METHODS AND MEANS FOR PROTECTING THE SKIN AGAINST PATHOGENIC MICROORGANISMS
20170202889 · 2017-07-20
Inventors
- Christine Lang (Berlin, DE)
- Andreas Heilmann (Berlin, DE)
- Markus Veen (Berlin, DE)
- Eckhard Budde (Berlin, DE)
- Mewes Boettner (Berlin, DE)
- Andreas Reindl (Mannheim, DE)
- Rolf Knöll (Laudenbach, DE)
Cpc classification
A61K2035/11
HUMAN NECESSITIES
A61P31/00
HUMAN NECESSITIES
A61K9/0014
HUMAN NECESSITIES
A61P17/16
HUMAN NECESSITIES
International classification
A61K9/00
HUMAN NECESSITIES
A61Q17/00
HUMAN NECESSITIES
Abstract
Described are microorganisms which are, in a first aspect, able to stimulate the growth of microorganisms of the resident skin microbial flora and which do not stimulate the growth of microorganisms of the transient pathogenic micro flora. In a second aspect microorganisms are described which are able to inhibit the growth of microorganisms of the transient pathogenic skin micro flora and which do not inhibit the growth of microorganisms of the resident skin micro flora. Also described are compositions comprising such microorganisms as well as the use of such microorganisms in cosmetic, prophylactic or therapeutic applications.
Claims
1. A microorganism which is able to stimulate the growth of one or more microorganisms of the resident skin microbial flora and which does not stimulate the growth of microorganisms of the transient pathogenic micro flora.
2. The microorganism of claim 1, wherein said microorganism: i) is able to stimulate the growth of Staphylococcus epidermidis; ii) is able to stimulate growth of Staphylococcus epidermidis in vitro; iii) is able to stimulate growth of Staphylococcus epidermidis in an in situ skin assay; iv) does not stimulate the growth of Staphylococcus aureus; v) is a microorganism belonging to the genus of Lactobacillus; vi) is Lactobacillus paracasei, Lactobacillus brevis or Lactobacillus fermentum; vii) Lactobacillus paracasei ssp. Paracasei; or viii) is selected from the group consisting of Lactobacillus paracasei, Lactobacillus brevis or Lactobacillus fermentum having DSMZ accession number DSM 17248, accession number DSM 17247, accession number DSM 17250 and accession number DSM 17249 or a mutant or derivative thereof, wherein said mutant or derivative retains the ability to stimulate the growth of at least one microorganism of the resident skin microbial flora and does not stimulate the growth of microorganisms of the transient pathogenic micro flora.
3-9. (canceled)
10. An inactive form of the microorganism of claim 1, which is able to stimulate the growth of one or more microorganisms of the resident skin microbial flora but which does not stimulate the growth of microorganisms of the transient pathogenic micro flora.
11. The inactive form of claim 10, which is thermally inactivated or lyophilized
12. A composition comprising the microorganism of claim 1 or an inactive form of the microorganism of claim 1 which is able to stimulate the growth of one or more microorganisms of the resident skin microbial flora but which does not stimulate the growth of microorganisms of the transient pathogenic micro flora.
13. The composition of claim 12, wherein said composition is: i) a cosmetic composition optionally comprising a cosmetically acceptable carrier or excipient; or ii) a pharmaceutical composition optionally comprising a pharmaceutically acceptable carrier or excipient.
14. (canceled)
15. A method for the preparation of a cosmetic or pharmaceutical composition for protecting skin against pathogenic bacteria comprising preparing a cosmetic or pharmaceutical composition which comprises the microorganism of claim 1 or an inactive form of the microorganism of claim 1 which is able to stimulate the growth of one or more microorganisms of the resident skin microbial flora but which does not stimulate the growth of microorganisms of the transient pathogenic micro flora.
16. A method for the prophylaxis or treatment of dermatitis comprising preparing a pharmaceutical composition which comprises the microorganism of claim 1 or an inactive form of the microorganism of claim 1 which is able to stimulate the growth of one or more microorganisms of the resident skin microbial flora but which does not stimulate the growth of microorganisms of the transient pathogenic micro flora, and utilizing the composition for the prophylaxis or treatment of dermatitis.
17. The method of claim 16, wherein the dermatitis is atopic dermatitis, psoriasis, poison-ivy dermatitis, eczema herpeticum, kerion or scabies.
18. A method for the production of a composition comprising the step of formulating the microorganism according to claim 1 or an inactive form of the microorganism of claim 1, which is able to stimulate the growth of one or more microorganisms of the resident skin microbial flora but which does not stimulate the growth of microorganisms of the transient pathogenic micro flora, with a cosmetically or pharmaceutically acceptable carrier or excipient.
19. A microorganism which is able to inhibit the growth of one or more microorganisms of the transient pathogenic skin micro flora and which does not inhibit the growth of microorganisms of the healthy normal resident skin micro flora.
20. The microorganism of claim 19, wherein said microorganism: i) is able to inhibit the growth of Staphylococcus aureus; ii) is able to inhibit the growth of Staphylococcus aureus in vitro; iii) is able to inhibit the growth of Staphylococcus aureus in an in vitro liquid assay; iv) is able to inhibit the growth of Staphylococcus aureus in an in situ skin assay; v) does not inhibit the growth of Staphylococcus epidermidis; vi) is a microorganism belonging to the genus of Lactobacillus; vii) Lactobacillus buchneri, or Lactobacillus delbrckii; viii) Lactobacillus delbrckii ssp. Delbrckii; ix) is selected from the group consisting of Lactobacillus buchneri and Lactobacillus delbrckii ssp. delbrckii having DSMZ accession number DSM 18007, and accession number DSM 18006 or a mutant or derivative thereof, wherein said mutant or derivative retains the ability to inhibit the growth of one or more microorganisms of the transient pathogenic skin micro flora and which does not inhibit the growth of microorganisms of the healthy normal resident skin micro flora.
