Use of bipolymer for reducing the formation of a biofilm

Abstract

The present invention relates to the use of a biopolymer for reducing the formation of a biofilm, preferably on a substrate, whereby the biopolymer is preferably a polypeptide, such as recombinant spider silk polypeptide. The present invention further relates to methods for producing a substrate which reduces the formation of a biofilm on a surface of said substrate.

Claims

1. A method for reducing the formation of a biofilm on a substrate, comprising the step of: applying a composition consisting essentially of a silk polypeptide and a solvent on the surface of a substrate to form a silk polypeptide coating on the surface or producing a substrate from a composition consisting essentially of a silk polypeptide and a solvent, thereby reducing the formation of a biofilm on the substrate.

2. The method of claim 1, wherein the biofilm is an aggregate of microorganisms in which cells adhere to each other and/or to the surface of the substrate, wherein the microorganisms are selected from the group consisting of bacteria, fungi, algae, protozoa, and any combination thereof.

3. The method of claim 1, further comprising drying the surface of the substrate after the composition is applied on the surface of the substrate.

4. The method of claim 1, wherein the substrate is a device.

5. The method of claim 4, wherein the device is selected from the group consisting of a medical device, a wastewater treatment device, a heating device, a ventilation device, and an air condition device.

6. The method of claim 1, wherein the substrate is selected from the group consisting of a synthetic inert substrate, an inorganic inert substrate, and a naturally occurring substrate.

7. The method of claim 6, wherein the (i) synthetic inert substrate is selected from the group consisting of polyester, polystyrene, polyamide (PA), polyaramid, polytetrafluorethylene (PTFE), polyethylene (PE), polypropylene (PP), polyurethane (PU), silicone, a mixture of polyurethane and polyethylenglycol (elastane), ultra high molecular weight polyethylene (UHMWPE), and high-performance polyethylene (HPPE), (ii) inorganic inert substrate is selected from the group consisting of, glass, carbon, ceramic, metal, sapphire, diamond, and semiconductor, or (iii) the naturally occurring substrate is selected from the group consisting of keratin, collagen, cellulose, teeth, bone, skin, and tissue.

8. The method of claim 1, further comprising drying the substrate after the substrate is produced from the composition.

9. The method of claim 1, wherein the silk polypeptide is a recombinant silk polypeptide.

10. A method for producing a substrate which reduces the formation of a biofilm on a surface of said substrate comprising the steps of: (i) providing a composition consisting essentially of a silk polypeptide and a solvent, and (ii) coating the composition onto the surface of said substrate to form a silk polypeptide coating on the surface.

11. The method of claim 10, wherein the method further comprises the step of: (iii) drying the surface of the substrate coated with the composition.

12. A method for producing a substrate which reduces the formation of a biofilm on a surface of said substrate comprising the steps of: (i) providing a composition consisting essentially of a silk polypeptide and a solvent, and (ii) forming said substrate from the composition.

13. The method of claim 12, wherein the method further comprises the step of: (iii) drying the substrate formed from the composition.

14. A method for reducing the formation of a biofilm on a substrate, comprising the step of: applying a composition consisting of a silk polypeptide and a solvent on the surface of a substrate to form a silk polypeptide coating on the surface or producing a substrate from a composition consisting of a silk polypeptide and a solvent, thereby reducing the formation of a biofilm on the substrate.

Description

BRIEF DESCRIPTION OF THE FIGURE

(1) FIG. 1 shows the reduction of biofilm formation due to the coating with a biopolymer. It particularly shows the reduction of biofilm formation on different substrates (polystyrene, rough textured silicone, polytetrafluorethylene (PTFE) and stainless/chrome steel) coated with an aqueous silk solution, a silk hydrogel, or a formic acid silk solution. A reduction of biofilm formation of 75% (polystyrene coated with a formic acid silk solution), 35% (polystyrene coated with a silk hydrogel), 41% (rough textured silicone coated with an aqueous silk solution), 13% (polytetraflourethylene (PTFE) coated with a formic acid silk solution), 40% (polytetraflourethylene (PTFE) coated with an aqueous silk solution) and 66% (stainless steel coated with a formic acid silk solution) was achieved. To determine the reduction of the biofilm formation, the mean value cell number of 16 samples of each experiment (polystyrene, rough textured silicone, polytetrafluorethylene (PTFE) and stainless steel substrates coated with a C.sub.16 protein aqueous solution, C.sub.16 protein formic acid solution and C.sub.16 protein hydrogel) was determined and calculated against the mean value cell number of the non-coated substrates. Non-coated substrate corresponds to normal biofilm formation (value of 0%). A reduction of biofilm formation of e.g. 75% is shown as a value of 75%.

