WO2003092382A1

WO2003092382A1 - Composition and method for controlling microbial adhesion and biofilm formation of surfaces

Info

Publication number: WO2003092382A1
Application number: PCT/DK2003/000165
Authority: WO
Inventors: Lone Kirsten Gram; Birte Fonnesbech Vogel; Dorthe Bagge-Ravn
Original assignee: Danish Institute For Fisheries Research
Priority date: 2002-04-30
Filing date: 2003-03-14
Publication date: 2003-11-13
Also published as: AU2003218628B2; AU2003218628A1

Abstract

The present invention describes how coating of surfaces with an extract, particularly a fish extract, can significantly reduce microbial adhesion, attachment, colonization and biofilm formation on surfaces. Such reduction of microbial adherence, attachment and colonization will be applicable in a large range of areas. The reduced numbers of adhered, attached or colonized microbial organisms is not due to a general growth inhibitory effect and therefore the anti-adhesive effect may not be caused by the presence of antimicrobials (antibiotics or non-antibiotics) in the fish extract.

Description

COMPOSITION AND METHOD FOR CONTROLLING MICROBIAL ADHESION AND BIOFILM FORMATION OF SURFACES

FIELD OF THE INVENTION

The present invention relates in general to the field of controlling adhesion and biofilm formation of microorganisms on a surface. Specifically there is provided a novel method and a novel composition comprising an extract obtainable from a fish, capable of controlling adhesion and/or biofilm formation of microorganisms onto surfaces.

TECHNICAL BACKGROUND AND PRIOR ART

Bacteria and other microorganisms (fungi, larvae) will rapidly adhere to surfaces, as any surface at a water-solid, water-air or solid-air interface typically is richer in nutrients than the surrounding environment.

Biofilm formation and microbial biofouling is, as described, a problem of enormous magnitude. For instance in the situation where the microbial adhesion, attachment or biofilm formation is present on surfaces of objects which come in contact with epithelial cells of an individual, it may cause a subsequently microbial adhesion or attachment to epithelial cells which may be the initial step in an infection process.

In the adhered state, microorganisms will change physiological and typically become more resistant to antimicrobial compounds such as disinfectants and antibiotics relative to their sensitivity when sustained in a planktonic state.

If supplied with a minimum of nutrients and water, the microorganisms adhering to inert surfaces will proliferate and secrete polymers in which they become embedded to provide a biofilm and this biofilm formation offers an even higher degree of protection of the microorganisms against adverse conditions.

Adhesion of microorganisms to solid surfaces and the subsequent formation of microbial biofilm is a phenomenon of immense importance to mankind. In particular, a great number of problems are created by adhering organisms.

On ships, biofouling causes an increase in surface resistance and therefore an enormous increase in fuel consumption. Until recently, ship paints were supplemented with a range of toxic compounds to repell microorganisms. Due to environmental concerns, these compounds have been prohibited and alternatives need to be developed.

• In the food and pharmaceutical industry, microorganisms will adhere to surfaces and hide in biofilms and be able to survive cleaning and disinfecting regimes. Subsequently, such organisms which may either constitute a health risk to the consumer or be able to spoil the product, can re-contaminate products during production. Several examples exist of such problems; thus several cases of listeriosis, which is a very serious food borne infection resulting in 20-40% fatality, were traced to butter and subsequently the clone of Listeria monocytogenes was traced to the processing equipment in the butter processing plant where it had persisted for several years.

• Also, biofilm may easily be formed on filters used e.g. for sterile filtration and result in clotting and standstill of production.

• Perhaps the most detrimental effects of microbial biofilm formation are seen in the clinical sector. For instance, the Pseudomonas aeruginosa infection that accompanies cystic fibrosis consists of a thick alginate containing biofilm in the lungs of CF patients. The biofilm state of P. aeruginosa renders it almost completely resistant to antiobiotic treatment.

• The widespread ability of microorganisms to adhere to surfaces makes catheters, pacemakers or any other prosthetic device extremely likely to carry microorganisms which subsequently infect the patient. Ear infection (otitis media) is particularly serious in children with ear drain where the microorganism gain access to the interior by adhering to the ventilation tube.

• In total, it is estimated that 60-80% of all hospital acquired infections are caused by adhesion of microorganisms and biofilm formation. Also, dental plaque results when cariogenic bacteria (eg Streptococcus mutans) collect in colonies and form deposits (biofilms) on tooth surfaces. Adhered bacteria constitute a major problem for users of contact lenses where infectious keratitis remains a serious complication affecting approx. 25 of every 10,000 users.

Many approaches and techniques have been developed to prevent microbial adhesion. These techniques are, in principle, divided into two major groups. The first group is a technique in which antimicrobial compounds (antibiotics or non-antibiotic factors) may be included in surfaces or a surface coating preventing bacteria from attaching and subsequently colonising and these compounds either have a growth inhibitory or a killing effect on the microorganisms. The second group is a technique where the surface is altered to repell microorganisms and no compound for inhibiting microbial growth or killing microorganisms is used. This other technique of repelling microorganisms is provided by chemical modification of the surface or by coating with repelling compound(s).

Examples of the first technique used to repel microorganisms include incorporation of antibiotics such as bacteriocin, nisin or ciprofloxacin in surface gels, organo tin compounds in ship paints, silver oxide ions in stainless steel or polymers or chitosan in food wrapping materials.

