CA2228521A1 - Protection against pathogenic microorganisms - Google Patents

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

1 Protection Aqainst Pathoqenic Microorqanisms 3 The present invention relates to a nutritionally and 4 prophylactically valuable product to improve the gut microbiological flora in mammals, including man, and in 6 fish, shellfish and mollusces.

8 I_ is recognised that the status of the microbiological 9 flora in the gut of an animal may have a profound effect on the wellbeing of the animal. Poor status of 11 the gut microflora may result in less than optimal 12 utilization of food and poor growth rate, in lower 13 production of the lower quality of products such as 14 milk, eggs, hide and carcass and/or in greater susceptibility of the animal to disease which may be of 16 longer duration or of greater severity when the gut 17 microflora is poor.

19 There has therefore been considerable interest in studying the gut microflora of animals, in ways of 21 establishing and maintaining a beneficial microflora 22 and in the mechanisms behind the observed benefits 23 conferred by a gut microflora of the correct status.
24 These investigations have concentrated on humans and on livestock of commercial significance. Thus the farmed CA 02228~21 1998-02-03 1 species that have been studied most are those such as 2 chickens and pigs, which commonly suffer from 3 intestinal diseases that may be treated or prevented by 4 intervention aimed at improving or modifying the gut microflora. Even in the absence of disease, 6 commercially significant improvements in performance 7 may be achieved by manipulating, or changing, the gut 8 microflora.

The type of intervention to alter the gut microflora 11 may take several forms. Treatment with antibiotics is 12 often practised to eliminate pathogenic microbes from 13 the gut. This is usually accompanied by a reduction of 14 those naturally resident microbes considered neutral or beneficial. Another form of intervention is to ensure 16 that food contains constituents that promote the growth 17 of beneficial microbes. Yet anther form of 18 intervention is to deliberately treat the animals with 19 a live population of beneficial microbes, usually by including such microbes in the food or drinking water.

22 Treatment with live microbes is increasingly practised.
23 The objective is to ensure that an adequate microflora 24 of the desired microbes is established in the gut at an early age of the animal or introduce into a potentially 26 disrupted gut ecosystem. The microflora becomes 27 established within the gut by association with the gut 28 mucosa and by colonizing the lumenal contents. Some 29 microbes may adhere directly to the mucosal epithelium while others may be resident within the mucilage that 31 lines the gut. The interactions between the microbes 32 and the host are complex but some detailed 33 understanding of microbe-microbe interactions and of 34 the surface interactions between gut microbes and mucosal cells is emerging.

CA 02228~21 1998-02-03 1 The detailed mode of action of the gut microflora is 2 not well understood, either with respect to the 3 biochemical reactions mediated by the whole population 4 or with respect to the activities of the individual S microbes within the population. This is particularly 6 true when considering effects on digestion and uptake 7 of nutrients. There is, however, more information 8 concerning the effects of gut microflora organisms on 9 health aspects. Such organisms have been studied with respect to their activation of dietary, especially 11 xenobiotic, compounds to carcinogens, and 12 detoxification activity by desirable microbes: the 13 potentiation of both non-specific and specific 14 immunological defence mechanisms by desirable microbes:
and the antagonism against pathogenic microbes by 16 desirable microbes.

18 The effects of gut microbes on enteric pathogens such 19 as Salmonella, Clostridium, and E. coli have been studied. Beneficial microbes can suppress or prevent 21 the colonization of the intestine by pathogens. The 22 mechanisms known or inferred are varied. There may be 23 very specific mechanisms such as the production of a 24 specific antimicrobial substance, such as bateriocin, by the beneficial microbes(s). There may be a 26 production of broad range antimicrobial such as 27 reuterin active against many bacteria, yeast and fungi.
28 Other chemical inhibitors of pathogens produced by 29 beneficial microbes include organic acids (in particular lactic acid) and hydrogen peroxide.
31 Pathogens may also be selected against by the physical 32 conditions of pH and redox potential controlled by 33 beneficial microbes in the gut. Beneficial microbes may 34 also prevent pathogens from becoming established in the gut by superior competition for nutrients or by 36 occupying sites on the gut mucosa or within the mucus CA 02228~21 1998-02-03 1 overlying the epithelial cells that would be required 2 by the pathogens for their colonization. A number of 3 these actions are encompassed within the term 4 "competitive exclusion".
6 Background to the present invention 8 In the studies of adjustment of the gut microflora in 9 humans and animals such as chickens, pigs and other effective treatments have been shown to consist of 11 administration of live cultures of microbes which 12 consist of or include bacteria belonging to the group 13 described as "lactic acid bacteria". Such cultures are 14 often termed "probiotic" preparations. The administration method is usually to provide the culture 16 in the food or drinking water although spraying of 17 newly hatched chicks and their surroundings has also 18 been effective. Successful administration may be 19 monitored by assessing the establishment of the supplied microbe(s) in the animal's digestive tract.

