aaggaacgcg aagattcaat tagaaaacaa attgaagaat caacttcaga cttcgacaag 1080 aagaagttac aagaacgtct tgcaaaactt actggtggtg tggctgttat ccacgtaggt 1140 gctgctactg aaactgaact taaggaacgt cgttacagaa tcgaagatgc tttgaactca 1200 actcgtgccg ctgttgatga aggctacgtt gccggtggtg gtactgcatt agttgatgtt 1260 gaaaaggcaa ttaaggacct taagggtgaa acatcagatg aacaaactgg tatcaacatt 1320 gttttaagag cattatcagc acctgtacgt caaattgctg aaaacgctgg taaagacggt 1380 gcagttgtat tgaacaagct tgaaagccaa gaaaacgaaa ttggttacaa tgctgcaact 1440 gataagtggg aaaacatggt tgaagctggt atcatcgacc caactaaggt aactcgtact 1500 gctttgcaaa atgctgcttc aattgctgct cttcttctta caactgaagc tgttgttgca 1560 gatattcctg aagataagcc agaagctcct caagcaggtg cagctggtgc tccaggtatg 1620 ggtatgtaa 1629 <210> 3 <211> 1050 <212> DNA <213> Lactobacillus acidophilus <220> <223> Lactobacillus acidophilus NCIMB43030 - pheS gene <400> 3 atggacttat ttgataagtt aaaagagctt catgaagaag gacttaagca aatcagtaaa 60 gctactgatg aaaagacttt gaatgaagta agagtcgaac ttgttggtcg taagggtgaa 120 ttaactaaga ttttgcactc aatgcgtgat gttgcaccag aaaatagacg tgaagtgggt 180 caaaaagtta atgaattgcg tgatttgttt aatgcccaat tagacgaagc aaaggaaaac 240 attgttaaag cagttttagc taaacgactt gaagaagaaa aaattgatgt tactttgcca 300 ggtcgtgaag ggcatttggg ctccaagcac ccaattaata tcatcttgga tgatcttgaa 360 agttatttca ttggtatggg ttacaaagtt gtacaaggtc cagaaattga aacagatcac 420 tatgtctttg aaatgatgaa cttaccaaag gatcacccag ctcgtgatat gcaagctact 480 ttctacatta atgatgaaaa cttgcttaga tctcaaactt caggtgacca agctcgtgtg 540 cttgaaaagc atgacttctc aaagggtcct cttaagatgg ttggtcctgg taaggtatac 600 cgtcgtgatg acgatgatgc gactcactct caccaattta tgcaaatgga agggttagtt 660 attgacaagc acgttactat gagcgattta aagggtactt tggaaatgat cgctaagcac 720 gtatttgggc aagatagagc aacccgttta cgtccaagtt atttcccctt cactgaacca 780 tctgtagaaa tggatgtatc ttgttttaat tgtgatggta aaggttgtcc aatttgtaaa 840 tacactggtt ggattgaagt attaggtgct ggtatggttc accctaatgt tttagaaaat 900 gctggtgttg attcaaacgt ttacggtggt tttgccttcg gtgtaggact tgatcgtttc 960 gcaattttga aatacggcat tgatgatatt cgtgacttct acacaaacga tgttcgtttc 1020 ttagaacaat tccgcaagga ggaaaagtaa 1050 <210> 4 <211> 553 <212> DNA <213> Lactobacillus plantarum <220> <223> Hypervariable regions V1-V3 of the 16S rRNA coding gene of Lactobacillus plantarum NCIMB43029 <400> 4 gtattaccgc ggctgctggc acgtagttag ccgtggcttt ctggttaaat accgtcaata 60 cctgaacagt tactctcaga tatgttcttc tttaacaaca