WO2004005292A1 - Novel antibiotics against vibrio anguillarum and the applications thereof in cultures of fish, crustaceans, molluscs and other aquaculture activities - Google Patents

Abstract

The invention relates to novel antibiotics against Vibrio anguillarum and the applications thereof in cultures of fish, crustaceans, molluscs and other aquaculture activities. Five diketopiperazines with activity against Vibrio anguillarum have been discovered, namely compounds: cyclo(D)-proline-(D)-phenylalanine (Z59), cyclo(D)-proline-(D)-isoleucine (Z56), cyclo(D)-proline-(D)-valine (Z57), cyclo(D)-proline(D)-leucine (Z54) and cyclo(D)-hydroxyproline-(D)-phenylalanine (B717), which are obtained from two marine bacteria, Roseobacter gallaeciensis (CECT 5719) and Roseobacter sp. (CECT 5718), which are isolated from larval cultures of Pecten maximus. The addition of said compounds at a concentration of 0.5 mg/mL to cultures of fish, crustaceans and molluscs increases the survival capacity thereof with values varying between 12 % and 33 %. The inhibiting activity thereof is comparable to that of antibiotics used at present in aquaculture.

Description

 TITLE

New active antibiotics against Vibrio anguillarum and its applications in fish, crustaceans, molluscs and other aquaculture activities.

DESCRIPTION Vibriosis is one of the diseases that causes the most economic losses in marine aquaculture (Toranzo and Barja. 1990; Myhr et al. 1991). This disease is caused by different bacterial species belonging to the genus Vibrio, being the species Vibrio anguillarum the main responsible for epizootics in a wide variety of fish, crustaceans and mollusks. In Galicia (NW of Spain), vibriosis has been a continuous and limiting problem in marine fish farming over the years. Since 1985 Vibrio anguillarum has been the main causative agent of vibriosis in turbot fry, being sporadically isolated from salmon and rainbow trout (Toranzo et al. 1987b; Toranzo and Baija. 1990). In the same way, this Vibrio has been the cause of mortalities in marine fish, molluscs and crustaceans from other geographical areas (Masumura et al. 1989; Baticados et al.

1990; Lavilla-Pitogo et al. 1990; Paillard and Maes. 1990; Myhr et al. 1991; Castro et al. 1992).

In view of the problems described above, which reduce productivity in aquaculture facilities, the addition of antibiotics to the culture medium has been systematically used. The main chemotherapeutic agents so far used in marine aquaculture are oxytetracycline, classical quinolones (oxolinic acid, flumechin) and fluorinated (enrofloxacin), and nitrofurans. Oxolinic acid and flumequine have become obsolete due to the emergence of resistant strains, while enrofloxacin and florfenicol are being used despite not being approved for use in fish due to the possible occurrence of resistant strains while no new chemotherapeutic agents are found. effective. For its part, the use of nitrofurans has been banned in aquaculture. In the specific case of scallop larval crops (pectén maxima) and other bivalves susceptible to aquaculture, it has been proven that the use of filtered and irradiated seawater with ultraviolet light, and frequent changes of water during cultivation, do not they are sufficiently effective measures, being necessary the use of antibiotics to prevent bacterial mortalities that usually appear spontaneously after a week or two of culture (Alderman and Michel. 1992; Nicolás et al. 1995).

Therefore, the search for new active antibiotics in the marine environment, capable of protecting crops in aquaculture facilities and not generating pollution, is a matter of great importance and for which new solutions are necessary (Douillet et al. 1993 , 1994; Nogami et al. 1992). DETAILED EXPLANATION OF THE INVENTION Bacterial strains:

155 bacterial strains _^ were introduced from the larval culture water of Pectén maximus _^ which were introduced into Petri dishes with a basal agar-marine medium (5 g / L bacteriological peptone, yeast extract: 1 g / L, ferric citrate strokes and Bacteriological agar: 14 g / L) pH 7.6 characterized by its saline content, and incubated at a temperature of 23 ° C in both liquid and plate cultures.