One well-known example of this first approach is the incorporation of the antibacterial compound triclosan into cutting boards, garments, floor coatings etc.

Similar approaches have been used in dental care where tooth coating waxing materials may include antibacterial compounds such as 5-amino-l,3-bis(2-ethylhexyl)-5- methylhexa-hydropyrimidine) and bacterial attachment may be reduced by 90% (1 log unit). Further, covalent binding of antimicrobials, such as peptides (e.g. bacteriocins), tertiary amines or N-halamine polymers, to surfaces can also act as "contact biocides".

The second technique used to repel microorganisms includes surface modifications and coating of surfaces.

Surface modifications

Polymer modifications, e.g. glow discharge treatment of polyethylene, polypropylene or ionozation of silicone tubings can reduce bacterial adherence.

Coating of surfaces

Coating of surfaces with detergents, wetting agents and other surface active compounds will cause a decrease in in vitro bacterial adsorption to surfaces with approx. 1 log unit.

Also, coating with chelating agents such as a mixture of monocycline/disodium EDTA (M- EDTA) reduce adhesion and giycoccalyx formation of Gram-positive cocci. Coating with polyglycol fatty esters or other fatty acid derivatives also act as antifouling agents.

Other studies have shown that using particularly globular proteins for coating of surfaces with food related materials might alter adhesion of bacteria to surfaces. This is true for both inert inorganic surfaces as well as organic surfaces such as epithelial cells. A protein such as fibronectin increases adhesion whereas albumin may prevent attachment. A coating with bovine serum albumin has shown to reduce the number of adhered Staphylococcus epidermidis by approximately 90%. Also ultrahigh temperature treated skimmed milk has shown to be effective against adhesion of microorganisms and the anti-adhesive effect occur when alphas-, beta- or kappa-casein or lactalbumin is used as coatings and believed to be due to protein chain mobility and steric exclusion.

Coatings of surfaces with non-specified conditioning films, like saliva, have also been shown to reduce the number of adhering bacteria on voice prostheses.

Extracts of various plant materials, such as tea (Rosaceae) prevent adhesion of perodontitis causing Porphyromonas gingivalis and extracts from cranberry and other plants and berries containing pro-anthocyanidin and similar compounds prevent adhesion of E.coii (with type 1 and type P fimbriae) causing urinary tract infections.

US 5,607,741 disclose the use of eelgrass as the source for providing non-sulphate phenol compounds which shows to have an anti-fouling activity. The compound in eelgrass characterised by having the primary anti-fouling activity is zosteric acid.

DE 196 46 324 discloses the use of a preparation of one or more secondary metabolites from the marine water organism or the freshwater organism which has an anti-fouling activity. The organisms disclosed are invertebrate organisms such as bacteria and algae.

In WO 01/22973 Al there is disclosed the use of a positively charged carbohydrate polymer like chitosan, for manufacturing of a medicament. Chitiosan may be prepared from chitin and this chitin is common in the marine environment and occurs in the skeleton of crustaceans such as shrimp and crab. WO 01/22973 Al discloses the reduction of viral adhesion to mammalian cells if they are coated with chitosan. One disadvantage of the use of chitosan is that chitosan has been shown to inhibit the growth of microorganisms and may, when used excessively, cause mutations and resistance of the microorganisms.

SUMMARY OF THE INVENTION

As described above, reduction or control of microbial adhesion, attachment, colonisation and biofilm formation on surfaces may be a potentially useful approach in many different applications. Thus, it is a requirement of such an approach to:

• be cheap,

• be able to display a high efficiency for repelling microorganisms from adhesion to surfaces, • be able to maintain a high repelling efficiency during extended time of exposure to microorganisms,

• be effective against a range of different microorganisms, and

• provide an extract or a composition which does not exhibit bactericidal or biocidal activity and does not kill the microbial organisms in order to avoid mutations or resistance of the exposed microorganisms.

Accordingly, the present invention provides a novel suitable composition and a novel suitable method for reduction of microbial adhesion, attachment and biofilm formation on surfaces, comprising a fish extract meeting the above mentioned requirements.

Thus, it is an object of the present invention to provide composition for controlling microbial organism adhesion, attachment, colonisation and/or biofilm formation on a surface, said composition comprising an extract of a marine water organism and/or a freshwater organism, such as a fish.

The composition may be contacted with a surface in order to increase the efficiency of repelling microorganisms from the surface.

It is a further object of the present invention to provide a method of controlling microbial organism adhesion, attachment, colonisation and/or biofilm formation on a surface, said method comprising the steps of:

(i) preparing an extract from a marine water organism and/or a freshwater organism, and

(ii) contacting the surface with an effective amount of said extract.

It is also an object of the present invention to use an extract obtained from a marine water organism and/or a freshwater organism to increase the efficiency of repelling microorganisms from the surface.

DETAILED DISCLOSURE OF THE INVENTION Thus, in the broadest aspect of the present invention a method and a composition for controlling microbial organism adhesion, attachment, colonisation and/or biofilm formation on a surface, comprising an extract of a marine water organism and/or a freshwater organism is provided. In the present context the terms "adhesion" and "attachment" relates to the connection between microbial organisms and a surface either as a single cell or as a group of cells. These terms also relates to any connection between a microbial organism and a surface after any kind of surface treatment like heating, coating or chemical modification of the surface or biocidal treatment of the surface or any combination thereof.

