WO1997006811A1

WO1997006811A1 - Protection against pathogenic microorganisms

Info

Publication number: WO1997006811A1
Application number: PCT/GB1996/001936
Authority: WO
Inventors: Staffan Kjelleberg; Patricia Conway; Roger Hammond; Anna Joborn; Christer Olsson; Allan Westerdahl
Original assignee: Ewos Ab; Bowman, David, Scott
Priority date: 1995-08-11
Filing date: 1996-08-08
Publication date: 1997-02-27
Also published as: JPH11511966A; GB2319728A; NO980550L; CA2228521A1; NO980550D0; GB9801913D0; AU6706096A; SE9502809D0

Abstract

The present invention relates to a bacteriostatic and bactericidal Carnobacterium. In particular one having the accession number DSM 10087 as deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH and/or an inhibitory compound produced by said strain.

Description

Protection Against Pathogenic Microorganisms

The present invention relates to a nutritionally and prophylactically valuable product to improve the gut microbiological flora in mammals, including man, and in fish, shellfish and mollusces.

It is recognised that the status of the microbiological flora in the gut of an animal may have a profound effect on the wellbeing of the animal. Poor status of the gut microflora may result in less than optimal utilization of food and poor growth rate, in lower production of the lower quality of products such as milk, eggs, hide and carcass and/or in greater susceptibility of the animal to disease which may be of longer duration or of greater severity when the gut microflora is poor.

There has therefore been considerable interest in studying the gut microflora of animals, in ways of establishing and maintaining a beneficial microflora and in the mechanisms behind the observed benefits conferred by a gut microflora of the correct status. These investigations have concentrated on humans and on livestock of commercial significance. Thus the farmed species that have been studied most are those such as chickens and pigs, which commonly suffer from intestinal diseases that may be treated or prevented by intervention aimed at improving or modifying the gut microflora. Even in the absence of disease, commercially significant improvements in performance may be achieved by manipulating, or changing, the gut microflora.

The type of intervention to alter the gut microflora may take several forms. Treatment with antibiotics is often practised to eliminate pathogenic microbes from the gut. This is usually accompanied by a reduction of those naturally resident microbes considered neutral or beneficial. Another form of intervention is to ensure that food contains constituents that promote the growth of beneficial microbes. Yet anther form of intervention is to deliberately treat the animals with a live population of beneficial microbes, usually by including such microbes in the food or drinking water.

Treatment with live microbes is increasingly practised. The objective is to ensure that an adequate microflora of the desired microbes is established in the gut at an early age of the animal or introduce into a potentially disrupted gut ecosystem. The microflora becomes established within the gut by association with the gut mucosa and by colonizing the lumenal contents. Some microbes may adhere directly to the mucosal epithelium while others may be resident within the mucilage that lines the gut. The interactions between the microbes and the host are complex but some detailed understanding of microbe-microbe interactions and of the surface interactions between gut microbes and mucosal cells is emerging. The detailed mode of action of the gut microflora is not well understood, either with respect to the biochemical reactions mediated by the whole population or with respect to the activities of the individual microbes within the population. This is particularly true when considering effects on digestion and uptake of nutrients. There is, however, more information concerning the effects of gut microflora organisms on health aspects. Such organisms have been studied with respect to their activation of dietary, especially xenobiotic, compounds to carcinogens, and detoxification activity by desirable microbes: the potentiation of both non-specific and specific immunological defence mechanisms by desirable microbes: and the antagonism against pathogenic microbes by deεirable microbes.

The effects of gut microbes on enteric pathogens such as Salmonella, Clostridium, and E. coli have been studied. Beneficial microbes can suppress or prevent the colonization of the intestine by pathogens. The mechanisms known or inferred are varied. There may be very specific mechanisms such as the production of a specific antimicrobial substance, such as bateriocin, by the beneficial microbes(s). There may be a production of broad range antimicrobial such as reuterin active against many bacteria, yeast and fungi. Other chemical inhibitors of pathogens produced by beneficial microbes include organic acids (in particular lactic acid) and hydrogen peroxide. Pathogens may also be εelected against by the physical conditions of pH and redox potential controlled by beneficial microbes in the gut. Beneficial microbes may alεo prevent pathogens from becoming established in the gut by superior competition for nutrients or by occupying siteε on the gut mucosa or within the mucus overlying the epithelial cells that would be required by the pathogens for their colonization. A number of these actions are encompassed within the term "competitive exclusion".

