FIELD OF THE INVENTION

The present invention is focused on the biotechnology area, particularly in fermentation processes for obtaining antagonistic activity from microorganisms belonging to the so- called group of Aerobic Endospore Forming Bacteria (AEFBs) from BaciUaceae and Paenibacillaceae families, under the presence of an inducer agent.

BACKGROUND

Over the history of humankind, disease control in both animals and plants has been mainly supported on the use of natural products (NPs) or products derived from natural sources. Antibiotics and pesticides are among these NPs, mainly used to combat infections of bacterial origin and to control fungal or bacterial plant diseases, respectively. The indiscriminate, excessive and inappropriate use of these natural compounds have rendered several problems though one in particular outstands: resistance or loss of sensibility of the pathogens to these molecules, leaving some of them out of the market, due to the large amounts that must be used to achieve control, and reducing the number of useful molecules to fight infections and diseases. Some of the most aggressive pathogens have evolved into strains which are resistant to many of the known antibiotics, as are the cases for Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus [1] posing a major threat in case of appearance of a massive infection or outbreak.

For instance, control and productivity enhancement methods in agriculture, to avoid the spread of phytopathogens, have been based for many years in strong chemical pesticides and fertilizers, as well as on synthetic antibiotics on minor cases. Use of antibiotics for controlling vegetable diseases on crops has been employed for many years, with the United States having implemented this technique for at least 50 years mainly on pear, apple, peach and nectarine plantations. As much of food availability and public health relies on the use of NPs and the mentioned problems are arising from its misuse, a solid need for research on new classes of antimicrobial compounds is evidenced, to respond to the high frequency of occurrence of resistance to all major classes of known antibiotics [2]. The investment in discovery and development of new bioactive compounds is crucial for maintaining the improvement of human, other animals and crops health avoiding the loss of lives due to infectious diseases [3], as well as rising our crop's productivity. This demand appears even more important when during the last decade natural products discovery programs have been gradually declining [4].

New molecular entities (NMEs) have been greatly obtained from natural products throughout history, shaping the landscape of new bioactive molecules discovered. According to the FDA, natural products derivates represent over one-third of all NMEs. From this pool of natural products, one-quarter comes from microbes and over time, this fraction has become enriched with more microbial natural products, now representing a significant portion of approved antibiotics, including more than two-thirds of all antibacterial NMEs. Since the 1930s, the total fraction of natural products has diminished, whereas semisynthetic and synthetic natural product derivatives have increased, evidencing the imminent decline on natural products discovery that has impacted the pipeline of NMEs from specific classes. Without the specific investment in the pursuit of novel natural products this trend is likely to continue [5].

Accordingly, the investment in research and development of novel and naturally originated molecules or compounds controlling the spread of infectious agents of both animals and plants is a matter of urgency [6,7] as well as their later formulation into bioproducts. It is estimated that about 30% of the worldwide sales of drugs are based on natural products and many of them, despite having been introduced several years ago, are still used today in clinical practice [2, 4] , leaving evidence of the impact that discoveries in this area have for public health and agriculture through time. Besides human and animal infectious diseases treatment, agricultural productivity, drugs development [8], waste water recovery and food industry are among the disciplines that would be highly beneficiated with the discovery of new bioactive molecules and their derived bioproducts. As many of the discovery processes of bioactive natural compounds and several other studies in natural products chemistry have indicated, microorganisms, mainly bacteria and fungi, are prolific sources of structurally unique, highly bioactive, and biomedical useful secondary metabolites [9]. Besides, microbial metabolites can be easily produced at quantities of up to kilograms by fermentation technology [4], facilitating their availability.

Among the species and taxonomical families on which numerous studies on microbial bioactive compounds have been done across the decades, the Bacillus spp. genus has had an important place as an outstanding member of the AEFB group. The production of antimicrobial metabolites by Bacillus spp. strains has been a highly studied subject. Moreover, since Bacillus spp. are characteristically omnipresent in soils, they exhibit high thermal tolerance and rapid growth in liquid culture, they readily form resistant spores and are considered to be a safe biological agent, their potential as biocontrol agents is considered high [10].

As regards to the future active NPs to be isolated from microbial strains, the appearance of a new set of genomic and metabolomic tools to determine cryptic or silent genes opens an exciting technological challenge that remains to be discovered, and it may be predicted that future exploration of novel gene clusters will expand the already remarkable spectrum of secondary metabolites isolated from the microbes. Therefore, Bacillus, which is a source of numerous antibiotics and other drugs, could give rise to a surprising array of new metabolites not yet discovered by activating silent gene clusters. One approach to activate silent genes is the incorporation of environmental signals (or inducers) that activate these regulatory systems.

Though research regarding the induction of silent or unidentified biosynthetic gene clusters, is increasing, specifically for AEFB strains it hasn't been largely explored, indicating that there is still a large potential of novel bioactive compounds that may lie undiscovered in the genomes of microorganism belonging to families such as Bacillaceae and Paenibacillaceae. Over the course of history, the most notable NPs, which have been obtained from Bacillus sp., are Cry proteins (crystal delta-endotoxins) from Bacillus thurigieins (Bt), lipopetides (surfactines, iturines, fengycines, kurstatines) mainly from B. subtilis and B. amyloliquefaciens, lantibiotics, from several species of the Bacillus genus and poliketides, also produced by several species. All of them have been mostly produced in traditional culture media, which does not require the addition of an inducer.

