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K. Schacht, U. Slotta, M. Suhre

Prevention of Biofilm Formation with Vegan Silk Polypeptides

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biofilm

Biofilms – Good and Bad

Bacteria are ubiquitous in nature – mostly living in groups and often in close association with each other as well as with sur-faces of any kind. These entities are commonly called biofilms and play an important role in digestive processes or in pre-serving health of higher animals by symbiotic living on skin, the large intestine and the gut (Fig. 1). Almost any environ-mental niche is occupied by biofilm forming bacteria. They are even found in acidic or alkaline hot springs and frozen glaciers. Besides these commonly occurring and usually de-sired biofilms, also undesired forms exist.

In personal care and hygiene, certain diseases of skin and mouth are closely linked to harmful bacterial biofilms as such films are predicted to play a role in many common dermatoses, damage of teeth as well as gingiva by form-ing dental plaque [8]. For hundreds of years, scientists and dentists have been looking for efficient methods to control oral diseases [6]. The development of suitable countermea-sures such as biofilm preventing surface modifications and antiplaque agents coupled with more effective delivery sys-tems for targeting specific bacteria and for improving the

retention of agents in the mouth are necessary [8]. Additionally, bacteria such as Staphylococcus species are responsible for contaminations of surfaces of medical de-vices or materials used in food industry [7]. Meth-icillin-resistant Staphy-lococcus aureus (MRSA) bacteria cause clini-cal-acquired diseases and Staphylococcus epider-midis can cause severe in-fections in immune-sup-pressed patients as well as in those with venous catheters, among others [9]. A novel approach for controlling biofilm forma-tion with a broad applica-bility is covering surfaces with innovative vegan silk polypeptides (Fig. 2).

Fig. 1 There is a number of mechanisms enabling bacteria to come into close contact with a surface, attach firmly to it, promote cell-to-cell interactions and grow as complex structure. The process of biofilm formation begins with an initial reversible attachment of microorganisms to a surface (1). This adhesion is initiated by pre-conditioning macromolecules present in the bulk liquid. In a second stage, cell adhesion becomes irreversible and attached bac-teria start to alter their surface structure for example by losing flagella (2). The first layers allow the attachment of further bacteria. The production of cell-to-cell signaling molecules promotes the differentiation and specialization of bacterial cells (3). The biofilm maturates, bacteria grow and the matrix is further stabilized (4). In fully established biofilms, some bacteria disperse and recover their free-floating, planktonic status (5) [10].

Prevention of Biofilm Formation with Vegan Silk PolypeptidesK. Schacht, U. Slotta, M. Suhre*

The prevention of undesired biofilm formation is one of the emerging tasks in personal care/oral care, medical technology, food applications and in the textile industry, among others. Today, there is no suitable technique available to prevent for-

mation of such biofilms except by heavy treatment with biocides and/or antibiotics. Here, a novel and promising approach by employing biofilm-preventing surfaces using innovative vegan silk polypeptides, available as powders (Silkbeads), aqueous solutions, hydrogels (Silkgel) and even in form of Biosteel® fibers, is presented.

abstract

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Strategies to Control Biofilm Formation

Common strategies to prevent biofilm formation or remove already established biofilms are often accompanied by em-ployment of antibiotics, biocides or ion coatings (e. g. silver). Moreover, several plant, microbial and milk polysaccharides possess the ability to block lectins from human pathogenic bacteria known to facilitate attachment and adherence of bacteria to surfaces. Also, polyethylene glycol (PEG) coatings show the ability to decrease biofilm formation with a clear re-lationship between the molecular size of the PEGs and the ef-ficiency to prevent biofilms – the bigger, the better. All these methods prevent biofilm formation by interfering with the attachment and expansion of immature biofilms [16].Other biofilm inhibition strategies employ anti-biofilm en-zymes, chelating agents and antimicrobial peptides or an-ti-adhesion agents such as pilicides. The latter inhibit the for-mation of bacterial pili and thereby biofilm formation up to 50 % at concentrations of only 3 µM [1].Ideally, preventing biofilm formation would be a more straightforward option than removal of already existing bio-films. Unfortunately, up to now there is no technique available to successfully prevent or control the formation of unwanted biofilms without causing adverse side effects, e.g. the em-ployment or release of toxic substances. Currently, the main strategy to prevent biofilm formation is to clean and disinfect regularly. Anti-biofilm surfaces, however, exist in principle (i.e. anti-microbial silver coatings), but those are only effective in certain circumstances and still under discussion in regard to toxicity and environmental burden [4].Besides treatment with toxic chemicals, biofilm formation can be influenced by various parameters such as texture/rough-

ness, hydrophobicity, surface chemistry or charge. Especially the physical and chemical properties of surfaces influence the adhesion of free-floating, planktonic bacterial cells, where textured and hy-drophobic surfaces with high roughness gener-ally promote the adhesion of bacteria. Thus, it is desirable to create/maintain a smooth surface on products that may come in contact with bacteria. Additionally, as soon as the substrate surface is oppositely charged to the cell surface, adhesion is also intensified.Here, we present data for the influence of vegan silk polypeptides on biofilm formation, applied as aqueous solutions or hydrogels (Silkgel), rendering this material highly interesting for applications in the personal care field, for medical devices as well as for the textile segment.

