WO2008040516A2

WO2008040516A2 - Microbiological composition and use thereof

Info

Publication number: WO2008040516A2
Application number: PCT/EP2007/008521
Authority: WO
Inventors: Maria Uphoff; Heinrich Josef Uphoff
Original assignee: Georg Fritzmeier Gmbh & Co. Kg
Priority date: 2006-10-02
Filing date: 2007-10-01
Publication date: 2008-04-10
Also published as: WO2008040516A3

Abstract

A composition is described which comprises a mixture of magnetotactic and phototrophic microorganisms. The composition is suitable as an agent against pathogenic microorganisms and for the prevention and treatment of skin changes.

Description

description

Microbiological composition and its use

The present invention relates to a composition with biocidal, antibiotic and antimycotic action and their use for the prevention and treatment of changes in the skin of humans and animals.

The surface of the skin (epidermis) in humans and animals is not completely smooth, but locally furrowed with furrows, wrinkles and wrinkles. The skin relief is particularly characteristic on the fingers, palms and soles of the feet.

The skin is regarded primarily as protection of the body, which in itself has various mechanisms for its own protection and can also activate accordingly. However, there are often pathological changes in the skin, such as scars, warts, acne, dandruff, lichen, psoriasis, eczema, skin cancer and other skin conditions or dermatoses. Often these are due to environmental influences. Often also occur bacterial infections on the skin surface, in addition to the infestation with fungi and extremely unpleasant. It comes here to a strong itching and thus to a painful scratching of the skin.

Morbid lesions occur in both humans and animals. In the latter not only pets such as cats or dogs are endangered, but also livestock stocks such as dairy cattle, horses and pigs. In particular, various types of dermatitis, in particular infectious diseases such as digital and papillomatous digital dermatitis (Mortellaro's disease) in dairy cattle, are involved In fact, the claw diseases caused by these diseases, if left untreated, can lead to hoof cancer at a later stage.

Antifungals and antibiotics are commonly used to combat fungal and bacterial infections. Also, most infectious claw diseases in dairy cattle are treated with topical antibiotics (spray, ointment) and many reports show a clinical response to antibiotics in various forms with or without bandage. Prevention and control strategies include footbaths containing various components. While foot baths are minimal in cost, as opposed to individualized treatment, the milking routine is minimally or not at all disturbed, however, there is little evidence that they are effective for the treatment of digital dermatitis, and they can be environmentally harmful. Products can lower prevalence and keep it at an acceptable level, but they are not effective against new wounds and they can not eradicate the disease in one plant. Heavier digital dermatitis wounds require frequent individual treatment with or without bandage.

The antibiotics and antimycotics used to treat skin diseases are generally synthetic pharmaceutical products that are known to cause significant side effects. Often, for example, with frequent use of these agents, there is no longer a reaction since, for example, fungi and bacteria have become resistant. In addition, the use of antibiotics in livestock is severely restricted in many countries. WO-A-03/047533 describes a skin surface treatment composition containing a mixture of photosynthetic microorganisms and luminescent bacteria which can treat diseases such as atopic dermatitis. However, the relatively large molecules of the microorganisms contained in the mixture are hardly absorbed by the skin.

DE _^ A-10 2006 037 632 describes a microbiological mixture for skin treatment, the piezoelectric nanostructures reductive / oxidative micro-organisms, a conjugated biopolymer and / or 5-aminolevulinic acid includes, and the effectiveness of the microorganism improved in the mixture, and a method of Apply this mixture to the skin.

Successes in treating digital dermatitis in dairy cattle with non-antibiotic agents have also been described (Kofier et al., J. Vet. Med. 2004, 51, 447-452; Brydl, E. et al., Treatment of DD without using of antibiotics - a clinical trial 13 ^th International Symposium and Conference Proceedings Maribor, Slovenia 2004; Manske, T. et al, Preventative Veterinary Medicine 2002, 53: 215-231; Gradle, CD, et al, Treatment...... .. of digital dermatitis lesions in dairy cows with novel nonantibiotic formulation in a foot bath 12 ^th International Symposium on lameness in ruminants Orlando, Florida 2002. Moore, DA et al, J. Amer Vet Med Assn 2001..... 219: 1435-1438, Seymore, J. et al., Proc. Amer Assoc. Bovine, Practitioner 2001, 34: 200; Britt, JS et al., J. Amer. Vet. Med. Assn. 1996, 209: Britt and McClure, The bovine practitioner 1998, 32: 25-28; Hernandez, J. et al., J. Amer., Vet. Med. Assn., 1999, 214: 688-690; Read, DH et al. , J. Vet. Diagnostic Invest. 1998, 10: 67-76). Many of these formulations contain soluble copper with peroxide, formic, acetic and propionic acids, copper, aluminum and zinc sulphate and benzalkonium chloride. However, the studies often included only individual farms, a small number of animals and / or short treatment periods, so that it can not be reliably concluded that these remedies are effective.

There is therefore a need for an agent for the prevention and treatment of skin lesions, in particular pathological skin changes in humans and animals, which represent a natural alternative to the established pharmaceutical preparations and which can be used without the risk of side effects. Another object of the invention is to provide a composition which improves the permeability of the skin to the molecules contained in the mixture.

This object is achieved with a microbiological composition according to claim 1. It has surprisingly been found that such a mixture has biocidal, in particular antibiotic and antifungal properties and can be used to treat changes in the skin.

The subject of the present invention is therefore a composition which is characterized in that it comprises at least one magnetotactic microorganism and at least one phototrophic microorganism.

The present invention further relates to the use of this composition as a cosmetic preparation, as a human or veterinary medicament, for example as an antibiotic or antimycotic, and as biocidal agent. The present invention furthermore relates to the use of the composition according to the invention for the production of a medicament for the prevention and treatment of skin changes, for example dermatoses.

An essential feature of the present invention is that the composition contains two groups of microorganisms, namely, magnetotactic microorganisms and phototrophic microorganisms.

The magnetotactic microorganisms used in the invention are preferably magnetotactic bacteria, i. mobile, anaerobic or microaerophilic bacteria containing ferromagnetic iron oxide (magnetite) or iron sulfide (geigerite) in crystalline form, so-called magnetosomes whose diameter is about 40 to 90 nm. These magnetosomes give the bacteria the ability to orient themselves according to the earth's magnetic field. Preferred representatives of the magnetotactic bacteria according to the invention are bacteria of the genus Spirillum, for example Aquaspirillum or Magnetospirillum, for example Magnetospirillum magnetotacticum and, preferably, Magnetospirillum gryphiswaldense.

The phototrophic microorganisms used according to the invention comprise both obligate and optionally phototrophic microorganisms. Optionally phototrophic microorganisms are preferably used, ie microorganisms that can grow in the dark both under anaerobic conditions in the light and under aerobic conditions. The microorganisms according to the invention can be photolithotrophic as well as photoorganotrophic. The phototrophic bacteria which can be used in the present invention include Gram-negative aerobic, rod-shaped and circular bacteria, as well as Gram-positive circular bacteria. These may have endospores or be present without spores. Phototrophic bacteria suitable according to the invention are, for example, purple bacteria, green sulfur bacteria, green non-sulfur bacteria, cyanobacteria, prochlorophytes, and bacteria of the family Heliobacteriaceae. Purple bacteria which can be used according to the invention are both sulfur purpuric bacteria, for example bacteria of the genera Allochromatium, Amoebobacter, Chromatium, Ectothiorhodospira, Halochromatium, Halorhodospira, Isochromatium, Lamprobacter, Lamprocystis, Marichromatium, Rhabdochromatium, Thermochromatium, Thiocapsa, Thiococcus, Thiscystis, Thiodictyon, Thiohalocapsa, Thiolamprovum, Thiopedia, Thiorodococcus , Thiorhodovibrio and Thiospirillum, as well as Nichtschwefpurpurbakterien, for example bacteria of the genera Rhodobacter, Rhodocyclus, Rhodoferax, Rhodomicrobium, Rhodopila, Rhodopseudomonas, Rhodoplanes, Rhodobium, Rhodospirillum, Rhodovibrio, Roseospira, Rhodospira Rhodovulum and Rubrivivax. Examples of green sulfur bacteria that can be used in the composition according to the invention are bacteria of the genera Chlorobium, Prosthecochloris and Chlorobaculum. Examples of green non-sulfur bacteria which can be used in the composition according to the invention are bacteria of the genera Chloroflexus, Oscillochloris and Heliothrix. Examples of cyanobacteria which can be used in the composition according to the invention are bacteria of the genus Anabaena and spirulina. Examples of prochlorophytes which can be used in the composition according to the invention are bacteria of the genera prochlorone, prochlorothrix, prochlorococcus. Suitable members of the family of Heliobacteriaceae are, for example, bacteria of the genera Heliobacterium, Heliophilum and Heliobacillus.