If the microbial organism is attached to a surface, it might be able to colonise on the surface. In the present context the terms "colonise" and "colonisation" relates to the multiplication of the microbial organism after it has attached to either host tissue or to other surfaces.

Colonisation may follow microbial invasion of a cell or microbial adhesion or attachment on a surface in the presence of nutrients and appropriate environmental conditions like temperature, pH and reduction potential in the host cell.

In the case microbial organisms are allowed to adhere, attach or colonise on a surface, a biofilm may be formed. A biofilm formation is the formation of a slimy, slippery coat formed by the microorganism when it is adhered or attached to or colonised on a surface. If supplied with a minimum of nutrients and water, the microorganisms adhering to a surface will proliferate and secrete polymers in which they become embedded to form a biofilm. This biofilm may offer an even higher degree of protection of the microorganisms against adverse conditions.

In accordance with the present invention, adhesion, attachment, colonisation or biofilm formation may be significantly reduced or substantially avoided on surfaces contacted with the extract of the marine water organism and/or freshwater organism relative to a corresponding non-coated surface or relative to a corresponding surface coated with other organic conditioning substances.

In the present context the term "contacting" or "contacted" relates to the treatment of the surface with the extract. This contact may be performed in two ways either by coating the extract onto the surface or by incorporating the extract into the surface, or a combination thereof.

In the present context the term "significantly reduced" relates to a high efficiency in reducing the number of microbial organisms adhering or attaching to colonising or biofilm forming on a surface where the number of microbial organisms adhering, attaching, colonising or biofilm forming has been reduced by a factor above 10, such as by a factor above 15, e.g. by a factor above 20, such as by a factor of above 25, e.g. by a factor above 30, such as by a factor above 40, e.g. by a factor above 50, such as by a factor above 100, e.g. by a factor above 150, such as by a factor above 200, e.g. by a factor above 300 relative to a corresponding non-coated surface.

In the present context the term "substantially avoided" relates to a high efficiency in reducing the number of microbial organisms adhering or attaching to colonising or biofilm formationon a surface where the number of microbial organisms adhering, attaching, colonising or biofilm forming has been reduced by a factor above 10, such as by a factor above 15, e.g. by a factor above 20, such as by a factor of above 25, e.g. by a factor above 30, such as by a factor above 40, e.g. by a factor above 50, such as by a factor above 100, e.g. by a factor above 150, such as by a factor above 200, e.g. by a factor above 300 relative to a corresponding non-coated surface.

Accordingly, in a preferred embodiment of the present invention the number of microbial organisms adhering, attaching, colonising and/or biofilm forming may be significantly reduced or substantially avoided on a surface by a reduction by 10-100%, such as 50- 100%, e.g. 75-100% such as 90-100% relative to a corresponding non-coated surface or relative to a corresponding surface coated with other organic conditioning substances.

In a prefered embodiment of the present invention, the surface may be found in the food industry, technical industry or the pharmaceutical industry or any combination thereof and the surface is selected from the group consisting of medical devices, living tissues, clinical devices, food packing devices, food handling devices, surgical devices, device for intra- body placements, pacemakers, catheters, epithelial cells, fermentors, filters, beakers, marine equipment, marine machinery and paper mills.

The microbial organism capable of introducing adverse effects relates to any microbial organism which is not intended to be present on the surface and has the capability of causing adverse effects due to the attachment or biofilm formation or a combination thereof. Therefore a complete list of organisms in specific areas will not be provided but the present invention will be considering microbial organisms in general. Thus, in the present context the term "microbial organism" relates to any organism which is capable of adhering attaching, colonising and/or forming a biofilm on a surface. In a preferred embodiment of the present invention, the microbial organism is selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, yeast, virus and fungi.

In the present context the term "marine water" relates to saltwater, seawater, brine water or water with a salinity of at least 30 ppt, such as at least 35 ppt, e.g. at leats 40 ppt, such as at least 45 ppt, e.g. at least 50 ppt, such as at least 100 ppt or any combination thereof.

In the present context the term "freshwater" relates to water found in all continental aquatic systems such as rivers and lakes or water with a salinity less than 30 ppt such as less than 25 ppt, e.g. less than 20 ppt, such as less than 15 ppt, e.g. less than 5 ppt or any combination thereof.

The composition of the present invention comprises an extract and this extract is obtainable from any marine water organism or a freshwater organism and may be prepared by cutting said organism into pieces prior to extraction. The sizes of the pieces may be in the range from one piece comprising the entire organism to fines of the organism such as blended or very fine cutted pieces. In an embodiment of the present invention the size of the pieces is approximately 1 cm³, such as approximately 4 cm³, e.g. approximately 8 cm³, such as approximately 10 cm³, e.g. approximately 15 cm³, such as approximately 20 cm³.

In a preferred embodiment of the present invention a high efficiency of the extract may be maintained for at least 20 hours, such as for at least 30 hours, e.g. for at least 2 days, such as at least 3 days, e.g. at least 4 days, such as for at least 5 days, e.g. at least 6 days, such as at least 8 days, e.g. for at least 10 days, such as at least 15 days, e.g. at least 25 days, such as at least 50 days, e.g. at least 75 days,such as for at least 100 days.

To prepare an extract as described, the marine water organism or the freshwater organism is extracted with a medium selected from the group consisting of water, buffer medium, acidic medium, alkaline medium, organic medium and other kinds of extraction media or any combination thereof.