21-28. (canceled)
29. An inactive form of the microorganism of claim 19, which is able to inhibit the growth of one or more microorganisms of the transient pathogenic skin micro flora and which does not inhibit the growth of microorganisms of the healthy normal resident skin micro flora.
30. The inactive form of claim 29, which is thermally inactivated or lyophilized.
31. A composition comprising the microorganism of claim 19 or an inactive form of the microorganism of claim 19 which is able to inhibit the growth of one or more microorganisms of the transient pathogenic skin micro flora and which does not inhibit the growth of microorganisms of the healthy normal resident skin micro flora.
32. The composition of claim 31, wherein said composition is: i) a cosmetic composition optionally comprising a cosmetically acceptable carrier or excipient; or ii) a pharmaceutical composition optionally comprising a pharmaceutically acceptable cattier or excipient.
33. (canceled)
34. A method for protecting skin against pathogenic bacteria comprising preparing a cosmetic or pharmaceutical composition which comprises the microorganism of claim 19 or an inactive form of the microorganism of claim 19 which is able to stimulate the growth of one or more microorganisms of the resident skin microbial flora but which does not stimulate the growth of microorganisms of the transient pathogenic micro flora, and utilizing the composition for protecting skin against pathogenic bacteria.
35. A method for the prophylaxis or treatment of dermatitis comprising preparing a pharmaceutical composition which comprises the microorganism of claim 19 or an inactive form of the microorganism of claim 19 which is able to stimulate the growth of one or more microorganisms of the resident skin microbial flora but which does not stimulate the growth of microorganisms of the transient pathogenic micro flora, and utilizing the composition for the prophylaxis or treatment of dermatitis
36. The method of claim 35, wherein the dermatitis is atopic dermatitis, psoriasis, poison-ivy dermatitis, eczema herpeticum, kerion or scabies.
37. A method for the production of a composition comprising the step of formulating the microorganism according to claim 19 or an inactive form of the microorganism of claim 19, which is able to stimulate the growth of one or more microorganisms of the resident skin microbial flora but which does not stimulate the growth of microorganisms of the transient pathogenic micro flora, with a cosmetically or pharmaceutically acceptable carrier or excipient.
Description
[0503]
[0504]
[0505]
[0506]
[0507] The second aspect of the invention is illustrated by
[0508]
[0509]
[0510]
[0511]
[0512]
[0513]
[0514]
[0515] The first aspect of the invention is illustrated by the following Examples 1 to 4:
EXAMPLE 1
Growth Stimulation of S. epidermidis in an In-Vitro-Hole Plate Assay
[0516] Specific lactic acid bacteria have been identified that are able to stimulate the growth of Staphylococcus epidermidis on agar plates in an in-vitro-hole plate assay. These lactic acid bacteria are described herein. To test this effect, precultured lactic acid bacteria have been filled into pre-cutted holes and a growth stimulation of the Indicator strain S. epidermidis has been observed. To advance the visual effect of growth stimulation Tellurite has been used. Tellurite specifically stains staphylococci. Stimulance was defined as the formation of a black ring around the hole the lactic acid bacterium was pipetted in and an increase of the colony count. Data are shown in
Cultivation and Preparation of Lactobacilli:
[0517] Lactic acid bacteria were cultivated from a 80 C. freezing culture in 1 ml MRS broth in Eppendorf tubes. The tubes were closed and cultivated for 2 days at 37 C. 10 l of this preculture were transferred to the main culture consisting of 7 ml MRS broth in Falcon tubes. The culture was incubated for two days. After cultivation cells were harvested by centrifugation (15 min, 4000g). The cell pellet was washed two times with K/Na-buffer (1 ml each). The cells were resuspended in 200 l K/Na buffer.
Cultivation and Preparation of the Indicator Strain:
[0518] The indicator strain was Staphylococcus epidermidis (DSM20044). 20 ml BHI broth in a shaking glass flask were inoculated with 15 l of a 24 h preculture. The indicator strain was cultivated for 24 h at 37 C. An aliquot was diluted to an optical density OD.sub.595 nm of 0.025-0.05 in BHI-broth and 800 l were spread on indicator plates (BHI/Tellurite). The agar was stamped using a cork borer. The holes were filled with the pre cultured lactic acid bacteria.
Media and Buffer:
[0519]
TABLE-US-00001 BHI-Agar Difco Agar 1.8%; 20 ml per plate BHI-Medium Difco BHI/Tellurite-Agar like BHI-Agar, after cooling to 50 C. 1 ml of a sterile filtered 1% potassium-Tellurite solution are transferred to 100 ml BHI-Medium; 20 ml per plate MRS-broth Difco, 150 l/well K/Na-buffer Kster Thiel, pH 7.0, autoclaved 0.066M Na.sub.2HPO.sub.4 2H.sub.2O 61.2 ml 0.066M KH.sub.2PO.sub.4 38.8 ml
EXAMPLE 2
Growth Stimulation of Staphylococcus epidermidis in an In-Situ-Skin Assay
[0520] Probiotic lactic acid bacteria have been identified that are able to stimulate the growth of Staphylococcus epidermidis directly on the skin.
