Patent Application
20210379122
The present disclosure relates to the isolation, identification and characterization of novel sporeforming probiotic strain(s) with NSPase (Non-Starch Polysaccharides-active hydrolases) activity isolated from fish gut microbiota, methods and uses thereof.
The sporeforming probiotic strain(s) with NSPase activity now disclosed are able of producing carbohydrate-active enzymes (CAZymes) that hydrolyse non-starch polysaccharides (NSPs) and accesses their potential as probiotics (PRO) for use in particular in aquafeeds.
The gastrointestinal microbial community plays a critical role on vertebrates' health and metabolism, impacting host metabolism, immune status and health/disease balance. In the last decade, this relationship has received increased attention particularly in humans, where it is known to control local (at the gut level) health status as well as systemic health. The gut microbiota of vertebrates, ranging from mammals to teleost fish, is involved in host appetite control and obesity development [1], protection against pathogens, immunity enhancement or inflammatory processes [2]. Additionally, gut microorganisms respond to a wide range of factors, including dietary composition, and harbor a relevant and diversified enzymatic repertoire that might interfere with host metabolism [3, 4]. This is particularly important in fish nutrition, because fish do not possess all of the necessary enzymes to cope with the current aquaculture dietary challenges [4].
A main difficulty within fish nutrition is its dependence on fish meal (FM), an unsustainable commodity and a source of organic pollutants. The most obvious sustainable alternatives to fish meal are plant feedstuffs, but their nutritive value is limited by the presence of high levels of non-starch polysaccharides (NSPs) which are not metabolized by fish.
These facts are disclosed to illustrate the technical problem addressed by the present disclosure.
Aquaculture output is growing rapidly and has already surpassed fisheries in terms of providing food to meet the growing human population [5]. Aquaculture is greatly dependent on FM, an unsustainable commodity and a source of organic pollutants, almost exclusively provided by fisheries. This is particularly obvious in carnivorous fish production due to their high dietary protein requirement (40-50%), which is mainly provided by FM. Plant feedstuffs (PF) are sustainable alternatives to FM, and among them, soybean meal (SBM), rapeseed meal (RSM), and sunflower meal (SFM), have been acknowledged as the most promising due to their high protein level, world-wide availability, and reasonable price. However, the nutritive value of PF is limited by the presence of several anti-nutritional factors, including high levels of non-starch polysaccharides (NSPs) which are not digested by fish [6]. NSPs content in SBM, RSM, and SFM averages 22-24% and the major NSPs components are pectic polysaccharides with arabinose, galactose, and xylose residues predominating. Yet, the proportion of these sugar residues varies between PF with galactose being predominant in SBM, arabinose in RSM, and xylose in SFM.
In fish, the carbohydrate-active enzymes (CAZymes) able to hydrolyze the β-glycosidic bonds of NSPs are scarce or non-existent. Thus, dietary NSPs remain indigestible and cannot be used as energy source. Moreover, indigestible NSPs might have detrimental effects on fish performance and nutrient digestibility and on fish health [7]. These adverse effects are associated with the viscous nature of NSPs and their interaction with gut epithelium, mucus, and microbiota, which ultimately result on physiological and inflammatory imbalances [7]. Additionally, and contrary to other animal species, such as pigs and poultry, the supplementation of PF based diets with exogenous carbohydrases does not necessarily translate into an effective strategy for improving NSPs utilization, as diverging results on their impact on fish growth performance and feed utilization have been reported. Therefore, gut microorganisms characterized by a rich secretome are a potential source of in loco carbohydrases that may help fish to overcome the mentioned constraints.
Live microorganisms that confer a health benefit to the host when administered in adequate amounts are denominated probiotics (PRO) [8]. In particular, PRO decrease the incidence of diseases by competing with pathogens for adhesion sites/nutrients; produce natural antimicrobial compounds that inhibit pathogens growth; contribute to a balanced gut microbiota; improve host growth; enhance host immune system and gastrointestinal histomorphology. PRO have also been implicated in bioremediation and water quality improvement by reducing antibiotic usage, contributing to aquaculture sustainability.
Among the bacterial species currently used as PRO, sporeformers show critical advantages: bacterial spores are remarkably resistant dormant structures [9], permitting good shelf-storage; spores are easily produced in large scale and can be dehydrated, facilitating feed incorporation without losing characteristics. Importantly, spores survive gut transit since they are acid and bile tolerant and become successfully established in the gut [10]. In particular, Bacillus subtilis spores, which enjoy GRAS (Generally Regarded As Safe) status from the U.S. Food and Drug Administration (FDA) and are included in the European Food Safety Authority (EFSA) list of Qualified Presumption of Safety (QPS) [11], experience exponentially growing applications in biomedicine and biotechnology (as oral vaccines, disinfectants, PRO or display systems) [12]. In fact, different sporeformers are nowadays used as human and animal PRO [12-13], but within European Union (EU) just one PRO has been authorised for use in aquaculture (Bactocell®, LALLEMAND Inc., Canada).