In a preferred embodiment of the present invention, the ratio between the marine water organism or the freshwater organism or any combination thereof to be extracted and the extraction medium is in the range of 50: 1 to 1: 50, such as in the range of 5: 1 to 1:5, e.g. in the range of 4: 1 to 1:4, such as in the range of 3: 1 to 1 :3, e.g. in the range of 1:2 to 2:1.

After the extract of the marine water organism or the freshwater organism has been prepared, any present insoluble particles may be separated from the extract by a process selected from the group consisting of heating, filtration, centrifugation and sedimentation. In the present context the term "insoluble particles" relates to any particles selected from the group consisting of fat, protein and pieces of the organism to be extracted which have not been dissolved in the medium.

In yet another embodiment of the present invention, the extract will maintain the control of microbial organism adhesion, attachment, colonisation and/or biofilm formation on a surface after the extract has been digested by at least one enzyme and contacted with a surface simultaneously or sequentially. In another embodiment of the present invention, the enzyme used for digestion of the extract is a protease. In a further embodiment of the present invention, the protease is selected from the group consisting of an alkalase and a neutrase.

Contrary to most other products or prior art, the extract of the present invention is capable of controlling microbial adhesion, attachment, colonisation and/or biofilm formation substantially without killing the microbial organism, without using an extract exhibiting biocidal activity or substantially or without inhibiting microbial growth or any combination thereof and thereby decreasing the risk of generating new resistant microorganisms which might be pathogenic and introduce adverse diseases.

To increase the applicability of the present invention, a surface may be contacted with the extract according to the invention, and the extract-manipulated surface may then be treated to provide a substantially dry surface where microbial adhesion, attachment, colonisation and/or biofilm formation is controlled.

In the present context the term "a substantially dry surface" relates to a surface obtained after the surface has been contacted with the extract where the repelling effect of the extract is received or absorbed by the surface and the remaining matrix comprising the extract may be removed or disappears.

In the present context the term "matrix" relates to a composition containing the repelling effect of the extract. In a preferred embodiment of the present invention, the matrix can be selected from the group consisting of liquid, water, oil, cream or any combination thereof.

In a preferred embodiment of the present invention, the surface will remain the increased repelling effect after it has been subjected to friction, contact to fluids, sun, light, cold, heat, vind or any combination thereof. Thus, the decrease of the controlling effect may be due to wear of the surface or naturally decomposition due to time. In a preferred embodiment of the present invention the marine water organism or the freshwater organism is selected from the group consisting of vertebrate organisms such as fish, crustaceans such as shellfish, bivalves molluscs, algae, sea weed, sea wrack, gobbles and the like. The fish may further be selected from the non-limiting group consisting of salmon, cod, herring, shark, whale, trout, perch, tuna and mackerel. The crustaceans may further be selected from the non-limiting group consisting of shrimp, crayfish, lobster and crab. The bivalves molluscs may further be selected from the non-limiting group consisting of oysters, mussels and clams. In the present specification the terms used for specific marine water organisms or the freshwater organisms are used for illustrating the invention and not intended to be limiting for the scope of the protection as defined in the appended claims.

The present invention describes how coating of surfaces with an extract, particularly a fish extract, can significantly reduce microbial adhesion, attachment, colonisation and biofilm formation on surfaces. Such reduction of microbial adherence, attachment and colonisation will be applicable in a large range of areas. Thus, the extract of the invention may reduce biofouling in the aquatic environment (ship hulls, nets, wood and steel constructions), adhering spoilage and pathogenic bacteria in the food industry, in the pharmaceutical industry and in the technical industry (e.g. paper mills). Further, the prevention of adhering microorganisms will have usage in the clinical sector, reducing bio-adhesion on various devices such as catheters, pacemakers, ventilation tubing etc.

The reduced numbers of adhered, attached or colonised microbial organisms is not due to a general growth inhibitory effect and therefore the anti-adhesive effect is not caused by the presence of antimicrobials (antibiotics or non-antibiotics) in the fish extract.

In an embodiment of the present invention, the extract may maintain the efficiency for controlling microbial organism adhesion, attachment, colonisation and/or biofilm formation on a surface after the extract has been subjected to increased temperatures or proteolytic digestion or a combination thereof.

In addition to the above mentioned characteristics of the extract of the present invention, the inventors also surprisingly found that besides showing no biocidal activity or substantially no inhibition of microbial growth, the extract possesses an increased effectivity to control microbial organism adhesion, attachment, colonisation and/or biofilm formation on a surface relative to other biofouling systems or biofouling components.

In a preferred embodiment of the present invention the surface treated with the extract of the present invention possesses a long lasting efficiency of repelling microbial organisms. According to the present invention the extract has an increased efficiency for controlling adhesion, attachment, colonisation and/or biofilm formation on a surface for a range of different microbial organisms.

In a preferred embodiment of the present invention any surface may be applicable upon which microbial organism adhesion, attachment, colonisation and/or biofilm formation is controlled by coating the surface with an extract according to the present invention.

In another preferred embodiment of the present invention an extract may be used for controlling microbial organism adhesion, attachment, colonisation and/or biofilm formation on a surface.

In a further preferred embodiment of the present invention, the extract according to the present invention may be applied in various cases:

• On ships, because biofouling causes an increase in surface resistance and therefore, an enormous increase in fuel consumption. Furthermore, does the extract not show any adversely environmental effects.