[0521] A culture of Staphylococcus epidermidis was diluted and directly applied to the skin and air dried. Afterwards an aliquot of the lactic acid bacterium was applied punctual on this skin area. The indicator strain Staphylococcus epidermidis can be stimulated directly on the skin by the lactic acid bacterium. After incubation the staphylococci were transferred from the skin to an agar plate using an adhesive tape. The agar plate was incubated at 37 C. An increased colony count indicates a growth stimulation of the indicator strain on the skin (
Cultivation and Preparation of Lactobacilli:
[0522] Lactic acid bacteria were cultivated from a 80 C. freezing culture in 1 ml MRS broth in Eppendorf tubes. The tubes were closed and cultivated for 2 days at 37 C. 10 l of this preculture were transferred to the main culture consisting of 7 ml MRS broth in Falcon tubes. The culture was incubated for two days. After cultivation cells were harvested by centrifugation (15 min, 4000g). The cell pellet was washed two times with K/Na-buffer (1 ml each). The cells were resuspended in 200 l K/Na buffer.
Cultivation and Preparation of the Indicator Strain:
[0523] The indicator strain was Staphylococcus epidermidis (DSM20044). 20 ml BHI broth in a shaking glass flask were inoculated with 15 l of a 24 h preculture. The indicator strain was cultivated for 24 h at 37 C. An aliquot was diluted to an optical density OD.sub.595 nm of 0.025-0.05 in BHI-broth. This solution was diluted again (1:100).
Media and Buffer:
[0524]
TABLE-US-00002 BHI-Agar Difco Agar 1.8%; 20 ml per plate BHI-Medium Difco MRS-broth Difco, 150 l/well K/Na-buffer Kster Thiel, pH 7.0, autoclaved 0.066M Na.sub.2HPO.sub.4 2H.sub.2O 61.2 ml 0.066M KH.sub.2PO.sub.4 38.8 ml
Application of S. epidermidis on the Forearm:
[0525] 400 l of a 1:100 dilution of the prepared indicator strain Staphylococcus epidermidis was spread evenly on a defined skin area (10 cm3 cm) and air dried.
Application of Lactobacilli on the S. epidermidis Inoculated Skin Area:
[0526] 10 l of prepared lactobacilli were punctually applied to the S. epidermidis pre-inoculated skin area. The arm was incubated for two hours in a normal environment.
Reisolation of Microorganisms from the Skin:
[0527] After 2 h the four upper skin layers were transferred to a BHI-agar plate using adhesive tape stripes. By this the isolated skin bacteria were transferred to the agar plate. The agar plates were incubated for 24 h at 37 C.
EXAMPLE 3
No Growth Stimulation of Staphylococcus aureus in an In-Situ-Skin Assay
[0528] Using this assay it is possible to check whether unwanted bacteria of the transient, pathogenic microbial flora are not stimulated by lactic acid bacteria that are able to stimulate bacteria of the protecting resident skin microbial flora.
[0529] For this purpose the indicator strain Staphylococcus aureus was highly diluted and applied to the skin in the same manner as Staphylococcus epidermidis (see Example 2). Again the stimulating activity of lactic acid bacteria was tested. A stimulation of Staphylococcus aureus by the described lactic acid bacteria could not be observed. The lactobacilli strains of the present invention, in particular those deposited with the DSMZ, did not show stimulation of Staphylococcus aureus. Data are presented in
Cultivation and Preparation of Lactobacilli:
[0530] Lactic acid bacteria were cultivated from a 80 C. freezing culture in 1 ml MRS broth in Eppendorf tubes. The tubes were closed and cultivated for 2 days at 37 C. 10 l of this preculture were transferred to the main culture consisting of 7 ml MRS broth in Falcon tubes. The culture was incubated for two days. After cultivation cells were harvested by centrifugation (15 min, 4000g). The cell pellet was washed two times with K/Na-buffer (1 ml each). The cells were resuspended in 200 l K/Na buffer.
Cultivation and Preparation of the Indicator Strain:
[0531] The indicator strain was Staphylococcus aureus (DSM346). 20 ml BHI broth in a shaking glass flask were inoculated with 15 l of a 24 h preculture. The indicator strain was cultivated for 24 h at 37 C. An aliquot was diluted to an optical density OD.sub.595 nm of 0.025-0.05 in BHI-broth. This solution was diluted again (1:100).
Media and Buffer:
[0532]
TABLE-US-00003 BHI-Agar Difco Agar 1.8%; 20 ml per plate BHI-Medium Difco MRS-broth Difco, 150 l/well K/Na-buffer Kster Thiel, pH 7.0, autoclaved 0.066M Na.sub.2HPO.sub.4 2H.sub.2O 61.2 ml 0.066M KH.sub.2PO.sub.4 38.8 ml
Application of Staphylococcus aureus on the Forearm:
[0533] 400 l of a 1:100 dilution of the prepared indicator strain Staphylococcus aureus was spread evenly on a defined skin area (10 cm3 cm) and air dried.
Application of Lactobacilli on the S. aureus Inoculated Skin Area:
[0534] 10 l of prepared lactobacilli were punctually applied to the S. aureus pre-inoculated skin area. The arm was incubated for two hours in a normal environment.