Study of antibacterial activity:

For the study of the antibacterial activity of these strains against Vibrio anguillarum, antagonism tests were used by the method of discs and microplates.

For the tests of antibacterial activity by the method of the discs sterile filter paper discs, with a diameter of 5 mm, were impregnated with the supernatant, and were deposited on agar plates previously seeded in striations with the Vibrio anguillarum and subsequently incubated for 24-48 h at 23 ° C. The activity was determined by the inhibition halo.

Microplate assays in the marine broth were carried out with pure cultures of the different bacterial and coculture strains of the chosen bacterial strain, plus the control strain. To each well of a 96-well microplate, 200 μl of marine broth and 10 μl of supernatants from different strains were added. Finally, they were added to each well

- ₃ 20 μl of the control strain {Vibrio anguillarum) diluted to 10.

As controls, six wells were maintained with 200 μl of marine broth, three of them inoculated with 10 μl of the corresponding bacterial strain and the other three with 20 μl of the

-3 control strain diluted to 10. The microplates were incubated for 24-72 h at a temperature of 23 ° C and the degree of inhibition was determined by an Elisa reader from the different wells. The tests were done in triplicate.

Location of antibacterial activity in pure and coculture cultures:

From the study of the activity of all the bacterial strains studied in this work, it can be deduced that only the two strains named CF-20 (Spanish Crop Type CECT 5719 Collection) and C-148 (CECT 5718), are capable of inhibiting the growth of the Vibrio anguillarum and therefore we continue our work only on these two strains.

In marine broth (bacteriological peptone 5 g / L, yeast extract: 1 g / L, traces of ferric citrate and bacteriological agar: 14 g / L) pure cultures of the strains were carried out

C-148, and CF-20 and co-cultures of C-148 and CF-20 with V. anguillarum. After growing up under stirring, for 48 h, the cultures and cocultures were centrifuged at 2724 G for 10 min at 4 ° C. Once the supernatant was separated from the precipitate, the cells were resuspended in a known volume of seawater, broken by ultrasound (3x1 min, 20 K z) and washed three times with sterile seawater. The broth supernatants and pellet wash and lysates were tested with the disc methods and the microplate method to locate the antagonistic effect. bacterial identification:

The identification of the bacterial strains under study was determined by determining their phenotypic characteristics, API-20E and API-20NE multi-test systems and genetic [genomic DNA extraction, phylogenetic analysis of 16S rRNA sequences, RAPD and hybridization assays with probes: DNA / DNA (DOT BLOT)]. Preparation of bacterial cultures of CF-20 and C-148

A colony of bacterial strains CF-20 or C-148 and another of V. anguillarum were seeded and grown in marine agar, inoculated in coculture in flasks of 1.8 L capacity with 1200 mL of marine broth each and were incubated at 23 ° C with orbital shaking at 140 rpm for 24 h.

After that time, when the bacterial growth is in exponential phase, the broth is centrifuged at 2724 G at 4 ° C, and the pellets are sonicated and washed with seawater. The inhibitory capacity of the supernatants and lysates was checked by the method of the discs, using as filter controls impregnated in marine broth. Isolation and identification of active substances: Extraction and isolation:

Following the culture procedure described above {Preparation of bacterial cultures of CF-20 and C-148), the 45 L of supernatant from the marine broth from the co-culture of CF-20 with V. anguillarum are combined with the supernatant of the lysates of the corresponding pellets, and the total volume is extracted 3 times with the mixture C1 ₂ CH ₂ : MeOH 75:25. The extracts were concentrated and partitioned between water and CH ₂ C1 ₂ and the concentrated organic phase was subjected to silica gel column chromatography eluting with hexane mixtures: AcOEt. For the localization of the inhibitory activity to V. anguillarum the isolation and separation processes were followed by the methods of discs and microplates, dissolving 1 mg of each fraction in 100 μl of ethanol.