22 The use of lactic acid bacteria as probiotic microbes 23 stemmed from the early work of Metchnikoff with human 24 infants and this group of bacteria has featured in much of the later work in both humans and animals. It 26 appears that many mammals do have lactic acid bacteria 27 as beneficial microbes in their digestive tract but 28 other microbes, such as Bacillus, and yeast and fungi 29 can be effective. In avian species, lactic acid bacteria are also important in probiotics but obligate 31 anaerobic bacteria are also claimed to be necessary for 32 protection against salmonellosis. In other cases a 33 mixed culture of relatively few (less than 10) types of 34 related microbes may be necessary; in yet other cases a complex mixed culture containing many tens (perhaps 36 100) of different types of microbes may be effective.

CA 02228~21 1998-02-03 1 The culture used in this last case may approximately 2 the entire resident beneficial microflora in the gut of 3 the target animal.

The source of probiotic microbes can be food such as 6 fermented milk products, like yoghurt, in the case that 7 it is desired to established or improve the population 8 of particular lactic acid bacteria in the gut, or 9 deliberately isolated cultures from the intestines of the target animal. In the latter instance, successful 11 probiotic samples have been isolated from faecal 12 samples but the microflora of faeces represents largely 13 the transient microbial population in the gut whereas 14 it is often desired or advantageous to employ a culture that is representative of the resident microbial 16 microflora in the gut in order to ensure that isolation 17 of the potential probiotic strain takes place from the 18 resident microflora, it is necessary to carry out 19 isolations from the alimentary canal directly, often from a location where it is desired to encourage the 21 probiotic strain to reside. Thus scrapings of the 22 internal intestinal mucosa of recently sacrificed, 23 healthy animals may be a source for isolation of 24 suitable organisms for testing as probiotics.
26 It is observed that the distribution of the gut 27 microflora in both qualitative and quantitative aspects 28 is not uniform along the length of the alimentary 29 canal. Frequently, the greatest and most varied populations are found in the lower intestine. It is 31 here that the gut microflora probably exerts its major 32 effect on digestion, nutrient uptake and also 33 intestinal colonization by pathogens.

After a population of the resident gut microflora has 36 been isolated, it may be used as such to inoculate an CA 02228~21 1998-02-03 1 animal with an inadequate gut microflora or it may be 2 resolved into individual microbial strains for 3 reintroduction as single strains or as simplified mixed 4 populations. In the case that it is desired or necessary to separate a mixed population into its 6 individual microbial strains, the techniques commonly 7 employed in microbiology may be used. In particular 8 separations may be made based upon the morphology of 9 colonies on various solid and liquid media grown with different carbon and energy sources, with different 11 nitrogen sources, under different conditions of gas 12 supply (aerobic and anaerobic), at different pH values 13 and other conditions known to those skilled in the art.

If individual microbial stains are to be selected from 16 a mixed population for use as probiotic strains it is 17 necessary to apply some practical criteria for their 18 selection. These criteria are partly dependent on the 19 required attributes of a probiotic strain and include:
21 - origination from the animal species in question;
22 - sufficient stability to digestive conditions 23 (acid, bile, enzymes) to allow survival;
24 - ability to colonize the animal species in question under practical conditions. This may include the 26 ability to adhere to the intestinal cells although 27 effective strains may be able to reside within the 28 intestinal mucus or lumenal contents without 29 direct contact with the intestinal mucosal cells;
30 - antagonism against potential microbial pathogens.
31 This may include the production of general or 32 specific antimicrobial substances by the selected 33 strain.
34 - safety in use. This includes the demonstration that the selected strain is not itself a pathogen 36 causing a clinical disease.