Background to the present invention

In the studies of adjustment of the gut microflora in humans and animals such as chickens, pigs and other effective treatments have been shown to consist of administration of live cultures of microbes which consist of or include bacteria belonging to the group described as "lactic acid bacteria". Such cultures are often termed "probiotic" preparations. The administration method is usually to provide the culture in the food or drinking water although spraying of newly hatched chicks and their surroundings has also been effective. Successful administration may be monitored by assessing the establishment of the supplied microbe(s) in the animal's digestive tract.

The use of lactic acid bacteria as probiotic microbes stemmed from the early work of Metchnikoff with human infants and this group of bacteria has featured in much of the later work in both humanε and animalε. It appears that many mammals do have lactic acid bacteria as beneficial microbes in their digestive tract but other microbes, such as Bacillus, and yeast and fungi can be effective. In avian species, lactic acid bacteria are also important in probiotics but obligate anaerobic bacteria are alεo claimed to be necesεary for protection againεt salmonellosiε. In other cases a mixed culture of relatively few (less than 10) types of related microbes may be neceεεary; in yet other caεeε a complex mixed culture containing many tens (perhaps 100) of different types of microbes may be effective. The culture used in this last case may approximately the entire resident beneficial microflora in the gut of the target animal.

The source of probiotic microbes can be food such as fermented milk products, like yoghurt, in the case that it is desired to established or improve the population of particular lactic acid bacteria in the gut, or deliberately isolated cultures from the intestines of the target animal. In the latter instance, successful probiotic samples have been isolated from faecal samples but the microflora of faeces represents largely the tranεient microbial population in the gut whereaε it is often desired or advantageous to employ a culture that is representative of the resident microbial microflora in the gut in order to ensure that isolation of the potential probiotic strain takes place from the resident microflora, it is necessary to carry out isolations from the alimentary canal directly, often from a location where it is desired to encourage the probiotic strain to reside. Thus scrapings of the internal intestinal mucosa of recently sacrificed, healthy animals may be a source for isolation of suitable organisms for testing as probiotics.

It is obεerved that the diεtribution of the gut microflora in both qualitative and quantitative aspects is not uniform along the length of the alimentary canal. Frequently, the greatest and most varied populations are found in the lower intestine. It is here that the gut microflora probably exerts its major effect on digeεtion, nutrient uptake and also intestinal colonization by pathogens.

After a population of the resident gut microflora has been isolated, it may be used as εuch to inoculate an animal with an inadequate gut microflora or it may be resolved into individual microbial strains for reintroduction as single εtrainε or as simplified mixed populations. In the case that it is desired or necessary to separate a mixed population into its individual microbial strains, the techniques commonly employed in microbiology may be used. In particular separations may be made based upon the morphology of colonies on various solid and liquid media grown with different carbon and energy sources, with different nitrogen sources, under different conditions of gas supply (aerobic and anaerobic) , at different pH values and other conditions known to those skilled in the art.

If individual microbial stains are to be selected from a mixed population for use as probiotic strains it is necessary to apply some practical criteria for their selection. These criteria are partly dependent on the required attributes of a probiotic strain and include:

- origination from the animal species in question; - sufficient εtability to digestive conditions (acid, bile, enzymes) to allow survival; - ability to colonize the animal species in question under practical conditions. This may include the ability to adhere to the intestinal cells although effective strains may be able to reside within the intestinal mucus or lumenal contents without direct contact with the intestinal mucosal cells; - antagonism against potential microbial pathogens. Thiε may include the production of general or εpecific antimicrobial εubstances by the selected strain. - safety in use. Thiε includeε the demonstration that the selected strain is not itself a pathogen causing a clinical disease. It will be apparent that individual microbial strains isolated from a population originating from resident gut microflora of a healthy animal εhould meet many of these criteria^{^} y, definition: including origination from the species in question and sufficient stability and ability to colonize the animal gut. In terms of selective criteria for choosing a probiotic strain, the ability to adhere to intestinal mucosal cells may be applied in the case where it is known that the microbe must adhere to such cellε, for example, when the strain is to be applied to new born or newly hatched infants which have a naked, or nearly so, gut mucosa. The inability of a strain to adhere to the gut mucosa, does not, however, indicate that the strain is without utility as a probiotic.