For instance, the insecticidal substances from the family of crystal delta-endotoxins and other exotoxins obtained generally from Bacillus thurigieins but also from B. sphaericus (Bs) strains, have been produced through a large number of processes and strains variations and are protected under several patents (i.e. WO 1994/12642, which discloses process details). Said process evidence that toxins are produced during the spore formation process of the bacterium, specifically in the sporulation phase when each cell liberates one spore and a protein toxin crystal, and that both liquid and solid state fermentations can be successfully employed for their production [11].

For the production of lipopetides from Bacillus spp. or Paenibacillus spp, several media have been optimized either to increase the yield of this family of active compounds [12], by adding large quantities of carbon source (up to 20 g/L of glucose) and by notably increasing the nitrogen source by adding both peptone and yeast extract to the media composition; or depending on the specific family of lipopeptides to be obtained during the fermentation, as the one done by Mosquera et al [13] which optimizes the production of metabolites from the surfactin and fengycin lipopeptide families, specially the production of the newly discovered isoform fengycine C. These lipopeptide producing fermentations are mostly done submerged or in liquid state.

Besides culture media compositions, traditional fermentations of AEFB strains are done in submerged fashion in liquid media, kept at 30°C or under mesophile temperatures between 20-40°C, aerated in order to keep a constant oxygen exchange, under neutral pH conditions close to 7.0, though a range between 5.5-8.5 can be kept, and agitated at 100- 200 rpm. But the absence of inducers or chemical signals in the mentioned fermentations for the production of specific groups of natural products from Bacillus sp., suggests that the genes responsible for the production of those compounds are constitutive, and thus expressed under standard and traditional culture conditions for microorganisms but do not belong to the group of biosynthetic silent gene clusters. Therefore, the use of inducer agents, of natural o synthetic nature, for activating silenced genes which could potentiate or express novel antimicrobial activity in strains from the families Bacillaceae and Paenibacillaceae is still very deficient and more research, methods and technologies are needed in order to broaden the number of original antimicrobial substances described for these metabolically rich bacterial families.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG 1. Inhibition zones of the gram-negative pathogen R. solanacearum produced by strains of AEFB in absence / presence (50 ppm) of TTC in solid BGA medium.

FIG 2. Inhibition zones of the gram-negative pathogen R. solanacearum produced by a strain of B. cereus EA-CB 1047 (A) / and one of B. pumilus EA-CB0009, at different inducer concentrations, ranging from 0-400 ppm (B) in solid culture. FIG 3. Growth curves of B. cereus EA-CB 1047, in the presence of different inducer concentrations in liquid BG medium: A) Time course growth profile, B) Viable cell concentration (CFU/mL) after 68 h of culture.

FIG 4. Growth curves of B. pumilus EA-CB09, in the presence of different inducer concentrations in liquid BG medium: A) Time course growth profile, B) Viable cell concentration (CFU/mL) after 68 h of culture.

FIG 5 Inhibition zones of the gram-negative pathogen R. solanacearum produced by strains of AEFB in absence/presence of different antioxidants in solid BGTA medium. FIG 6. Inhibition zones of other gram-negative and gram-positive pathogens in the presence and absence of TTC in solid BGA medium.

FIG 7. Dual plate assay photograph of cell-free supernatants (CFS) of a monoculture of B. cereus 1047 under the presence/absence of TTC.

FIG 8. Photograph of inhibition zones of R. solanacearum after growing B. cereus 1047 in a solid BGTA monoculture.

FIG 9. Growth kinetics of the liquid co-culture of B. cereus EA-CB1047 and R. solanacearum EAP09, in the presence of 50 ppm of TTC. FIG 10. Differential transcriptomic profile of B. subtilis NCIB 3610 strain under the presence and absence of inducer agent, obtained through Nanostring™ technology.

FIG 11. Table of values from the HPLC/MS profiles of active methanolic extracts (+1/ first box) and negative control extracts (-I/second box)

FIG 12. HPLC/MS profiles of active methanolic extracts (+1 /first box) and negative control extracts (-I/second box).

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to a method for the production of active compounds from AEFBs, specifically from Bacillaceae and Paenibacillaceae families. The method comprises the cultivation of these microorganisms under appropriate conditions and a suitable culture medium comprising at least one inducing agent and extract the active compounds. By this method, said microorganisms produce active compounds that can be used for controlling crop pests causing agents and other animal and human pathogens. DETAILED DESCRIPTION

Bacillaceae and Paenibacillacea families are part of the order Bacillales. The distinguishing feature of a large number of species of both families is the production of endospores, which are round, oval, or cylindrical highly refractive structures formed within bacterial cells. Spores are heat resistant and the developmental cycle of spore formers enables them to switch between vegetative cell to spore and spore to vegetative cell.

Members of these families are ubiquitous in ecosystems, the vast majority of them are non-pathogenic, of aerobic nature, which may be strict or facultative, have a rod shape and present catalase production. Their ability to produce a great variety of bioactive natural products is well known, since they demonstrate great catabolic and biosynthetic versatility. It is only recently, however, that significant progress has been made in understanding how synthesis of enzymes is regulated, with most studies having been concentrated on B. subtilis and closely related species [14].