EXAMPLES

(2) To determine the formation of a biofilm on a surface of a substrate, cell count measurements were applied. The surface of different substrates (polystyrene, polytetrafluorethylene, silicone, and stainless steel) was coated with a silk biopolymer. The surface of said substrates was then contacted with a microbial solution. As a control, an uncoated substrate was used. The coated substrate and the uncoated substrate (control or blank) were incubated with the microbial solution between 5 to 10 hours at 30 C., allowing the formation of a biofilm. After the removal of the microbial solution from the coated substrate and the uncoated substrate (control or blank), the number of microorganisms grown on the surface of the coated substrate was determined and compared to the number of microorganism grown on the surface of the uncoated substrate (control or blank).

Example 1: Preparation of the Coating Solution/Hydrogel

(3) The C.sub.16 protein was prepared as described in WO 2006/008163.

(4) a) Preparation of a C.sub.16 Protein Aqueous Solution:

(5) C.sub.16 protein was dissolved in 6 M GdmSCN, diluted with 5 mM Tris/HCl pH 8.5 to 0.6 M GdmSCN. The solution was cross-filtrated against 4 M Urea (10-15) and then cross-filtrated against 5 mM Tris/HCl pH 8.5 (Crossflow; Vivaflow 10-20) until no urea was detectable.

(6) Alternatively, C.sub.16 protein was dissolved in 6 M GdmSCN, diluted with 5 mM Tris/HCl pH 8.5 to 0.6 M GdmSCN. The protein solution was cross-filtrated against 5 mM Tris/HCl pH 8.5 (Crossflow Vivaflow 10-20) until no GdmSCN was detectable.

(7) 14 ml of C.sub.16 protein aqueous solution (C16 protein solution in 5 mM Tris, pH 8.5; protein concentration 12.5 g/l) were filled up to a final volume of 17 ml with deionized water to final protein concentration of 10 g/l.

(8) b) Preparation of a C.sub.16 Protein Hydrogel:

(9) 30 ml of a C.sub.16 protein solution (C.sub.16 protein solution in 5 mM Tris, pH 8.5; protein concentration 12.5 g/l) were filled up with 7.5 ml of 5 mM Tris to final protein concentration of 10 g/l. The C.sub.16 protein solution was then autoclaved in 250 ml Schott-flask (autoclave Systec VX-100) to form a hydrogel.

(10) c) Preparation of C.sub.16 Protein Formic Acid Solution:

(11) 212.9 mg of C.sub.16 protein (-sterilized) were mixed with 21.2 ml of formic acid 98% (Carl-Roth, #4742.2, Lot 42422005) by rotation for a period of 30 min at room temperature resulting in a final protein concentration of 10 g/l.

(12) Alternatively 100 mg of C.sub.16 protein were dissolved in 10 ml formic acid 98% (Carl-Roth, #4742.2, Lot 42422005) by rotation for a period of 30 min at room temperature resulting in a final protein concentration of 10 g/l.

Example 2: Preparation of the Substrates

(13) The different substrates were prepared as follows:

(14) a) Preparation of the polytetrafluorethylene (PTFE) substrates: PTFE patches with a diameter of 5 mm were punched out using a commercial perforator and glued to the bottom of a 96 well plate using Sylgard 184 silicone (Swiss-composite).

(15) b) Preparation of the stainless steel substrates: stainless steel patches (Product Nr. FK110250/1, 300649549, GoodFellow) with a diameter of approx. 5 mm were punched out using a commercial perforator, flattened with a hammer and glued to the bottom of a 96 well plate using Sylgard 184 silicone (Swiss-composite).
c) Preparation of polystyrene substrates: the untreated wells of a polystyrene 96-well plate were used as substrate.