The active fractions obtained from the column were chromatographed by HPLC on a normal phase serrüpreparative column (μ-porasil; hexane: AcOEt 2: 3; flow: 3 mL / min) providing 3.5 mg of Z54 with a t _R of 22 min; 4 mg of Z56 with a t _R of 29 min; 3 mg Z57 with a t _R of 37 and 5 mg of Z59 with a t _R of 44 min. On the other hand, 45 L of culture supernatant of strain C-148, were extracted and chromatographed as before. The active fractions eluted from the chromatography column provided an impure compound that was purified by HPLC on a reverse phase column (μ-Bondapak C-18; MeOH: H ₂ O 95: 5; flow: 2 mL / min) giving 3 mg of pure active B717 with a t _R of 17 min and a mixture that subjected to HPLC in a semi-preparative column of normal phase (μ-porasil; hexane-AcOEt 2: 3; flow: 3 mL / min) provided important active compounds against to Vibrio anguillarum 4 mg of Z54; 3.5 mg of Z56; 3 mg of Z57 and 2.5 mg of Z59. All compounds showed optical activity (Table 1) and were identified as diketopiperacins.

The structural elucidation of these compounds was carried out using techniques of

13 1

NMR (experiments C, H and DEPT), mass (e.; (+) FAB; low and high resolution) mainly, as well as by direct comparison with authentic samples obtained by synthesis. Table 1 shows the most relevant spectroscopic data ( ¹³ C, 1H and MS).

Table 1: Retention times t (min), {α} ^D and fragments in antibiotic mass spectrometry.

Synthesis of diketopiperacins:

In order to have authentic samples of the diketopiperacins for direct comparison with the natural ones, for antibiotic activity tests and for the determination of the structural characteristics linked to that activity, they were prepared by known procedures (described later: Preparation of diketopiperacins LL and DD) and from the corresponding α-amino acids, a wide series of diketopiperacins of the LL and DD series that were then epimerized to obtain the corresponding dipetopiperacins LD and DL. Preparation of diketopiperacins LL and DD:

Protected dipeptides such as BOC (butyloxycarbonyl) and methyl ester were first prepared and cycled by heating in formic acid / BuOH / Toluene. Thus, the LL and DD diketopiperacins of Table 2 were prepared, except for B717 which was obtained by coupling between Cbz- (D) -trans-4-hydroxy- (D) -proline with (D) -phenylalanine methyl ester followed of N-deprotection by catalytic hydrogenation and cyclization to give compound B717.

Preparation of diketopiperacins LD and DL by epimerization:

The procedure described by Ott et al. 1963 and Adamczeski et al. 1995, with slight modifications as described below:

In a small glass vial, 20 mg of diketopiperacin DD or LL are dissolved in 0.3 rnL of sodium hydroxide (0.5 mL of 0.5 N NaOH in H ₂ O: MeOH, 1: 1) and kept for 15 min at room temperature. A few drops of 15% hydrochloric acid are added and left overnight. The solution is concentrated in vacuo and the residue is extracted with methylene chloride, the solvent is concentrated and crystallized with ethyl acetate.

Minimum inhibitory concentration (MIC) of isolated antibiotics:

The minimum inhibitory concentration (Civil) of the isolated active substances Z54, Z56, Z57, Z59 and B717 was determined by the agar dilution method described in the NCCLS guide (1997), with slight modifications in the composition of the medium, time of incubation and temperature. Solutions of the active substances were prepared at different concentrations (0.005; 0.05; 0.1; 0.2; 0.3; 0.4; 0.5; 0.7; 0.8 and 5 μg / mL) , and suspensions of V. anguillarum at different dilutions (from 10 to 10 cells / mL). Different concentrations of the active substances on Petri dishes previously seeded with V. anguillarum are then added and the inhibition halos measured after 24-48 hours. .

In vivo activity of active substances:

In vivo studies of the antibiotic effect of these isolated substances Z54, Z56, Z57, Z59 and B717 were also carried out in various crops of interest in aquaculture such as those of Dicentrarchus labrax, Sparus aurata, Psetta maxim, Maja squiado, Ruditapes decussatus and Ostrea Edulis Those carried out with Ruditapes decussatus and Ostrea edulis, which are representative, are described below.