gagttttacg agccgaaacc 120 cttcttcact cacgcggcgt tgctccatca gactttcgtc cattgtggaa gattccctac 180 tgctgcctcc cgtaggagtt tgggccgtgt ctcagtccca atgtggcyga ttaccctctc 240 aggtcggcta cgtatcattg ccatggtgag ccgttacccc accatctagc taatacgccg 300 cgggaccatc caaaagtgat agcygaagcc atctttcaar ctcggaccat gcggtccaag 360 ttgttatgcg gtattagcat ctgtttccag gtgttatccc ccgcttctgg gcaggtttcc 420 cacgtgttac tcaccagttc gccactcact caaatgtaaa tcatgatgca agcaccaatc 480 aataccagag ttcgttcgac ttgcatgtat taggcacgcc gccagcgttc gtcctgagcc 540 aggatcaaac tct 553 <210> 5 <211> 1626 <212> DNA <213> Lactobacillus plantarum <220> <223> Lactobacillus plantarum NCIMB43029 – groEL gene <400> 5 atggctaaag aattaaagtt ctctgaagat gcacgttcag cgatgctaaa aggtgtcgat 60 caattagctg acacagttaa gtcaacgtta ggtcctaagg gtcgcaacgt tgttttggaa 120 caatcatatg gttcaccaac aattactaat gatggtgtaa cgattgctaa ggcgatcgaa 180 ttagacgatc atttcgaaaa catgggtgct aagttagttt ctgaagttgc ttcaaagact 240 aatgacatcg ctggtgatgg gacgactact gcaacggtct taacacaatc aatcgttaat 300 gaaggtatga agaacgttac ggccggtgct aaccctgttg gcattcgtcg tgggattgaa 360 gaagctacta agacggcggt tgactcatta cacgctatgg cacacgaagt taagacgcaa 420 gaagatattg cgcaaatcgc ttctgtatct tcagcaagtg aagaaactgg taaattgatt 480 gccgaagcca tggaaaaagt tggtcatgac ggtgttatca cgattgaaga atcacgtggt 540 gttgatacta gcttagacgt tgttgaaggg atgcaattcg accgcggcta cttatcacaa 600 tacatggtta ctgataatga taagatggaa gcggatcttg acaatccata tatcttaatt 660 actgataaga agatttcaaa cattcaagat atcttaccac tattacaatc catcgttgaa 720 caaggcaagc cattgttgat cattgctgat gacatttctg gtgaagcttt accaacctta 780 gtcttgaaca agatgcgtgg gacgtttaac gttgtcgccg ttaaggcacc cggttttggt 840 gatcggcgta aggaacaatt acaagatatc gctatcttaa ctggcgggac ggttatcact 900 gacgaccttg gccttgaatt gaaggacacg accatcgatc aattaggtca agccaacaaa 960 gttacggtta ctaaggataa caccaccatt gttgaaggcg ctggttccaa ggatgctatc 1020 tcagaacggg ttgaatttat ccgtaaccaa atcggtgaaa caacttctga ctttgacaaa 1080 gaaaagttac aagaacgttt agctaaatta gctggtgggg ttgccgttgt tcgtgtcggt 1140 gccgctactg aaactgaatt gaaggaacgt aaataccgga ttgaagatgc tttgaacgca 1200 actcgggccg ccgttgaaga aggctttgtt gctggtggtg gtactgcttt gattaacgtt 1260 atcaaagatg ttgctgcatt gaaggaaact ggtgacgttc aaactgggat caacattgtt 1320 aaacgtgctt tggaagaacc agttcgccaa atcgctgaaa atgctggttt ggaaggctct 1380 gttatcgttg aaaaaatgaa ggaacaaaag ccaggtgttg gtttcaacgc cgcaactgat 1440 gaatgggttg