Preferably, the composition according to the invention contains a mixture of optionally phototrophic microorganisms, the two

Cyanobacteria, green sulfur bacteria, purple bacteria, green non-sulfur bacteria such as Chloroflexus and Heliobacteriaceae such as Heliobacillus. According to a preferred embodiment of the invention, there are three, four, five or six of said bacterial forms in the mixture.

The light which drives the photosynthesis may be either external light, but is preferably derived from a microorganism of the group of luminescent bacteria contained in the composition according to the invention and providing the light required for photosynthesis. Luminescent bacteria are understood to mean all naturally occurring or recombinant bacteria capable of emitting light. The light emission of these luminescent bacteria, which is also referred to as bioluminescence, is usually carried out in the oxidation of luciferins or long-chain aldehydes under the action of luciferases.

In a preferred embodiment, the luminescent bacteria contained in the inventive composition are luminescent bacteria of the genus Photobacterium, Vibrio, Shewanella, Photohabdus, Pseudomonas or Beneckea, which can be used alone or in a mixture. Examples of suitable luminescent bacteria are Photobacterium phosphoreum, Vibrio fischeri, Vibrio harveyi, Pseudomonas lucifera. The interplay between the phototrophic microorganisms and the luminescent bacteria causes the phototrophic microorganisms are excited by the luminescent bacteria for photosynthesis. The microorganisms operate photosynthesis with hydrogen sulfide and water as adduct and release sulfur or oxygen. They can also bind nitrogen as well as phosphate and degrade organic and inorganic matter.

Furthermore, other microorganisms which have a positive effect on the composition according to the invention may also be present. Preference is given to using lactic acid bacteria, sulfate-reducing bacteria, archaea, fungi, such as mold and yeast fungi, and algae. Examples of lactic acid bacteria suitable according to the invention are those of the genus Lactobacillus, preferably of the species Lactobacillus casei, Lactobacillus plantarum, Lactobacillus delbrückii, such as Lactobacillus delbrückii subsp. lactis and bulgaricus, Lactobacillus fructosum, and Lactobacillus sakei. Examples of suitable sulfate-reducing bacteria according to the invention are those of the genera Desulfobacter and Thermodesulfobacterium. Examples of archaea suitable according to the invention are methanogenic bacteria such as those of the genus Methanospirillum, for example Methanospirillum hungatei. Examples of fungi suitable according to the invention are those of the genera Kluyveromyces and Penicillium, such as Kluyveromyces marxianus and Penicillium roqueforti. Examples of algae according to the invention are, for example, freshwater algae such as those of the genus Chlorella.

The above-mentioned microorganisms are predominantly present in the composition as living microorganisms, usually in an amount of about 1 x 10 to 1 x 10 ¹⁰ CFU / l.

The composition according to the invention preferably additionally comprises a biopolymer, for example a polysaccharide. Preferably, the polysaccharide is an aminopolysaccharide, preferably a chitin-based aminopolysaccharide, for example chitosan, obtainable by the action of alkalis on chitin, or a chitosan derivative, such as chitosan-lactate. The proportion of chitin-based polysaccharide in the composition is advantageously between 3 and 30% by weight, preferably between 5 and 15% by weight, based on the total weight of the composition.

In order to optimize the composition according to the invention, in particular to improve the odor, herbal extracts may furthermore be present. In principle, any mixture of herbs can be used here, as long as the effect of the composition according to the invention is not impaired. However, it has turned out that a herbal extract of plantain, hawthorn, sage, nettle, wild berry, birch or mint is particularly supportive. Of course, mixtures of the said herbs can be used, in which case at least two are present.

The composition according to the invention may contain as further active components such as enzymes, in particular proteases. It has been found that preferably such enzymes or enzyme mixtures are suitable as can be obtained from pineapple comosus, carica papaya and / or pancreas. Examples are papain, trypsin, chymotrypsin, bromelain and to name other proteases. In particular, a mixture containing at least two of said enzymes is added. Such preparations are commercially available, for example, under the trade name Wobenzym.

To stabilize, in particular, the microbiological mixture, customary media additives, such as trace elements, vitamins and salts, may be present in the composition according to the invention. These include, preferably, sodium, manganese, copper, boron, zinc, iron, sulfur, magnesium, potassium, calcium, phosphate or mixtures thereof. Suitable vitamins are commercially available (for example K3129, Sigma Aldrich). Suitable salts are, for example, salts of alkali metals, alkaline earth metals and other metals. The anions used are preferably the chlorides, bromides, fluorides, iodides, carbonates, bicarbonates, sulfates and hydrogen phosphates of these metals. As examples of alkaline earth metals, alkali metals and other metals which are in cationic form, mention may be made of calcium, magnesium, sodium, calcium, iron, ammonium, strontium and manganese.

If necessary, the composition of the invention may also contain silica.

In a preferred embodiment, the photosynthetic microorganisms and luminescent bacteria are present in a ratio of 1:10 to 1: 500, preferably 1: 100, in the composition of the invention. The concentration of the bacteria, based on the composition, preferably in a range of 2-3% by weight. Preferably, the composition of the invention contains photosensitizers or precursors thereof which can additionally assist the action of the composition by a photodynamic process. These photosensitizers may be released by microorganisms contained in the composition or added directly to the composition. The photosensitizers or their precursors are taken up by the diseased cells of the organism to be treated and accumulate in them. By exposure to light, these photosensitizers are activated and lead to the formation of singlet oxygen or other reactive substances such as radicals that damage the cells of the affected area of the skin. The cells damaged in this way are destroyed by necrosis or apoptosis. Examples of suitable photosensitizers are bacteriochlorophyll, porphyrins and their precursors, such as 5-aminolevulinic acid, the pigment released by Monascus purpurus (Pigment 3658) and the pigment released by Chlorobium limicola nadson (Cell dye 2145). Accordingly, the composition according to the invention preferably contains microorganisms which release photosensitizers or their precursors, such as Monascus purpurus, Chlorobium limicola or bacteria of the genera Erythrobacter and Pseudomonas, for example Erythrobacter litoralis and Pseudomonas fluorescens, usually in the amounts indicated above. The composition according to the invention particularly preferably contains 5-aminolevulinic acid.

Furthermore, other supporting components may also be included in the present biological composition. These include, for example, alginates, such as alginates used for O ₂ production, conjugated polymers and piezoelectric materials, and anti-inflammatory agents such as rutoside.

It has been found that the combination according to the invention of magnetotactic and phototrophic microorganisms, preferably in combination with luminescent bacteria and aminopolysaccharides, has a biostatic and / or biocidal, in particular an antibiotic and antifungal activity. The composition according to the invention can therefore be used as a biostatic and / or biocidal agent, for example as a disinfectant. Furthermore, it has been found that the composition according to the invention is suitable for the prevention and treatment of skin diseases such as dermatoses and for promoting wound healing. The composition according to the invention is also outstandingly suitable as an antibiotic or as an antimycotic and for controlling pathogenic microorganisms, for example spirochetes such as Trepanosoma. As a biological product, the composition of the invention is excellently accepted by the skin and shows no side effects.

Dermatoses that can be treated with the composition according to the invention are, for example, various types of dermatitis, ie inflammatory skin diseases caused by bacteria, viruses, parasites or fungi, such as yeasts, allergic or autoimmune skin diseases, by physical or chemical damage Conditional skin diseases, benign and malignant skin neoplasms, skin disorders due to disorders of the skin gland function and exanthematic skin diseases as a result of infectious diseases. Examples include furunculosis, anthrax, herpes, for example, herpes simplex and herpes zoster, scabies, trichophytosis, microspore, thrush, eczema, hives, burns, sunburn, acid burn, fibroma, melanoma, skin cancer, acne, seborrhea and psoriasis.