CA 02228~21 1998-02-03 1 It will be apparent that individual microbial strains 2 isolated from a population originating from resident 3 gut microflora of a healthy animal should meet many of 4 these criteria~by definition: including origination from the species in question and sufficient stability 6 and ability to colonize the animal gut. In terms of 7 selective criteria for choosing a probiotic strain, the 8 ability to adhere to intestinal mucosal cells may be 9 applied in the case where it is known that the microbe must adhere to such cells, for example, when the strain 11 is to be applied to new born or newly hatched infants 12 which have a naked, or nearly so, gut mucosa. The 13 inability of a strain to adhere to the gut mucosa, does 14 not, however, indicate that the strain is without utility as a probiotic.

17 In the case that one objective of using a probiotic is 18 to combat pathogenesis via the gut, selection based on 19 demonstrated antagonism towards likely or actual pathogens in the gut of the species concerned is 21 indicated. Numerous in vitro methods of showing and 22 quantifying such antagonism are known to those skilled 23 in the art of selecting microbes producing antibiotics 24 and may be applied here, is recognized, however, that antagonism, or the extend of antagonism, may vary 26 depending on the in vitro methods used. It is also 27 recognized that it may be difficult to show in vivo the 28 same antagonism which can be demonstrated in vitro, 29 partly because it is difficult to reproduce exactly the in vivo conditions in the laboratory experiments.
31 However, the effectiveness of the probiotic may be 32 readily demonstrated by subjecting the animal treated 33 with the probiotic strain to challenge with a disease 34 causing microbe.
36 Since the gut microflora may harbour pathogenic CA 02228~21 1998-02-03 1 microbes in the carrier state and hence there are no 2 signs of clinical disease, it is clearly necessary to 3 exclude such pathogens from the selected population of 4 probiotic strains. Suitable tests to establish the non-pathogenicity of a test probiotic strain include 6 deliberate injection into the animal. One route for 7 injection in such tests is into the peritoneal cavity.
8 Observation of lack of disease symptoms and inability 9 to isolate live microbes of the test strain from the target organs indicates lack of pathogenicity.

12 It is known that fish harbour bacteria with inhibiting 13 activity against pathogens in their gut microbial 14 flora. Thus Westerdahl, A., et al, Appl Environm.
Microbiol. 572223-2228 (1991), and Olsson. J. C., et 16 al, APPl En-vironm, Microbiol. 58:551-5556 (1992) 17 discloses isolation and characterization of turbot 18 associated bacteria with inhibitory effects against 19 Vibrio anquillarum.
21 US-A-4,657,762 discloses a composition useful in the 22 treatment of disturbances in the normal intestinal 23 flora of poultry, whereby the composition contains 24 anaerobic bacteria of intestinal origin.
26 Austin B., et al, J. Fish Disease 15:55-61 (1992) 27 discloses inhibition of bacterial fish pathogens by 28 Tesraselmis suecica by administering supernatants and 29 extracts from heterotrophically grown Tetraselmis suecica which inhibit different prawn pathogenic 31 vibrios.

33 Robertson, B., et al, J Fish Diseases 13:291-400 (1990) 34 discusses enhancement of non-specific disease resistance in Atlantic salmon, Salmo salar L., by a 36 glucan from SaccharomYces cerevisiae cell walls when CA 02228~21 1998-02-03 1 injected intraperitoneally.

3 Smith, P., et al, J. Fish Diseases, 16:521-524 (1993) 4 discloses evidence for the competitive exclusion of Aeromonas salmonicida from fish with stress-inducible 6 furunculosis by a fluoroscent pseudomonad isolated from 7 gill and gill mucus of brown salmon, Slamo trutta L, 8 which strain had been isolated for its ability to 9 inhibit Aeromonas salmonicida.

11 Douillet, P, A., et al, Aquaculture, 119:25-40 (1994) 12 discloses the use of probiotic for the cultures of 13 larvae of the Pacific oyster (Crassostrea gigas 14 Thunberg) whereby addition of strain CA2 as a food supplement to xenic larval cultures of the oyster 16 Crassostrea gigas enhanced growth of the larvae.