Reisolation of Microorganisms from the Skin:
[0535] After 2 h the four upper skin layers were transferred to a BHI-agar plate using adhesive tape stripes. By this the isolated skin bacteria were transferred to the agar plate. The agar plates were incubated for 24 h at 37 C. The data are shown in
EXAMPLE 4
No Growth Stimulation of S. aureus in an In-Vitro-Hole Plate Assay
[0536] Specific lactic acid bacteria have been identified that are able to stimulate the growth of Staphylococcus epidermidis on agar plates in an in-vitro-hole plate assay but not the representative of the transient microbial skin flora Staphylococcus aureus. To test this effect, precultured lactic acid bacteria that are able to stimulate Staphylococcus epidermidis have been filled into pre-cutted holes and absence of growth stimulation of the indicator strain S. aureus has been observed. To advance the visual effect of growth stimulation tellurite has been used. Tellurite specifically stains staphylococci. Stimulance was defined as the formation of a black ring around the hole containing the lactic acid bacterium and an increase of the colony count. The lactobacilli strains of the present invention, in particular those deposited with the DSMZ did not show stimulation of Staphylococcus aureus. Data are shown in
Cultivation and Preparation of Lactobacilli:
[0537] Lactic acid bacteria were cultivated from a 80 C. freezing culture in 1 ml MRS broth in Eppendorf tubes. The tubes were closed and cultivated for 2 days at 37 C. 10 l of this preculture were transferred to the main culture consisting of 7 ml MRS broth in Falcon tubes. The culture was incubated for two days. After cultivation cells were harvested by centrifugation (15 min, 4000g). The cell pellet was washed two times with K/Na-buffer (1 ml each). Cells were resuspended in 200 l K/Na buffer.
Cultivation and Preparation of the Indicator Strain:
[0538] The indicator strain was Staphylococcus aureus (DSM346). 20 ml BHI broth in a shaking glass flask were inoculated with 15 l of a 24 h preculture. The indicator strain was cultivated for 24 h at 37 C. An aliquot was diluted to an optical density OD.sub.595 nm of 0.025-0.05 in BHI-broth and 800 l were spread on indicator plates (BHI/Tellurite). The agar was stamped using a cork borer. The holes were filled with the pre cultured lactic acid bacteria.
Media and Buffer:
[0539]
TABLE-US-00004 BHI-Agar Difco Agar 1.8%; 20 ml per plate BHI-Medium Difco BHI/Tellurite-Agar like BHI-Agar, after cooling to 50 C. 1 ml of a filter sterilized 1% potassium-Tellurite solution are transferred to 100 ml BHI-Medium; 20 ml are distributed per plate MRS-broth Difco, 150 l/well K/Na-buffer Kster Thiel, pH 7.0, autoclaved 0.066M Na.sub.2HPO.sub.4 2H.sub.2O 61.2 ml 0.066M KH.sub.2PO.sub.4 38.8 ml
[0540] The second aspect of the invention is illustrated by the following Examples 5 to 13:
EXAMPLE 5
Growth Inhibition of S. aureus in an In Vitro Hole Plate Assay
[0541] Specific lactic acid bacteria have been identified, that are able to specifically inhibit the growth of Staphylococcus aureus on agar plates in an in vitro hole plate assay. To test this effect, pre cultured lactic acid bacteria have been filled into pre-cutted holes and a growth inhibition of the indicator strain S. aureus has been observed. Data are shown in
Cultivation and Preparation of Lactobacilli:
[0542] Lactic acid bacteria were cultivated (OB-LB-Sa3; DSM 18006) from a 80 C. freezing culture in 1 ml MRS broth in eppendorf tubes. Tubes were closed and cultivated for 2 days at 37 C. 10 l of this pre culture was transferred to the main culture consisting of 7 ml MRS broth in falcon tubes. The culture was incubated for 2 days. After cultivation cells were harvested by centrifugation (15 min, 4000g). The cell pellet was washed two times with K/Na-buffer (each 1 ml). Cells were resuspended in 200 l K/Na buffer.
Cultivation and Preparation of the Indicator Strain:
[0543] The indicator strain was Staphylococcus aureus (DSM346). 20 ml BHI broth in a shaking glass flask were inoculated with 15 l of a 24 h pre culture. The indicator strain was cultivated for 24 h at 37 C. An aliquot was diluted to an optical density OD.sub.595 nm of 0.025-0.05 in BHI-broth and 800 l spread on indicator plates (BHI). The agar was stamped using a cork borer. The holes were filled with 5 l or 10 l of the pre cultured lactic acid bacteria.
Media and Buffer:
[0544]
TABLE-US-00005 BHI-Agar Difco Agar 1.8%; 20 ml per plate BHI-Medium Difco MRS-broth Difco K/Na-buffer according to Kster Thiel, pH 7.0, autoclaved 0.066M Na.sub.2HPO.sub.4 2H.sub.2O 61.2 ml 0.066M KH.sub.2PO.sub.4 38.8 ml
EXAMPLE 6
Growth Inhibition of S. aureus in an In Vitro Liquid Assay
[0545] Specific lactic acid bacteria have been identified, that are able to specifically inhibit the growth of Staphylococcus aureus in liquid medium in an in vitro liquid assay. To test this effect, pre cultured lactic acid bacteria have been co-incubated with the indicator strain S. aureus in liquid cultivation medium, optimized for the growth of Staphylococci. The degree of inhibition was quantified by counting the colony forming units of the indicator strain in comparison to the control without lactic acid bacteria. Data are shown in
Cultivation and Preparation of Lactobacilli:
[0546] Lactic acid bacteria were cultivated (OB-LB-Sa3; DSM 18006) from a 80 C. freezing culture in 1 ml MRS broth in eppendorf tubes. Tubes was closed and cultivated for 2 days at 37 C. 10 l of this pre culture was transferred to the main culture consisting of 7 ml MRS broth in falcon tubes. The culture was incubated for 2 days. After cultivation cells were harvested by centrifugation (15 min, 4000g). The cell pellet was washed two times with K/Na-buffer (each 1 ml). Cells were resuspended in 200 l K/Na buffer with 250 mM glycerol and incubated for 17 h.