In animals, such as livestock, the composition according to the invention has proven to be extremely effective, for example, for the treatment of digital and papillomatous digital and interdigital dermatitis as well as of hoof cancer.

A decrease in the symptoms, such as the itching, and a significant improvement in the structure of the skin surface, such as the departure of dandruff and redness, are already observed after a short time, usually after a few days.

The composition according to the invention as a cosmetic preparation or as a medicament can be applied in any dosage form which is suitable for application to the skin. Preferably, the composition according to the invention is provided in aqueous form, for example in the form of a tincture, an ointment, a cream, a gel or a lotion. However, the shape of a plaster or spray is possible. The composition of the invention may also be present as a bath or shower additive. For all application forms, the composition according to the invention can be processed with the auxiliaries and additives customary in this field and suitable for the mentioned forms of application. The composition according to the invention preferably has a pH in the range of the physiological pH, for example a pH of between 5.5 and 8.0.

It is possible that the user himself may freshly prepare the composition according to the invention. So can the Composition of the invention also be present as a set. In a container, for example, the microorganisms, the herbal extracts and the aminopolysaccharide may be included. In two other containers, the salt and the enzyme or the enzyme mixture may be filled. The user can then make his own composition for the treatment of the skin surface by means of a suitable instructions for use and immediately apply it to the affected skin.

It is believed that it could also play a role for the beneficial effect of the composition according to the invention in addition to the biocidal antibiotic and antifungal effect that by the presence of magnetotactic microorganisms in the composition according to the invention, for example a gel such as a contact gel on the skin electrical or magnetic fields can be generated, which additionally promote the formation of protective substances in the skin.

The present invention will now be described in more detail in the following examples, which are given by way of illustration only and are not intended to limit the invention in any way.

Examples

example 1

Preparation of a composition according to the invention

A composition was prepared having the following ingredients:

For this purpose, the microorganisms used (commercially available or from the German Collection of Microorganisms and Cell Cultures (DSMZ), Inhoffenstraße 7 B, 38124 Braunschweig, Germany) and algae were grown in suitable media and combined in the amounts indicated above. After addition of vitamin solution, aminolevulinic acid and chitosan lactate was made up to 1 1 with a mixture of the culture media used for cultivation and stored in the incubator at 39 ⁰ C.

1 L of this stock solution was mixed with 80 g of chitosan lactate and allowed to swell for 1 day. Then, the mixture was treated several times for 5-10 minutes at 39 to 40 ⁰ C in an ultrasonic bath until a viscous mass formed. After addition of herbal extract (sage - white thorn nettle concentrate) the mass was passed through a sieve, allowed to cool and bottled.

The ointment composition thus obtained, hereinafter also referred to as RC, referred reacre ^® or reacre Agricura, as described below for the treatment of dermatitis in dairy used.

Example 2

Treatment of digital dermatitis in dairy cattle

1. Animals, material and methods

1.1 Farms, animals and their management

The study was conducted in 2005 and in spring 2006 on 5 very productive dairy farms in the federal state of Saxony, Germany. 103 cows with a total of 114 acute or chronic digital dermatitis (DD) wounds on one or more toes were included in the study. Two groups were established, the first group (number of Cows = 94, number of wounds = 105) was treated with the biological ointment prepared in Example 1 and the second group (number of cows = 9, number of wounds = 9) was treated with oxytetracycline spray (OTC) (Blauspray, Germany).

Operation 1

The first farm included a herd of 600 dairy cows, most of them Holstein-Friesian cattle. The cows were kept in a free stall, depending on the stage of lactation and partly on age. They were fed a mixed ration based on corn silage and milked three times a day in a carousel milking parlor. The feet of the cows were carved once a year by professional hoof care workers, but during the year the farm was visited every two weeks by a hoof-care worker who examined and treated lame cows. For the present study, the farm was visited twice a week (Tuesday and Friday) in July 2005, when the hoof curlers performed their routine work. Cows of different milking groups and of different ages were examined while being worked on hoof care. When DD was diagnosed, the cow was included in the study. 24 cows with a total of 25 wounds were included in the study. A cow was taken out because it was expelled from the herd before the completion of the 10-day study. Therefore, 23 cows with 24 wounds remained.

Operation 2

The second farm included a herd of 1000 dairy cows, most of them cattle of the Holstein-Friesian breed. Depending on the lactation stage, the cows were in a freewheeling stable system held. They were fed a mixed ration based on corn silage and milked three times a day in a herringbone milking parlor. The claws of the cows were checked twice a year by professional hoof care workers

(Genossenschaft Klauenpfleger eG Sachsen). For the remainder of the year, the farm was regularly visited by hoof carers who examined and treated lame cows. In August 2005, the company was visited twice a week

(Monday and Thursday) when the hoof workers did routine work. First calving heifers were examined in this herd, and if DD was diagnosed, the heifer was included in the study. 24 heifers with a total of 25 wounds were included in the study.

Operation 3

Plant 3 included a herd of about 600 dairy cows, mostly black and white. The cows were kept in a free-range stable system depending on the stage of lactation. They were fed a mixed ration based on corn silage and milked twice a day in a carousel milking parlor. Lame cows were kept in a separate group. The hooves of the cows were cut twice a year by professional hoof care workers (Genossenschaft Klauenpfleger eG Sachsen). Lame cows were examined and treated in this establishment by a trained employee. The farm was visited twice a week (Tuesday and Friday) in November 2005, when the company worker was treating the sluggish cows. Cows of different milking groups and of different ages were examined, and if DD was diagnosed, the cow was included in the study. 12 cows with a total of 13 wounds were included in the study.

Operation 4

Plant 4 included a herd of about 200 dairy cows, mostly black and white. The cows were kept in a free-range stable system. Cows that were difficult to become pregnant were kept separate, and dry cows were kept in large deep-freeze pens. The heifers were raised in another farm and brought back to the main farm shortly before the first insemination. The cows were fed a mixed ration based on corn silage and milked twice daily in a herringbone milking parlor. The hooves of the cows were treated twice a year by professional hoof care workers (Genossenschaft Klauenpfleger eG Sachsen). As in the third farm, lame cows were examined and treated by a trained farm employee. The plant was visited twice a week (Monday and Thursday) in November 2005, when the company worker was treating the lame cows. Cows of different milking groups and different ages were examined, and if DD was diagnosed, the cow was included in the study. 23 cows with a total of 30 wounds were included in the study.

Operation 5

Plant 5 included a herd of about 800 dairy cows, mostly of the Holstein-Friesian breed. The cows were kept in a free-range stall system, depending on the stage of lactation. The cows were fed a mixed ration based on corn silage and three times a day in a carousel milking parlor milked. The hooves of the cows were treated twice a year by professional hoofcutters (Genossenschaft Klauenpfleger eG Sachsen). For the remainder of the year, the farm was regularly visited by hoof carers who examined and treated lame cows. In April 2006, during one of the two annual hoof care periods, the farm was visited twice a week. Cows of different meik groups and different ages were examined when they were processed in hoof care. When DD was diagnosed, the cow was included in the study. 21 cows with a total of 22 wounds were included in the study.

1.2 Selection criteria for digital dermatitis and examination procedures

1.2.1 Selection criteria for digital dermatitis

Digital dermatitis was diagnosed when wounds on the palmar / plantar or dorsal skin immediately above the coronary margin define the clinical definition of digital dermatitis ("Delineated superficial ulceration of the bordering skin with white epithelial margin and chronic dermatitis.] Obviously often contagious.

Different, often severe lameness ") and papillomatous digital (verrucous) dermatitis (" Chronic proliferation of the dorsal and / or plantar / palmar skin, initially moist and later developing into wart-like tissue growths, mild lameness ") according to Espinasse et al. (Editions du Point Veterinaire 1984, 14-17, 1984). 1.2.2 Investigation procedure

In both groups, the claws were expertly cut before treatment. After the treatment, each cow was examined four times within two weeks. The wounds were measured by a modified ABC scoring system (Bergsten, C. Descriptive nomenclature and scoring of foot lesions at hoof trimming, work shop report part II ll ^th International Symposium on Disorders of the Ruminant Digit Parma, Italy;.. 2000) as digital Dermatitis (DD) or papillomatous digital dermatitis (PDD). Localization of the wound (dorsal, palmar and / or interdigital) and severity were classified as. The appearance of these wounds was documented in each examination with a digital camera, Konica Minolta, Dimage X21. Scaling was used on all photos to facilitate comparison and to more accurately calculate the wound score. Before the examination and treatment, the claws were washed with tap water, cleaned and dried with a cotton sponge. The wounds were treated with the composition of Example 1 and bandaged. The results were evaluated by two independent examiners and the mean scores were documented.