Species belonging to the Bacillaceae and Panibacillaceae families are both taxonomically and metabolically diverse, and they exhibit immense metabolic capabilities and versatile biochemistry through the production of structurally diverse bioactive chemical structures. Although they mainly synthesize peptides, antibiotics belonging to other chemical classes as polyketides and terpenoids, are also produced by these microorganisms, from which determination of their chemical structure and mechanism of biological action are of fundamental and practical interest [9]. Yet, there is one famous family of potent antimicrobial compounds, which though produced by a wide variety of bacteria, is mainly synthetized by Bacillus and Paenibacillus spp. These compounds are lipopeptides, which evidence potent antimicrobial activity. In recent years, several lipopeptide antibiotics have gained FDA approval for the treatment of multidrug-resistant infections, including both daptomycin and polymyxin B, and others are in various stages of clinical and preclinical trials, taking into account that only two new structural types of antibiotics have entered the market in the last 40 years, these being linezolid and the bacterial lipopeptide daptomycin [15]. Besides Bacillus genus, other microbial species, as are the ones belonging to the phylum actinobacteria, have been the main focus of a lot of research in antimicrobial drug discovery. The actinomyces have been studied as a natural compounds' source because of their great metabolic diversity and their long association with the environment. They have provided about two-thirds (more than 4000) of the naturally occurring antibiotics discovered, including many of those important in medicine, such as aminoglycosides, anthracyclines, chloramphenicol, β-lactams, macrolides and tetracyclines.

Approaches to search and discover new antibiotics are generally based on the screening of naturally occurring microorganisms, or on biotechnological manipulation of the known antibiotic producing microorganism. However, current efforts to find new antibiotics are faced with the difficulty that the probability of discovering these unknown molecules is declining as the odds of discovering or re-isolating already known molecules are increasing. It is therefore important to devise methodologies to enhance the probability of discovering new antibiotics from these two genera and other microbial families, knowing that their capability for antibiotics production is not species specific but strain specific and that taxonomical classification may not be useful in the prediction of their antibiotics production capability; however, specific genotypes conferring strain-specific antibiotic production have been observed. Such specific genotypes might correlate with specific gene clusters for biosynthetic enzymes as reported recently for Streptomyces sp. strains that produce actinorhodin, streptomycin, methylenomycin and erythromycin [16]. Nature has developed strategies for the assembly of structurally diverse natural products derived from biosynthetic pathways in microbial sources, thus new strategies to isolate novel species thought to be uncultivable as well as synthetic biology approaches ranging from genome mining of microbial strains for cryptic biosynthetic pathways to their heterologous expression have been emerging [2].

There have been numerous reports on the discovery of novel natural products from sequenced microbes by genomics-guided approaches [9]; as an example in Bacillus genus, some studies evidence that when mining these complex biosynthetic pathways in Bacillus genomes, a greater biosynthetic potential for novel natural products than anticipated is revealed [9]. Consequently, is it certain that one important challenge that lies ahead is the development of general methods for activating the expression of silent or cryptic biosynthetic gene clusters [17], perhaps using inducers that trigger their expression, as the one stated for the antagonism-inducing fermentation conditions in the present invention.

AEFB strains in which the phenomenon described on the present invention was first observed belong to a registered culture collection with registration number 191 of the Von Humboldt Institute, established by EAFIT University and AUGURA's (the Colombian banana producers trade association) research center, CENIBANANO. Most of them belong to the genus Bacillus spp. and Paenibacillus spp. This collection of microorganisms counts with the permission No. 89 for accessing the genetic resource, issued by Colombia's competent authority (MINAMBIENTE), which allows both Institutions (EAFIT University and AUGURA) to handle the comprised genetic resources for research purposes. These microorganisms were isolated either from the phyllosphere or the rhizosphere of banana and plantain plants belonging to commercial plantations located in the region of Uraba, Colombia.

The culture medium for potentiating the production of active compounds with antimicrobial activity in strains from several genera of AEFB, like Bacillus spp. and Paenibacillus spp, comprises one inducer agent, in optimal concentrations for potentiating their antagonistic behavior. When these AEFB are grown in the suitable medium containing an inducer agent, bacterial strains belonging to different plant and animal pathogenic species are inhibited, indicating the production of antibacterial compounds. When the inducer agent is removed from the culture medium or replaced by a chemically similar one, little or no inhibition is observed.

B. cereus and related species as B. thurigiensis, as well as B. pumilus strains are the AEFB species evidencing the largest antimicrobial activity against the sensitive strains tested, through the dual plate technique in a suitable medium containing an inducer agent such as triphenil tetrazolium chloride (TTC) salt. B. amyloliquefaciens, B. subtilis, B. megaterium, P. pasadenensis, B. altitudinis, B. simplex, B. licheniformis, B. coagulans, B. firmus, Aeribacillus palidus, Marinibacillus marinus are among other tested species which evidence induction of antimicrobial activity in the presence of TTC salt.

The following section enunciates the correct method for obtaining inducible active compounds with antimicrobial activity, from AEFB strains, preferably from the genus Bacillus spp., Paenibacillus spp., Marinibacillus spp and Aeribacillus spp in presence of inducer agent. In order to obtain the optimal induction of the antimicrobial activity, the steps and conditions which must be followed while preparing and handling the fermentations in the culture medium containing the inducer agent, from the inoculum preparation until the time of recovery of such compounds from the medium, are comprised in the present invention.

The antagonism assay for testing a strain's susceptibility against the inducible antagonism produced by AEFB in presence of the inducer agent must be performed in a culture medium suitable for the growth of both the tested and the antagonism inducible AEFB strains, which contains TTC salt in a concentration ranging from > 0 to 400 ppm. A dual plate technique assay is performed in which a cellular suspension of the sensitive strain (between 1 x 10 ⁴ to 1 x 10 ⁷ CFU/mL) is plated to the medium prior to adding a plug or a suspension of the AEFB strains in a well.

After the adequate incubation period, depending on the tested species (from 24 to 72 hours for some species), large inhibition zones of the tested strain are observed, produced around the AEFB strains. For instance, when testing the inducible antagonism of AEFB against the phytopathogenic bacterium R. solanacearum, dual plate assays are performed in BGTA medium [18] (special peptone (Oxoid®), glucose (Merck®), yeast extract (Oxoid®), casaminoacids (BD), TTC salt (Merck ®)), since it is the optimal medium for growing this bacterial species. Media can be either solid or liquid, and antimicrobial activity can also be recovered from monocultures of the AEFB producing strain.