(16) For coating of the polytetrafluorethylene, stainless steel and polystyrene substrates with a C.sub.16 protein hydrogel, 75 l of a 1% C.sub.16 protein hydrogel were pipetted into the wells of a 96-well plate and dried overnight. The next day, 50 l of methanol were pipetted in each well and dried at 60 C. in oven. The resulting wells were homogeneously coated with a thin film. Untreated wells were used as control.

(17) For coating of the polytetrafluorethylene, stainless steel and polystyrene substrates with a C.sub.16 protein aqueous solution, 120 l of 1% C.sub.16 protein aqueous solution were pipetted into the wells of the plate and dried overnight. The next day, 50 l of methanol were pipetted in each well and dried at 60 C. in oven. The resulting wells were homogeneously coated with a transparent thin film. Untreated wells were used as control.

(18) For coating of the polytetrafluorethylene, stainless steel and polystyrene substrates with a C.sub.16 protein formic acid solution, 70 l of 1% C.sub.16 protein formic acid solution were pipetted into the wells of a 96-well plate and dried overnight. The resulting wells were homogeneously coated with a transparent thin film. Untreated wells were used as control.

(19) d) Preparation of silicone substrates (rough-textured):

(20) For the coating of textured silicone patches with a C.sub.16 protein aqueous solution, silicone patches were cut out from a textured silicone foil to a diameter fitting into the wells and subsequently washed with ethanol. The silicone patches were attached to a glass slide and dip-coated into the C.sub.16 protein aqueous solution. Therefore the glass slides with attached silicone patches were dipped into the 1% C.sub.16 protein aqueous solution for a period of 2 min and subsequently dried for a period of 5 min. This step of dipping and drying was repeated three times. The glass slides with attached silicone patches were dried for a period of 5 min at 60 C. Then the glass slides with attached silicone patches were dipped into phosphate buffer (0.5 M Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 for a period of 30 s and dried for a period of 30 s. After a washing step with deionized H.sub.2O (10 s) and a drying step (30 s) the silicone substrates were removed from the glass slides and glued to the bottom of the wells of a polystyrene 96-well plate using Sylgard 184 silicone (Swiss-composite). Untreated silicone patches served as a control.

(21) For the coating of textured silicone patches with a C.sub.16 protein formic acid solution, silicone patches were attached to a glass slide and dip-coated into the C.sub.16 protein formic acid solution. Therefore the glass slides with attached silicone patches were dipped into the 1% C.sub.16 protein formic acid solution for a period of 2 min and subsequently dried for a period of 5 min. This step of dipping and drying was repeated three times. The glass slides with attached silicone patches were dried for a period of 5 min at 60 C. Then the silicone substrates were removed from the glass slides and glued to the bottom of the wells of a polystyrene 96-well plate using Sylgard 184 silicone (Swiss-composite). Untreated silicone patches served as a control.

(22) For the coating of textured silicone patches with a C.sub.16 protein hydrogel, silicone patches were cut out from a textured silicone foil to a diameter fitting into the wells and subsequently washed with ethanol. The silicone patches were glued to the bottom of the wells of a polystyrene 96-well plate using Sylgard 184 silicone (Swiss-composite). The silicone patches were coated by pipetting 75 l of a 1% C.sub.16 protein hydrogel onto a silicone patch and dried overnight. The next day, 50 l of methanol were pipetted on each patch and dried in oven at 60 C. The resulting wells were homogeneously coated with a thin film. Untreated silicone patches glued to the bottom of the 96-well plate served as a control.

Example 3: Determination of the Biofilm Formation on Different Coated Substrates

(23) In order to test the biofilm formation, polystyrene 96-well plates (Greiner bio-one, PS, flat bottom) assembled with different substrates (polystyrene, rough textured silicone, polytetrafluorethylene (PTFE) and stainless steel) were coated with the silk biopolymer according to example 2. As a control, uncoated substrates were used. The 96-well plates were incubated with an adherent bacterial culture of biofilm-forming bacteria (Staphylococcus aureus). After incubation, the number of bacterial cells grown on the 96 well plates was determined and compared to the number of bacterial cells grown on the respective substrates without silk biopolymer coating.