18 Description of Present Invention It has now surprisingly been shown that a bacterium 21 found within a bacterial population isolated from one 22 Atlantic salmon, Salmo salar, exhibits strong 23 inhibitory effects against bacterial fish pathogens, 24 Vibrio anquillarum, (vibriosis), Vibrio ordalli (vibriosis), Aeromonas salmonicida, (furunculosis), and 26 others. The strain which has been denoted strain K in 27 the following has been provided the accession number 28 DSM 10087 as deposited with the Deutsche Sammiung von 29 Mikroorganismen und Zellkulturen GmbH on 6 July 1995 under the Budapest Treaty.

32 The strain K, as hitherto isolated from an Atlantic 33 salmon, in accordance with Olsson, J. C., et al, Appl 34 Environm. Microbiol. 58:551-556 (1992) (enclosed herein as a reference) proved to be a motiel, Gram-positive 36 pleomorphic, facultative anaerobic rod. The CA 02228~21 1998-02-03 1 antibacterial activity of the strain K was analysed and 2 the results suggested that multiple, broad range 3 antibacterial compounds are released in the surrounding 4 medium during the logarithmic phase of growth in TSBS
(Tryptic Soya Broth supplemented with Salt, NaCl 2%
6 w/v) medium. The inhibitory compounds were also 7 produced when the strain K was grown in diluted 8 intestinal mucus, which suggests that the strain K
9 bacterium will proliferate and produce the antibacterial substance in the gut. The antibacterial 11 compounds were heat labile, and were initially 12 determined to have a molecular weight of about 140-150 13 dalton by gel filtration. The antibacterial compounds 14 have been found to have an inhibitory activity against both Gram-negative and Gram-positive bacteria, but not 16 against yeast. The antibacterial compounds are 17 bacteriostatic at low concentrations but bactericidal 18 at higher concentrations. The activity is maintained 19 when the compounds are stored in frozen state, but is lost when maintained at 23 C.

22 Strain K shows the following phenotypic 23 characterization:

The major fatty acids are 16:0 (31.1~); 16:1 (24.2%);
26 and 18:1 cis 9 (23.4~). The remaining fatty acids are:
27 18:2 cis 9, 12; 18:0A (10.8%); 14:0 (5.4%) and 18:0 28 (3.5%). Strain K can utilize the following carbon 29 sources: sucrose, maltose, trehhalose, mannitol, ribose, B-D-glucopyranoside. It was not able to 31 produce acids from the following carbon sources: cyclo-32 dextrin, tagotose, D-arabitol, L-arabinose, melezitose, 33 melibiose, pullulan, glycogen, raffinose, lactose, and 34 sorbitol. It did not produce acetoin. It was able to hydrolyse hippurate. Beta-Glucosidase activity was 36 demonstrated. No activity of the following enzymes CA 02228~21 1998-02-03 1 were detected: urease, betagalactosidase, beta-2 glucuronidase, alfa-galactosidase, arginin dihydrolase, 3 alanylphenylalanyl-prolin arylamidase, acide 4 pryoglutamique arylamidase, N-acetyl-beta-glycosaminidase.
7 Strain K is sensitive to gentamycin, erythromycin, 8 rifampicin, tetracyclin, ampicillin, and kanamycin. It 9 is sensitive to a lesser extent to neomycin and nalidixic acid. Strain K does not harbour any 11 detectable plasmids.

13 A 2.3 fold diluted of a cell-gree culture supernatant 14 provides a total growth inhibition of Vibrio anquillarum (HT11360). Aeromonas salmoncida (ATCC
16 14174) is more sensitive than the Vibrio anquillarum 17 strain, and a 10 times dilution results in total growth 18 inhibition during a 24 hours test period. Any dilution 19 of the TSBS does not interfere with these results.
21 The active compounds loses its activity gradually at 22 4 C and cannot be detected after 8 weeks. When frozen 23 the full activity remains for at least 12 months.

The activite compound of strain K inhibits a large 26 number of bacteria, whereby no difference is seen 27 between Gram-negative bacteria. All pathogens tested 28 were sensitive, whereby StaPhYlococcus aureas and 29 Proteus vulqaris CCUG 6327 proved to be the most sensitive and E. Coli Av24 and Pseudomonas aeruqinosa 31 the least. Yeast is not inhibited.