For testing antimicrobial activity on a solid monoculture fermentation, the AEFB strain must be placed over a membrane of 0.2 μπι pore size, remove it after 48 h of incubation and then plate the suspension of the sensitive strain, observing inhibition halos around the place where the AEFB grew. On a liquid monoculture, the active cell-free supernatant (CFS) can be recovered and tested against the sensitive strain on a dual plate assay. Recovering inducible antimicrobial activity is thus achievable from AEFB fermentations both in liquid and solid state.

Preferred media composition for observing the inducible antagonism in AEFB strains: composition and inducer concentrations

The culture medium can be liquid or solid and should be preferably composed of special peptone 10 g/L (Oxoid®), glucose 5 g/L (Merck®), yeast extract 1 g/L (Oxoid®), casaminoacids 1 g/L (BD®), bactoagar 18 g/L (BD®), TTC salt 50 ppm (Merck ®), namely BGTA medium. Liquid medium does not contain agar. TTC salt constitutes the inducer agent, essential for observing the inducible antagonism. Optimum concentrations of this inducer agent, which triggers the antagonistic behavior of AEFB, range between >0-400 ppm (mg/L) depending on the AEFB strain, being lower for Paenibacillus spp. strains (in the range of >0-50 ppm) and higher for Bacillus spp. strains, with an optimum between 50-100 ppm for B. cereus, of 100-200 ppm for B. pumilus strains and of 25-200 ppm for B. subtilis.

Any suitable media for the normal growth of AEFB strains, if performing a mono-culture, as are Luria-Bertani (LB), tryptic soy broth/agar (TSB/TSA) or nutrient broth/agar (NB/NA) or any suitable media for the growth of both the sensitive and inducible AEFB strain if performing a co-culture, can account for a variation of the preferred media composition. For the specific case of R. solanaceraum strains, a suitable variation consists of TSA (Triptic Soy Agar) diluted to the 10% strength + 0.1% sacarose, 1.8% agar and the optimum concentration of TTC salt.

The environmental conditions for incubation can range from 20 to 40°C, be cultivated either in dark or light conditions, with or without agitation. Even though, the optimal results have been obtained incubating at 37°C and 150 rpm, protected from light for liquid co-cultures and monocultures, and incubating at 37°C, also protected from light for solid co-cultures and monocultures. Under any case, the preferred media are BGTA and 10% TSA variation with the presence of the inducer agent at the optimal concentration for the specific AEFB. The optimal pH for obtaining inducible antagonistic activity from AEFB strains depends on the strain species, with some of them like B. cereus performing better at a basic pH while others, as B. pumilus rather acid pH values. Although, AEFB fermentations pH under the presence of the inducer does not need to be constantly controlled, just monitored, an initial pH value between 5.5 and 8.5 is preferred for most AEFB species.

The inducer agent which has been discovered to induce and potentiate the antimicrobial activity in AEFB strains and which is stated in the present invention belongs to a group of compounds known as tetrazolium salts, which change from colorless or weakly colored aqueous solutions when oxidized, to brightly colored derivatives known as formazans when reduced. They have been used as vital dyes in redox histochemistry and in biochemical applications.

Apparently, there are not existing publications or patents related to the induction of antimicrobial activity in bacterial strains by means of using tetrazolium compounds or derivatives thereof. The general structure of tetrazolium salts is as follows:

Were the difference between figures I, II and III lays in the double bond and the quaternary nitrogen positions.

The tetrazolium salt preparation from formazans is described in patent FR1088230A, which is incorporated by reference. A wide variety of C-atoms substituents have been introduced chemically to tetrazoles, yielding tetrazolium salts when combined with a halogen atom. These halogen atoms can range from chlorine, to bromide or fluorine and R' substituents that have been reported in tetrazolium salts variation from TTC comprise different functional groups besides the phenyl, as are: cyano, dimethylthiazol, iodophenyl, nitrophenyl, 2-methoxy-4-nitro-5-sulfophenyl, sulfo or disulfophenyl, phenylamino-carbonyl, carboxymethoxyphenyl, among others. According to present invention, tetrazolium salts which can act as inducers are: 2,3,5- triphenyl-2H-tetrazolium chloride (TTC), 2-(4-iodophenyl)-3-(4- nitrophenyl)-5-phenyl- 2H- tetrazolium chloride (INT), 5-[3-(carboxymethoxy)phenyl]-3-(4,5-dimethyl-2- thiazolyl)-2-(4-sulfo-phenyl) - 2H tetrazolium inner salt (CTC), Nitrotetrazolium blue (NBT), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5-[3- (carboxymethoxy)phenyl]-3- (4,5-dimethyl-2-thiazolyl)-2-(4- sulfo-phenyl)-2H- tetrazolium inner salt (MTS). Specifically TTC, NBT and INT,are preferred inducer agents.

Most of the reported tetrazolium salts have aryl or chemical functional groups derived from an aromatic ring in this free C-position as well as in the other two ones from Nitrogen atoms [19]. Subsequently, the unique chemical and biological properties of tetrazolium salts that have led to their widespread application in histochemistry, cell biology, biochemistry and biotechnology depend on the positively charged quaternary tetrazole ring core containing four nitrogen atoms, or in the net charge of the moiety comprising this tetrazole ring [20].