1.3. disease parameters

1.3.1 Wound classification

Wound scores and inflammatory parameters were recorded. The severity of the wounds was determined by the modified ABC scoring system of Bergsten et al. (supra). The wounds were visually rated 0 to 4 according to size and severity as described in Table 1. The look The wounds were documented on each scan using digital photos (Dimage X21, Konica Minolta). At each wound, a scale was created to facilitate the measurement and allow further comparisons and evaluations of the wound.

Table 1. Modified ABC wound scoring system.

1 . 3. 2 pain

The onset or absence of pain was noted by touching the wound with a cotton sponge and observing the reaction. The pain was recorded as "yes" or "no".

1.3.3 Swelling and redness

Swelling and redness were also recorded as "yes" or "no".

1.3.4 Lame

The cows were examined by a veterinarian for lameness. A five-point scoring system of 1, normal to 5, strong lame, was used (Greenough P.R. et al., Cattle Lameess, Zinpro Corporation, Eden Prairie, MN 1997). In farm 1, the lameness was assessed as the cows ran on a yard with straw. In farms 2 and 3, the assessment was made when the cows were walking on a concrete slatted floor. In farm 4, the cows were examined while walking on concrete floor, and in operation 5 the lameness was assessed as the cows ran on solid concrete before the hoof care stand and on concrete slatted floor behind the hoof care stand.

1.4 Wound score measurement

1.4.1 Selection criteria

The selection of cows with digital dermatitis was as described above under Material and Methods. 103 cows in five Enterprises were included in the experiment. Most cows were identified in routine hoof care, but some cows were also identified when they were examined for lameness. The first time a suspicious wound was detected, the leg and wound were washed with tap water, cleaned and dried with a cotton cloth. When clinically confirmed to be a DD wound, the wound was classified as described above and the test results recorded. The wound was also photographed for further assessment. The wound was retreated and examined, classified and photographed as described below on days 3, 7 and 10 after the initial examination, ie four times in two weeks, and the results were recorded.

1.4.2 Treatment

After determining that the wound was a DD wound, the wound was cleaned as described above and treated with the ointment obtained in Example 1 by applying the ointment to the wound and bandaging the foot. This was repeated after examination of the wound on days 3 and 7.

1.4.3 Determination of wound area and circumference

The area and extent of the wounds were determined from the digital photos by scanning the photos using the Analysis 2.0 program (SIS Münster Germany 2002). Interdigital wounds were excluded regardless of their type or severity because they could not be accurately measured. Cows with fairly close wounds on the same leg were excluded as no statistical independence could be achieved. Every picture was taken Scaling was calibrated on the original images so that standardized comparable data could be measured and later used for statistical evaluation. In total, the measurement included 88 cows (23 wounds from farm 1, 12 from farm 2, 10 from farm 3, 24 from farm 4 and farm 19 from operation 5).

1.4.4 Analysis

From all studies a mean and a standard deviation (SD) were calculated. Furthermore, the difference between day 0 and day 3, 7 and 10 was calculated. With an SPSS statistical program the differences in the different study days (0 → 3, 0 → 7, 0-> 10, 3 → 7, 3 → 10, 7- → 10), in the five different farms, in the treatment forms and compared with the digital dermatitis form and the papillomatous digital dermatitis form.

Further, the severity levels visually observed by the panelists during operation and the actual size of the wound area / perimeter calculated by the Analysis 2.0 program were compared. It was investigated whether the "modified ABC scoring system" according to Bergsten is a scoring model suitable for the assessment of Mortellaro's disease.The migration results in the biopsies (see below) were compared with the wound area / wound circumference, and for operation 5 with the Analysis 2.0 program The microbiological results were also compared with measurements of wound area / circumference and the visual assessment of severity by the inspectors in the farms (see above) .All wounds also underwent a clustering program There is an increased risk for a wound within a holding if there is already a wound.

1.5 Microbiology

1.5.1 Histological and bacterial investigations

1.5.1.1 Tissue samples

For histological and bacterial examination 13 cows from farm 5 with 13 wounds were selected. The wounds included 3 wounds classified as ulcerative DD, grade 1 according to the modified ABC scoring system, two wounds classified as ulcerative DD, grade 2, two wounds classified as ulcerative DD, grade 3, two Wounds classified as grade 1 papillomatous DD and three wounds classified as grade 2 papillomatous DD (see Table 1).

On the first day of the study and on day 10, 13 biopsies were taken, using a sterile punch biopsy needle with a diameter of 0.6 mm (Kruuse, Hamburg, Germany). The punching station was marked and photographed for later comparison with a digital camera. The biopsies taken before treatment were taken in the diseased area at the border to healthy tissue. The biopsy punch after treatment was not in the same place as the pre-treatment biopsy. If there was still diseased tissue, the punch was made at this point. If no diseased tissue was seen on day 10, the biopsy was taken from where the wounds were before complete healing based on the information from the previously taken photos. 1.5.1.2 Processing Biopsy and Preparation for In Situ Fluorescence Hybridization (FISH)

The biopsies were immediately stored and fixed for at least 24 hours at 4 ^° C. with 3.7% (v / v) formaldehyde in PBS (pH 7.4) with 50% (v / v) ethanol. For embedding, cold-curing resin was used (Moter et al., Microbiology 1998, 144: 2459-2467). Histoblocks were cut with a rotary microtome (medium, type DDM 0036) with steel knives with carbide cutters. Tissue sections (5μm) were smoothed on sterile water, placed on silanized slides (Super Frost, Medium, 3-aminopropyltrimethoxysilane, Sigma) and stored in plastic containers at room temperature.

1.5.1.3 Bacterial strains

The following bacterial strains were used as control strains in all FISH experiments: E. coli (ATCC 25922), Treponema vincentii (ATCC 33580), Treponema brennaborense (DD 5/3) (DSM 12168), and Treponema denticola (ATCC 33521). The bacterial strains were treated with 3.7% formaldehyde in PBS containing 50% (v / v) ethanol (pH 7.4) (v / v), and stored at 4 ^0C.

1.5.1.4 Oligonucleotide probes

As oligonucleotide probes for FISH, synthetic oligonucleotide sequences labeled with a fluorescent dye such as FITC (fluorescein isothiocyanate) or cyanine dyes (Cy3, Cy5) were used. The oligonucleotide sequences were copies of the ribosomal sequence of the bacterium, but the probe were DNA sequences. The 16S rRNA gene provides the greatest accuracy in identifying most microorganisms.

The probe EUB338 (5 'GCTGCCTCCCGTAGGAGT 3'), which is specific for bacteria, was used to visualize all bacterial popuiacion in the biopsy slice. Probe TRE I (5 ^' ACGCAAGCTCATCCTCAAG 3') is specific for Group I oral Treponema. The TRE II and DDK2.4 probes were used to specifically detect the respective group of oral Treponema first described by Choi et al. (Choi et al., International Journal of Systemic Bacteriology 1997, 175-181). The probes were commercially synthesized (Biomers, Ulm, Germany) and labeled at the 5 'end with either fluorescein or fluorochrome Cy3 (indocarbocyanine) (kindly provided by A. Moter, Berlin, Germany).

1.5.1.5 In situ fluorescence hybridization (FISH)

The specificity of the oligonucleotide probes was optimized on bacterial control strains as positive or negative controls by varying the stringency of the hybridization buffer. The specific hybridization in each later experiment on the tissue sections was controlled by parallel hybridization with bacterial control strains. The best specificity was obtained with a hybridization buffer containing 0% formamide, 0.9 M NaCl, 20 mM Tris-HCl, pH 7.3, and 0.01% SDS. The hybridization buffer was mixed with 5 pmol of the respective probe and applied to the tissue section preheated. The slides were incubated for 2-3 hours in a dark humid chamber at 49 ° C, with rinsed in distilled water and air dried. Covering was done with a VECTASHIELD Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA ₇ USA). For all tissue sections, EUB338 was used. One sample of each wound scoring group (n = 5 groups) was further hybridized with TREI (Cy3) or DDK24 (Cy3) and EUB338 Cy5.