33 Strain K grows in intestinal mucus. Growth was 34 proceeded by a lag phase of at least 7.5 hours. This is comparable with the length of the lag phase that is 36 exhibited in TSBS by the same strain in the same CA 02228~21 1998-02-03 1 temperature. Strain K was found to produce substances 2 during growth in mucus that are inhibitory to growth of 3 the fish pathogens Vibrio anquillarum and Aeromonas 4 salmonicida. The inhibitors were detected in the mucus at the onset of the logarithmic growth (7.5 hours). An 6 increase in the inhibitory activity was observed 7 throughout the log phase and into stationary phase.
8 The growth of the pathogens was not inhibited in the 9 control culture with intestinal mucus without strain K.
The colonies on the TSBS plates were identified as 11 strain K, a Carnobacterium by biochemical tests. The 12 bacteria in the pinpoint colonies were found to be 13 motile, forming pairs or chains with four cells, Gram 14 positive, catalase and oxidase negative. Inhibition zones were around the colonies when tested against V.
16 anguillarum.

18 The colony forming units (CFU) were found to increase 19 approximately 3 log during 12.5 hours of growth in faeces suspension. No lag phase was seen during growth 21 in faeces suspension. After the culture had reached 22 stationary phase, a. 12.5 hours, no further increase of 23 inhibitory activity was detected.

Production of substances that inhibit the growth of the 26 fish pathogens, V. anquillarum and A. salmonicida, was 27 detected after 7.5 hours. The inhibitory activity 28 increased until 12.5 hours and then remained unchanged.
29 V. anquillarum and A. salmonicida grew in the faeces control. The identity of the strain K colonies was 31 confirmed as described in the previous section.

33 Neither strain K, nor its inhibiting compound (-s), is 34 toxic to fish. No fish in any tested group died or showed any external signs of disease during the 36 experiments in which fish were exposed to strain K.

CA 02228~21 1998-02-03 1 Spleens from both infected with strain K and control 2 fish were free from culturable bacteria.

4 Media diluent and culture conditions s 6 The media and diluent used with Tryptic Soya Broth 7 (TSB, Difco) and TSBS (TSB supplemented with 2% NaC);
8 TSA (TSB + 15% agar) and TSAS (TSA supplemented with 2%
9 NaCl); TSAS soft agar (TSBS + 5% agar); Marine agar (MA, Difco); Nutrient agar (NA, Difco); Brain heart 11 infusion (BHl, Difco); Rogosa (Difco); TCBS Cholera 12 Medium (Oxoid); Marine Minimal Medium (MMM, Neidhardt 13 et al, (1974)); VFI (peptone 1.0g, yeast extract 0.5 g, 14 glucose 0.5g, starch 0.5g; Salmon intestinal buffer (Hickman (1968)); NaHCO3 1.03g, NaCl 1.97g, KCl 0.16g, 16 CaCl2.2H20 2.07g, MgS04.7H20 21.95g, MgCl2.6H20 10.67g, 17 agar 15g distilled water 1000ml); VNSS agar (peptone 18 1.0g, yeast extract 0.5g, glucose 0.5g, starch 0.5g, 19 FeSO4.7H20 0.01g, Na2HPO4 0.01g, agar 15g in 1000ml NSS);
NSS (Nine Salt Solution: NaCl 17.6g, Na2S04 1.47g, NaHCO3 21 0.08g, KCl 0.25g, KBr 0.04g, MgCl2.6H.0 1.87g, CaCl2.2H20 22 0.41g, SrCl2.6H20 0.008g, H3B03 0.008g in 1000 ml double 23 distilled water). Horse-blood agar (HBA, TSA
24 supplemented with 5% horse blood.
26 Bioassay for the detection of inhibitory effect 28 i) Double-layer agar method.