Cultivation and Preparation of the Indicator Strain:
[0547] The indicator strain was Staphylococcus aureus (DSM346). 10 ml BHI broth in a shaking glass flask were inoculated with 15 l of a freezing culture for a 24 h pre culture. The culture was diluted with fresh BHI broth to a cell concentration of 2.510.sup.8 cells/ml.
Liquid Inhibition Assay
[0548] For the liquid assay 5 l of the freshly prepared lactic acid bacteria (out of 200 l) and 10 l of the pre cultured indicator strain S. aureus were inoculated for a co-cultivation in 10 ml of BHI broth. The culture was incubated for 7 h. Afterwards 100 l of a 1:10000 dilution was spread on a BHI agar plate for quantification of the colony forming units. The plate was incubated for 24 h hours and the colony forming units were counted.
Media and Buffer:
[0549]
TABLE-US-00006 BHI-Agar Difco Agar 1.8%; 20 ml per plate BHI-Medium Difco MRS-broth Difco K/Na-buffer according to Kster Thiel, pH 7.0, autoclaved 0.066M Na.sub.2HPO.sub.4 2H.sub.2O 61.2 ml 0.066M KH.sub.2PO.sub.4 38.8 ml
EXAMPLE 7
No Growth Inhibition of Staphylococcus epidermidis an In Vitro Liquid Assay
[0550] Using this assay it was possible to check whether selected lactic acid bacteria that were able to inhibit the growth of the pathogenic microorganism Staphylococcus aureus did not inhibit the major member of the commensal micro flora of the skin, Staphylococcus epidermidis in an in vitro liquid assay.
[0551] To test this effect, pre cultured lactic acid bacteria have been co-incubated with the indicator strain in a liquid culture. The degree of inhibition was quantified by counting the colony forming units of both indicator strains in comparison to the control without lactic acid bacteria. Data are shown in
Cultivation and Preparation of Lactobacilli:
[0552] Lactic acid bacteria were cultivated (OB-LB-Sa3; DSM 18006) from a 80 C. freezing culture in 1 ml MRS broth in eppendorf tubes. Tubes were closed and cultivated for 2 days at 37 C. 10 l of this pre culture was transferred to the main culture consisting of 7 ml MRS broth in falcon tubes. The culture was incubated for 2 days. After cultivation cells were harvested by centrifugation (15 min, 4000g). The cell pellet was washed two times with K/Na-buffer (each 1 ml). Cells were resuspended in 200 l K/Na buffer with 250 mM glycerol and incubated for 17 h.
Cultivation and Preparation of the Indicator Strain:
[0553] The indicator strain was Staphylococcus epidermidis (DSM20044). 20 ml BHI broth in a shaking glass flask was inoculated with 15 l of a freezing culture for a 24 h pre culture.
Liquid Inhibition Assay
[0554] For the liquid assay 5 l of the freshly prepared lactic acid bacteria (out of 200 l) and 10 l of the pre cultured indicator strain S. epidermidis were inoculated for a co-cultivation in 10 ml of BHI broth. The culture was incubated for 7 h. Afterwards 100 l of a 1:10000 dilution was spread on a BHI agar plate for quantification of the colony forming units. The plate was incubated for 24 h hours and the colony forming units were counted.
Media and Buffer:
[0555]
TABLE-US-00007 BHI-Agar Difco Agar 1.8%; 20 ml per plate BHI-Medium Difco MRS-broth Difco K/Na-buffer according to Kster Thiel, pH 7.0, autoclaved 0.066M Na.sub.2HPO.sub.4 2H.sub.2O 61.2 ml 0.066M KH.sub.2PO.sub.4 38.8 ml
EXAMPLE 8
Growth Inhibition of Staphylococcus aureus in an In Situ Skin Assay
[0556] Lactic acid bacteria have been identified that are able to inhibit the growth of S. aureus directly on the skin.
[0557] To test this effect, a culture of Staphylococcus aureus was diluted and directly applied to the skin and air dried. Afterwards an aliquot of the lactic acid bacterium was applied on this skin area. Thus the indicator strain Staphylococcus aureus was inhibited directly on the skin by the lactic acid bacterium. After incubation the staphylococci were transferred from the skin to an agar plate using in an adhesive tape. The agar plate was incubated at 37 C. A decreased colony count in comparison to the control without lactic acid bacteria indicates a growth inhibition of the indicator strain on the skin.
Cultivation and Preparation of Lactobacilli:
[0558] Lactic acid bacteria were cultivated (OB-LB-Sa3; DSM 18006) from a 80 C. freezing culture in 1 ml MRS broth in eppendorf tubes. Tubes were closed and cultivated for 2 days at 37 C. 10 l of this pre culture were transferred to the main culture consisting of 7 ml MRS broth in falcon tubes. The culture was incubated for 2 days. After cultivation cells were harvested by centrifugation (15 min, 4000g). The cell pellet was washed two times with K/Na-buffer (each 1 ml). Cells are resuspended in 200 l K/Na buffer.
Cultivation and Preparation of the Indicator Strain:
[0559] The indicator strain was Staphylococcus aureus (DSM346). 20 ml BHI broth in a shaking glass flask were inoculated with 15 l of a 24 h pre culture. The indicator strain was cultivated for 24 h at 37 C.
Media and Buffer:
[0560]
TABLE-US-00008 BHI-Agar Difco Agar 1.8%; 20 ml per plate BHI-Medium Difco MRS-broth Difco K/Na-buffer Kster Thiel, pH 7.0, autoclaved 0.066M Na.sub.2HPO.sub.4 2H.sub.2O 61.2 ml 0.066M KH.sub.2PO.sub.4 38.8 ml
Application of S. aureus on the Forearm:
[0561] 400 l of an 1:100 dilution of the prepared indicator strain Staphylococcus aureus was spread consistently on a defined skin area (10 cm3 cm) and air dried.