In the case that one objective of using a probiotic is to combat pathogenesiε via the gut, selection based on demonstrated antagonism towards likely or actual pathogens in the gut of the specieε concerned is indicated. Numerous in vitro methods of showing and quantifying such antagoniεm are known to thoεe εkilled in the art of εelecting microbes producing antibiotics and may be applied here, is recognized, however, that antagonism, or the extend of antagonism, may vary depending on the in vitro methods used. It is also recognized that it may be difficult to show in vivo the same antagonism which can be demonstrated in vitro. partly because it is difficult to reproduce exactly the in vivo conditionε in the laboratory experimentε. However, the effectiveneεε of the probiotic may be readily demonεtrated by εubjecting the animal treated with the probiotic εtrain to challenge with a diεeaεe causing microbe.

Since the gut microflora may harbour pathogenic microbes in the carrier state and hence there are no signs of clinical disease, it is clearly necessary to exclude such pathogens from the selected population of probiotic strains. Suitable tests to establish the non-pathogenicity of a test probiotic strain include deliberate injection into the animal. One route for injection in such tests is into the peritoneal cavity. Observation of lack of disease symptoms and inability to isolate live microbes of the test strain from the target organs indicates lack of pathogenicity.

It is known that fish harbour bacteria with inhibiting activity againεt pathogens in their gut microbial flora. Thus Westerdahl, A., et al, Appl Environm. Microbiol. 572223-2228 (1991, , and Olsεon. J. C. , et al, Appl En-vironm, Microbiol. 58:551-5556 (1992) discloses isolation and characterization of turbot aεsociated bacteria with inhibitory effects against Vibrio anquillarum.

US-A-4 , 657, 762 discloses a composition uεeful in the treatment of disturbances in the normal intestinal flora of poultry, whereby the compoεition contains anaerobic bacteria of intestinal origin.

Austin B., et al, J. Fish Diεeaεe. 15:55-61 (1992) discloses inhibition of bacterial fish pathogenε by Teεraεelmis suecica by administering supernatantε and extracts from heterotrophically grown Tetraselmiε εuecica which inhibit different prawn pathogenic vibrios.

Robertson, B. , et al, J Fish Diseases 13:291-400 (1990) discuεεeε enhancement of non-specific diseaεe reεiεtance in Atlantic salmon, Salmo salar L. , by a glucan from Saccharomyces cereviεiae cell wallε when injected intraperitoneally.

Smith, P., et al, J. Fish Diεeaεes. 16:521-524 (1993) discloses evidence for the competitive exclusion of Aeromonas salmonicida from fish with stresε-inducible furunculoεis by a fluoroscent pseudomonad isolated from gill and gill mucus of brown salmon, Sla o trutta L, which strain had been isolated for its ability to inhibit Aeromonas salmonicida.

Douillet, P, A., et al, Aquaculture. 119:25-40 (1994) discloεeε the uεe of probiotic for the cultures of larvae of the Pacific oyster (Crasεoεtrea gigas Thunberg) whereby addition of strain CA2 as a food supplement to xenic larval cultures of the oyster Crassoεtrea gigas enhanced growth of the larvae.

Description of Present Invention

It has now surprisingly been shown that a bacterium found within a bacterial population isolated from one Atlantic salmon, Sal o salar. exhibitε εtrong inhibitory effectε against bacterial fiεh pathogens, Vibrio anquillaruro. (vibriosis) , Vibrio ordalli (vibriosis) , Aeromonas salmonicida. (furunculosis) , and others. The strain which has been denoted strain K in the following has been provided the accession number DSM 10087 as deposited with the Deutsche Sammiung von Mikroorganismen und Zellkulturen GmbH on 6 July 1995 under the Budapest Treaty.