acatgatcaa agctggtatc gtggacccaa ctaaggtaac gcgttctgct 1500 ttacaaaatg ccgcttctgt ttcagccctt ctcttaacga ctgaagccgt tgtcgctgaa 1560 aaacctgaag aaaatgcacc agctgcacca gccgcaccaa acccaggtat gggcggtatg 1620 atgtaa 1626 <210> 6 <211> 1047 <212> DNA <213> Lactobacillus plantarum <220> <223> Lactobacillus plantarum NCIMB43029 – pheS gene <400> 6 atgagtttac aagatcgatt aaccgaatta cgcgatcaag gcttggccga tattaaatcc 60 gccgatgttt tgaaaaaggt taaccaagtc aaagttgatt tgcttggtaa aaagggtccg 120 attacagaag tattgcgcgg aatgcgcgac ttaagcccgg aagaacggcc aaaggtgggc 180 gcttatgcca acgaagttcg tgaccggatt caggctgcga ttgatgaacg ccgtgaagaa 240 ctggaacaag cagccgttaa tgagcaattg gctgccgaaa aactggacgt gacgttaccg 300 ggtcgggaag ttccacaagg tcagcctcac gtgattaccc agattattac tgaattggaa 360 gatctattta tgggaatggg ctatcaaatt gttgatggtg atgaagttga agaagattac 420 tacaactttg aacggttgaa cttaccgaag gaccatcccg cccgtgacat gcaagacacg 480 ttctatatta ccaaagacgt gctactacgc acgcagacgt ctgctgatca gccgcggtca 540 cttgaaaatc acgatttttc taaaggaccg ctgaaggtct tgtcacctgg ccgcgtttat 600 cggcgtgata cggatgatgc aacccattcc catcaatttc atcaaattga agggttagtc 660 gtggacaagc atattacgat ggctgatttg aagggcacct taattctggt tgccaagact 720 ttgtttggcg atcaattcga tgttcggcta cggccaagct tctttccatt cacggaacca 780 tccgtagaag ctgatgtaac ttgctttaat tgcaatggca agggctgtgc aatctgtaag 840 caaacgggtt ggatcgaagt actgggtgcc ggcatggttc acccccacgt gttagaaatg 900 tctggcattg atccagaaga atatggtggt tttgcctttg ggttaggacc agaccgcttt 960 gcaatgttga aatacggtgt tgacgatatc cgcaacttct acttgaatga cgtgcggttc 1020 ttgtcacagt tctataagaa aggttag 1047 <210> 7 <211> 403 <212> DNA <213> Streptococcus thermophilus <220> <223> Hypervariable regions V1-V2 of the 16S rRNA-encoding gene of Streptococcus thermophilus <400> 7 cggcgttgct cggtcagggt tgcccccatt gccgaagatt ccctactgct gcctcccgta 60 ggagtctggg ccgtgtctca gtcccagtgt ggccgatcac cctctcaggt cggctatgta 120 tcgtcgccta ggtgagccat tacctcacct actagctaat acaacgcagg tccatcttgt 180 agtggagcaa ttgccccttt caaataaatg acatgtgtca tccattgtta tgcggtatta 240 gctatcgttt ccaatagtta tcccccgcta caaggcaggt tacctacgcg ttactcaccc 300 gttcgcaact catccaagaa gagcaagctc ctctcttcag cgttctactt gcatgtatta 360 ggcacgccgc cagcgttcgt cctgagccag gatcaaactc tca 403 <210> 8 <211> 1140 <212> DNA <213> Streptococcus thermophilus <220> <223> Streptococcus thermophilus – recA gene <400> 8 gtggctaaga aaacaaagaa aacagaagaa atcacaaaga