• In the food and pharmaceutical industry, less microbial organisms will adhere to surfaces and form biofilms and subsequently survive cleaning and disinfecting regimes when applying the extract of the present invention.

• For decreasing biofilm formation on filters used e.g. for sterile filtration.

• For decreasing the adhesion, attachment, colonisation and/or biofilm formation of microbial organisms in the clinical sector. For instance, for the Pseudomonas aeruginosa infection that accompanies cystic fibrosis consists of a thick alginate containing biofilm in the lungs of CF patients.

• For decreasing the ability of microbial organisms to adhere to surfaces such as catheters, pacemakers or any other prosthetic devices which are likely to carry microorganisms which subsequently infect the patient.

To decrese the tendency of infections related to various drains, e.g. ear infections (otitis media), particularly serious in children with ear drain where the microorganism gains access to the interior by adhering to the ventilation tube, may be obtainable by the extract of the present invention.

• For decreasing hospital acquired infections caused by adhered microbial organisms. It is estimated that hospital acquired infections caused by adhesion of microbial

5 organisms and biofilm formation may be reduced by at least 30%, such as at least

40%, e.g. at least 50%, such as at least 60%, e.g. at least 75%, such as 100% by using the presently described invention.

• For decreasing dental plaque as a results of cariogenic bacteria (eg Streptococcus 10 mutans) which collect in colonies and form deposits (biofilms) on tooth surfaces.

• For decreasing eye infections caused by bacteria adhered to contact lenses constitute a major problem for the users of contact lenses where infectious keratitis remains a serious complication affecting approx. 25 of every 10,000 users.

15

The present invention will be further illustrated in the following figures and non-limiting examples.

Fig. 1 shows an experimental set-up for the study of microbial adhesion: glass beaker with 20 steel disks positioned in a holder.

Fig. 2 shows the adhesion of Pseudomonas strain DB82 suspended in PBS buffer to stainless steel surfaces coated with 1/7 TSB, cod extract, 1/7 cod extract supplemented with 0.25% glucose or cold-smoked salmon extract. The experiments were conducted at 25 25°C and bacteria quantified using an indirect conductance measurement.

Fig. 3 shows the adhesion of Shewanella strain A2 suspended in PBS buffer to stainless steel surfaces coated with 1/7 TSB (squares), cod extract (circles) and salmon extract (triangles). The experiment was conducted at 25°C and bacteria quantified using an 30 indirect conductance measurement.

Fig. 4 shows the adhesion of Listeria monocytogenes strain V517a suspended in PBS buffer to stainless steel surfaces coated with 1/7 BHI (squares) or with salmon extract (circles). The experiment was conducted at 15°C and bacteria quantified using an indirect 35 conductance measurement.

Fig. 5 shows the adhesion of Candida lipolytica strain B-114-3 suspended in PBS buffer to stainless steel surfaces coated with 1/7 TSB (squares) or with cod extract (circles). The experiment was conducted at 25°C and bacteria quantified using an indirect conductance measurement.

Fig. 6 shows the adhesion of Pseudomonas strain DB82 suspended in PBS buffer to stainless steel surfaces coated with 1/7 TSB (squares), 1/7 TSB with 1% BSA (upward triangles), 1% BSA (downward triangles) or cod extract (circles). The experiment was conducted at 25°C and bacteria quantified using an indirect conductance measurement.

Fig. 7 shows growth of a) Shewanella strain A2, b) Pseudomonas strain DB82 and c) Listeria monocytogenes strain V517a in fish extract (squares), trypticase soy broth (circles) and 1/7 trypticase soy broth (triangles) at 25°C.

EXAMPLES

Example 1 Microorganisms. Adhesion and attachment studies were conducted with several microbial organisms; both Gram-negative and Gram positive bacteria as well as yeasts. Pseudomonas spp. (strain DB82) was isolated from a processing plant producing cold-smoked salmon. Shewanella spp. (strain A2) was isolated from vacuum-packed trout stored at chill temperatures. A yeast, Candida lipolytica. (strain B114-3) was isolated from a plant producing semi- preserved herring. Listeria monocytogenes (strain V517a) was isolated from a processing plant producing cold-smoked salmon.

Substrates and growth media.

The Gram-negative microorganisms were grown in Tryptone Soya Broth (TSB; Oxoid CM 129) for 24 hours with agitation at 25°C. The bacteria were harvested at 3000 x g for 10 min, and resuspended in Phosphate Buffered Saline (0.8% NaCI, 0.02% KCI, 0.144% Na₂HP0₄, 0.024% NaH₂P0₄ (PBS), pH = 7,4). Yeast was grown in MYGP broth (3 g malt extract Dicfo 0186-17-7, 3 g yeast extract, Difco 0127-17-9, 5 g bacto peptone, Difco 0118-17-0, 10 g glucose D(+) Merck, 1,08337 per liter deionised water) for 24 hours and L. monocytogenes was grown in Brain Heart Infusion broth (BHI; CM225 Oxoid) for 4 days at 15°C whereafter the cells were harvested at 3000 x g for 10 min and resuspended in PBS. Purity of all strains were checked by streaking on Trypticase Soy Agar (TSA; CM131 Oxoid), Iron Agar (IA; CM964 Oxoid), MYGP-agar or on BHI-agar.

Fish extract and other conditioning (coating) liquids.