The strain K, aε hitherto iεolated from an Atlantic salmon, in accordance with Olsson, J. C, et al, Appl Environm. Microbiol. 58:551-556 (1992) (enclosed herein as a reference) proved to be a motiel, Gram-positive pleomorphic, facultative anaerobic rod. The antibacterial activity of the strain K was analysed and the results suggested that multiple, broad range antibacterial compounds are released in the surrounding medium during the logarithmic phase of growth in TSBS (Tryptic Soya Broth supplemented with Salt, NaCl 2% w/v) medium. The inhibitory compounds were also produced when the strain K was grown in diluted intestinal mucus, which suggests that the strain K bacterium will proliferate and produce the antibacterial substance in the gut. The antibacterial compounds were heat labile, and were initially determined to have a molecular weight of about 140-150 dalton by gel filtration. The antibacterial compounds have been found to have an inhibitory activity againεt both Gram-negative and Gram-positive bacteria, but not against yeast. The antibacterial compounds are bacteriostatic at low concentrationε but bactericidal at higher concentrationε. The activity is maintained when the compounds are εtored in frozen εtate, but iε loεt when maintained at 23 ^*C.

Strain K εhowε the following phenotypic characterization:

The major fatty acids are 16:0 (31.1%); 16:1 (24.2%); and 18:1 cis 9 (23.4%) . The remaining fatty acids are: 18:2 cis 9, 12; 18:0A (10.8%); 14:0 (5.4%) and 18:0 (3.5%). Strain K can utilize the following carbon sources: sucroεe, maltoεe, trehhalose, mannitol, ribose, B-D-glucopyranoside. It was not able to produce acids from the following carbon sourceε: cyclo- dextrin, tagotose, D-arabitol, L-arabinose, melezitose, melibiose, pullulan, glycogen, raffinose, lactose, and sorbitol. It did not produce acetoin. It was able to hydrolyse hippurate. Beta-Glucosidase activity waε de onεtrated. No activity of the following enzymes were detected: ureaεe, betagalactoεidase, beta- glucuronidase, alfa-galactosidaεe, arginin dihydrolaεe, alanylphenylalanyl-prolin arylamidase, acide pryoglutamique arylamidase, N-acetyl-beta- glycosaminidase.

Strain K is sensitive to gentamycin, erythromycin, rifampicin, tetracyclin, ampicillin, and kanamycin. It is sensitive to a lesser extent to neomycin and nalidixic acid. Strain K does not harbour any detectable plasmids.

A 2.3 fold diluted of a cell-gree culture supernatant provides a total growth inhibition of Vibrio anquillarum (HT11360) . Aeromonas salmoncida (ATCC 14174) is more εenεitive than the Vibrio anquillarum εtrain, and a 10 timeε dilution reεults in total growth inhibition during a 24 hours test period. Any dilution of the TSBS does not interfere with these resultε.

The active compounds loses its activity gradually at 4'C and cannot be detected after 8 weeks. When frozen the full activity remains for at leaεt 12 months.

The activite compound of εtrain K inhibits a large number of bacteria, whereby no difference is seen between Gram-negative bacteria. All pathogens tested were sensitive, whereby Staphylococcus aureaε and Proteus vulgaris CCUG 6327 proved to be the most sensitive and E. Coli Av24 and Pseudomonas aeruginosa the least. Yeast is not inhibited.

Strain K grows in intestinal mucus. Growth waε proceeded by a lag phaεe of at least 7.5 hours. This is comparable with the length of the lag phase that is exhibited in TSBS by the same strain in the same temperature. Strain K was found to produce substanceε during growth in mucus that are inhibitory to growth of the fish pathogens Vibrio anquillarum and Aeromonas salmonicida. The inhibitors were detected in the mucus at the onset of the logarithmic growth (7.5 hours) . An increase in the inhibitory activity was observed throughout the log phase and into stationary phase. The growth of the pathogens was not inhibited in the control culture with intestinal mucus without strain K. The colonies on the TSBS plates were identified as strain K, a Carnobacterium by biochemical tests. The bacteria in the pinpoint colonies were found to be motile, forming pairs or chains with four cellε, Gram positive, catalase and oxidase negative. Inhibition zones were around the colonies when tested against V. anguillarum.