Larval cultures:

The larval cultures of Ruditapes decussatus and Ostrea edulis were kept in the laboratory at a controlled temperature of 20 ° C, the water being renewed and the bacterial flora in general and the V. anguillarum analyzed every two days. Two types of Experiments: The first in 100 mL tubes to make an approximate determination of the dose of each of the active substances that provides the best growth and / or survival of the larvae in relation to the control, and the second in 70 L tanks for confirm the most appropriate activity and dose, and approach the operating conditions in aquaculture.

EXPERIMENT 1:

For these experiments, 100 mL pyrex tubes were used, containing 90 mL of seawater, filtered at 1 μm and a larval density of 5-7 larvae ^" for Ruditapes decussatus and 1-2 larvae for Ostrea edulis. Age of the larvae at the beginning of the experiments was 3 to 5 days.The cultures were maintained with aeration throughout the experiment, and fed with a mixture of the microalgae Isochrysis galbana (T-iso), Chaetoceros calcitrans, Pavlova Lutheri and Tetraselmis Suecica (20: 10: 20: 5 cells. μL ^"1 , respectively).

To these cultures were added different concentrations (0.005; 0.05; 0.1; 0.2; 0.3; 0.4; 0.5; 0.7; 0.8 and 5 μg / mL) of each of the active substances, and the dose of each substance was determined, which allows the best growth and / or survival of the larvae, measured 22 days after the start of the culture. EXPERIMENT 2:

Once the appropriate dose of each substance was determined according to the method described in experiment 1, the effect of the active substances in larval cultures contained in 70 L tanks was checked, maintaining the same experimental conditions of experiment 1, except that the counting of The larvae were performed 11 days after the addition of the substances.

Mortality, larval growth and bacterial flora:

For the determination of mortality and larval growth after the addition of the isolated antibiotic substances (Z54, Z56, Z57, Z59 and B717), the larvae from the previous experiments were collected in sieves, and suspended in a small volume of seawater, from which three 100 μL aliquots were taken and examined for the estimation of the average number of larvae and the calculation of survival. To analyze the evolution of the vibrios and bacterial flora in general, after the addition of the antibiotic substances, samples of 1 mL of culture were taken that were diluted with sterile seawater to a dilution of 10 ^" . 40 mL aliquots of these dilutions were seeded in duplicate on Z2216 marine agar (Difco) and Thiosulfate-Citrate-Bile- agar Sucrose (TCBS) (Oxoid). The marine agar culture plates were incubated at 23 ° C, for 2-4 days, and those of TCBS for 2 days. After this time, the viable units (CFU: Colony Forming Units) were calculated by multiplying the average number of colonies by the inverse of the dilution factor. From the study of the activity of all bacterial strains present in scallop, sea bream and flat oyster cultures, it can be deduced that only the two strains called CF-20 and C-148 are capable of inhibiting the growth of Vibrio anguillarum. In addition, it is verified that this activity only occurs when either of these two bacterial strains is in direct contact with V. anguillarum. When separate assays for the pellet and culture supernatant of CF-20 and C-148 were carried out the activity results clearly indicate that antibiotic substances are present in the supernatant and in the cell pellet.

The study of its phenotypic characteristics, multipmeba API-20E and API-20NE and genetic systems, identify CF-20 strains as Roseobacter galleaciensis sp and C-148 as Roseobacter sp.

Cultures of CF-20 and C-148 strains:

In order to isolate and identify the active substances against Vibrio anguillarum, and obtain them in sufficient quantity, a series of small-scale cultures were first carried out to determine the best experimental conditions for the cultivation of the active strains and the production of antibiotics as well as the moment in which the concentration is maximum. From the results, it was deduced that the culture in marine broth (peptone: 5 g / L, yeast extract: 1 g / L and ferric citrates) at 23 ° C, with orbital agitation (140 rpm) for 24 hours and in coculture of the CF-20 or C-148 strain and Vibrio anguillarum were the most favorable conditions. With this information, co-cultures of strains CF-20 and C-148 were established with the