As indicated, the steel disks were coated with a liquid before microbial adhesion was allowed to take place. Conditioning liquids included various fish extracts as well as several meat and vegetable based broths normally used for microbial growth (Table 1).

Fish extract from fresh cod and salmon was prepared by chopping fresh fish fillets into pieces and water was added in a ratio of 2 parts of fish to 1 part water (weight: weight basis). The fish soup was boiled for 5 minutes and the liquid pressed through a strainer. Thereafter, the extract was boiled again without stirring. After simmering for 5 minutes, the extract is cooled for five minutes and filtered through standard coffee-filters. The fish extract may or may not be buffered with phosphate buffer to restore the buffering capacity to the level of the raw fish. The extract was then sterilised at 100°C for 30 minuntes. Trimethylamine oxide and cysteine may be added to the cod juice. Extract from cold- smoked salmon was prepared in a similar manner. Manipulations of fish extract.

Due to the observed reduction in microorganisms adhering to surfaces coated with fish extracts, attempts were made to manipulate the extract to abolish the adhesion repelling effect. The extracts were diluted (1/7) to determine if less nutrients would change the effect and samples of the extract were supplemented with both glucose, lectin, and mannan to evaluate the effect of increased amount of nutrients. The latter components being polymers typically involved in microbial adhesion.

Adhesion experiments. The system used for studying microbial adhesion to surfaces was set up where stainless steel (AISI 316, unpolished) disks of 2x1 cm were cut and cleaned by autoclaving in detergent. After cleaning, the disks were defatted using acetone and after drying they were clamped vertically in a circular rack placed in a glass beaker (Figure 1). The rack allows a magnetic stirrer to be placed in the middle ensuring slight agitation of the suspension of microorganisms. The whole system was sterilised at 121°C for 15 minuntes. The steel disks were submerged in the liquid used for conditioning for half an hour and finally a suspension of microorganisms was added to the beaker. Light stirring was applied to avoid settling of the organisms and the system was placed at 25°C or at 15°C.

Enumeration of microorganisms on surfaces.

Steel disks were removed at regular intervals, rinsed in PBS, dubbed carefully on clean paper and the number of microorganisms adhering to the surface enumerated by submerging the whole surface in TSB substrat (Pseudomonas, Shewanella) or BHI (Listeria) or yeast (MYGP) in Malthus^® conductance tubes. Numbers of organisms adhering were estimated using the Malthus^® indirect conductance measurement. Conductance detection times from surface measurements were prepared to a standard curve prepared for each organism for each experiment using the PBS suspended cells in a 10-fold serial dilution.

Results

Microorganisms adhered readily and rapidly to the coated steel surfaces. In 1-2 hours, 10³ - 10⁵ cfu/cm² were measured, and in some experiments, significant numbers were adhering after 15 minutes. Over 24 hours a slight increase was seen in numbers of adhering microorganisms. Thus, the experiments demonstrated the universal ability of microorganisms to adhere to surfaces (Table 1).

On surfaces coated with cod juice, Pseudomonas strain DB82 adhered in lower numbers than on surfaces coated wih e.g. TSB, 1/7 TSB or any other commercial microbiological substrate (Figure 2). Thus, approx. 10⁶ cfu adhered per cm² when coated with TSB or 1/7 TSB and only 10⁴ cfu adherede per cm² when coated with fish extract. Coating the surface with cod or salmon extract (Figure 3) also significantly reduced the adhesion of Shewanella strain A2. Salmon coating also reduced adhering numbers of Listeria monocytogenes (Figure 4) as well as cod extract coating reduced numbers of yeast adhering (Figure 5). Preparing extracts from a cold-smoked salmon (containing salt) and using this for conditioning caused a similar reduction in adhering numbers (Figure 2). Neither the addition of glucose, lipid or polymers like lectin and mannan had any significant influence on the anti-adhesive effect of fish extracts. A summary of all treatments and an average of the reduction on microbial adhesion (measured as log (cfu/cm²) is shown in Table 1. In general, coating with fish extracts caused a 10-100 fold lowering of the microbial numbers, i.e. a 90-99% reduction.

Table 1. Overview of combinations of conditioning films and microorganisms for which adhesion was quantified and the reduction in cfu/cm² adhering

Organism Conditioning film Supplements Log (cfu/cm²) Log(cfu/cm²) after 6 h reduction¹

Pseudomonas DB82 Cod none 10⁴ 1.5 - 2

+ lectin 10⁵ 1

+ mannan 5 x 10⁴ 1 - 1.5

1/7 cod none 10⁴ 1.5 - 2

+ 0.25% glucose 10⁴ 1.5 - 2

Cod, dried film 10⁴ 1.5 - 2

Cold-smoked 5 x 10³ 2 - 2.5 salmon juice

Lectin 3 x 10⁵ 0.5 - 1

Mannan 3 x 10⁵ 0.5 - 1

BSA 3 x 10⁵ 0.5 - 1

Cooked milk 3 x 10^s 0.5 - 1

TSB 10⁶ 0

1/7 TSB none 10⁶ 0

+ BSA 10⁵ 0

1/7 LB 10⁶ 0

1/7 BHI 10⁶ 0

Shewanella A2 Cod 10⁴ 1 - 2

Salmon 10⁴ 1 - 2

1/7 TSB 5 x 10⁵ 0

1/7 LB 5 x 10⁵ 0

1/7 BHI 5 x 10⁵ 0 Organism Conditioning film Supplements Log(cfu/cm²) Log(cfu/cm²) after 6 h reduction¹

L. monocytogenes Salmon - lipid 10^J 2.0-3.0 V517a + lipid 10⁴ 1.5-2.0 1/7 BHI 10^b 0

Candida B 114-3 Cod 10^J 1 1.5 1/7 TSB 5 xl0⁴ 0

1) "Reduction" as compared to adhering numbers with TSB, 1/7 TSB or BHI as conditioning film

5 Example 2

To evaluate if the anti-adhesive effect observed from the fish extract was due to simple growth inhibition, the proliferation of microorganisms in fish extract, in Trypticase Soy Broth and in 1/7 Trypticase Soy Broth was determined. 10

The substrates and the growth media were the same as used in example 1 and the fish extract was prepared as described in example 1.