Application of Lactobacilli on the S. aureus Inoculated Skin Area:
[0562] 10 l of prepared lactobacilli was applied to the S. aureus pre-inoculated skin area. The arm was incubated for six hours in a normal environment.
Reisolation of Microorganisms from the Skin:
[0563] After 6 h the four upper skin layers were transferred to a BHI-agar plate using adhesive tape stripes. Thus the isolated skin bacteria were transferred to the agar plate. Agar plates were incubated for 24 h at 37 C.
EXAMPLE 9
No Growth Inhibition of Staphylococcus epidermidis in an In Situ Skin Assay
[0564] Lactic acid bacteria have been identified that inhibit the growth of Staphylococcus aureus, while the growth of Staphylococcus epidermidis is not affected directly on the skin.
[0565] Using this assay it was possible to check if the commensal microorganism Staphylococcus epidermidis of the healthy normal skin flora was not inhibited by lactic acid bacteria that are able to inhibit Staphylococcus aureus.
[0566] Therefore the indicator strain Staphylococcus epidermidis was applied highly diluted to the skin in the same manner as Staphylococcus aureus. Again the inhibiting activity of lactic acid bacteria was tested. An inhibition of Staphylococcus epidermidis has not been observed with the described lactic acid bacteria.
Cultivation and Preparation of Lactobacilli:
[0567] Lactic acid bacteria were cultivated (OB-LB-Sa3; DSM 18006) from a 80 C. freezing culture in 1 ml MRS broth in eppendorf tubes. Tubes were closed and cultivated for 2 days at 37 C. 10 l of this pre culture was transferred to the main culture consisting of 7 ml MRS broth in falcon tubes. The culture was incubated for 2 days. After cultivation cells were harvested by centrifugation (15 min, 4000g). The cell pellet was washed two times with K/Na-buffer (each 1 ml). Cells were resuspended in 200 l K/Na buffer.
Cultivation and Preparation of the Indicator Strain:
[0568] The indicator strain was Staphylococcus epidermidis (DSM20044). 20 ml BHI broth in a shaking glass flask were inoculated with 15 l of a 24 h pre culture. The indicator strain was cultivated for 24 h at 37 C.
Media and Buffer:
[0569]
TABLE-US-00009 BHI-Agar Difco Agar 1.8%; 20 ml per plate BHI-Medium Difco MRS-broth Difco K/Na-buffer Kster Thiel, pH 7.0, autoclaved 0.066M Na.sub.2HPO.sub.4 2H.sub.2O 61.2 ml 0.066M KH.sub.2PO.sub.4 38.8 ml
Application of Staphylococcus epidermidis on the Forearm:
[0570] 400 l of a 1:100 dilution of the prepared indicator strain Staphylococcus epidermidis was spread consistently on a defined skin area (10 cm3 cm) and air dried.
Application of Lactobacilli on the S. epidermidis Inoculated Skin Area:
[0571] 10 l of prepared lactobacilli were applied to the S. epidermidis pre-inoculated skin area. The arm was incubated for six hours in a normal environment.
Reisolation of Microorganisms from the Skin:
[0572] After 6 h the four upper skin layers was transferred to a BHI-agar plate using adhesive tape stripes. Thus the isolated skin bacteria are transferred to the agar plate. Agar plates are incubated for 24 h at 37 C.
EXAMPLE 10
No Growth Inhibition of Micrococcus luteus in the In-Vitro-Liquid Assay
[0573] The selected lactic acid bacteria that are able to inhibit the growth of the pathogenic microorganism Staphylococcus aureus do not inhibit the relevant member of the commensal micro flora of the skin, Micrococcus luteus in an in vitro liquid assay. To test this effect, pre cultured lactic acid bacteria have been co-incubated with the indicator strain in a liquid culture. The degree of inhibition was quantified by counting the colony forming units of both indicator strains in comparison to the control without lactic acid bacteria. Data are shown in
Cultivation and Preparation of Lactobacilli:
[0574] Lactic acid bacteria were cultivated (OB-LB-Sa3; DSM 18006 and OB-LB-Sa16; DSM 18007) from a 80 C. freezing culture in 1 ml MRS broth in eppendorf tubes. Tubes were closed and cultivated for 2 days at 37 C. 10 l of this pre culture was transferred to the main culture consisting of 7 ml MRS broth in falcon tubes. The culture was incubated for 2 days. After cultivation cells were harvested by centrifugation (15 min, 4000g). The cell pellet was washed two times with K/Na-buffer (each 1 ml). Cells were resuspended in 200 l K/Na buffer with 250 mM glycerol and incubated for 17 h.
Cultivation and Preparation of the Indicator Strain:
[0575] The indicator strain was Micrococcus luteus. 20 ml BHI broth in a shaking glass flask was inoculated with 15 l of a freezing culture for a 24 h pre culture.
Liquid Inhibition Assay:
[0576] For the liquid assay 5 l of the freshly prepared lactic acid bacteria (out of 200 l) and 10 l of the pre cultured indicator strain M. luteus were inoculated for a co-cultivation in 10 ml of BHI broth. The culture was incubated for 7 h. Afterwards 100 l of a 1:1000 dilution was spread on a BHI agar plate for quantification of the colony forming units. The plate was incubated for 24 h and the colony forming units were counted.