Difference between Silk Proteins and Vegan Silk Polypeptides

Silk webs fascinated humans for thousands of years, mostly due to their toughness and ductility,

but also because silk webs seem not to cause inflammation and allergic reactions [2].Moreover, bacteria and fungi are not able to grow on silk webs. Unfortunately, this well-developed natural substance was not available for technical applications for a long time.AMSilk´s silk polypeptides are inspired by this natural high-per-formance material and differ significantly from commonly used silkworm silk hydrolysates. The silk polypeptides are manufactured biotechnologically without the use of any kind of animal derived substances, rendering them 100 % vegan. As they are not hydrolyzed, their functionality persists. The biotechnological manufacturing process enables a high and consistent purity which cannot be reached by natural pro- ducts (Fig. 3) [5]. Furthermore, vegan silk polypeptides can be processed into a variety of morphologies, reaching from

Fig. 2 Vegan silk polypeptides, applied from aqueous solutions or hydrogels (Silkgel), are an efficient protection against bacterial adhesion and mitigate bio-film formation on substrates such as teeth, medical devices, technical surfaces, as well as on textiles.

Fig. 3 Differences between hydrolyzed natural silk proteins and vegan silk polypeptides.

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aqueous silk solutions and hydrogels (Silkgel) to powdery forms (Silkbeads) or fibers with extraordinary physical and chemical stability as well as superior mechanical properties. Textiles made of the high-performance silk fiber Biosteel® are 100 % biodegradable, lightweight and deliver silky touch, smoothness and moisture management.

Methods and Results

Silk as Protective Agent against Bacterial Adhesion

Vegan silk polypeptides can be applied to surfaces in manifold ways. Basically, these polypeptides are available as powders (Silkbeads), aqueous solutions or hydrogels (Silkgel), which in the latter cases can be used as surface coating. Silkgel used in cosmetic formulations is easily mixable with nearly any substance and creates a thin film on surfaces upon ap-plication, for example by spraying, dip coating or applying cosmetic formulations containing volatile substances or com-ponents that are absorbed into the skin. Moreover, vegan silk polypeptides are available as (Biosteel®) fibers which can be processed into textiles. To determine the biofilm formation on Biosteel® textiles in comparison to commonly used polyes-ter textiles, a modified adhesion test according to ISO 22196 (QualityLabs BT GmbH, Nuremberg) was performed (Fig. 4A). To analyze the anti-adhesive activity on the fabrics incubation with Staphylococcus epidermidis DSM 18857 was performed for 24 hours. Staphylococcus epidermidis is one of the most frequent causes of biofilm-associated infections, due to the fact that staphylococci are frequent commensal bacteria on the human skin and mucous surfaces [17].After 24 hours of incubation of the fabrics in the bacteria suspension, the samples were washed and adherent bacte-ria were detached by sonication. Afterwards, a dilution se-ries was plated on agar and the colony forming units were determined. A significant reduction of biofilm formation by 99.9 % was observed on Biosteel® fabrics when compared to polyester.An alternative in vitro test using Staphylococcus arlettae shows similar results. Polyester and Biosteel® fabrics were incubated for five to six hours in the bacterial suspension, washed with phosphate buffered saline (PBS) and incubated on CASO agar plates at 37 °C for two days. Afterwards, the textiles were removed and a dense bacterial lawn was ob-served on polyester, whereas almost no bacterial growth was detected on plates with Biosteel® fabrics (Fig. 4B). In addition to the in vitro results, in vivo tests were performed to assess whether Silkgel-treated synthetic textiles show similar effects. To this end, uncoated and Silkgel-coated polyester samples were incubated on human skin for six hours. Afterwards, the textiles were incubated on CASO agar plates for two days. Impressively, even low concentrations of silk polypeptides within a Silkgel-coating of polyester textiles reduce biofilm formation significantly (data not shown). One possible expla-nation for the anti-biofilm effect of silk polypeptides as pure

protein fiber and as coating is that teichoic acids (present in gram-positively staphylococcus species), an anionic polymer, linked to peptidoglycans or to the underlying plasma mem-brane, lead to a negative charge of the bacterial cell wall. The silk protein is also negatively charged and thus bacterial ad-hesion is not favored due to electrostatic repulsion. This effect could also be relevant for malodor of clothes which often is produced by bacteria transforming perspiration into odorous substances (e.g. butyric acid). Here, Biosteel® textiles as well as transient silk coating of textiles may result in reduced mal-odor due to the prohibited bacterial adhesion. Additionally, silk as softener or special treatment in addition to detergents could cause reduced bacterial adhesion on clothes or even within washing machines.In summary, pure vegan silk polypeptides provide an efficient protective layer against bacteria thereby preventing biofilm formation.