Shewanella strain A2 and Pseudomonas strain DB82 were pre-cultured in TSB overnight at 15 25°C and L. monocytogenes strain V517a was pre-cultured in BHI. Pseudomonas DB82 were diluted 1000-fold and the other organisms were diluted 10, 000-fold in sterile physiological saline. Flasks with 50 ml of each growth medium were inoculated with 1 ml of the diluted bacterial suspension. Colony counts were performed after 0, 5 and 24 hours incubation at 25°C. Shewanella and Pseudomonas counts were enumerated on TSA and 20 Listeria counts were enumerated on BHI-agar.

Results

All the microorganisms grew very well in the different kinds of fish extracts and no growth inhibitory effect was noted as compared to the standard substrates (Figure 7). 25

Example 3

Adhesion or attachment to fish extract coated surfaces were compared to surfaces coated 30 with Bovine Serum Albumin (BSA) since its globular nature may reduce adhesion as described by An et al. 1996. The microorganisms, the substrates, the growth media and the preparation of the fish extract were the same as described in example 1.

As fish extracts mainly consist of amine compounds such as amino acids, peptides and proteins, the fish extracts were digested with proteases (Neutrase (at pH 6.6.) and Alkalase (at pH 8.0)).

Two sets of experiments were conducted. In the first experiment, surfaces were coated with BSA and BSA was added to 1/7 TSB to determine if an anti-adhesive effect similar to the fish extracts could be seen. In the second experiment, manipulation of the protein fraction of the fish extracts was obtained by digestion with proteases. The extract was digested for 30 or 60 minutes with Neutrase (at pH 6.6) or with Alkalase (at pH 8.0).

The adhesion experiments and the enumeration of the microorganisms on surfaces were the same as described in example 1.

Results

The first experiment showed that the number of microorganisms adhering to BSA coated surfaces were lower than number of microorganisms adhering to 1/7 TSB coated surfaces, but the BSA reduction did not reach the same level as when the surfaces were coated with cod extract (Figure 6).

The second experiment showed that coating of surfaces with protease digested fish extracts resulted in the same numbers adhering as on non-digested fish extracts.

Discussion and Conclusions of the experiments

The experiments above demonstrate that coating surfaces with extracts made from fish

(such as cod, salmon, cold-smoked salmon) cause a dramatic and a long lasting reduction in the number of microorganisms adhering, attaching to the surface. This effect was observed for both Gram-positive, Gram-negative bacteria as well as for pathogenic bacteria and food spoiling bacteria and yeasts. The lowering of numbers adhering were also shown neither to be due to a general growth inhibitory effect nor the presence of antimicrobials (antibiotics or non-antibiotics) in the fish extract. REFERENCES

• WO 01/22973 Al

• An, Y.H., G.W. Stuart, S.J. McDowell, S.E. McDaniel, Q. Kang and R.J. Friedman 1996. Prevention of bacterial adherence to implant surfaces with a crosslinked albumin coating in vitro. Journal of Orthopaedic Research 14:846-849.

Claims

1. A method of controlling microbial organism adhesion, attachment, colonisation and/or biofilm formation on a surface, said method comprising the steps of:

(i) preparing an extract from a marine water organism and/or a freshwater organism selected from the group consisting of vertebrate organisms, crustaceans and bivalves molluscs, and

(ii) contacting the surface with an effective amount of said extract.

2. A method according to claim 1, wherein the extract in step (ii) is treated to provide a substantially dry surface where microbial adhesion, attachment, colonisation and/or biofilm formation is controlled.

3. A method according to any of the claims 1 or 2, wherein the controlling of microbial adhesion, attachment, colonisation and/or biofilm formation is obtained substantially without killing the microbial organism and/or without using an extract exhibiting biocidal activity and/or substantially without inhibiting microbial growth.

4. A method according to any of the preceding claims, wherein the extract is prepared by extracting the marine water organism and/or the freshwater organism with a medium selected from the group consisting of water, buffer medium, acidic medium, alkaline medium, organ medium and other kind of extraction medium or any combination thereof.

5. A method according to claim 4, wherein the marine water organism and/or the freshwater organism is cut into pieces prior to extraction.

6. A method according to any of the preceding claims, wherein the ratio between the marine water organism and/or the freshwater organism to be extracted and the extraction medium is in the range of 50: 1 to 1:50, such as in the range of 5: 1 to 1:5, e.g. in the range of 1:2 to 2: 1.

7. A method according to any of the preceding claims, wherein any present insoluble particles is separated from the extract by a process selected from the group consisting of heating, filtration, centrifugation and sedimentation.