The plates were screened for inhibition by a 31 modified double-layer agar method described by 32 McLeod and Govnlock (1921). Macrocolonies of the 33 inhibitory bacteria were obtained by spot seeding 34 on agar plates (10 ~l of a liquid culture in log phase) and incubating the plates for 18 hours at 36 23 C. The macro-colonies were treated with CA 02228~21 1998-02-03 1 chloroform vapour for 30 min. The pathogen (100~1 2 of a 10 x diluted liquid culture) was then seeded 3 into a tube of melted (temperature to 45 C) TSAS
4 soft agar (3ml) mixed and then poured onto the top of the plates. After incubation for 18 hours the 6 zones of growth inhibition created by the 7 producing colonies were measured as the distance 8 between the edge of the macro colony and the edge 9 of the clearing zone.
11 ii) Liquid bioassaY in microtitre wells 13 The inhibitory effect was determined as changes in 14 the optical density (OD6~(,), using microtitre spectrophotometer (Bio Tech. Biokinetics). The 16 inhibition assay was performed in microtitre wells 17 (Nunc, 96 wells). The inhibitory bacterium was 18 grown in TSBS at 23 C. The cells were removed by 19 centrifugation and the supernatant was filter sterilized (MFS-25 cellulose acetate filter units, 21 0.2 ~m). The cell free supernatant (150 ~l) was 22 transferred to a microtitre well and an equal 23 volume of fresh TSBS (150 ~l) was added.
24 Thereafter the target microorganism (3 ~l from a logphase culture) was inoculated in the well and 26 the growth was monitored by measuring the optical 27 density at 610 nm for 24 hours. As a control a 28 dilution series with 2% NaCl solution and TSBS was 29 made to relate growth of the pathogen with the amount of medium added. To control for any auto-31 inhibition by the pathogen, cell-free culture 32 supernatant (from a culture of the pathogens) was 33 treated in the same way as the supernatant derived 34 from the inhibitory strain.

CA 02228~21 1998-02-03 1 Table 1 3 The double-layer agar method was used to test growth 4 inhibition of a wide range of bacteria, as well as salmon related yeasts by macro-colonies of isolated 6 strain K.

8 Orqanism GRAM Inhibition g +/- Zone 11 Strain K +
12 Vibrio anguillarum HTI 1360 + +++
13 V. anguillarum 2129 + +++
14 V. anguillarum + +++
V. ordalli NCMB 2127 + ++++
16 V. fisheri + ++++
17 Photobacterium angustum S14 - ++++
18 Aeromonas salmonicida ATCC 14174 ++++
19 A. hydrophila +++
A. hyrophila NCTC 8049 ++++
21 Escherichia coli B CCUG 214 +++
22 E. coli Av24 +
23 Vibrio sp. 4:44 +++
24 Salmon isolate +++
Vibrio sp. D2 +++++
26 Pseudomonas aeruginosa +
27 Staphylococcus aureus + +++++
28 Serratia marcescens CCUG 760 - +++
29 Micrococcus luteus + ++++
Proteus vulgaris CCUG 6327 _ +++++
31 Klebsiella oxytoca CCUG 383 - +++
32 Baciluus mageaterium CCUG 1817 + ++++
33 B. subtilis CCUG 163B + ++
34 Acinetobacter calcoaceticus CCUG 12864 - ++++
Streptomyces griseus CCUG 760 ++++
36 Citrobacter freundi CCUG 418 - ++++

CA 02228~21 1998-02-03 1 Marine yeast Sc18 (unidentified fish 2 isolate) 3 Marine yeast Sc3 Saccharomyces cerevisiae Debaryomyces hansenii HFI
6 S. cerevisiae CBS 7764 7 __ ___ 8 Inhibition zone radius (mm): 0 (-); 1-5 (+); 6-10 (++);
9 11-15 (+++); 16-20 (++++); >20 (+++++) 11 Table 2 13 Phenotype of strain K

15 ProPertY Strain K

17 Single rod ~, two polar flagella 18 Pleomorphic +
19 Motility +
20 Flagellation dipolar 21 Spores 22 Gram reaction +
23 Colony diameter <1 mm 24 Colony appearance on TSA Circular; entire;
semitranslucent; raised 26 Pigmentation Buff 27 Odour 28 Anaeorbic growth +
29 Temperature for growth 4-30 C
30 pH for growth 5.5 to 9 31 pH change during growth 7.2 to 6.8 32 Salinity for growth (NaCl) 0 to 6 33 Catalase 34 Oxidase 35 Haemolysis alfa 36 Urease 1 Hydrogensulphide production 2 Fermentative in Leifson weak acid, no gas 4 Strain K is thereby identified as a Carnobacterium and 5 was primarily characterized as Carnobacterium 6 alterfunditum. However, further investigations of 16S

7 rRNA demonstrates that Carnobacterium alterifunditum is 8 the closest related organism with a homology of 98.7%.