The colony forming units (CFU) were found to increase approximately 3 log during 12.5 hours of growth in faeces suspension. No lag phase was seen during growth in faeces suεpenεion. After the culture had reached εtationary phase, at 12.5 hours, no further increase of inhibitory activity was detected.

Production of substances that inhibit the growth of the fish pathogens, V. anquillarum and A. salmonicida. was detected after 7.5 hourε. The inhibitory activity increaεed until 12.5 hourε and then remained unchanged. V. anquillarum and A. salmonicida grew in the faeces control. The identity of the strain K colonies was confirmed as described in the previous εection.

Neither εtrain K, nor its inhibiting compound (-s) , is toxic to fish. No fiεh in any teεted group died or showed any external signs of diseaεe during the experimentε in which fiεh were expoεed to strain K. Spleens from both infected with strain K and control fish were free from culturable bacteria.

Media, diluent and culture conditions

The media and diluent used with Tryptic Soya Broth (TSB, Difco) and TSBS (TSB supplemented with 2% NaC) ; TSA (TSB + 15% agar) and TSAS (TSA supplemented with 2% NaCl) ; TSAS soft agar (TSBS + 5% agar) ; Marine agar (MA, Difco) ; Nutrient agar (NA, Difco) ; Brain heart infusion (BH1, Difco) ; Rogosa (Difco) ; TCBS Cholera Medium (Oxoid) ; Marine Minimal Medium (MMM, Neidhardt et al, (1974)); VFI (peptone l.Og, yeast extract 0.5 g, glucose 0.5g, starch 0.5g; Salmon intestinal buffer (Hickman (1968)); NaHC0₃ 1.03g, NaCl 1.97g, KCl 0.16g, CaCl₂.2H₂0 2.07g, MgS0₄.7H₂0 21.95g, MgCl₂.6H₂0 10.67g, agar 15g distilled water 1000ml) ; VNSS agar (peptone 1-Og, yeast extract 0.5g, glucose 0.5g, starch 0.5g, FeSO₄.7H₂0 O.Olg, Na₂HP0₄ O.Olg, agar 15g in 1000ml NSS); NSS (Nine Salt Solution: NaCl 17.6g, Na₂S0₄ 1.47g, NaHC0₃ 0.08g, KCl 0.25g, KBr 0.04g, MgCl₂.6H₂0 1.87g, CaCl₂.2H₂0 0.41g, SrCl₂.6H₂0 O.OOδg, H₃B0₃ 0.008g in 1000 ml double distilled water) . Horse-blood agar (HBA, TSA supplemented with 5% horse blood.

Bioasεay for the detection of inhibitory effect

i) Double-layer agar method.

The plateε were screened for inhibition by a modified double-layer agar method described by McLeod and Govnlock (1921) . Macrocolonies of the inhibitory bacteria were obtained by εpot seeding on agar plates (10 μl of a liquid culture in log phaεe) and incubating the plateε for 18 hourε at 23'C. The macro-colonieε were treated with chloroform vapour for 30 min. The pathogen (lOOμl of a 10 x diluted liquid culture) was then seeded into a tube of melted (temperature to 45°C) TSAS soft agar (3ml) mixed and then poured onto the top of the plates. After incubation for 18 hours the zones of growth inhibition created by the producing colonies were measured as the distance between the edge of the macro colony and the edge of the clearing zone.

Liquid bioassay in microtitre wellε

The inhibitory effect waε determined as changes in the optical density (OD₆₁₀) , using microtitre spectrophotometer (Bio Tech. Biokinetics) . The inhibition assay was performed in microtitre wells (Nunc, 96 wells) . The inhibitory bacterium was grown in TSBS at 23 °C. The cells were removed by centrifugation and the supernatant was filter εterilized (MFS-25 celluloεe acetate filter unitε, 0.2 μm) . The cell free supernatant (150 μl) was transferred to a microtitre well and an equal volume of fresh TSBS (150 μl) was added. Thereafter the target microorganiεm (3 μl from a logphaεe culture) waε inoculated in the well and the growth waε monitored by measuring the optical density at 610 nm for 24 hours. As a control a dilution series with 2% NaCl solution and TSBS was made to relate growth of the pathogen with the amount of medium added. To control for any auto- inhibition by the pathogen, cell-free culture supernatant (from a culture of the pathogens) was treated in the same way as the supernatant derived from the inhibitory εtrain. Table 1

The double-layer agar method waε uεed to teεt growth inhibition of a wide range of bacteria, aε well aε salmon related yeaεtε by macro-colonies of isolated strain K.