agtttggtga tgagcgtcgc 60 aaagcactcg acgatgcatt aaaaaacatt gaaaaagatt ttggtaaggg tgcagttatg 120 cgtcttggtg agcgtgcaga gcaaaaagtt caggttatga gctcaggctc actagctttg 180 gatattgctc ttggtgcggg tggttaccct aaaggtcgta ttattgaaat ttacggacca 240 gaatcatcag gtaaaacaac tgttgccctt catgcagttg ctcagactca aaaagaaggt 300 ggcatcgcag cttttatcga tgccgagcat gctcttgacc ctgcgtatgc agcagctcta 360 ggtgttaata tcgatgagct tcttttgtcg cagcctgatt ctggtgagca aggtctcgaa 420 attgcaggta agctgattga ctctggtgca gtggatttag ttgttgttga ctcagttgcg 480 gccttggtac cacgtgcaga aattgatgga gatattggtg acagtcatgt aggacttcaa 540 gcgcgtatga tgagtcaagc catgcgtaaa ctttctgcat ctattaataa aacaaaaacg 600 attgctatct ttattaacca gttgcgtgaa aaagttggta tcatgtttgg taacccagag 660 actaccccag gtggacgtgc tttaaaattc tatgcatcag tacgtcttga tgtacgtggt 720 aatacacaaa ttaaaggaac cggtgacaaa aaggaccaaa atgttggtaa ggaaaccaag 780 attaaggttg ttaaaaacaa agttgctcca ccatttaaag aagcttttgt tgaaattatg 840 tatggcgaag gaatttcaca aaccggtgaa cttgtaaaaa ttgcaagtga tataggcatt 900 attcagaaag ccggagcttg gttctcatat aatggggaga aaattggtca aggatctgaa 960 aatgctaaaa agtatttagc agatcaccct gagatttttg cagaaatcga tcataaagta 1020 cgcgtacact acggtctgat tgagctagat gaggacgatg ttgttgaaga tacacaagtt 1080 gaagacacgt ctgatgaact cattctagat cttgattcaa ccattgaaat cgaagaataa 1140 <210> 9 <211> 2550 <212> DNA <213> Streptococcus thermophilus <220> <223> Streptococcus thermophilus - secA gene <400> 9 atggcaaata tattacgcaa aatcattgaa aatgataagg gcgaaattaa aaaactagaa 60 aaaactgcca agaaagttga gagctatgct gatgcaatgg cggctctttc agatgaagaa 120 cttcaggcaa aaacagaaga atttaaacaa cgatatcaaa acggagaaag tctagatcag 180 ctcttgccgg aggcatttgc ggttgttcgt gagggagcga aacgtgtcct tggacttttc 240 ccatatcgtg tgcaaattat gggtggtatt gtgcttcact atggtgacgt agcggagatg 300 cgtacagggg aagggaaaac ccttactgcg acaatgcctg tctacttgaa tgctatttca 360 ggtgaaggtg tacacgttat caccgttaac gaataccttt cagagcgtga tgcgactgaa 420 atgggtgagc tttatagttg gctcggtttg tcagtaggga tcaacctttc atctaaatca 480 ccagctgaaa aacgtgaagc ttataattgt gacatcactt actcaaccag ctcagaggta 540 ggtttcgact atcttcgtga taatatggtt gttcgtaagg aaaacatggt acaacgccca 600 ttgaactttg ccttggtcga tgaggttgac tcggttctta ttgatgaagc tcgtacacca 660 ttgatcgtat cagggcctgt aagttcagaa actaatcagt tgtatcaccg tgcggatgct 720 tttgttaaga cattgactga agatgattat gcgattgata ttccaacaaa aacaattggt 780 ttgaatgact caggtattga caaggctgaa gagttcttca