Vibrio anguillarum in 1.8 L flasks that were used for the extraction of active substances against Vibrio anguillarum from the broth supernatant and cell lysates in which we had previously located the activity. Isolation, structure and synthesis of antibiotics: Isolation:

The extraction, separation and isolation processes were followed through the activity tests on disks and microplates. Thus, by selective extraction of the broth and lysates of the co-cultures of CF-20 and C-148, an active extract was obtained against Vibrio anguillarum whose fraction soluble in dichloromethane, was subjected to chromatographic separation on silica gel that provided several active fractions from which finally 3-5 mg of the active components were isolated by HPLC against the pure Vibrio anguillarum.

From the bacterial strain CF-20, the four compounds were obtained: Z54, Z56, Z57 and Z59, while extracts of strain C-148 provided in addition to those same four components, in similar amounts, a new active component B717.

The structural study of these compounds carried out by spectroscopic methods (NMR and MS mainly), show for Z54, Z56, Z57, Z59 the presence of signals in the proton and carbon NMR characteristics of diketopiperacins containing the Pro ring fused with Leu, He, Val and Phe. The low and high resolution electronic impact and FAB mass spectra confirm these structures. For its part, compound B717, has spectroscopic characteristics similar to those of Z59 but contains 4-hydroxyproline instead of proline. The spectroscopic data mentioned further indicate that the two asymmetric centers of the five diketopiperacins have the same configuration, (LL or DD) and are consistent with those described for cycle (L) -proline- (L) -Leucine (Pickenhagen et al. 1975 ; Paul et al. 1976; Schmitz et al. 1983; Adamczeski et al. 1995; Ginz et al. 2000), cycle (L) -proline- (L) -Isoucine (Adamczeski et al 1995; Ginz et al 2000), cycle (L) -proline- (L) -valine (Paul et al. 1976; Schmitz et al 1983; Adamczeski et al 1995 and Ginz et al 2000), cycle (L) -proline- (L) -phenylalanine (Pickenhagen et al. 1975; Paul et al. 1976; Adamczeski et al. 1995 and Ginz et al. 2000), and cycle (L) -trans-4-hydroxypropyl- (L) - phenylalanine (Adamczeski et al. 1989; Young et al 1976; Trigos et al. 1997; Jayatilake et al. 1996; Ginz et al. 2000), but the rotational power values of our compounds indicate that they are the corresponding enantiomers. Synthesis of isolated antibiotics: The corresponding diketopiperacins with stereochemistry LL and DD were prepared using common procedures (described above in Synthesis of diketopiperacins) from alpha amino acids. By epimerization of the above, the corresponding diketopiperacins with stereochemistry LD and DL were obtained. The direct comparison showed that the natural ones are the DD enantiomers. Thus Z54 is cycle (D) -proline- (D) -Isoleucma, Z56 is cycle (D) -proline- (D) -Leucine, Z57 is cycle (D) - proline- (D) -valine, Z59 is cycle (D) -proline- (D) -phenylalanine, and B717 is cyclo (D) -trans-4-droxypropyl- (D) -phenylalarine. STRUCTURES OF ISOLATED COMPOUNDS

Cycle (D-pro-D-phe): Z59

ciclo(D)-pro-(D)-Leu: Z54 ciclo(D)-pro-(D)-val: Z57

cycle (D) -pro- (D) -Leu: Z54 cycle (D) -pro- (D) -val: Z57

Cycle (D) -tr-ms-4-hydroxypropyll-D-phenylalanine

B717 cycle (D) -pro- (D) -Ile: Z56

Tables 1, 3 and 4 show the proton, carbon and E. mass NMR data of antibiotics isolated from bacterial strains.

Antibiotic activity of natural and synthetic diketopiperacins: The synthesis of diketopiperacins with diverse stereochemistry, from alpha amino acids, allowed us to have a wide collection of compounds that were subjected to tests of their antibiotic activity together with natural samples (Table 2).