Within the biochemistry of tetrazolium salts, the mechanism by which they are bioreduced has been perhaps the topic that has mostly grabbed the interest of researchers, together with their site of reduction, in order to assess novel applications in cell biology. Evidence has accumulated towards tetrazolium salts measuring the integrated pyridine nucleotide redox status of cells, as it is thought that the reduction of these compounds likely involves reaction with NADH or similar reducing molecules that transfer electrons to the tetrazole molecule [21]. The prototype tetrazolium salt compound is known as 2,3,5-triphenyltetrazoliumchloride (TTC), first synthesized more than a century ago [22]. It is a water-soluble compound when oxidized, but its formazan, namely Triphenil Formazan (TFP) is highly insoluble in aqueous solvents. This particular tetrazolium salt, has been modified in many ways over the years by adding nitro, iodo and methoxy groups to the phenyl rings, thus yielding a large diversity of tetrazolium salts available in the present-day [20], and it has been reported for having selective properties on gram negative bacterial strains. It has been observed that gram-positive bacteria are inhibited more readily by 2,3,5- triphenyltetrazoliumchloride than gram-negative organisms [23, 24]. In a preferred embodiment of the invention, the culture medium used in the method comprises TTC salt as inducer agent. If this compound is removed from the culture medium, the inhibition zones are no longer observed. If a chemically similar compound replaces TTC salt, named INT, which also belongs to the tetrazolium salts family, a inhibition is still observed, demonstrating that even though TTC is the most potent inducer for this group of inducible compounds, other members of the tetrazolium family of compounds can act as inducers as well. Inhibition in liquid media can be obtained by setting either a monoculture of only the AEFB or a liquid co-culture of both the AEFB and the sensitive strain. These fermentations can be obtained in BGT medium or in a suitable media amended with the optimal concentration of inducer agent. Monitoring of the producing strain populations in a monoculture, or of both strains populations in co-culture, can be easily achieved through colony counts and expressed in CFU/mL, counting the colonies in selective media for each bacterial strain. Following this procedure for the co-culture has evidenced a decrease of up to one order of magnitude (Log) in the sensitive strain population. Other techniques that can be employed to measure the susceptible strain population decrease are:

Randomly tagging the susceptible strain with the gfp operon and perform cell cytometry cell counts of both susceptible strain and AEFB every hour until for up to 24 hours.

Perform population counts every hour in a fluorescence microscope. This decrease is observed between 12-16 hours post co-inoculation of both strains, when initial concentrations of both are close to lxlO ⁶ CFU/mL and are inoculated into fresh medium in a 1 : 1 relation. If any of these concentrations are altered the decrease in the susceptible strain is indeed observed but the post co-inoculation time of occurrence of this inhibition is achieved earlier or further in the fermentation time course.

The detailed steps for correctly achieving AEFB strains inducible antagonistic behavior, either in solid or in liquid culture are stated below:

Monoculture fermentation: Liquid or solid state fermentation

For achieving a correct monoculture in liquid state fermentation, the following steps must be carried out: a) Select a desired fermentation volume, always keeping 70-90% of the fermentation recipient volume for air space and oxygenation if performing it in an erlenmeyer. If an aerated bioreactor is selected, an oxygen supply must be provided and up to

50-90% of the volume can be used. b) Activate the AEFB strains in any suitable agar medium for AEFB growth, preferably Luria-Bertani (LB) agar or TSA by incubating at 30-37°C in an incubator.

c) Prepare a pre-inoculum by transferring one or more colonies of the AEFB into a suitable media, preferably liquid LB or TSB containing the inducer agent in the same concentration as the final fermentation. Incubate at 30-37°C and 50-300 rpm, for 10-20 h, preferably at 37°C and 150 rpm for 15 h.

d) After incubating the pre-inoculum during 10-20 h, add approximately 1% of its volume to the recipient containing the final volume of the suitable fresh sterile medium, which must contain the optimum inducer concentration according to the AEFB species. An initial concentration of approximately lxlO ⁶ CFU/mL of the producing strain is always preferred.

e) Incubate the fermentation at 30-37°C, and aeration of 0.5-2.5 vvm and 50-300 rpm. Between 12 and 16 hours post-inoculation is when the antagonistic behavior takes place, reaching a maximum between 14 and 15 hours.

For achieving a correct monoculture in solid-state fermentation, the following steps must be carried out: a) Activate the AEFB strains preferably in LB agar or TSA, by incubating at 30- 37°C in an incubator.

b) A pre-inoculum must be obtained by inoculating one or more colonies of the AEFB in a suitable liquid medium, preferably liquid LB or TSA and incubating them for 12-15 h at 100-200 rpm and 37°C.

c) Make wells in the agar with a sterile microbiological drill or with a Pasteur pipette, without reaching the bottom of the plate.

d) Apply 25 μL· of the AEFB cell suspension from step b) to each well. Let it settle or dry and apply other 25 μL· of cell suspension, reaching a total of 50 μL·. In another embodiment of the invention, agar discs from step a) of the AEFB could also be applied to the suitable agar media with the inducer agent at its optimal concentration.

e) Incubate the solid-state fermentation at any temperature between 20-40°C, preferably at 30-37°C f) Detect and recover activity at 24-48 hours of incubation. Co-culture fermentation: Liquid or solid state fermentation

For achieving a correct co-culture in liquid state fermentation, the following steps must be carried out: a) Select a desired fermentation volume, always keeping 70-90% of the fermentation recipient volume for air space and oxygenation if performing it in an erlenmeyer. If an aerated bioreactor is selected, an oxygen supply must be provided and up to 50-90% of the volume can be used.

b) Activate the AEFB strains in any suitable agar medium for AEFB growth, preferably Luria-Bertani (LB) agar or TSA by incubating at 30-37°C in an incubator.