acttggaaaa tttgtacgat 840 attgacaatg ttgccttgac tcactatatt gacaatgccc ttcgtgccaa ctacattatg 900 ttgcgtgata ttgactacgt ggtaagtcct gagcaagaaa tccttattgt tgaccaattt 960 actggtcgta ccatggaagg tcgtcgtttt tcagatgggc tccaccaagc cattgaggct 1020 aaagaaggtg taccagtcca agaggaaacc aagacttctg cctcaatcac ttaccaaaat 1080 atgttccgta tgtacaagaa attgtcaggt atgactggta ctggtaagac tgaagaggat 1140 gaattccgtg aaatctataa catgcgagtc atcccaattc caacaaaccg tccaattcaa 1200 cgtattgacc atgatgacct tctgtactca actttggatg ctaaattccg tgctgtggta 1260 caagatgtta agcgtcgtta tgagaagggg caaccagttc ttattggtac ggttgccgtt 1320 gaaacatctg acttgatttc taagatgttg gttgacgcag gtattcctca cgaggtgctt 1380 aatgccaaga accacgaaaa agaagcgcat attatcatga atgcaggtca acgtggtgct 1440 gttacgattg cgaccaatat ggccggtcgt ggtactgaca tcaaacttgg tgaaggtgtt 1500 ctggagcttg gtggactttg tgtcattggt acagagcgtc atgaaagtcg tcgtatcgac 1560 aaccagttgc gtggtcgttc gggacgtcaa ggggatccag gggagtctca attctaccta 1620 tctcttgaag acgaattgat gcgtcgtttc ggttctgacc gtatcaagca tgtcttggaa 1680 cgtttgaacg ctgatgacga agatattgtt atcaaatcac gtatgttgac ccgtcaagtg 1740 gaatcagctc aaaaacgtgt cgaagggaat aactacgata ctcgtaaaca agttcttcag 1800 tacgatgacg ttatgcgtga acagcgtgaa atcatctacg ctgagcgtta tgatgttatt 1860 acagcagaac gtgaccttga acctgaaatc aaggctatga tcaagcgtac tattaaccgt 1920 acagcagaac gtgaccttga acctgaaatc aaggctatga tcaagcgtac tattaaccgt 1920 acggttgatg gacacagtcg taacgatcaa gaagaagctc ttaaaggtat cttgaacttt 1980 acggttgatg gacacagtcg taacgatcaa gaagaagctc ttaaaggtat cttgaacttt 1980 gcacgtcagg ccttggttcc tgaggatgcc atctcgcttg aagatctaaa agaagtggga 2040 gcacgtcagg ccttggttcc tgaggatgcc atctcgcttg aagatctaaa agaagtggga 2040 gaggtgacta aacgtagtgt taattatgat gctatcaagg tttatctgac tgagttagca 2100 gaggtgacta aacgtagtgt taattatgat gctatcaagg tttatctgac tgagttagca 2100 gataatgttt atgatcgtca aattaagaaa ttgcgttcag aggaggctat tcgcgaattc 2160 gataatgttt atgatcgtca aattaagaaa ttgcgttcag aggaggctat tcgcgaattc 2160 caaaaagtct tgattttgat ggttgttgat aataagtgga cagaccacat tgatgccctt 2220 caaaaagtct tgattttgat ggttgttgat aataagtgga cagaccacat tgatgccctt 2220 gatcaattac gtaacgccgt tggtatgcgt ggttatgcgc aaaacaaccc aatcgttgag 2280 gatcaattac gtaacgccgt tggtatgcgt ggttatgcgc aaaacaaccc aatcgttgag 2280 tatcaatctg aaagtttcaa gatgttccaa gatatgattg gtgctattga gtatgatgta 2340 tatcaatctg aaagtttcaa gatgttccaa gatatgattg gtgctattga gtatgatgta 