All natural antibiotics (Z54, Z56, Z57, Z59 and B717) isolated from both bacterial strains CF-20 and C-148 have shown strong activity against Vibrios and bacterial flora in general, which justifies the effect found in the initial larval cultures. In vitro tests show an activity (0.03 <MIC <0.07 Table 2), comparable or superior to that of some of the antibiotics currently used in aquaculture such as oxytetracycline (MIC: 0.5 μg.mL ^"1 ).

The systematic study of the antibiotic activity of the collection of synthesized and natural diketopiperacins, shows that the activity is essentially linked to the stereochemistry of the compound and that in general, the DD isomers are about 2 times more active than the LD and DL while that LL isomers are practically inactive.

Table 2: Mimic ima inhibitory concentration (MIC; μg.mL ^"1 ) against V. anguillarum of a selection of diketopiperacins with stereochemistry DD, LD and DL.

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When the active antibiotic substances were tested in vivo, it could be verified that this activity is maintained and that they produce a clear reduction of the total bacterial flora and that of the Vibrios and that they improve the survival of the species grown in the tank. As an example, Figure 3 shows the results of in vivo tests with larvae of Ruditapes decussatus mollusc, of the natural diketopiperacins isolated from strains CF-20 and C-148. In these experiments, it is observed that the survival of the larvae increases in values ranging from 12 to 33%, the most active compound being Z59 and the least active B717, there being no essential differences in the activity of the others that remain around 20%.

The high mortality in the control tanks clearly shows that the bacterial flora is the main cause of mortality of the larvae, as indicated by the values obtained for CFU and the observed changes in bacterial flora in general and in Vibrio.

In vivo essays

D survival

Z57 Z59 Z54 Z56 B717 (0.5) (0.5) (0.5) (0.5) (0.5) substances added (μg.mL ^"1 )

Graph 3: In vivo activity tests against Ruditapes decussatus

In fact, the antibiotic activity of these substances is so intense that in cultures treated with antibiotics at concentrations as low as 0.5 ppm., Vibrios are reduced by more than 70%. The protective effect that the addition of these substances has on larval cultures is clearly evidenced by the fact that the larvae of cultures not treated with antibiotics did not survive more than four days. In contrast to that described for commercial antibiotics such as chloramphenicol, florfenicol, flumequina and trimethoprim / sulfadiazine, our in vivo tests show that active diketopiperacins do not lose their activity due to changes in salinity and temperature (Christian. 2001) of the medium, associated with the passage from in vitro to in vivo assays. Procedure for the use of antibiotics:

Mollusc treatment:

A solution containing the antibiotic or mixture of the described natural and synthetic antibiotics is added to the tank at the beginning of the larval culture so that the final concentration is 0.5-1 μg / mL. After two days the water in the tank is renewed and the antibiotic solution is replenished, restoring the initial concentration. The process of water renewal and the addition of antibiotics is repeated up to a total of five times. After that time, (10 days) the treatment is terminated. Fish and crustacean treatment:

1) For crops with water circulation in a closed system, the water in the tank is reduced to the minimum level that does not affect the cultivated species, and the antibiotic is added until reaching the final concentration indicated above, leaving it to act for 30 min with stirring and after that time, the normal water level is restored. This treatment is repeated every two days for a total of 4-5 times (8-10 days of treatment).

2) For crops in which the water circulates in an open system, the water supply is stopped for 30 min, the level is lowered to the minimum that does not affect the cultivated species and the antibiotic is added as in the previous case. After 30 min the normal water circulation is restored. This process is repeated every two days up to a total of 4-5 times (8-10 days of treatment).