1.5.1.6 Microscopy

The visualization of bacteria in hybridized sections was performed on an epifluorescence microscope (Axioplan 2 Imaging MOT, Carl Zeiss, Jena, Germany). The microscope was equipped with a 100 W high pressure mercury lamp (HB103, Osram) and 10x, 40x and 100x objectives (Zeiss). Eng filter sets F31, HQ-F41-007 and HQ-F41-001 (AHF, Analysentechnik, Tübingen, Germany) were used to analyze the DAPI, FITC and Cy3 signals. Digital images were taken with an AxioCam HRc. Image acquisition and processing was done with a standard software package (Axiovision 4.1) supplied with the device. Z-stacks of interesting areas were further processed with the AxioVision Deconvolution Software Module (Zeiss).

1.5.1.7 Measuring migration and sensitivity of the bacteria to the ointment of the invention

The aim of this study was to measure the sensitivity of Treponema bacteria to ointment medication. Since most Treponema species are difficult to cultivate, the sensitivity to the medication, unlike other bacteria found in the biopsies, can not be tested by culture. With the FISH technology, the Bacteria are stained according to the group of oral Treponema to which they belonged. The combination of FISH and epifluorescence microscopy allowed overlapping digital images of the surface of the biopsy to be made on the slide. The invasion path of the bacteria into the epidermal tissue was determined with the measurement program of the standard software package Axiovision 4.1, and the SPSS statistics program compared the results before and after the treatment to determine if the Treponema bacteria were sensitive to the medication.

1.5.2 Examination of the bacteria and sensitivity test

1.5.2.1 Processing of biopsy and cultivation

A total of 13 biopsies were obtained from the same cows, using the procedure mentioned above in the section Microbiology Tissue Samples. The biopsies arrived in a PORT-T tube in the laboratory. On the day of arrival, the biopsies were cut and ground under sterile conditions in a mortar. The material was applied to the following growth media. Columbia Blood Agar, Boiled Agar, Endo Agar, CNA Agar, Columbia Agar with Kanamycin, BHI Broth and Thioglycollate Broth. Agar plates and liquid broth were cultured aerobically at 36 ° C for 24-48 hours, Schaedler agar and Columbia agar with kanamycin anaerobically at 36 ° C for 24-72 hours. The evaluation took place after 24 hours, 48 hours and 72 hours. 1 . 5. 2. 2 bacteria egg solates

The following bacterial species were isolated: Streptococcus spp. (non-hemolytic streptococcus), Staphylococcus spp (coagulase-negative staphylococcus), enterococcus, coryneform rods, enterobacteria, spores, fungi, yeast, Prevotella spp. (P. bivia, P. corodens, P. intermedia, P. loeschii, F. buccalis, P. melaninogenica), Peptostreptococcus spp. (Ps. Anaerobicus, Ps. Asacharolyticus), Porphyrmonas spp. (Po. gingivalis), Proteus, Veilonella spp, Propionibacterium acnes. Pure cultures were frozen in Cryobanch tubes.

1.5.2.3 Sensitivity tests (agar diffusion test).

A Brucella agar was prepared by adding 43g Brucella agar (powder), 1 ml hemin stock solution, 1 ml Vitamin K and 1 1 distilled water were mixed, were autoclaved and then cooled to 50 ⁰ C. Subsequently, 25 ml of sheep's blood were added.

Without RC (20 ml agar) agar mixture; sensitivity for the test, the following amounts of agar and inventive composition ^(® reacre, RC) have been used 125 μl RC and 19.875 ml agar; 250 μl RC and 19.75 ml agar; 500 μl RC and 19.50 ml agar;

1 ml RC and 19.00 ml agar; 2 ml RC and 18.00 ml agar; and 4 ml RC and 16.00 ml agar. The frozen bacterial cultures found in the biopsies were inoculated for the sensitivity test on the Brucella agar mixture.

2 μl of the inoculum was added to each plate. As controls, two plates were cultivated without RC under C0 ₂ atmosphere and anaerobically. The agar plate colonies were monitored at 24 and 48 h to determine if colony forming units (CFUs) were present or not. 1.5.3 Specific Treponema brennaborense on-examination

The purpose of this study was to determine whether the microbiological composition of the invention has an inhibitory effect on the growth of Treponema bacteria.

1.5.3.1 Bacterial strain

For this experiment, the strain Treponema brennaborense (DSM 12168) was chosen. This strain was first isolated from a cow suffering from acute digital dermatitis (Schrank, K., et al., Int. J. Systemic Bacteriology 1999, 49, 43-50). The strain for this experiment was kindly provided by the University Hospital Charite, Humboldt University in Berlin.

1.5.3.2 Cultivation procedure

The cultivation of the Treponema strain was carried out with several passages in an OMIZ-PAT medium (Wyss, C. et al., Int. J. Systemic Bacteriology 1996, 46: 745-752) and in a BSK-H medium (Sigma B 3528 ). This was followed by an in vitro incubation with various concentrations of the inventive composition (RC) prepared in Example 1. The following concentrations were used;

a) control culture without addition of RC, 900 μl Treponema culture and 100 μl culture medium, b) 900 μl Treponema culture and 100 μl RC, c) 900 μl Treponema culture, 50 μl RC and 50 μl culture medium, d) 900 μl Treponema culture , 25 μl RC and 75 μl culture medium. The cultures were incubated at 37 ° C under strictly anaerobic growth conditions. The samples were collected 1 h, 3 h, 12 h and 24 h after incubation and subjected to in situ hybridization. All steps were done twice.

1.5.3.3 Sample fixation

The Treponema cultures were washed several times with PBS, pH 7.4, and fixed at 4 ° C for 4 hours in 4% paraformaldehyde solution. A second series of samples was fixed in an ethanol solution. All samples were stored at -20 ° C.

1.5.3.4 In situ fluorescence hybridization

For the hybridization step, silanized 8-well slides were used. Standard hybridization buffer and wash buffer were added with 10 mM TRIS / HCl, pH 7.4, 0.9 M NaCl, 600 μl ddH ₂ O and 2 μl 10% SDS. The slides were incubated for 4 hours in a dark humidity chamber at 46 ^° C., rinsed with distilled water and air dried. For covering a mounting medium and a cover glass was used. For all tissue sections EUB-Mix, DDK5 / 3, and TRE I were used.

1.5.3.5 Microscopy

The bacteria in the hybridized sections were visualized on an epifluorescence microscope (Axiskop, Carl Zeiss, Jena, 100x / 01-Immersionsobj ektiv Neofluar, Germany). The microscope was equipped with a high pressure mercury lamp (HBO 50W, Osram) and a special filter for Cy3. 2 results

2.1 animals

A total of 103 cows with 114 wounds were included in the study. 13 cows had wounds on both hind legs at the same time. Two groups were formed, the first group (number of cows = 94, number of wounds = 105) was treated with the ointment according to the invention, the second group (number of cows = 9, number of wounds = 9) was treated with oxytetracycline spray (OTC spray) ( Blauspray, Germany).

The wounds were mainly on the hind legs (hind legs to front legs, 109/5). The distribution of wounds in groups 1 and 2 together was: left foreleg (LF) 2, right foreleg (RF) 3, left hind leg (LH) 56 and right hind leg (RH) 53. When considered separately, the distribution for group 1 was: LF 2, RF 2, LH 51, RH 50, and for Group 2: LF 0, RF 1, LH 5, RH 3.