2340 actcgtacga tgatgaaagc acaaatccac gaacaatctc gtgaacatgt taatgaacgt 2400 actcgtacga tgatgaaagc acaaatccac gaacaatctc gtgaacatgt taatgaacgt 2400 gtttcaacaa cagcaacagg caatattcaa gcacatcaag cagatgctaa cggtcaagag 2460 gtttcaacaa cagcaacagg caatattcaa gcacatcaag cagatgctaa cggtcaagag 2460 attgacttta gcaaggttgg tcgtaatgac ttctgtccat gtggctctgg taagaaattc 2520 attgacttta gcaaggttgg tcgtaatgac ttctgtccat gtggctctgg taagaaattc 2520 aaaaattgtc acggtcgtaa acagttttag 2550 aaaaattgtc acggtcgtaa acagttttag 2550 <210> 10 <210> 10 <211> 1438 <211> 1438 <212> DNA <213> Bacillus amyloliquefaciens <220> <223> Bacillus amyloliquefaciens - 16S rRNA coding gene <400> 1 gggggctgct aagctgcaag tcgagcgggc agatgggagc ttgctccctg atgttagcgg 60 cggacgggtg agtaacacgt gggtaacctg cctgtaagac tgggataact ccgggaaacc 120 ggggctaata ccggatggtt gtctgaaccg catggttcag acataaaagg tggcttcggc 180 taccacttac agatggaccc gcggcgcatt agctagttgg tgaggtaacg gctcaccaag 240 gcgacgatgc gtagccgacc tgagagggtg atcggccaca ctgggactga gacacggccc 300 agactcctac gggaggcagc agtagggaat cttccgcaat ggacgaaagt ctgacggagc 360 aacgccgcgt gagtgatgaa ggttttcgga tcgtaaagct ctgttgttag ggaagaacaa 420 gtgccgttca aatagggcgg caccttgacg gtacctaacc agaaagccac ggctaactac 480 gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt ccggaattat tgggcgtaaa 540 gggctcgcag gcggtttctt aagtctgatg tgaaagcccc cggctcaacc ggggagggtc 600 attggaaact ggggaacttg agtgcagaag aggagagtgg aattccacgt gtagcggtga 660 aatgcgtaga gatgtggagg aacaccagtg gcgaaggcga ctctctggtc tgtaactgac 720 gctgaggagc gaaagcgtgg ggagcgaaca ggattagata ccctggtagt ccacgccgta 780 aacgatgagt gctaagtgtt agggggtttc cgccccttag tgctgcagct aacgcattaa 840 gcactccgcc tggggagtac ggtcgcaaga ctgaaactca aaggaattga cgggggcccg 900 cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg aagaacctta ccaggtcttg 960 acatcctctg acaatcctag agataggacg tccccttcgg gggcagagtg acaggtggtg 1020 catggttgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc 1080 cttgatctta gttgccagca ttcagttggg cactctaagg tgactgccgg tgacaaaccg 1140 gaggaaggtg gggatgacgt caaatcatca tgccccttat gacctgggct acacacgtgc 1200 tacaatggac agaacaaagg gcagcgaaac cgcgaggtta agccaatccc acaaatctgt 1260 tctcagttcg gatcgcagtc tgcaactcga ctgcgtgaag ctggaatcgc tagtaatcgc 1320 ggatcagcat gccgcggtga atacgttccc gggccttgta cacaccgccc gtcacaccac 1380 gagagtttgt aacacccgaa gtcggtgagg taacctttag gagccagccg ccgaaggt 1438