Media and Buffer:
[0577]
TABLE-US-00010 BHI-Agar Difco Agar 1.8%; 20 ml per plate BHI-Medium Difco MRS-broth Difco K/Na-buffer according to Kster Thiel, pH 7.0, autoclaved 0.066M Na.sub.2HPO.sub.4 2H.sub.2O 61.2 ml 0.066M KH.sub.2PO.sub.4 38.8 ml
EXAMPLE 11
No Growth Inhibition of Escherichia coli in the In-Vitro-Liquid Assay
[0578] The selected lactic acid bacteria that are able to inhibit the growth of the pathogenic microorganism Staphylococcus aureus do not inhibit other human relevant microorganisms, e.g Escherichia coli in an in vitro liquid assay.
[0579] To test this effect, pre cultured lactic acid bacteria have been co-incubated with the indicator strain in liquid culture. The degree of inhibition was quantified by counting the colony forming units of both indicator strains in comparison to the control without lactic acid bacteria. Data are shown in
Cultivation and Preparation of Lactobacilli:
[0580] Lactic acid bacteria were cultivated (OB-LB-Sa3; DSM 18006 and OB-LB-Sa16; DSM 18007) from a 80 C. freezing culture in 1 ml MRS broth in eppendorf tubes. Tubes were closed and cultivated for 2 days at 37 C. 10 l of this pre culture was transferred to the main culture consisting of 7 ml MRS broth in falcon tubes. The culture was incubated for 2 days. After cultivation cells were harvested by centrifugation (15 min, 4000g). The cell pellet was washed two times with K/Na-buffer (each 1 ml). Cells were resuspended in 200 l K/Na buffer with 250 mM glycerol and incubated for 17 h.
Cultivation and Preparation of the Indicator Strain:
[0581] The indicator strain was Escherichia coli. 20 ml BHI broth in a shaking glass flask was inoculated with 15 l of a freezing culture for a 24 h pre culture.
Liquid Inhibition Assay:
[0582] For the liquid assay 5 l of the freshly prepared lactic acid bacteria (out of 200 l) and 10 l of the pre cultured indicator strain E. coli were inoculated for a co-cultivation in 10 ml of BHI broth. The culture was incubated for 7 h. Afterwards 100 l of a 1:1000 dilution was spread on a BHI agar plate for quantification of the colony forming units. The plate was incubated for 24 h and the colony forming units were counted.
Media and Buffer:
[0583]
TABLE-US-00011 BHI-Agar Difco Agar 1.8%; 20 ml per plate BHI-Medium Difco MRS-broth Difco K/Na-buffer according to Kster Thiel, pH 7.0, autoclaved 0.066M Na.sub.2HPO.sub.4 2H.sub.2O 61.2 ml 0.066M KH.sub.2PO.sub.4 38.8 ml
EXAMPLE 12
Degree of Growth Inhibition of S. aureus in an In-Vitro-Hole Plate Assay in Comparison to Bacitracin and Erythromycin
[0584] Specific lactic acid bacteria have been identified, that are able to specifically inhibit the growth of Staphylococcus aureus on agar plates in an in-vitro-hole plate assay. This effect has been compared to commercial antibiotic cream preparations of bacitracin and erythromycin. To compare this effect, both antibiotics have been filled into pre-cutted holes at different concentrations and a growth inhibition of the indicator strain S. aureus has been observed (calibration curves in
Cultivation and Preparation of Lactobacilli:
[0585] Lactic acid bacteria were cultivated (OB-LB-Sa3; DSM 18006) from a 80 C. freezing culture in 1 ml MRS broth in eppendorf tubes. Tubes were closed and cultivated for 2 days at 37 C. 10 l of this pre culture was transferred to the main culture consisting of 7 ml MRS broth in falcon tubes. The culture was incubated for 2 days. After cultivation cells were harvested by centrifugation (15 min, 4000g). The cell pellet was washed two times with K/Na-buffer (each 1 ml). Cells were resuspended in 200 l K/Na buffer.
Cultivation and Preparation of the Indicator Strain:
[0586] The indicator strain was Staphylococcus aureus (DSM346). 20 ml BHI broth in a shaking glass flask were inoculated with 15 l of a 24 h pre culture. The indicator strain was cultivated for 24 h at 37 C. An aliquot was diluted to an optical density OD.sub.595 nm of 0.025-0.05 in BHI-broth and 800 l spread on indicator plates (BHI). The agar was stamped using a cork borer. The holes were filled with 5 l or 10 l of the pre cultured lactic acid bacteria or corresponding volumes of commercial antibiotic preparations.
Media and Buffer:
[0587]
TABLE-US-00012 BHI-Agar Difco Agar 1.8%; 20 ml per plate BHI-Medium Difco MRS-broth Difco K/Na-buffer according to Kster Thiel, pH 7.0, autoclaved 0.066M Na.sub.2HPO.sub.4 2H.sub.2O 61.2 ml 0.066M KH.sub.2PO.sub.4 38.8 ml
EXAMPLE 13
Protease Stability of Lactobacillus Inhibitory Substance
[0588] Specific lactic acid bacteria have been identified, that are able to specifically inhibit the growth of Staphylococcus aureus on agar plates in an in-vitro-hole plate assay. The antimicrobial activity of selected lactobacilli has been characterized concerning digestibility by proteinase K, proteas from Streptomyces griseus, chymotrypsin and trypsin. Cell free preparations of Lactobacillus supernatants have been prepared and incubated with different proteases for 1 h at 37 C. Afterwards these preparations have been tested for their ability to inhibit the growth of the indicator strain S. aureus. The diameter of the inhibition zones has been measured and the area of inhibition has been calculated thereof (see
Cultivation and Preparation of Lactobacilli:
[0589] Lactic acid bacteria were cultivated (OB-LB-Sa3; DSM 18006) from a 80 C. freezing culture in 7 ml MRS broth in falcon tubes. Tubes were closed and cultivated for 2 days at 37 C. 7 ml of this pre culture was transferred to the main culture consisting of 40 ml MRS broth in flasks. The culture was incubated for 2 days. After cultivation cells were harvested by centrifugation (15 min, 4000g). The cell pellet was washed two times with K/Na-buffer (each 2 ml). Cells were resuspended in 10 ml BHI medium and incubated for 6 h at 37 C. Cells were harvested by centrifugation (15 min, 4000g) and the supernatant was used for protease incubation. In detail, 150 l of the supernatant was incubated with 15 l of a 10 mg/ml protease solution at 37 C.