c) Activate the sensitive strain in a suitable medium and incubate at its optimal temperature.

d) Prepare a pre-inoculum by transferring one or more colonies of the AEFB into a suitable media, preferably liquid LB or TSB containing the inducer agent in the same concentration as the final fermentation. Incubate at 30-37°C and 50-300 rpm, for 10-20 h, preferably at 37°C and 150 rpm for 15 h.

e) Follow the same previous step for the sensitive strain, incubating the preinoculum in a suitable medium and under the same concentration of the inducer as the final fermentation is intended to have.

f) Prepare cell suspensions of both the producing and sensitive strains by adjusting optical density (O.D) to a value corresponding to a final concentration of between lxlO ⁷ to lxlO ⁸ CFU/mL. For AEFB this O.D is between 0.8 and 1.

g) Co-inoculate both strains onto the fresh sterile liquid medium, which must contain the optimum inducer concentration according to the AEFB species, applying a volume of each cell suspension in order to reach a 1 : 100 dilution of each microorganism, for an initial concentration of approximately lxlO ⁶ CFU/mL. h) Incubate the fermentation at 30-37°C, 50-300 rpm and under aerated conditions.

Between 12 and 16 hours post-inoculation is when the antagonistic behavior takes place, reaching a maximum between the 14 and 15 hours. i) Measure the sensitive strains population decrease or recover active CFS during this specific time frame.

For achieving a correct co-culture in solid-state fermentation, the following steps must be carried out:

For achieving a correct co-culture in solid-state fermentation, any device that can contain solid media (such as agar) under sterile or axenic conditions is suitable for observing the inducible antagonism and recovering the active metabolites. Even though, for achieving this, a standard method comprising 90 mm diameter agar plates is recommended, following the details below. a) Activate the AEFB strains preferably in LB agar or half strength TSA, by incubating at 30-37°C in an incubator.

b) Activate the sensitive strain in its suitable medium, and incubate at a temperature and time optimum for the strain in an incubator. For R. solanacearum, BGT agar,

30°C and 48 hours is preferred.

c) Prepare a cell suspension of the sensitive strain to a final concentration of between 1x10 ^s to lxlO ⁶ CFU/mL. For R. solanacearum lxlO ⁶ CFU/mL is preferred. d) Plate 100 μΕ of the sensitive strain cell suspension onto a petri dish containing the suitable agar media with the inducer agent at its optimal concentration for the

AEFB strain. For R. solanacearum and Bacillus sp., the preferred agar is BGT with TTC concentrations between 25 to 200 ppm.

e) Make wells in the agar with a sterile microbiological drill or with a Pasteur pipette, without reaching the bottom of the plate.

f) Prepare a cell suspension of the AEFB strain to a final concentration of between lxlO ⁸ to lxlO ⁹ CFU/mL, either by suspending colonies onto sterile distilled water or by adjusting the concentration of a liquid pre-inoculum.

g) Apply 25 μL· of the AEFB cell suspension from step f) to each well. Let it settle or dry and apply another 25 μL· of cell suspension, reaching a total of 50 μί. In another embodiment of the method of invention, agar discs of the AEFB could also be applied to the suitable agar media with the inducer agent at its optimal concentration. h) Incubate the solid-state fermentation between 22-37°C, preferably at the optimal growth temperature of the sensitive strain.

i) Detect and recover activity at 24-72 hours of incubation, depending on the species of the susceptible strain. For R. solanacearum and Bacillus sp., 72 h of incubation is preferred.

Extraction of active compounds

For obtaining and recovering active compounds of both liquid and solid state fermentations the following steps must be followed: a) For a liquid fermentation, recover active cell-free supernatants (CFS) by collecting the desire volume of fermentation at 12-16 hours post-inoculation, centrifuging to remove biomass and filtering through a sterile 0.2 or 0.45 μπι pore- size membrane o syringe filter. b) For solid state fermentations, cut a certain weight of the inducible halos containing the compounds and the producing strain, using a sterile scalpel and cut them into approx. 2 mm squares and mix them in a flask with a volume of an organic solvent (i.e. methanol, acetone, ethyl acetate) corresponding to 4X the weight of the halos obtained. Macerate the agar cubes in the solvent and leave in extraction for 2-4 h under agitation at 150 rpm and room temperature. Afterwards, centrifuge the extraction solvent to remove cell debris and agar residues, then evaporate the liquid under vacuum at 45-50°C. Recover the residue from evaporation by suspending it in 2 mL of solvent. c) To further purify the cell free extracts, either from solid or liquid fermentations, use a solid-phase extraction (SPE) C-18 column. Add the cell free extract to the column, and do a step elution: first with water and then with the solvent. The active metabolites elutes in the organic fraction. Vacuum manifold can be used to accelerate the elution process. d) Save the purified extract in an amber glass vial for other purification steps by HPLC or for any antagonism trials or final application.

The following examples illustrate the invention, without the inventive concept being restricted thereto

EXAMPLES

Example 1. Inducible antimicrobial activity in solid co-cultures Sixteen (16) different AEFB strains, each representative of a different species from the BaciUaceae family and one belonging to the Paencibacillus spp. genus, some of them registered in the collection No 191 of the Humboldt Institute in Colombia and all identified to the species level by analysis of the 16S rDNA sequence, were tested against the phytopathogenic bacterium R. solanacearum by the dual plate assay.