Claims

1. A Lactobacillus plantarum strain with the accession number 43029, deposited at the NCIMB Ltd. depository on April 20, 2018 (Lactobacillus plantarum NCIMB 43029), wherein the Lactobacillus plantarum strain comprises the hypervariable regions V1-V3 of the 16S rRNA encoding gene of SEQ ID NO:4, the groEL gene of SEQ ID NO:5, and the pheS gene of SEQ ID NO:

6.

2. A Lactobacillus acidophilus strain with the accession number 43030, deposited at the NCIMB Ltd. depository on April 20, 2018 (Lactobacillus acidophilus NCIMB 43030), wherein the Lactobacillus acidophilus strain comprises the hypervariable regions V1-V3 of the 16S rRNA encoding gene of SEQ ID NO:1, the groEL gene of SEQ ID NO:2, and the pheS gene of SEQ ID NO:

3.

3. Use of the Lactobacillus plantarum strain NCIMB 43029 according to claim 1 and the Lactobacillus acidophilus strain NCIMB 43030 according to claim 2 in the preparation of a medicament for treating wounds or injuries with an antibacterial re-epithelializing agent, wherein the wound or injury is not a wound or injury of the gastrointestinal mucosa.

4. The Lactobacillus plantarum strain NCIMB 43029 according to claim 1, the Lactobacillus acidophilus strain NCIMB 43030 according to claim 2, and a Streptococcus thermophilus strain comprising the hypervariable regions V1-V2 of the 16S rRNA encoding gene of SEQ ID NO:7, the recA gene of SEQ ID NO:8, and the secA gene of SEQ ID NO:9 in the preparation of a medicament for treating skin injuries or wounds by activating or inducing iNOS expression.

5. Use of the Lactobacillus plantarum strain NCIMB 43029 according to claim 1, the Lactobacillus acidophilus strain NCIMB 43030 according to claim 2, and the Streptococcus thermophilus strain according to claim 4 in the preparation of a medicament for treating wounds or injuries with an antibacterial re-epithelializing agent, wherein the wound or injury is not a wound or injury of the gastrointestinal mucosa.

6. A composition having antibacterial and re-epithelializing effects, which comprises Lactobacillus plantarum and Lactobacillus acidophilus, wherein Lactobacillus plantarum is the Lactobacillus plantarum strain NCIMB 43029 according to claim 1 and Lactobacillus acidophilus is the Lactobacillus acidophilus strain NCIMB 43030 according to claim 2, and wherein the Lactobacillus plantarum and the Lactobacillus acidophilus are live bacteria or non-viable bacteria, which are used as a medicament in the re-epithelialization process of an injury or a wound, wherein the injury or wound is not an injury or wound of the gastrointestinal mucosa.

7. The composition according to claim 6, further comprising the Streptococcus thermophilus strain according to claim 4, wherein the weight percentage of the Streptococcus thermophilus strain in the composition ranges from 0.5 wt% to 20 wt%, and wherein the Streptococcus thermophilus strain is viable bacteria or non-viable bacteria.

8. The composition according to claim 6 or 7, which is for oral or topical use.

9. The composition according to claim 6, which is in the form of powder, granules, tablets for gums or vagina, or capsules, or gels.

10. The composition according to claim 7, which is in the form of powder, granules, tablets for gums or vagina, or capsules, or gels.

11. The composition according to claim 8, which is in the form of powder, granules, tablets for gums or vagina, or capsules, or gels.

12. The composition according to claim 6, wherein the weight percentage of Lactobacillus plantarum in the composition ranges from 1 wt% to 40 wt% based on the total weight of the composition.

13. The composition according to claim 7, wherein the weight percentage of Lactobacillus plantarum in the composition ranges from 1 wt% to 40 wt% based on the total weight of the composition.

14. The composition according to claim 8, wherein the weight percentage of Lactobacillus plantarum in the composition ranges from 1 wt% to 40 wt% based on the total weight of the composition.

15. The composition according to claim 9, wherein the weight percentage of Lactobacillus plantarum in the composition ranges from 1 wt% to 40 wt% based on the total weight of the composition.

16. The composition according to claim 10, wherein the weight percentage of Lactobacillus plantarum in the composition ranges from 1 wt% to 40 wt% based on the total weight of the composition.

17. The composition according to claim 11, wherein the weight percentage of Lactobacillus plantarum in the composition ranges from 1 wt% to 40 wt% based on the total weight of the composition.

18. The composition according to claim 6, wherein the weight percentage of Lactobacillus acidophilus in the composition ranges from 1 wt% to 40 wt% based on the total weight of the composition.

19. The composition according to claim 7, wherein the weight percentage of Lactobacillus acidophilus in the composition ranges from 1 wt% to 40 wt% based on the total weight of the composition.

20. The composition according to claim 8, wherein the weight percentage of Lactobacillus acidophilus in the composition ranges from 1 wt% to 40 wt% based on the total weight of the composition.

21. The composition according to claim 9, wherein the weight percentage of Lactobacillus acidophilus in the composition ranges from 1 wt% to 40 wt% based on the total weight of the composition.

22. The composition according to claim 10, wherein the weight percentage of Lactobacillus acidophilus in the composition ranges from 1 wt% to 40 wt% based on the total weight of the composition.