(24) In particular, an inoculum of 200 l of Staphylococcus aureus in culture medium (0.5% TSB (tryptic soy broth) with yeast peptone dextrose) was added to the 96-well plates. Culture medium (0.5% TSB (tryptic soy broth) with yeast peptone dextrose without Staphylococcus aureus was used as control. After incubation on a microtiter plate shaker (100 rpm, 6 h), the supernatant was removed and the wells were carefully washed three times with physiological NaCl solution. Following the removal of the supernatant, 150 l of NaCl solution were added to each well and the plate was mixed thoroughly (10 min, Vortex) to remove the adherent bacterial cells. After a dilution series the cells were spotted on tryptic soy agar square plates and grown for a period of 16 to 24 hours at 33 C. The determination of cell numbers was made by counting the colonies with the help of a binocular.

(25) To determine the reduction of the biofilm formation, the mean value cell number of 16 samples of each experiment (polystyrene, rough textured silicone, polytetrafluorethylene (PTFE) and stainless steel substrates coated with a C.sub.16 protein aqueous solution, C.sub.16 protein formic acid solution and C.sub.16 protein hydrogel) was determined and calculated against the mean value cell number of the non-coated substrates. FIG. 1 shows the reduction of biofilm formation on different substrates (polystyrene, rough textured silicone, polytetrafluorethylene (PTFE) and stainless steel) coated with an aqueous silk solution (aqueous solution), a silk hydrogel (hydrogel) and a formic acid silk solution (formic acid). A reduction of biofilm formation of 75% (polystyrene coated with a formic acid silk solution; column 1), 35% (polystyrene coated with a silk hydrogel; column 2), 41% (rough textured silicone coated with an aqueous silk solution; column 3), 13% (polytetrafluorethylene (PTFE) coated with a formic acid silk solution; column 4), 40% (polytetrafluorethylene (PTFE) coated with an aqueous silk solution; column 5) and 66% (stainless steel coated with a formic acid silk solution; column 6) was achieved.

Example 4: Determination of Biofilm Formation on a Biopolymer Textile Substrate Compared to a Polyester Textile Substrate and a Silk-Coated Silicone Foil Compared to a Non-Coated Silicone Foil

(26) In this example, biofilm formation on a biopolymer textile substrate compared to a polyester textile substrate was determined. The biopolymer textile substrate was a silk biopolymer textile substrate. The silk biopolymer was composed of 100% C.sub.32NR4 silk protein. The silk protein was prepared as described in WO 2006/008163. The protein was then processed into fibers as described in WO 2014/037453. Three multifilaments were twisted into a yarn using a ring twisting machine. This yarn was taken for the kitting process. A 2D-pattern was knitted out of this yarn material. The resulting silk biopolymer textile was cut into three 5 cm5 cm samples and sterilized by autoclaving.

(27) In addition, biofilm formation on a silk-coated silicone foil compared to a non-coated silicone foil was determined. Three samples of silicone foil (5 cm5 cm) were coated with 1% C.sub.16 silk hydrogel. Therefore, the samples sterilized by autoclaving were incubated in 20 ml 1% C.sub.16 silk hydrogel for 5 minutes. The silk hydrogel was prepared as described in example 2. After incubation, the samples were dried over night at RT under sterile conditions. Three samples of uncoated silicone foil (5 cm5 cm sterilized by autoclaving) were used as control.

(28) Three samples of uncoated polyester were used as PES test textile. Therefore, three polyester multifilaments (TWD Fibres GmbH, Deggendorf) were processed into a knitted fabric. The resulting polyester textile was cut into three 5 cm5 cm samples.