Organism GRAM Inhibition

+/-. Zone

Strain K + —

Vibrio anguillarum HTI 1360 + +++

V. anguillarum 2129 + +++

V. anguillarum + +++

V. ordalli NCMB 2127 + ++++

V. fiεheri + ++++

Photobacterium anguεtum S14 ++++

Aeromonas salmonicida ATCC 14174 ++++

A. hydrophila +++

A. hyrophila NCTC 8049 ++++ Escherichia coli B CCUG 214 +++ E. coli Av24 + Vibrio sp. 4:44 +++ Salmon isolate +++ Vibrio sp. D2 +++++ Pseudomonas aeruginosa + Staphylococcus aureus + +++++ Serratia marcescenε CCUG 760 - +++ Micrococcus luteus + ++++ Proteus vulgaris CCUG 6327 _ +++++ Klebεiella oxytoca CCUG 383 - +++ Baciluus mageateriu CCUG 1817 + ++++

B. subtilis CCUG 163B + ++ Acinetobacter calcoaceticus CCUG 12864 — ++++ Streptomyces griseus CCUG 760 ++++ Citrobacter freundi CCUG 418 — ++++ Marine yeast Scl8 (unidentified fish isolate)

Marine yeaεt Sc3 Saccharomyceε cereviεiae

CBS 7765

Debaryomyces hansenii HFI

S. cerevisiae CBS 7764

Inhibition zone radius (mm) : 0 (-) ; 1-5 (+) ; 6-10 (++) ; 11-15 (+++) ; 16-20 (++++) ; >20 (+++++)

Table 2

Phenotype of εtrain K

Property Strain K

Single rod +, two polar flagella

Pleomorphic +

Motility +

Flagellation dipolar

Spores

Gram reaction +

Colony diameter <1 mm

Colony appearance on TSA Circular; entire; semitranslucent; raiεed

Pigmentation Buff

Odour

Anaeorbic growth +

Temperature for growth 4-30°C pH for growth 5.5 to 9 pH change during growth 7.2 to 6.8

Salinity for growth (NaCl) 0 to 6%

Catalase

Oxidase

Haemolysis alfa

Urease Hydrogenεulphide production Fermentative in Leifεon weak acid, no gaε

Strain K is thereby identified as a Carnobacterium and was primarily characterized as Carnobacterium alterfunditum. However, further investigations of 16S rRNA demonεtrateε that Carnobacterium alterifunditum iε the cloεest related organism with a homology of 98.7%. However, according to previouε publicationε (Collins et al, 1987) this would justify describing the isolate as a new species of the genus. Thus the εtrain K iε a new εtrain, whereby it will be named more εpecifically later on.

The inhibitory compound(s) was partly purified by fist removing the bacteria cells from a TSBS culture by entrifugatino in the early stationary growth phaεe (13000 x g for 10 min) . The sample was then kept at 4'C during the subεequent purification εtepε. The cell-free culture supernatant was fractionated by pasεing it through a 500 dalton cut-off filter (Amicon, Difaflo YC05) . Further purification was performed by gelfiltration using a G10 Sephadex (Pharmacia, Sweden) in a XK26/40 column. PBS (2mM) waε used as a effluent buffer. The ultra-filtrated supernatant was applied to the coloumn and eluated at a flow rate of 44 ml/h. Fractions (2.9 ml) were collected and examined for antimicrobial activity by the liquid bioassay described above. The apparent size of the inhibiting compound(- s) was determined using a standard curve including NADH (709 D) , N-formyl-Met-Leu-Phe-Phe (584.7 D, Sigma), Tyr-Gly-Gly (295.3 D, Sigma) , L-tryptophan (204.23 D) and tyroεine (181.19 D) as markers. Blue dextran waε uεed to determine the void volume of the coloumn.