Silk Coating Reduces Biofilm Formation on Hydrophobic Surfaces

It is well documented that biofilm has become a problem in food industries and medicine as it renders its inhabitants resistant to antimicrobial agents and cleaning. The contam-inations of food may occur at any stage in the process from food production to consumption [3, 14]. Biofilm forma-tion on surgical instruments or implanted medical devices by Staphylococcus epidermidis or Staphylococcus aureus represents one of the major problems as they cause hospi-tal-acquired infections and infections on indwelling medi-cal devices. Due to the fact that staphylococci are frequent

Fig. 4 Biofilm formation on Polyester and Biosteel® fabrics. In vitro tests with Staphylococcus epidermidis (A) and Staphylococcus arlettae (B).

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commensal bacteria on the human skin, they contaminate medical devices when being inserted during surgery. Since equipment in food industries and medical devices are made of different types of materials, e.g. Teflon®, stainless steel and polymers, early-stage biofilm formation (6 hours incu-bation) by Staphylococcus aureus on different silk-coated and uncoated surfaces was determined (Swissatest Testma-terialien AG, St. Gallen, Fig. 5).Silk coating mediates a reduced bacterial adhesion on all sur-faces with slight differences between the chosen materials. Silk coating on polystyrene shows the highest reduction of bacterial adhesion by 75 %, whereas silk coating on Teflon® results in a reduction of biofilm formation by 18 % and on stainless steel by 53 %. The hydrophobicity of surfaces also in-fluences the biofilm formation. Bacterial adhesion is preferred on hydrophobic surfaces [12]. Therefore, the hydrophobicity of the tested surfaces was determined by contact angle mea-surements (Fig. 6).Vegan silk coating of the surfaces reduces the hydrophobicity which results in a reduced biofilm for-mation. Silk-coated Teflon® still shows a contact angle of 112 °. The higher hydrophobicity com-pared to coated polystyrene and stainless steel causes a diminished biofilm reduction.Another hydrophobic material, sil-icone, is frequently used in medi-cal applications. The major cause of complications with silicone implants in particular is capsular fibrosis which can in some cases eventually lead to deformation of the breast accompanied by pain and, at worse, to reoperation. A close relation between capsular fibrosis and biofilm formation on implants has been described: the

better the bacterial adhesion, the higher the risk of biofilm generation, the higher the risk of capsular fibrosis and reop-eration [15]. To investigate the influence of vegan silk on biofilm formation on silicone substrates, the adhesion of two different staphy-lococcus species on textured silicone surfaces was analyzed (Fig. 7).Staphylococcus aureus was incubated for six hours on un-coated as well as coated silicone surfaces to determine the formation of biofilms. The bacterial adhesion of Staphylococ-cus aureus was reduced by 39 % on silk-coated silicone sur-faces compared to uncoated silicone. Staphylococcus epider-midis, however, was incubated for 24 hours on the silicone surfaces and the biofilm formation was reduced by 93 % on silk-coated silicone. In this case, the surface morphology is critical for the observed anti-adhesive effect, since rough sur-faces are more prone to biofilm formation and maturation

Fig. 5 Biofilm formation of Staphylococcus aureus ATCC 20231 on different surfaces (polystyrene, Teflon® and stainless steel), either uncoated or silk-coated after 6 hours of incubation.

Fig. 6 Contact angles of uncoated and silk-coated surfaces (image: polystyrene surface). Generally, if the water contact angle is larger than 90 °, the surface is considered hydrophobic and if the water contact angle is smaller than 90 °, the surface is considered hydrophilic. The silk coating increases the hydrophilicity.

Polystyrene Teflon® Stainless steel

Contact angle ( °)

Uncoated surfaces93

(hydrophobic)119

(hydrophobic)98

(hydrophobic)

Silk-coated surfaces51

(hydrophilic)112

(hydrophobic)44

(hydrophilic)

Fig. 7 Biofilm formation on uncoated and silk-coated silicone surfaces after incubation of Staphylococcus aureus (6 h, Swissatest Testmaterialien AG, St. Gallen) and Staphylococcus epidermidis (24 h, QualityLabs BT, Nuremberg) on surfaces.