8. A method according to any of the preceding claims, wherein the microbial organism is selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, yeast, and fungi.

9. A method according to any of the preceding claims, wherein the number of microorganisms adhered to the surface coated with the extract prepared in step (i) are significantly reduced relative to a corresponding non-coated surface.

10. A method according to any of the preceding claims, wherein the number of microorganisms adhered to the surface are reduced by 5 - 500 fold relative to a corresponding non-coated surface.

11. A method according to any of the preceding claims, wherein the adhesion, attachment, colonisation and/or biofilm formation of a microbial organism on a surface is controlled in such a way that the adhesion, attachment, colonisation and/or biofilm formation is reduced by 10-100%, preferably by 50-99%, relative to a corresponding non-coated surface.

12. A method according to any of the preceding claims, wherein the surface to be contacted with the extract of step (i) is selected from the group consisting of clinical devices, food packing devices, food handling devices, surgical devices, device for intra- body placements, pacemakers, catheters, epithelial cells, fermentors, filters, beakers, marine equipment, marine machinery and paper mills.

13. A method according to any of the preceding claims, wherein the extract prepared in step (i) is digested by at least one enzyme.

14. A method according to claim 13, wherein the enzyme is a protease.

15. A method according to claim 14, wherein the protease is selected from the group consisting of an alkalase and a neutrase.

16. A method according to any of the preceding claims, wherein the marine water organism and/or the freshwater organism is selected from the group consisting of fish and shellfish.

17. A method according to claim 16, wherein the fish is selected from the group consisting of salmon, cod, herring, shark, whale, trout, perch, tuna and mackerel.

18. A method according to any of the claims 1-15, wherein the crustaceans is selected from the group consisting of shrimp, crayfish, lobster and crab.

19. A method according to any of the claims 1-15, wherein the bivalves molluscs may further be selected from the non-limiting group consisting of oysters, mussels and clams.

20. A composition for controlling microbial organism adhesion, attachment, colonisation and/or biofilm formation on a surface, said composition comprising an extract of a marine water organism and/or a freshwater organism.

21. A composition according to claim 20, wherein the controlling of microbial adhesion, attachment, colonisation and/or biofilm formation is obtained substantially without killing the microbial organism and/or without using an extract exhibiting biocidal activity and/or substantially without inhibiting microbial growth.

22. A composition according to claim 20 or 21, wherein the extract is prepared by extracting the marine water organism and/or the freshwater organism with a medium selected from the group consisting of water, buffer medium, acidic medium, alkaline medium, organic medium and other kinds of extraction media or any combination thereof.

23. A composistion according to claim 22, wherein the marine water organism and/or the freshwater organism is cut into pieces prior to extraction.

24. A composition according to any of claims 20-23, wherein the ratio between the marine water organism and/or the freshwater organism to be extracted and the extraction medium is in the range of 50: 1 to 1:50, such as in the range of 5: 1 to 1:5, e.g. in the range of 1:2 to 2: 1.

25. A composition according to any of claims 20-24, wherein any present insoluble particles are separated from the extract by a process selected from the group consisting of heating, filtration, centrifugation and sedimentation.

26. A composition according to any of claims 20-25, wherein the microbial organism is selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, yeast, and fungi.

27. A composition according to any of claims 20-26, wherein the number of microorganisms adhered to the surface coated with the extract is significantly reduced relative to a corresponding non-coated surface.

28. A composition according to any of claims 20-27, wherein the number of microorganisms adhered to the surface are reduced by a factor above 10 relative to a corresponding non-coated surface.

5

29. A composition according to claim 27, wherein the adhesion, attachment, colonisation and/or biofilm formation is controlled in such a way that adhesion, attachment, colonisation and/or biofilm formation on the surface is reduced by 10-100%, such as 50- 100%, e.g. 75-100% such as 90-100% relative to a corresponding non-coated surface or

10 relative to a surface coated with other organic conditioning substances.

30. A composition according to any of claims 20-29, wherein the surface to be contacted with the extract is selected from the group consisting of clinical devices, food packing devices, food handling devices, surgical devices, device for intra-body placements,

15 pacemakers, catheters, epithelial cells, fermentors, filters, beakers, marine equipment, marine machinery and paper mills.

31. A composition according to any of claims 20-30, wherein the extract is digested by at least one enzyme.

20

32. A composition according to claim 31, wherein the enzyme is a protease

33. A composition according to claim 31, wherein the protease is selected from the group consisting of an alkalase and a neutrase.

25

34. A composition according to any of claims 20-33, wherein the marine water organism and/or the freshwater organism is selected from the group consisting of fish and shellfish.

35. A composition according to claim 34, wherein the fish is selected from the group 30 consisting of salmon, cod, herring, shark, whale, trout, perch, tuna and mackerel.

36. A composition according to any of the claims 20-33, wherein the crustaceans is selected from the group consisting of shrimp, crayfish, lobster and crab.

35 37. A composition according to any of the claims 20-33, wherein the bivalves molluscs may further be selected from the non-limiting group consisting of oysters, mussels and clams.

38. A surface upon which microbial organism adhesion, attachment, colonisation and/or biofilm formation is controlled by using a method according to any of the claims 1-19 and/or by coating the surface with a composition according to any of the claims 20-37.

39. Use of a composition according to any of claims 20-37 for controlling microbial organism adhesion, attachment, colonisation and/or biofilm formation on a surface.