Table 3: ¹³ C-NMR (CDC1 ₃ , 300 MHz) data of antibiotics isolated from bacterial strains: CF-20 and C-148

Table. 4: 1H-NMR data (CDC1 ₃ , 300 MHz) of antibiotics isolated from bacterial strains: CF-20 and C-148:

Table. 4 (Continued):

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Claims

1.- New active antibiotics, and their synthetic analogues, against Vibrio anguillarum obtained from cultured bacterial strains, and their applications in fish culture, crustaceans, molluscs and other aquaculture activities. 2.- The bacterial strains CF-20 {Roseobacter galleaciensis sp) and C-148 {Roseobacter sp.), Deposited in the Spanish Type Culture Collection with the number CECT 5719 and CECT 5718, respectively, and characterized by: - Be bacilli with brown colonies, Gram negative, and mobile; - Its RAPD profile and its hybridization with DNA of other species of the genus Roseobacter, - Not presenting, arginine dehydrolase activity, lysine decarboxylase, or gelatinase; - Be the strain C-148 sensitive to Ampicillin, LincoSpectin and Norfloxacin. 3. Method of cultivation of the bacterial strains CF-20 and C-148, according to claim 2, characterized by the pure culture of the strains and in coculture of these strains with V. anguillarum in 1.8 L flasks with compound marine broth of yeast extract, peptone and traces of ferric citrate, at temperatures between 4 ° and 35 ° C and pH between 4 and 10, with orbital agitation for 24 hours. 4. Procedure for obtaining antibiotics, from the cultivated strains, according to claim 3, characterized by: - Selective extraction of marine broth and lysates from CF-20 and C-148 cultures. - Obtaining the active extract by using organic solvents of different polarities. - Chromatographic separation in silica gel of the active extract that provides several active fractions. - Isolation by HPLC in amounts of mg of the pure active components. 5- Method of obtaining antibiotics from strain CF-20, according to claim 4, characterized in that the compounds have a diketopiperazine structure, corresponding to: Z54: cycle (D) -proline- (D) -Isoleucin; Z56: cycle (D) -proline- (D) -Leucine; Z57: cycle (D) -proline- (D) -valine; Z59: cycle (D) -proline- (D) -phenylalanine. 6. Method for obtaining antibiotics from strain C-148, according to claim 4, characterized in that the compounds have a diketopiperazine structure, corresponding to: Z54: cycle (D) -proline- (D) -Isoleucin ; Z56: cycle (D) -proline- (D) -Leucine; Z57: cycle (D) -proline- (D) -valine; Z59: cycle (D) -proline- (D) -phenylalanine; and B717: cycle (D) - trans-4-hydroxypropyl- (D) -phenylalanine. 7. New natural antibiotics, according to claims 5 and 6, corresponding to the diketopiperazines with structure I: Z54, Z56, Z57, Z59 and B717 isolated from marine bacteria CF-20 and C-148. 8.- Use in aquaculture of diketopiperacins with structure I, and absolute stereochemistry DD, DL and LD, derived from natural and synthetic D and L-α-amino acids, and diketopiperacins with absolute stereochemistry D, derived from glycine, and any of the other natural or synthetic D-α-amino acids, where Ri and R ₂ may be the same or different and correspond to the side chains of the α-amino acids of natural or synthetic origin, including 4-hydroxyproline, hydroxylysine, demosin, isodemosin, 3-methylhistidine, norvaline, homocysteine citrulline, homocysteine, homoserine, ornithine and methionine sulfone, cis and trans-α- (carboxycyclopropyl) glycine, α- (methylenecyclopropyl) glycine, and the radicals R ₃ and R-ι, the same or different, they can be H, alkyl, alkenyl, alkynyl, alkylaryl, aryl, acyl, carboxyalkyl, carboxyalkenyl, carboxyalkynyl, carboxyaryl, carboxyalkylaryl, as antibiotics against Vibrio anguillarum and bacterial flora in c Ultimate mollusks, crustaceans and fish.

I 9.- Use in aquaculture of compounds with structure I, such as Z54, Z56, Z57, Z59 and B717, as antibiotics against Vibrio anguillarum and bacterial flora in shellfish, crustacean and fish crops. 10. Use of antibiotics, according to claims 1, 4-9, alone or in combination with other agents, in the preparation, of any kind of pharmaceutical form, of food additives, of products with microalgae, of artemia, of rotifers of feed for the treatment of diseases in fish culture, crustaceans and molluscs and aquaculture activities.