2.2 Investigation procedure

2.2.1 Erosive and papillomatous digital dermatitis

A total of 84 wounds were classified as erosive digital dermatitis and 30 wounds as a papillomatous form. Table 2a, b, c below shows the location of the wounds on the claws. Table 2a. Localization of the wounds on the claws

DD = the erosive form and PDD = the papillomatous form

Table 2b. Localization of the wounds on the claws treated with test ointment (reacre®)

DD = the erosive form and PDD = the papillomatous form

Table 2c. Localization of the wounds on the claws treated with OTC spray

DD = the erosive form and PDD = the papillomatous form

2.3 Disease parameters

2.3.1 Severity of the wounds

A total of 84 erosive DD wounds and 30 PDD wounds were registered. 76 DD 29 and PDD wounds were treated with the ointment according to the invention obtained in Example 1 (reacre ^®) treated (group 1) and 8 DD-1 wounds and PDD-wound treated with OTC Spray (group 2). The data in Table 3a, b, c show the change in wound score from day 0 to day 10 of the treatment. The results suggest that most often seen is an improvement of one to two degrees of severity.

The table shows the total number of wounds included in this clinical study and their visible change in wound score from day 0 (onset of clinical trial) to day 10 (end of clinical trial).

DD = erosive form of digital dermatitis;

PDD = papillomatous form of digital dermatitis;

Number = number of wounds;

Grade = severity of wounds, classification from 0 (cured) to 4 (very severe).

Table 3b. Change in wound grade from day 0 to day 10 when treated with reacre®

DD = erosive form of digital dermatitis;

PDD = papillomatous form of digital dermatitis;

Number = number of wounds;

Grade = severity of wounds, classification from 0 (cured) to 4 (very severe).

Table 3c. Change in wound grade from day 0 to day 10 when treated with OTC spray

DD = erosive form of digital dermatitis;

PDD = papillomatous form of digital dermatitis;

Number = number of wounds;

Grade = severity of wounds, classification from 0 (cured) to 4 (very severe).

The results show that in Group 1, 97 wounds (92.4% of the wounds) improved by one or more degrees of severity depending on the form, of which 52 (49.5%) completely healed, in 7 (6.7%) cases the wound score remained unchanged and in one (1%) patients the severity of the wound worsened from day 0 to day 10.

Thirty cases (total number of wounds = 76) of erosive digital dermatitis completely healed, 8 wounds had score 3 on day 0, 10 wounds had score 2, 8 wounds had score 1, and 4 had wound scores 0.5 on day 0.

22 cases (total number of wounds = 29) of papillomatous digital dermatitis completely healed, 5 wounds had score 3 on day 0, 10 had score 2, and 7 had score 1.

Of the 52 healed wounds that completely healed, 8 wounds healed after one treatment, 23 wounds healed after two treatments and 21 wounds healed after three treatments.

2.3.2 pain

In group 1, 47% of the wounds showed a decrease in the pain response, 16% showed no change in the pain response, 1% of the patients increased in pain, and 36% were completely free of pain from the 0th to the 14th day. The pain response was generally the first or second Dressing change negative. Pain-free wounds were more commonly observed in operations 3 and 4. This is probably due to the fact that the wounds on both farms were smaller than those of the first two farms. In group 2, the decrease in pain response was 56%. 44% of the patients showed no pain during the entire experiment.

2.3.3 Swelling and redness

Irrespective of the type, no redness was registered in any of the 114 patients with digital dermatitis. In 10% of the wounds (12 cows) the swelling decreased from the first day of treatment to the last day of treatment. In these 8 cows, the swelling disappeared in the first three or four days of treatment. In this group, 2 patients were also diagnosed with Limax, and one patient had limax and a sole ulcer at that time, so the result could be false-positive.

1 cow (1%) had swelling throughout the experiment. The leg was swollen to the tarsal root and a fetal joint infection was diagnosed. In addition, Limax was also diagnosed in this patient, which could give a false positive result. In 1 patient (1%) the swelling increased. In 88% of the cows, no swelling was observed. In group 2, no swelling was observed in 100% (9 wounds) during the experiment.

2.3.4. lameness

Lameness decreased in 59% of the cases, in 10% the lameness did not change, 3% showed increasing lameness and 28% of the lameness Cows were lame free throughout the experiment. Lameness wounds were usually observed on the second or third visit. Limax was also diagnosed in 11 cases, which could provide a false-positive degree of lameness. In 4 of these 10 patients, Limax did not occur with Mortellaro's disease. The other 7 patients had both Limax and Mortellaro's disease. All wounds were classified as papillomatous dermatitis. Which disease is primary and which is secondary, can not be determined in this experiment. In 2 cases there was also a sole ulcer, which could give a false positive lame response. In group 2, lameness decreased in 89% of cases and 11% was lame-free throughout the experiment.

2.5 Microbiology

The identification of bacteria in digital dermatitis wounds and their migration depth was carried out by hybridization with fluorescently labeled oligonucleotides and in situ fluorescence hybridization (FISH). FISH enables the visualization, identification, numbering and localization of organisms in tissue and their phylogenetic identification by means of oligonucleotide probes directed against the 16S rRNA, and makes it possible to test the sensitivity of the bacteria to the composition according to the invention.

2.5.1 Histological examination and examination of the bacteria

In order to determine the migration of Treponemas into the tissue, the spatial distribution of spirochetes in the Epidermis examined and measured the migration depth of the bacteria. Good tissue resolution provided accurate histological orientation in the tissue. A high signal-to-noise ratio and autofluorescent background allowed the localization of bacteria in the epidermis. All 26 biopsies went through the treatment steps. The visual macroscopic wounds were typed and classified as follows: three wounds were designated DD with severity 1, three wounds as DD with severity 2, two wounds as DD with severity 3, two wounds as PDD with severity 1 and three as PDD with severity 2 classified. Due to the three-dimensional localization of the papillae relative to the tissue orientation, it was impossible to measure the migration of the bacteria into the tissue in papillomatous digital dermatitis, therefore these ten samples were excluded from the study (Figures 13a, b). The remaining 16 samples were included in the result and statistically examined.

2.5.1.1 Measurement of Migration and Sensitivity of Bacteria to the Composition of the Invention

Table 4 shows the scheme used to study the digital dermatitis wounds, as well as the non-parametric statistical assessment. Hybridization with EUB 338 and FISH revealed that a variety of different microorganisms were found mainly in the debris at the surface of the wounds (data not shown). At pre-treatment biopsies, the surface of the tissue appeared to be very irregular after regular biopsy after treatment. Autofluorescent erythrocytes were also commonly found in the debris, especially in the ulcerous form. In the papillomatous form, the erythrocytes were not found regularly. The Autofluorescence of the drug was very evident in the post-treatment biopsies. It was also impossible to distinguish the bacteria of the drug from the bacteria that might be left over from the infection.

Simultaneous hybridization with probe DDK 2.4 showed that a large number of spirochetes were in the debris, but the majority was localized in the tissue (results not shown). In the ulcerous form, the spirochaetes appeared to migrate from the surface to the dermis as a wedge. Furthermore, the bacteria appeared to be in bundles around the tissue cells. However, there were no spirochetes in the cells. In the papillomatous form, the spirochete bundles appeared to be in the middle of the papilla. In both forms, the bacteria in the tissue sometimes appeared in a ring with no connection to the surface.

Further analyzes were done by simultaneously using different combinations of specific oligonucleotides. This showed a stratification of various treponemal phylotypes in the tissue. Members of group I and II oral treponema are regularly found on the biopsies. Group I and Group II spirochaetes are among the bacteria that try to invade, but they are not the only precursors. Individual spirochaetes are the deepest in the tissue, but it is not yet known to which Treponema species they belong. Table 4. Penetration depth of the treponema in the tissue before and after treatment with ointment under dressing

The visual clinical score is also shown.

* indicates a significant improvement.

Cow # = number of the subject. Degree = degree of visual

Wound cores. (SD) = standard deviation

2.5.2 Bacterial examination and sensitivity test

2.5.2.1 Biopsy treatment, cultivation and bacterial isolate

In order to examine the bacterial sensitivity found in the biopsies against the composition tested (reacre® agricura), the bacteria were cultured. Microscopic examination before incubation revealed many cells, Gram-positive cocci, Gram-positive and Gram-negative rods, and sometimes fungi and coagulase-negative Staphylococcus. An evaluation after 24 h under aerobic incubation showed mostly no growth. In four samples, growth of Enterococcus, spores, coryneform rods, coagulase-negative Staphylococcus, and Enterobacteriaceae was reported see. An evaluation after 24 h under anaerobic conditions gave 11 samples without growth, and in three samples Peptostreptococcus spp. (Anaerobicus), coryneform rods, coagulase-negative staphylococcus, spores and fungi.