(29) In order to determine the reduction of the biofilm formation, a thin liquid film (400 l) containing the bacteria Staphylococcus epidermidis DSM 18857 (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures) (1.2510.sup.4 cells/cm.sup.2) were applied to four different substrate samples (5 cm5 cm, in triplicates): polyester uncoated (PES test textile), silk biopolymer textile, textured silicone foil uncoated, textured silicone foil coated with silk biopolymer. The substrate samples coated with the liquid film containing the bacteria were subsequently covered with a foil (Stomacher-Bags) to prevent desiccation. Immediately after application of the liquid film containing the bacteria, a zero sample was collected from the surface of the substrates and the covering foil by vortexing or sonification in PBS to determine the bacterial count (CFU) (tovalue). The zero samples and the substrate samples (polyester uncoated, biopolymer textile, textured silicone foil uncoated, textured silicone foil coated with silk biopolymer) were further incubated in the liquid film containing the bacteria under humid conditions at 37 C. After 24 h, the bacteria were collected from the surface of the substrate samples and the covering foil by vortexing or sonification in PBS. The bacterial cells were plated to determine the bacterial count (CFU) (t.sub.24-value). The results are shown in table 1.

(30) TABLE-US-00001 TABLE 1 t.sub.0 t.sub.24 Sample [cells/cm.sup.2] [cells/cm.sup.2] Polyester uncoated 1.0 10.sup.4 1.2 10.sup.4 1.2 10.sup.4 1.6 10.sup.3 2.1 10.sup.3 1.2 10.sup.3 Silk biopolymer textile <1 10.sup.1 <1 10.sup.1 <1 10.sup.1 Silicone foil uncoated 1.0 10.sup.4 1.0 10.sup.4 1.0 10.sup.4 1.1 10.sup.3 1.3 10.sup.3 7.9 10.sup.2 Silicone foil coated 9.6 10.sup.1 4.8 10.sup.1 7.2 10.sup.1 with silk biopolymer

(31) Table 1 shows that the biopolymer textile results in a reduction of biofilm formation of more than 99.99% compared to uncoated polyester. In other words, the formation of a biofilm on the biopolymer textile was almost completely avoided. With the silk coated silicone foil, a reduction of biofilm formation of 93.16% could be determined compared to uncoated silicone foil.

Example 5: Determination of the Biofilm Formation on Cotton Fabric and Polyester Fabric Compared to Silk Biopolymer Fabric (In Vitro and In Vivo)

(32) In this example, the biofilm formation on cotton fabric and polyester fabric compared to silk biopolymer fabric (in vitro and in vivo) was determined. The silk biopolymer fabric sample was composed of 100% C.sub.32NR4 silk protein. The silk protein was prepared as described in WO 2006/008163. The protein was then processed into fibers as described in WO 2014/037453. Three multifilaments were twisted into a yarn using a ring twisting machine. This yarn was taken for the weaving process. The yarn was used in direction of warp and weft while weaving a fabric size of A4. The fabric was cut into 1.5 cm1.5 cm samples.

(33) In order to determine the reduction of the biofilm formation on cotton fabric and polyester fabric compared to biopolymer fabric in vitro, two samples of each fabric (1.5 cm1.5 cm, sterilized by autoclaving) were incubated in 10 ml (4.310.sup.4 cells/ml in CASO-broth bacterial solution containing Staphylococcus arlettae DSM 30634 (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures) at room temperature (RT) for 5-6 hours with agitation. After incubation, the fabric samples were washed twice in PBS (first washing step: 10 ml PBS with agitation at 60 rpm for 5 min, second washing step: 10 ml PBS with agitation for 1 min). After the first and second washing step, PBS was removed from the samples by dabbing the fabric samples on sterilized tissue paper. The fabric samples were applied to CASO agar plates. After incubation of the plates for 48 h at 37 C. and for 36 h at RT (room temperature), the number of bacterial colonies was determined by optical inspection. The sample with the highest bacterial growth was set 100%. The sample with the lowest/no bacterial growth was set 0%. Fabric samples sterilized by autoclaving were used as a negative control. The results are shown in Table 2.