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than smooth surfaces. The silk coating on textured silicones lead to a surface smoothening, thereby reducing biofilm for-mation.Taken together, on all tested surfaces silk coatings led to re-duced biofilm formation. In general, bacterial attachment will most readily occur on surfaces which are rougher, more hydrophobic and positively charged. Vegan Silk Polypeptides, applied as aqueous solution or Silkgel, render hydrophobic surfaces more hydrophilic, rough surfaces smoother and pos-itively charged surfaces more negatively charged. All these features combined lead to a reduced bacterial adhesion and decreased biofilm formation. Therefore, the employment of toxic substances or even antibiotics can be reduced and may-be completely avoided. Furthermore, silk coatings could re-duce or substitute the employment of harsh disinfectants to protect sensitive materials.

Summary

AMSilk’s innovative vegan silk polypeptides are available as Biosteel® fibers, powders (Silkbeads), aqueous solutions and hydrogel (Silkgel). The latter provide an efficient protective layer against bacteria and prevent biofilm formation. In ad-dition to its other beneficial properties such as breathable shield against irritants and against negative environmental influences [11, 13] these renders this unique material highly interesting for various applications in personal care, medi-cine as well as food and textile industries. For example, veg-an silk polypeptides can be used as tool to prevent biofilm formation in oral care, on silk coated implants, in technical equipment as well as on textiles. By preventing biofilm for-mation adverse side effects, e.g. caused by the use of toxic substances or by formation of bacterial resistances can be mitigated.

References

[1] Abdel-Aziz, S. M., & Aeron, A. (2014). Bacterial Biofilm: Dispersal and Inhibi-tion Strategies. SAJ Biotechnology, 1.

[2] Bon, M. (1710). A discourse upon the usefulness of the silk of spiders. Philo-sophical Transactions, 27, pp. 2–16.

[3] Bryers, J. D. (2008). Medical Biofilms. Biotechnol Bioeng., pp. 1-18.

[4] Cloutier, M., Mantovani, D., & Rosei, F. (2015). Antibacterial Coatings: Chal-lenges, Perspectives, and Opportunities. Trends in Biotechnology, 33(11), pp. 637-652.

[5] Heidebrecht, A., & Scheibel, T. (2013). Recombinant Production of Spider Silk Proteins. Advances in Applied Microbiology, 82, pp. 115-153.

[6] Huang, R., Li, M., & Gregory, R. L. (2011). Bacterial interactions in dental biofilm. pp. 435-444.

[7] Jefferson, K. K. (2004). What drives bacteria to produce a biofilm? FEMS Microbiology

[8] Marsh, P. D. (2004). Dental Plaque as Microbial Biofilm. Caries Res(38), pp. 204-210.

[9] Otto, M. (2008). Staphylococcal Biofilms. Curr Top Microbiol Immunol(322), pp. 207-228.

[10] Sauer, K. (2003). The genomic and proteomic of biofilm formation. Genome Biology, 4(6).

[11] Schlay, S., & Slotta, U. (2016). Efficient Skin Protection Against Negative Envi-ronmental Influences by Breathable, Vegan Silk Polypeptides. SOFW, pp. 14-19.

[12] Shi, L., Ardehali, R., Caldwell, K. D., & Valint, P. (2000). Mucin coating on polymeric material surfaces to suppress bacterial adhesion. Colloids and Surfaces B: Biointerfaces, pp. 229-239.

[13] Slotta, U., Rüther, L., Mehrwald, R., & Römer, L. (2015). Breathable Shield Against irritants - Effective Protection for Stressed Skin with Functional Silk Polypeptides. SOFW, pp. 26-31.

[14] Srey, S., Jahid, I. K., & Ha, S.-D. (2013). Biofilm formation in food industries: A food safety concern. Food Control, pp. 572-585.

[15] Steiert, A., Boyce, M., & Sorg, H. (2013). Cap-sular contracture by silicone breast implants: possible causes, biocompatibility, and prophy-lactic strategies. Medical Devices, pp. 211-218.

[16] Thebault, P., & Jouenne, T. (2015). Antibacte-rial coatings. In The Battle Against Microbial Pathogens: Basic Science, Technology Advances and Educational Programs (pp. 483-489). A. Méndez-Vilas.

[17] Vuong, C., & Otto, M. (2002). Staphylococcus epidermidis infections. Microbes Infect, pp. 481-489.

*contact

Dr. Kristin SchachtDr. Ute Slotta

Dr. Michael Suhre

AMSilk GmbHAm Klopferspitz 19 im IZB

82152 Planegg/MunichGermany

www.amsilk.com