The inhibitorswas extracted using ethyl acetate at pH 2.5 and εubεequently purified on TLC using silica gel. The inhibitors are stable for at least 24 hours at a pH of 2 to 11.

The partly purified inhibitory compound(-s) was subjected to heat treatment, various enzyme treatments, metaperiodate treatment, εtability tested, and culture media dependency.

The action of the inhibiting compound(-s) was determined as follows. Aeromonas salmonicida from a culture in log phase was inoculated into freεh TSBS to a denεity of about 1 x 10₆ cellε/ml. The culture was incubated for 30 min prior to starting the experiment. To 100 ml culture flasks, 10 ml of the Aeromonas salmonicida culture and a mixture (10 ml final volume) of the partly purified inhibitor εupernatant, and NSS (pH 7.2) waε tranεferred such that a series of concentrations of the inhibitor was obtained. The cultures were slowly shaken at 23'C and εampleε in triplicate were taken to determine the number of colony forming units (CFU) during at time period of 26 hours.

A 4 fold dilution of the cell-free supernatant resulted in total growth inhibition of Vibrio anguillarum (H111360) . Aeromonas salmonicida (ATCC 14174) was found to be more sensitive than the Vibrio anguillarum strain and a 10 fold dilution still resulted in a total growth inhibition during the 24 hourε teεt period. The dilution of TSBS did not interfere with the results.

The action of the inhibitory compound (-s) against Aeromonas salmonicida using different dilutions is shown in the Table 3 below. Table 3

Dilution CPU.ml after

0 1 2 3 4 5 6 7 8 9 10 11 hrs

2 fold 6.4 1.0 0.2 0.2 0.1 0.05 0.0 0.0 0. xlO⁵ 5 5 0

4 fold 6.4 4.4 2.0 1.0 0.8 0.2 0.1 0.0 xlO³

6 fold 6.4 5.4 3.0 1.9 1.4 1.0 0.8 0.2 0. 0.0 0. xlO* 1 5 0

The action of the inhibitory compound(-s) against Vibrio anguillarum as determined as the growth of strain K in TSBS at 23'C as and expressed as the increase in optical denεity at 610 nm over time will be given below. Further the minimal dilution of free cell culture εupernatant cauεing total inhibition of Vibrio anguillarum. The reεults are combined in Table 4 below.

Table 4

Incubation Time (hrs) Increase . in.0 ptical Minimal dilution deαsity at 610 nm arbitrary units

5.5 0.025 30

6 0.05

6-5 0.08 50

7 0.12

7.5 0.15 70

8 0.18

8.5 0.21 80

9 0.23

9.5 0.25 80

10 0.25

10.5 0.25 80

16 0.25 80

The present strain K the other strains capable of producing the active compound or chemically related compounds or the active compound derived therefrom and chemically related compounds and derivatives thereof can, in particular, be used in the prophylactic or therapeutic treatment of fish infected by fish pathogens, whereby an amount of the strain K that provides an inoculum allowing the colonization of the fish intestine by the εtrain K or an amount of the εtrain K that provideε an active amount of the ^• inhibiting compound, is administered to the fish, or an active amount of the inhibiting compound as εuch is administered to the fish for prophylactic and/or therapeutic treatment of fish susceptible to fiεh pathogenε. Hereby all types of fish are encompassed, to which strain K iε not pathogenic, harmful or deleteriouε, but in particular εalmonids, turbot, yellow tail, seabasε/seabream and other types of farmed fish and other aquaculture species, such as shellfish (prawns and shrimps) and mollusceε.

It might be εo that strain K is pathogenic, harmful or otherwise deleterious as such in some of the organism to which it is administered although this is not foreseen. However, the active compound(-s) therefrom might each be so, but can be administered instead for obtaining a bacteriostatic or bactericidal effect.