(34) TABLE-US-00002 TABLE 2 Biofilm formation on different fabric samples: Sample 1 Sample 2 Cotton 4 4 Polyester 4 4 Silk Biopolymer 0 0 Negative Control 0 0

(35) Table 2 shows that no biofilm formation could be detected on silk fabric, whereas cotton fabric and polyester fabric exhibit strong biofilm formation. The following symbols represent the biofilm formation: 4: 100%, 3: 75%, 2: 50%, 1: 25%, 0: 0% (no biofilm formation)

(36) In order to determine the reduction of the biofilm formation on cotton fabric and polyester fabric compared to silk biopolymer fabric in vivo, five samples of each fabric (1.5 cm1.5 cm, sterilized by autoclaving) were applied to human skin. Therefore, the fabric samples were fixed to the human skin of five different test persons with the aid of a plaster for period of 8 hours. After removing the plasters with the fabric samples from the skin of the test persons, the fabric samples were washed twice in PBS (first washing step: 10 ml PBS with agitation at 60 rpm for 5 min, second washing step: 10 ml PBS with agitation for 1 min). After the first and second washing step, PBS was removed from the samples by dabbing samples on sterilized tissue paper. The skin facing side of the fabric samples were placed onto CASO agar plates. After incubation at RT for 36 h, the number of bacterial colonies was determined by optical inspection. The sample with the highest bacterial growth was set 100%. The sample with the lowest/no bacterial growth was set 0%. Fabric samples sterilized by autoclaving were used as a negative control. The results are shown in Table 3.

(37) TABLE-US-00003 TABLE 3 Biofilm formation on different subjects (in vivo): Subject 1 Subject 2 Subject 3 Subject 4 Subject 5 Cotton 3 1 3 4 2 Polyester 4 3 1 2 3 Silk 0 0 0 0 0 Biopolymer Negative 0 0 0 0 0 control

(38) Table 3 shows that no biofilm formation after application to human skin could be detected on silk biopolymer fabric, whereas cotton fabric and polyester fabric exhibit weak to strong biofilm formation. The following symbols represent the biofilm formation: 4: 100%, 3: 75%, 2: 50%, 1: 25%, 0: 0% (no biofilm formation)

Example 6: Determination of the Biofilm Formation on Non-Coated Polyester Fabric Compared to Polyester Fabric Coated with Silk Biopolymer

(39) In this example, the biofilm formation on non-coated polyester fabric was compared to polyester fabric coated with silk biopolymer. The polyester fabric samples used for the comparative test were coated with 1% C.sub.16 silk hydrogel. Therefore, the samples sterilized by autoclaving were incubated in 20 ml 1% C.sub.16 silk hydrogel for 5 minutes. The silk hydrogel was prepared as described in example 2. After dipping, the samples were dried over night at RT under sterile conditions.

(40) In order to determine the reduction of the biofilm formation on non-coated polyester fabric compared to polyester fabric coated with biopolymer in vivo, two samples of uncoated polyester fabric (1.5 cm1.5 cm, sterilized by autoclaving) and two samples of silk-coated fabric were applied to human skin. Therefore, the fabric samples were fixed to the human skin of four different test persons with the aid of a plaster for period of 8 hours. After removing the plasters with the fabric, samples from the skin of the test persons the fabric samples were washed twice in PBS (first washing step: 10 ml PBS with agitation at 60 rpm for 5 min, second washing step: 10 ml PBS with agitation for 1 min). After the first and second washing step, PBS was removed from the fabric samples by dabbing the samples on sterilized tissue paper. The skin facing side of the fabric samples were placed onto CASO agar plates. After incubation for 48 h at 37 C. and for 36 h at RT, the number of bacterial colonies was determined by optical inspection. The sample with the highest bacterial growth was set 100%. The sample with the lowest/no bacterial growth was set 0%. Fabric samples sterilized by autoclaving were used as a negative control. The results are shown in Table 4.

(41) TABLE-US-00004 TABLE 4 Biofilm formation on non-coated polyester fabric compared to polyester fabric coated with biopolymer (in vivo): Subject 1 Subject 2 Subject 3 Subject 4 Polyester uncoated 4 3 1 2 Polyester coated 0 0 0 0 Negative control 0 0 0 0

(42) Table 4 shows that no biofilm formation on silk coated polyester fabric after application to human skin could be detected, whereas uncoated polyester fabric exhibit weak to strong biofilm formation. The following symbols represent the biofilm formation: 4: 100%, 3: 75%, 2: 50%, 1: 25%, 0: 0% (no biofilm formation).