23. The composition according to claim 11, wherein the weight percentage of Lactobacillus acidophilus in the composition is in the range of 1% to 40% by weight of the total weight of the composition.

24. The composition according to claim 6, comprising: 10% to 90% by weight of Lactobacillus plantarum, and 90% to 10% by weight of Lactobacillus acidophilus, both based on the total weight of the composition.

25. The composition according to claim 6, comprising: 20% to 80% by weight of Lactobacillus plantarum, 40% to 10% by weight of Lactobacillus acidophilus, and further comprising 40% to 10% by weight of Streptococcus thermophilus, both based on the total weight of the composition, wherein the Streptococcus thermophilus is the Streptococcus thermophilus strain according to claim 4.

26. The composition according to claim 6, which comprises at least one pharmaceutically acceptable excipient selected from the group consisting of: seaweed, bee pollen, honey, aerated clay, and zeolite.

27. The composition according to claim 7, which comprises at least one pharmaceutically acceptable excipient selected from the group consisting of: seaweed, bee pollen, honey, aerated clay, and zeolite.

28. The composition according to claim 7, which is used as a medicament in the re-epithelialization process of an injury or wound, wherein the injury or wound is not an injury or wound of the gastrointestinal mucosa, and wherein the re-epithelialization process occurs by activating or inducing iNOS expression.

29. The composition according to claim 28, wherein the wound is selected from the group consisting of chronic wounds, acute wounds, burn wounds, post-surgical wounds, traumatic wounds, dermabrasion, diabetic ulcers, and pressure ulcers, and the wound is not a wound of the gastrointestinal mucosa.

30. The composition according to claim 28, which is used in the re-epithelialization process of an injury or wound of the skin, oral cavity, or vaginal mucosa.

31. The composition according to claim 29, which is used in the re-epithelialization process of an injury or wound of the skin, oral cavity, or vaginal mucosa.

32. The composition according to claim 28, wherein when the composition is used in the re-epithelialization process of an injury or wound of the skin, oral cavity, vaginal mucosa, diabetic ulcer, or pressure ulcer, the composition is for topical use in the form of granules or powder, tablets for the gums, or tablets for the vagina.

33. The composition according to claim 29, wherein when the composition is used in the re-epithelialization process of an injury or wound of the skin, oral cavity, vaginal mucosa, diabetic ulcer, or pressure ulcer, the composition is for topical use in the form of granules or powder, tablets for the gums, or tablets for the vagina.

34. The composition according to claim 30, wherein when the composition is used in the re-epithelialization process of an injury or wound of the skin, oral cavity, vaginal mucosa, diabetic ulcer, or pressure ulcer, the composition is for topical use in the form of granules or powder, tablets for the gums, or tablets for the vagina.

35. The composition according to claim 28, wherein the injury or wound presents a biofilm comprising: Enterococcus faecalis, Candida albicans, and Gram-negative bacteria, wherein the Gram-negative bacteria include Klebsiella pneumoniae and Proteus mirabilis.

36. The composition according to claim 29, wherein the injury or wound presents a biofilm comprising: Enterococcus faecalis, Candida albicans, and Gram-negative bacteria, wherein the Gram-negative bacteria include Klebsiella pneumoniae and Proteus mirabilis.

37. The composition according to claim 30, wherein the injury or wound presents a biofilm comprising: Enterococcus faecalis, Candida albicans, and Gram-negative bacteria, wherein the Gram-negative bacteria include Klebsiella pneumoniae and Proteus mirabilis.

38. A bandage comprising the composition according to any one of claims 6 to 27.

39. The bandage according to claim 38, which is used as a medicament.

40. The bandage according to claim 39, which is used as a medicament in the re-epithelialization process of an injury or wound.

41. A kit comprising a sterile compressible container or a single-dose or multi-dose pump, which comprises the composition according to any one of claims 6 to 27 and corresponding package insert.

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