Briefly, 100 \\L of R. solanacearum suspension containing 10 ^Λ 6 CFU/mL was spread on BG agar [18] with and without TTC salt at 50 ppm. Afterwards a 5 mm disc of each AEFB, grown in TSA for 48 h at 30 °C, was transferred into BG agar and incubated at 22 °C. Inhibition of R. solanacearum growth was assessed by measuring the thickness (mm) of the growth inhibition zone after incubation for 72 h. As shown in Figure 1, when in the presence of 50 ppm of TTC salt (inducer agent), all bacteria generated larger inhibition halos of the gram negative sensitive, something that was not observable when the inducer was totally absent from the medium. Thus, activity is only observable when TTC is present in the medium, acting as an inducer of antimicrobial compounds production.

Figure 1 evidences what is observed when the assessment of the inducible antimicrobial activity of the sixteen AEFB was performed under 0 and 50 ppm of the inducer agent. B. cereus EA-CB1047, B. pumilus EA-CB0009,B. subtilis NCIB 3610, Aeribacillus palidus, B. licheniformis and B. coagulans are species that evidenced higher inducible antimicrobial behavior. Hence, specific trials were done for the first three strains, varying the concentration of the inducer agent, which is summarized in example 2 (Figure 2).

Example 2. Inducible antagonism in co-culture under different inducer concentrations

Figure 1 evidences what is observed when the assessment of antimicrobial activity of the sixteen AEFB was performed under 0 and 50 ppm of the inducer agent. B. cereus EA- CB 1047 and B. pumilus EA-CB0009 are the strains that evidenced the higher inducible antimicrobial behavior. Hence, specific trials were done for each strain, varying the concentration of the inducer agent by the dual plate assay described in example 1 , which is summarized in Figure 2, in order to establish if the inducible activity was dose dependent and therefore determine an optimal concentration of the inducer agent under which the antagonistic behavior takes place.

As shown in figure 2, the inhibition halo generated by B. cereus against R. solanacearum is increased in a concentration dependent manner, reaching its maximum at 50 to 100 ppm (Figure 2A). Similar behavior, but more attenuated, is observed with B. pumilus (Figure 2B), for which TTC concentrations are optimal between 50-200 ppm and for B. subtilis (Figure 2C), which evidences high inducible antimicrobial activity under concentrations between 25-200 ppm of the inducer agent. These results suggest that TTC induces the expression of genes involved in the production of active metabolites in a dose dependent manner or that TTC affects growth of the AEFB. Therefore, growth curves of B. cereus and B. pumilus at different TTC concentrations were conducted in liquid culture in microplates, observing that its growth is not affected at concentrations between 0 to 100 ppm but at 200 and 400 ppm B. cereus growth is reduced (Figure 3A and B). As for B. pumilus, the more attenuated impact in its growth under higher doses of TTC is observable in Figure 4A and B.

We can conclude from these observations that TTC induces the production of active metabolites in a dose dependent manner, and the optimal concentration is species or strain specific and must be determined in each AEFB strain before producing a fermentation in order to obtain the optimal inducible antimicrobial activity.

Example 3. Inducible antimicrobial activity in a solid co-culture produced in other suitable media

In order to establish the culture variations that retain the ability to induce the antagonistic compounds production, co-culture fermentations by dual plate assay of AEFB and R. solanacearum were performed in different media: TSA10 (10% TSA, 1% sucrose, 1.5% agar), BGTA with the addition of each one of four different antioxidant compounds ( - Tocopherol (vitamin E), L-ascorbic acid (Vitamin C), Glutathione and Uric acid), and BGTA as a positive control for inducible antagonism production. The media always had the optimal TTC concentration according to the producer AEFB strain.

Figure 5 illustrates part of the results, in which no difference was observed between de positive control and the antagonism trials done in the different alternative media that contained the antioxidant compound, demonstrating that as long as the inducer agent is present at the optimal concentration in the medium, the inducible antagonism occurs. The same result was obtained when the antagonistic activity was tested in TSA 10: the activity in presence of the inducer agent was always retained.

Example 4. Other sensitive strains to the inducible antimicrobial compounds

The inducible antimicrobial compounds were tested for their activity against other gram-positive and gram-negative pathogenic bacteria, in a solid co-culture with the AEFB strain being B. cereus 1047 and the inducer agent at 50 mg/L BGTA medium. Escherichia coli, R. eutropha, Serratia marcescens and Staphylococcus aureus at lxlO ⁶ CFU/mL inoculums were prepared and 100 μΕ of each one were plated over the plate surface. 50 uL of a B. cereus cell suspension were inoculated in wells done with a sterile Pasteur pipette and the plates were incubated at 30°C during 48 h, after which the inhibition zones were measured. Figure 6 summarizes the results, in which the addition of TTC salt to the medium generated inhibition halos of the pathogens that were not observed in the absent of the inducer.

Example 5. Inducible antimicrobial activity of B. cereus EACB-1047 in the presence of INT salt Antagonism trials in plates containing INT as the inducer tetrazolium salt were performed using R. solanacearum as the sensitive strain. Halos of about 8 mm were formed in the presence of 80 mg/L of INT. This result is an indicator of other tetrazolium salts also acting as inducer agents of antimicrobial activity on strains belonging to the families Bacillaceae and Paenibacillaceae.

Example 6. Antimicrobial activity in a liquid monoculture

In order to determine the activity obtained in a monoculture of B. cereus EA-CB1047 in the presence of TTC, a liquid culture in BG medium was established. Briefly, the culture was set up in Erlenmeyers flasks of 1000 mL with 150 mL of BG medium with or without TTC salt and incubated at 30°C, 150 rpm for 20 hours. Two mL samples of the liquid monoculture were taken every two hours and the samples were centrifuged at 14.000 rpm and 4°C during 10 minutes, to remove the cells. The supernatant was recovered and filtered through a sterile syringe filter or sterile membrane of 0.2 μπι of pore size, obtaining a cell-free supernatant (CFS). These CFS were tested for their antagonistic activity as indicated in the detailed description.