The strain K or active inhibiting compound derived therefrom can be administered orally as such or via the feed, which is the best mode, bathing of young or older fish, single inoculation of young or older fish to establish the εtrain K in the gut, or repeated inoculation of young or older fish. When the active compound as such iε adminiεtered together with the feed, one has to consider the lability of the compound, if it iε to be incorporated into the feed before the hydrothermal forming of pellets. A suitable meanε to avoid thermal deεtruction of the active compoundε is to add them to the feed pellets after their information and cooling.

The strain K or its active inhibitory compound(-s) can be administered in different ways εuch aε via food, feed-stuff including drinking water, as a compoεition as εuch containing the εtrain. Further it can be added via spraying the animals, including fishes, by immersion of the animals, in particular when fish is concerned, by injection into the gut, or via inhalation.

Claims

1. Use of a probiotic for treating mammals, including man, fish, shellfish and mollusceε, compriεing at leat one microbial εtrain isolated from the resident gut microflora of healthy fish and selected by methods known per se to be capable of establishing itεelf at an effective level in the inteεtine of the animal treated, whereby the strain is a bacteriostatic and bactericidal Carnobacterium.

2. The use aε in Claim 1 of a Carnobacterium having the acceεεion number DSM 10087 as deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH.

3. The use of said strain of claims 1-2 for the prophylactic treatment of mammals, including man, fish and other aquatic animalε.

4. The use of said strain of Claims 1-2 for the therapeutic treatment of mammals, including man, fish and other aquatic animals.

5. The use of the probiotic of Claims 1 to 4 , whereby εaid εtrain iε adminiεtered by immerεion of the subject in a liquid container εaid probiotic.

6. The uεe of the probiotic of Claimε 1 to 4, whereby εaid εtrain iε adminiεtered via the food/feed including drinking water, εupplied to the subject.

7. The use of the probiotic of Claims 1 to 4, whereby said strain is adminiεtered via εpraying onto the εubject.

8. The uεe of the probiotic of Claims 1 to 4, whereby the said stain is administered via injection into the subject.

9. The uεe of the probiotic of Claimε 1 to 4, whereby the said strain is administered via inhalation into the subject.

10. The use according to Claims 2 to 9, whereby said strain is used in the treatment of infections caused by Gram-positive and/or Gram-negative bacteria, such as Vibrio anguillarum, Vibrio ordalii, Vibrio fischeri, Aeromonaε salmoncida, Photobacterium angustum, Aeromonas hydrophila, Staphylococcuε aureuε, Bacilluε megaterium, Acinetobacter calcoaceticuε, Serratia marceεceneε, Micrococcuε luteus, Proteuε vulgaris.

11. A microbe inhibiting active compound derived from strain of Claim 1.

12. The use of a microbe inhibiting active compound of Claim 11 in the prophylactic treatment of mammals, including man, fish, εhellfiεh and molluεces.

13. The use of a microbe inhibiting active compound of Claim 11 in the therapeutic treatment of mammals, including man, fiεh and mollusces.

14. The uεe of a microbe inhibiting compound of Claim 12 or 13 for inhibiting the growth of Gram- positive and/or Gram-negative bacteria, such as Vibrio anguillarum, Vibrio ordalii, Vibrio fischeri, Aeromonas salmoncida, Photobacterium angustum, Aeromonas hydrophila, Staphylococcus aureuε, Bacillus megaterium, Acinetobacter calcoaceticus, Serratia marceεcenε, Micrococcus luteus, Proteuε vulgaris.

15. A probiotic for treating mammals, including man, fiεh, εhellfiεh and ollusces comprising at least one microbial strain iεolated from the reεident gut microflora of healthy fish and selected by methods known per se to be capable of establishing itself at an effective level in the intestine of the animal treated, whereby the strain is a bacterioεtatic and bactericidal Carnobacterium.

16. A probiotic according to Claim 15, wherein the strain is a Carnobacterium having the accesεion number DSM 10087 aε depoεited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH in combination with other microbes isolated from a gut microflora of a healthy subject.

17. The use of a bacteriostatic and bactericidal compound expressed by said strain for the treatment of mammals, including man, fish, shellfiεh and molluεces in combination with other microbes isolated from a gut microflora of a healthy subject.