9 However, according to previous publications (Collins et 10 al, 1987) this would justify describing the isolate as 11 a new species of the genus. Thus the strain K is a new 12 strain, whereby it will be named more specifically 13 later on.

15 The inhibitory compound(s) was partly purified by fist 16 removing the bacteria cells from a TSBS culture by 17 entrifugatino in the early stationary growth phase 18 (13000 x g for 10 min). The sample was then kept at 19 4 C during the subsequent purification steps. The 20 cell-free culture supernatant was fractionated by 21 passing it through a 500 dalton cut-off filter (Amicon, 22 Difaflo YC05). Further purification was performed by 23 gelfiltration using a G10 Sephadex (Pharmacia, Sweden) 24 in a XK26/40 column. PBS (2mM) was used as a effluent 25 buffer. The ultra-filtrated supernatant was applied to 26 the coloumn and eluated at a flow rate of 44 ml/h.

27 Fractions (2.9 ml) were collected and examined for 28 antimicrobial activity by the liquid bioassay described 29 above. The apparent size of the inhibiting compound(-30 s) was determined using a standard curve including NADH

31 (709 D), N-formyl-Met-Leu-Phe-Phe (584.7 D, Sigma), 32 Tyr-Gly-Gly (295.3 D, Sigma), L-tryptophan (204.23 D) 33 and tyrosine (181.19 D) as markers. Blue dextran was 34 used to determine the void volume of the coloumn.
36 The inhibitorswas extracted using ethyl acetate at pH

CA 02228~21 1998-02-03 1 2.5 and subse~uently purified on TLC using silica gel.
2 The inhibitors are stable for at least 24 hours at a pH
3 of 2 to 11.

The partly purified inhibitory compound(-s) was 6 subjected to heat treatment, various enzyme treatments, 7 metaperiodate treatment, stability tested, and culture 8 media dependency.

The action of the inhibiting compound(-s) was 11 determined as follows. Aeromonas salmonicida from a 12 culture in log phase was inoculated into fresh TSBS to 13 a density of about 1 x 106 cells/ml. The culture was 14 incubated for 30 min prior to starting the experiment.
To 100 ml culture flasks, 10 ml of the Aeromonas 16 salmonicida culture and a mixture (10 ml final volume) 17 of the partly purified inhibitor supernatant, and NSS
18 (pH 7.2) was transferred such that a series of 19 concentrations of the inhibitor was obtained. The cultures were slowly shaken at 23 C and samples in 21 triplicate were taken to determine the number of colony 22 forming units (CFU) during at time period of 26 hours.

24 A 4 fold dilution of the cell-free supernatant resulted in total growth inhibition of Vibrio anguillarum 26 (H111360). Aeromonas salmonicida (ATCC 14174) was 27 found to be more sensitive than the Vibrio anguillarum 28 strain and a 10 fold dilution still resulted in a total 29 growth inhibition during the 24 hours test period. The dilution of TSBS did not interfere with the results.

32 The action of the inhibitory compound(-s) against 33 Aeromonas salmonicida using different dilutions is 34 shown in the Table 3 below.

CA 02228~21 1998-02-03 Table 3 Dilution CFU.ml after O 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. xlO' S S O
4 fold 6.4 4.4 2.0 1.0 0.8 0.2 0.1 0.0 xlO~
6 fold 6.4 5.4 3.0 1.9 1.4 1.0 0.8 0.2 0. 0.0 0. xlO' S O

1 The action of the inhibitory compound(-s) against 2 Vibrio anguillarum as determined as the growth of 3 strain K in TSBS at 23 c as and expressed as the 4 increase in optical density at 610 nm over time will be given below. Further the minimal dilution of free cell 6 culture supernatant causing total inhibition of Vibrio 7 anguillarum. The results are combined in Table 4 below.