The antagonism trial plate was set by plating 100 μL· of a cell suspension of the sensitive strain (R. solanacearum EAP09) at lxlO ⁶ CFU/mL on to dry, solid BGA medium with or without the inducer agent. The following steps were followed to apply the extracts: a) Wells in the agar were opened with a sterile microbiological drill or a Pasteur pipette.

b) 50 μL· of the cell free supernatant (CFS) were added to each well, letting them be absorbed by the agar and then adding another 50 μL·, reaching a final volume of 100 of CFS.

c) This procedure was repeated for samples from 13-16 hours post-inoculation, observing activity during all these fermentation times, corresponding to the transition from log-phase to stationary phase of the AEFB fermentation.

d) The antagonism trial plates were incubated at 25-30°C for 48 hours to read the inhibition halos produced by the inducible compounds on the sensitive strain. Figure 7, a photograph of the inhibition halos against R. solanacearum, produced by the SLC obtained at 14 h of fermentation, illustrates the results and evidences that an inhibition halo is only observable around the extract obtained under +1 condition, meaning under the inducer agent presence in the fermentation.

Example 7. Antimicrobial activity in a solid monoculture

Monocultures of B. cereus EA-CB 1047 were performed in BGTA medium, with TTC at 50 ppm. The strain was first grown on 50% TSA and plugs of it were obtained using a sterile Pasteur pipette. Sterile cellulose membranes, of 0.2 μπι of pore size were placed over dry, solid BGTA plates and the plugs of B. cereus were placed upside down (with the bacteria entering in contact with the membrane) over the sterile membranes. The plates were incubated at 37°C during 48 h. After the incubation of this monoculture, membranes with the cells were removed and a suspension of lxlO ⁶ CFU/mL of R. solanacearum was plated over the surface of the medium. As controls, other plates were set with monocultures of B. cereus with membranes but without the inducer agent, plates without the membranes, with and without TTC to observe the inhibition halos and monocultures with the sensitive strain R. solanacearum plated before placing the membranes, so that inhibition zones were observable with the B. cereus growing over them. No inhibition halo was observed when the pathogen was plated after removing B. cereus grown over the membranes without TTC in the medium. In the contrary, an inhibition halo of R. solanacearum is observable after 48 h of incubation at 30°C and under dark conditions, evidencing the secretion of the inducible antimicrobial compounds onto the solid medium in the presence of the inducer, and thus, the viability of a solid-state monoculture. This is also a strong indicator that inducible antimicrobial metabolites production from AEFB is independent of the presence of the sensitive strain. These results can be observed in Figure 8. Example 8. Liquid co-culture

Liquid co-cultures of both the AEFB and the gram-negative strain, R. solanacearum EAP09 for this case, were performed in order to assess the capacity of the induced antimicrobial activity acting in liquid medium. As described earlier in the text, BGT medium was inoculated in a 1 :1 relation of both strains and monitoring of the two microorganisms. When B. cereus enters the stationary phase, R. solanacearum concentration is reduced 10 times approximately, as can be observed in the curves presented in Figure 9.

Example 9. Active metabolites extraction and characterization

Agar extractions were performed from antagonism trials were the inducible compounds producing strain was B. subtilis NCIB 3610, against R. solanacerarum AW1 strain. The procedures followed were the ones stated in the detailed description for recovering antagonism compounds from methanolic extraction.

The active extracts plus a negative control coming from an extraction with 100% of agar pieces with both strains without the inducer, thus not presenting antagonism halos, were injected individually into an UHPLC-MS system, with the MS having an ESI ionization source. This metabolomics experiment gave as a result differential chromatograms, under which unique peaks are observed for the methanolic extract from halos produced under the inducer presence, as can be observed in Figure 11 and Figure 12. Inducible compounds are found to have monoisotopic masses ranging from 394.312 to 684.434.

A solid phase extraction (SPE) in CI 8 columns was also performed of these methanolic active extracts and active compounds were eluted the 100% methanol fraction, retaining the antagonistic activity. As a complementary technique to assess the chemical nature of the antagonistic compounds, RNA sequencing was performed using Next Generation Sequencing. Total RNA was isolated from liquid monocultures of B. subtilis NCIB 3610 strain under the presence and absence of the inducer agent. Then, cDNA synthesis was conducted from purified mRNA and RNA-seq libraries were constructed following Illumina recommendations. MiSeq Illumina platform was used to sequence all the treatments and replicates.

The raw data was analyzed first by quality check and then mapping the reads to the reference genomes. Three bioinformatics strategies to quantify abundances of genes were run using R package; EDGE-Pro, DEseq2 and Tuxedo Suite. Main results suggest than 8 genes are differentially expressed when comparing one condition to the other (+1/- I). The overexpressed genes are involved in nitrogen metabolism, specifically with the histidine biosynthetic pathway and the downregulated ones are related to the psiderophores and iron uptake metabolism.

Total RNA was also isolated from an inhibition trial on agar plates, both from strains growing under the presence and absence of the inducer agents. These total RNA isolated were hybridized to previously designed Nano-string ® chips, in order to obtain a transcriptomic profile under the two conditions and compare expression ratios. The results suggest that for the +1 condition, nitrogen metabolism related genes are up- regulated, specially the ones involved in purine and pyrimidine nucleotide salvage and interconversion, being coherent with the results obtained on the RNA-seq experiment. The Nano-string ® results from two independent experiments are summarized in FIG 10.

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