A 48 h evaluation revealed in all samples a mixed flora of aerobic microorganisms, including coryneform rods, Enterobacter, Enterococcus, spores, Streptococcus spp., Staphylococcus spp., Nonhemolytic Streptococcus spp., Prevotella spp. (Loeschii), Peptostreptococcus spp. (Ps. Anaerobicus, P. asacharolyticus), Proteus and coagulase-negative Staphylococcus. An evaluation after 48 h showed no growth in two samples, the remainder showed a mixed flora of anaerobic microorganisms; Porphyrmonas spp. (P. gingivalis), Prevotella spp. (Corodens, P. intermedia, P.loeschii, P. buccalis, P. melaninogenica), Peptostreptococcus spp. (P. anaerobicus, P. asacharolyticus), Veilonella spp, spores, Propionibacterium acnes.

2.5.2.2 Sensitivity test (agar diffusion test)

Sensitivity tests were done with the following bacteria; Streptococcus spp., Staphylococcus spp., Enterococcus, Prevotella spp. (P. intermedia, loeschii, buccae, corodens, bivia), Porphyrmonas spp. (P. gingivalis) and

Peptostreptococcus spp. (P. anaerobicus, asacharolyticus). There are microorganisms in the composition of the invention which grow after mixing with agar. The species to be tested sometimes have a slow one

Growth rate and should therefore be cultured for more than 24 h, which leads to a greater growth of microorganisms in the drug. Table 5 shows at which concentration of the tested composition (reacre®) inhibits the growth of the tested bacteria.

For all anaerobic bacteria tested, a 1 ml dose of medication inhibits the formation of colony-forming units (CFU) by 55%, 2 ml of CFU inhibition by 79%, and 4 ml of inhibition by 100%. Table 6 describes the inhibitory concentration for CFU for all bacteria tested.

Table 5. Anaerobic Sensitivity Test

+ = CFU, - = no CFU. Table 6. Anaerobic Sensitivity Test

+ = CFU, - = no CFU;

The above studies demonstrate the effect of the microbiological composition of the invention in the treatment of digital dermatitis in dairy cattle from five large dairy herds with digital dermatitis wounds, taking into account the type, size, location and severity of the wounds. Of the wounds treated with the ointment of the present invention, 49% completely healed within a two-week period, and 92% of all wounds improved one or more degrees of severity in the same time period. In an agar diffusion test, which examined the sensitivity of the bacteria found in the biopsies to the composition according to the invention, a marked biostatic / biocidal effect of the composition on the bacteria was shown. In situ fluorescence hybridization (FISH) of the biopsies showed a significant decrease in the migration depth of the bacteria after treatment with the composition of the invention. Overall, a 92% improvement in clinical disease pattern was seen with the composition of the present invention, and an improvement in bacterial migration into the tissue was seen in 38%. Example 3

Test for antimicrobial effect

3.1. Preparation of a composition according to the invention for detecting an antibiotic or antifungal activity

1 l of the stock solution obtained in Example 1 was heated in an ultrasonic bath at 39 - 40 ⁰ C, and then 20 g

®

Sea salt, 10 g of Epsom salt and 2 g Wobenzym stirred in succession stirred. After dissolving the additives, 15 ml of a 5% chitosan lactate solution and a few drops of herbal concentrate were added.

The antibiotic and antifungal activity of the composition according to the invention, also referred to below as RC2, was tested with the microorganisms indicated in Table 7 below.

Table 7

Table 7, cont.

* German collection of microorganisms

** Collection of the Institute of Hygiene and Environmental Medicine

The 5 fungal cultures are from the genera Cladosporum, Penicillium and Aspergillus and include molds that belong to the black spores or the highly sporulating, psychophilic and thermophilic Zellulosezersetzern. The bacterial cultures are the species Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus faecium and Rhodococcus rhodochrous.

3.2 Cultivation of the test organisms

The different bacterial strains were grown in 60 ml Caso broth (Merck 1.05459) 12 hours at 37 ⁰ C precultured. In each case 1 ml of the overnight culture (= working culture 1-5 x 10 ⁸ CFU / ml) was added in 150 ml pre-tempered (45 ° C +/- 1 ° C), still liquid Caso agar, which was used as a top layer after cooling for uptake the test piece served.

The yeast Candida albicans was spiked for 3 days on malt extract agar (MEA) consisting of 25.2 g malt agar; Difco 211220, 5.5 g Bacto agar; Difco 214010, 750 μl of trace eluent solution and 750 ml of double-distilled water.

The yeast suspension was adjusted to a Mc Farland standard of 0.5, which corresponds to a cell number of about 1.5 x 10 ⁸ CFU / ml.

The molds were also pre-cultivated on malt extract agar (MEA) based on the Swiss Standards Association (SNV 195921). From each fungal strain, one or more 8-day fungal cultures grown on slant agar tubes incubated at room temperature were washed off with 2 ml of sterile 0.9% NaCl solution and combined. After filling to 10 ml with NaCl solution, the suspension was centrifuged at about 3,000 g for 5 min, the supernatant was decanted and the spore pellet resuspended in 10 ml NaCl solution. The spore counts were counted microscopically in a counting chamber according to Thoma and adjusted after centrifuging again and decanting the supernatant by adding appropriate amounts of sterile 0.9% NaCl solution to a number of about 10 ⁷ spores / ml.

3.3 Investigation Methods

The inhibitor assay was performed in Petri dishes filled with 15 ml of agar medium.

For all studies initially largely nutrient-free water agar was used with an addition of dipotassium hydrogen phosphate 2.5 g / l, which should simulate a starvation of microorganism cultures, as it is given in the growth of indoor (wall) surfaces in nature. Due to the weak or absent growth of most bacterial cultures and yeast, these approaches were additionally repeated in a second series of tests under optimal nutrient conditions.

For this purpose, the Petri dishes were instead of Wasseragar with 15 ml of Caso agar medium from. Merck No.: 1.05458) with an addition of 2.5 g / l dipotassium hydrogen phosphate (Merck No .: 5104) and 2.5 g / l dextrose (Difco No. 0155-17-4), which also contained 10 ⁸ cells of the various test bacteria / fungi per 150 ml.

An antimicrobial effect is given when inhibition of bacterial growth takes place despite favorable growth conditions, both in gram-positive and in gram-negative bacterial strains and / or a fungal culture.

3.3.1 Module 1 Agar diffusion test

A modified agar diffusion test (Wallhäuser) was used to demonstrate the basic antimicrobial activity of the composition according to the invention. To this end, pure cultures of the test organisms listed in 3.1 are investigated in parallel experimental batches suspended in agar medium.

To take each test liquid, three holes (diameter 1 cm) were punched under aseptic conditions into each agar plate with a cork borer.

Each 400 μl of the composition of the invention to be tested was placed in these stirrers of the agar medium. When Controls were each a plate with sterile water as a test suspension and a plate without additives (growth control) and a sterile control.

The test organisms were light (L) at room temperature and incubated at 25 ⁰ C in the dark (D) in the incubator and the growth dsr bacterial and fungal cultures after 24 48- 96-, 144-, 216- and 336-hour Incubation time visually and microscopically examined and recorded. The investigation was carried out in the dark in order to prove the applicability of the composition according to the invention even without daylight, for example in basements.

3.3.2 Module 2 Germ carrier test

The germ carrier test was carried out in accordance with DIN EN ISO 20645: 2005. Petri dishes, which were suspended with 15 ml of water agar medium in the 10 cells of the various test bacteria / fungi per 150 ml, were also used to hold the germ carriers impregnated with 400 μl of test liquid (filter paper 3 × 3 cm ² ).

After solidification of the agar medium, one tissue specimen per Petri dish was placed with sterile forceps and incubated at room temperature in the light or at 25 ° C in the incubator (Figure 1).

As test organisms, the ten organisms listed under 3.1 were also used. The test organisms were incubated as above in light and in the dark, and after 24, 48, 96, 144, 216 and 336 hours of incubation, growth was visually and microscopically examined and recorded. in this connection particular attention was paid to inhibition sites and growth under the tissue specimen.

3.4 results

3.4.1 Agar diffusion test: growth under nutrient limitation

The results of the growth tests under conditions of nutrient limitation are summarized for the bacterial test organisms in Table 8 and for the fungi (yeast and molds) in Table 9.