CA 02228~21 1998-02-03 Table 4 T.~ ;o~ Tirne (hrs) Incre~se.in,Optical Minimal dilution density at 610 nm ' .,. ~ units 0.02~ 30 6 0.05 6.5 0.08 50 7 0.12 0.1~ 70 8 0.18 8.5 o 1 8 9 o 3 9.~ 0.25 80 0.2~
10.5 0.2s X0 16 01~ X0 1 The present strain K the other strains capable of 2 producing the active compound or chemically related 3 compounds or the active compound derived therefrom and 4 chemically related compounds and derivatives thereof can, in particular, be used in the prophylactic or 6 therapeutic treatment of fish infected by fish 7 pathogens, whereby an amount of the strain K that 8 provides an inoculum allowing the colonization of the 9 fish intestine by the strain K or an amount of the strain K that provides an active amount of the 11 inhibiting compound, is administered to the fish, or an 12 active amount of the inhibiting compound as such is 13 administered to the fish for prophylactic and/or 14 therapeutic treatment of fish susceptible to fish pathogens. Hereby all types of fish are encompassed, CA 02228~21 1998-02-03 1 to which strain K is not pathogenic, harmful or 2 deleterious, but in particular salmonids, turbot, 3 yellow tail, seabass/seabream and other types of farmed 4 fish and other aquaculture species, such as shellfish (prawns and shrimps) and mollusces.
~ 6 7 It might be so that strain K is pathogenic, harmful or 8 otherwise deleterious as such in some of the organism 9 to which it is administered although this is not foreseen. However, the active compound(-s) therefrom 11 might each be so, but can be administered instead for 12 obtaining a bacteriostatic or bactericidal effect.

14 The strain K or active inhibiting compound derived therefrom can be administered orally as such or via the 16 feed, which is the best mode, bathing of young or older 17 fish, single inoculation of young or older fish to 18 establish the strain K in the gut, or repeated 19 inoculation of young or older fish. When the active compound as such is administered together with the 21 feed, one has to consider the lability of the compound, 22 if it is to be incorporated into the feed before the 23 hydrothermal forming of pellets. A suitable means to 24 avoid thermal destruction of the active compounds is to add them to the feed pellets after their information 26 and cooling.

28 The strain K or its active inhibitory compound(-s) can 29 be administered in different ways such as via food, feed-stuff including drinking water, as a composition 31 as such containing the strain. Further it can be added 32 via spraying the animals, including fishes, by 33 immersion of the animals, in particular when fish is 34 concerned, by injection into the gut, or via inhalation.

Claims (17)

Claims

1. Use of a probiotic in the preparation of a medicament for treating mammals, including man, fish, shellfish and mollusces, comprising at least one microbial strain isolated from the resident 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 bacteriostatic and bactericidal Carnobacterium.

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

3. The use of said strain of claims 1-2 in the preparation of a medicament for the prophylactic treatment of mammals, including man, fish and other aquatic animals.

4. The use of said strain of Claims 1-2 in the preparation of a medicament 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 the medicament is administrable by immersion of the subject in a liquid containing said probiotic.

6. The use of the probiotic of Claims 1 to 4, whereby the medicament is administrable via the food/feed including drinking water, supplied to the subject.

7. The use of the probiotic of Claims 1 to 4, whereby the medicament is administrable via spraying onto the subject.

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

9. The use of the probiotic of Claims 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, Aeromonas salmoncida, Photobacterium angustum, Aeromonas hydrophila, Staphylococcus aureus, Bacillus megaterium, Acinetobacter calcoaceticus, Serratia marcescenes, Micrococcus luteus, Proteus 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, shellfish and mollusces.

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

14. The use 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 aureus, Bacillus megaterium, Acinetobacter calcoaceticus, Serratia marcescens, Micrococcus luteus, Proteus vulgaris.

15. A probiotic for treating mammals, including man, fish, shellfish and mollusces comprising at least one microbial strain isolated from the resident 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 bacteriostatic and bactericidal Carnobacterium.

16. A probiotic according to Claim 15, wherein the strain is a Carnobacterium having the accession number DSM 10087 as deposited 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, shellfish and mollusces in combination with other microbes isolated from a gut microflora of a healthy subject.