Cultivation and Preparation of the Indicator Strain:
[0590] The indicator strain was Staphylococcus aureus (DSM346). 20 ml BHI broth in a shaking glass flask were inoculated with 15 l of a 24 h pre culture. The indicator strain was cultivated for 24 h at 37 C. An aliquot was diluted to an optical density OD.sub.595 nm of 0.025-0.05 in BHI-broth and 800 l spread on indicator plates (BHI). The agar was stamped using a cork borer. The holes were filled with 5 l or 10 l of the pre cultured cells and was incubated with 15 l of a 10 mg/ml protease solution at 37 C. for 1 h. Afterwards 5 l or 10 l of the protease treated lactobacillus supernatant was used for the inhibition assay
Media and Buffer:
[0591]
TABLE-US-00013 BHI-Agar Difco Agar 1.8%; 20 ml per plate BHI-Medium Difco MRS-broth Difco K/Na-buffer according to Kster Thiel, pH 7.0, autoclaved 0.066M Na.sub.2HPO.sub.4 2H.sub.2O 61.2 ml 0.066M KH.sub.2PO.sub.4 38.8 ml
CITED REFERENCES
[0592] Aly, R., Maibach, H I., Shinefield, H R., Strauss, W G. (1972): Survival of pathogenic microorganisms on human skin. J Invest Dermatol. 58(4): 205-210. [0593] Bisno, A L. (1984): Cutaneous infections: microbiologic and epidemiologic considerations. Am J Med. 76(5A): 172-179. [0594] Brook, I. (2000): The effects of amoxicillin therapy on skin flora in infants. Pediatr Dermatol. 17(5): 360-363. [0595] Elek, S D. (1956): Experimental staphylococcal infections in the skin of man. Ann. NY Acad Sci. 65: 85-90. [0596] Feingold, D S. (1985): Cutaneous microbial flora. Cutis. 36(5A): 1. [0597] Gfatter, R., Hackl, P., Braun, F. (1997): Effects of soap and detergents on skin surface pH, stratum corneum hydration and fat content in infants. Dermatology. 195(3): 258-262. [0598] Gibbons, R J., Houte, J V. (1975): Bacterial adherence in oral microbial ecology. Annu Rev Microbiol. 1975; 29: 19-44. [0599] Hurst, V. (1959): Transmission of hospital staphylococci among newborn infants. Pediatrics 25: 204-214. [0600] Imokawa, G., Akasaki, S., Hattori, M., Yoshizuka, N. (1986): Selective recovery of deranged water-holding properties by stratum corneum lipids. J Invest Dermatol. 87(6): 758-761. [0601] Korting, H C. (1992): Einflu des pH-Wertes auf das Wachstum von Staphylococcus epidermidis, Staphylococcus aureus und Propionibacterium acnes in kontinuierlicher Kultur. Zbl. Hyg. 193: 78-90. [0602] Korting, H C., Hbner, K., Greiner, K., Hamm, G., Braun-Falco, O. (1990): Unterschiede des Hautoberflchen-pH-Wertes und der bakteriellen Mikroflora durch Langzeit-Anwendung synthetische Detergenz-Zubereitungen mit pH 5.5 und pH 7.0 in Acta Derm Venereol. 70: 429-457. [0603] Larson, E. (2001): Hygiene of the skin: when is clean too clean? Emerg Infect Dis. 7(2): 225-230. [0604] Leyden, J J., McGinley, K J., Nordstrom, K M., Webster, G F. (1987): Skin microflora. J Invest Dermatol. 88(3): 65-72. [0605] Lukas, A. (1990): Beeinflubarkeit des Wachstums wichtiger Bakterien der Residentflora in-vitro durch den pH-Wert. In: O. Braun-Falco, H C. Korting (Hrsg.): Hautreinigung mit Syndets, 104-112. [0606] Milyani, R M., Selwyn, S. (1978): Quantitative studies on competitive activities of skin bacteria growing on solid media. J Med Microbiol. 11(4): 379-386. [0607] Ohnishi, Y., Okino, N., Ito, M., lmayama, S. (1999): Ceramidase activity in bacterial skin flora as a possible cause of ceramide deficiency in atopic dermatitis. Clin Diagn Lab Immunol. 6(1): 101-104. [0608] Roth, R R., James, W D. (1988): Microbial ecology of the skin. Annu Rev Microbiol. 42: 441-464. [0609] Selwyn, S., Ellis, H. (1972): Skin bacteria and skin disinfection reconsidered. Br Med J. 1(793): 136-140. [0610] Sullivan, A., Edlund, C., Nord, C E. (2001): Effect of antimicrobial agents on the ecological balance of human micro flora. Lancet Infect Dis. 1(2): 101-114. [0611] Yosipovitch, G., Maibach, H I. (1996): Skin surface pH: A protective acid mantle in Cosmetics Toiletries magazine 111 (12): 101