Table 8

* Growth: ++ strong; + weak; - no growth; (-) not clear; (rcw) growth of microorganisms of the composition of the invention;

RC2 / L: Test of the composition according to the invention under light RC2 / D: Test of the composition according to the invention in the dark Table 9

* Growth: ++ strong; + weak; - no growth, - (-) unclear, -

(rcw) growth of microorganisms of the invention

Composition;

RC2 / L: Test of the composition according to the invention in light

RC2 / D: Test of the composition according to the invention in the dark 3.4.2 Test batch with test specimens: Growth time with nutrient limitation

The results of the growth tests under conditions of nutrient limitation are summarized for the bacterial test organisms in Table 10 and for the fungi (yeast and molds) in Table 11.

Table 10

* Growth: ++ strong; + weak; - no growth; (-) not clear;

(rcw) growth of microorganisms of the invention

Composition;

RC2 / L: Test of the composition according to the invention in light

RC2 / D: Test of the composition according to the invention in the dark

Table 11

* Growth: ++ strong; + weak; - no growth; (-) not clear;

(rcw) growth of microorganisms of the invention

Composition;

RC2 / L: Test of the composition according to the invention in light

RC2 / D: Test of the composition according to the invention in the dark

3.4.3 Growth with additional nutrient supply of the test organisms

For the bacteria and yeasts, the approaches were repeated with optimal nutrient supply of the test organisms. The growth of the bacterial and fungal cultures was visually and microscopically examined and recorded after 20, 48 and 96 hours of incubation. The results of the

Agar diffusion tests and the test on the specimens are summarized in Tables 12 and 13 below.

Table 12

* Growth: ++ strong; + weak; - no growth; (-) not clear;

(rcw) growth of microorganisms of the invention

Composition;

RC2 / L: Test of the composition according to the invention in light

RC2 / D: Test of the composition according to the invention in the dark Table 13

RC2 / L: Test of the composition according to the invention under light RC2 / D: Test of the composition according to the invention in the dark Examination of the antimicrobial composition according to the invention showed a marked antibacterial activity both in the suspension test and in the germ carrier test compared to the test organisms Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus faecium and Rhodococcus rhodochrous. Antifungal activity has been demonstrated in the molds Cladosporium cladosporioides, Penicillin ™, crysogenum, Aspergillus versicolor and Trichoderma harzianum. Both preparations had no influence on the growth of the yeast Candida albicans tested. Essentially, no influence of light could be detected; the batches incubated in the light at room temperature showed no different inhibitory effects compared to the batches incubated in the dark (incubator at 25 ° C).

It can also be seen from the experiments that the antimicrobial action is predominantly due to agar-diffusible, water-soluble substances which, in terms of their action, can be termed antibiotics (data not shown). In addition, it was found, in particular from the experiments with nutrient limitation in the molds investigated that a competition for the remaining nutrients affects the growth of the test organisms. This results, for example, that cladosporioides having at Cladosporium after 336 hours of incubation in an incubator at 25 ⁰ C, the growth control a stronger colony density to large short, no longer inhibited fungal spores while in the medium with the inventive composition with distance from the active ingredient, dark-colored colonies grow out (data not shown). In addition, the results show that at the prolonged incubation time (216, 336 hours) of the range of the Ausstichzylinder with the test liquid with nutrient limitation causally emanating from the inventive composition fungal growth (rcw). The identification of the fungi was found to be Fenicillium roquaforti and Paecilomyces variotil.

In the experiment under optimal nutrient conditions, it was also found that the growth of a yeast overgrowing the entire plate was very rapid from the test substrate.

Claims

claims

Composition, characterized in that it comprises at least one magnetotactic microorganism and at least one phototrophic microorganism.

2. The composition according to claim 1, characterized in that the at least one magnetotactic microorganism is selected from magnetotactic bacteria, in particular the genus Magnetospirillum.

3. The composition according to claim 1 or 2, characterized in that the at least one phototrophic microorganism is selected from purple bacteria, green sulfur bacteria, green non-sulfur bacteria, cyanobacteria, Prochlorophyten and bacteria of the family Heliobacteriaceae and mixtures thereof.

4. Composition according to one of claims 1 to 3, characterized in that the composition comprises at least one microorganism from the group of luminescent bacteria.

5. The composition according to claim 4, characterized in that the luminescent bacterium is selected from bacteria of the genera Photobacterium, Vibrio, Shewanella, Photohabdus, Pseudomonas or Beneckea or mixtures thereof.

Composition according to any one of Claims 1 to 5, characterized in that the composition comprises at least one aminopolysaccharide.

A composition according to claim 6, characterized in that the aminopolysaccharide is a chitin-based polysaccharide.

8. The composition according to claim 6 or 7, characterized in that the aminopolysaccharide is chitosan or a derivative thereof, in particular chitosan lactate.

Composition according to any one of Claims 1 to 8, characterized in that the composition contains a herbal extract.

10. The composition according to claim 9, characterized in that the herbal extract ribwort plantain, hawthorn, sage, nettle, wild berries, birch or mint or mixtures thereof.

11. The composition according to any one of claims 1 to 10, characterized in that the composition further comprises at least one enzyme, in particular from the group of proteases.

12. The composition according to claim 11, characterized in that the enzyme from pineapple comosus, Carica papaya and / or pancreas originate.

13. The composition according to claim 11 or 12, characterized in that the enzyme is selected from papain, trypsin, chymotrypsin, bromelain or mixtures thereof.

Composition according to any one of Claims 1 to 13, characterized in that the composition further comprises trace elements.

15. The composition according to claim 14, characterized in that the trace elements are selected from sodium, manganese, copper, boron, zinc, iron, sulfur, magnesium, potassium, calcium, phosphate or mixtures thereof.

Composition according to any one of Claims 1 to 15, characterized in that the composition additionally comprises salts.

17. The composition according to claim 16, characterized in that the salts are selected from metal salts, in particular alkali metal and alkaline earth metal salts.

18. A composition according to claim 16 or 17, characterized in that the salts are selected from the chlorides, bromides, fluorides, iodides, carbonates, bicarbonates, sulfates or hydrogen phosphates of the metals.

19. The composition according to any one of claims 1 to 18, characterized in that the phototrophic microorganisms and the luminescent bacteria in a ratio of 1: 10 to

1: 500, preferably 1: 100 are included.

Composition according to any one of Claims 1 to 19, characterized in that the total mass of microorganisms in the composition is in the range from 2 to 3% by weight, relative to the total weight of the composition.

21. A composition according to any one of claims 1 to 20, characterized in that they, in mixture with conventional Additives, in the form of a tincture, ointment, cream, lotion, a gel or a bath or shower additive is present.

Composition according to any one of Claims 1 to 21, characterized in that it has a content of photosensitizers.

23. The composition according to any one of claims 1 to 22 as a human or veterinary drug.

24. Use of a composition according to any one of claims 1 to 22 for the manufacture of a human or veterinary medicament for the prevention or treatment of dermatoses and / or for promoting wound healing.

25. The use according to claim 24, wherein the dermatoses are selected from bacterial, viral, parasitic or fungal inflammatory skin diseases (dermatitis), allergic or autoimmune related

Skin diseases, skin diseases caused by physical or chemical damage, benign and malignant skin neoplasms, skin disorders caused by disorders of the skin gland function and exanthematic skin diseases as a result of infectious diseases.

26. Use according to claim 24 or 25, wherein the dermatoses are selected from dermatitis, furunculosis, anthrax, herpes, for example herpes simplex and herpes zoster, scabies, trichophytia, microsporion, thrush, eczema, hives, burns, sunburn, acid burns, fibroma, Melanoma, skin cancer, hoof cancer, acne, seborrhea and psoriasis.

27. Use according to any one of claims 24 to 26, wherein the medicament is in a form suitable for topical application, in particular in the form of a tincture, an ointment, a cream, a lotion, a gel or a plaster.

28. Use of a composition according to any one of claims 1 to 22 for the preparation of an antibiotic and / or antimycotic.

29. Use of a composition according to any one of claims 1 to 22 as a cosmetic preparation.

30. Use of a composition according to any one of claims 1 to 22 as a biostatic and / or biocidal agent.