The role of gut microbiota in shaping human and animal health is well established, and the potential health benefit of manipulating the gut ecosystem using PRO is increasingly being accepted. In carnivorous fish, such as European sea bass, an ideal PRO should not only enhance resistance to pathogens, i.e. by competitive exclusion, the most common criteria for selection of PRO strains, but also help fish in their current dietary challenges, including the utilization of PF. In this disclosure, the application of a PF-based dietary pressure to modulate European sea bass gut microbiota composition and corresponding metabolic functions revealed to be a successful strategy to find carbohydrate-active bacteria with PRO potential. In particular, it was targeted and isolated spore-forming Bacilli, commonly used in PRO preparations, mainly due to their extreme resistance characteristics and indefinitely survival, advantageous for industrial applications [9-10, 12-13, 17].
The composition of the gut microbial communities of fish have been demonstrated to adapt when the host is fed different dietary ingredients [1, 3, 4]. Thus, a selective pressure of plant-based diets on fish gut microbiota, can be a beneficial strategy for an enrichment of bacteria with a secretome able to mobilize the dietary NSPs. By targeting bacterial sporulating isolates with diverse carbohydrase activities from the gut of European sea bass (Dicentrarchus labrax), isolates with high probiotic potential were obtained. By inferring the adaptive fitness to the fish gut and the amenability to industrial processing, the best two candidates were identified to become industrially valuable PRO for improvement of fish health and utilization of dietary NSPs, contributing for sustainable and more cost-effective aquaculture practices.
Thus, the present disclosure relates to screening fish gut microbiota for bacteria capable of producing extracellular digestive enzymes that hydrolyse NSPs present in PF, in particular mannans, glucans, xylans, arabinans, and galactans.
Gut microbiota isolates showing promising metabolic traits and absence of safety concerns can be used as PRO in cost-effective and environmental-friendly diets by allowing the host to obtain energy from otherwise indigestible dietary constituents. In fact, native bacteria with PRO potential will be more apt to become established and persist in the fish gut environment after withdrawal from the diet.
The present disclosure provides several advantages, namely: it solves the incapability of fish to efficiently digest and utilize PF as alternative protein source to FM; compared to exogenous purified enzymes (the only available technology, with poor results in fish), this disclosure has the added value of being also an autochthonous PRO that besides helping fish with the digestive challenges also contributes to fish health and welfare by antagonizing fish pathogens, having a dual positive effect on fish performance. Additionally, this product, by being a sporeformer, is more robust and resistant than the other PRO in EU market, allowing feed incorporation without losing characteristics and storage without refrigeration.
The present disclosure relates to ABP1 which was received on 8 Jun. 2018 and accepted for deposit for patent purposes at the Colección Española de Cultivos Tipo (CECT)—International Depositary Authority under the Budapest Treaty—under the accession number CECT 9675.
The present disclosure also relates to ABP2 which was received on 8 Jun. 2018 and accepted for deposit for patent purposes at the Colección Española de Cultivos Tipo (CECT)—International Depositary Authority under the Budapest Treaty—under the accession number CECT 9676.
The present disclosure therefore relates to a bacterial strain selected from ABP1 with a deposit under the accession number CECT 9675, of Aug. 6, 2018, at Colección Española de Cultivos Tipo, and/or from ABP2 with a deposit under the accession number CECT 9676, of Aug. 6, 2018, at Colección Española de Cultivos Tipo.
Furthermore, the present disclosure also relates to a composition for aquatic animal feed comprising a bacterial strain selected from ABP1 with a deposit under the accession number CECT 9675, of Aug. 6, 2018, at Colección Española de Cultivos Tipo, and/or from ABP2 with a deposit under the accession number CECT 9676, of Aug. 6, 2018, at Colección Española de Cultivos Tipo.
In an embodiment, said composition may comprise 1×105-1×1012 of colony forming units of the bacterial strain per gram of the composition, preferably said composition may comprise 1×107-1×1010 colony forming units of the bacterial strain per gram of the composition, more preferably said composition may comprise 2×109 colony forming units of the bacterial strain per gram of the composition.
In an embodiment, said composition may further comprise a preservative.
In an embodiment, said composition may be a granulate form, a powdered form or a pellet.
In an embodiment, said composition may be a granulate form wherein the granulate form is coated, in particular wherein the coating comprises a salt and/or wax and/or a flour.
The present disclosure also relates to a method for feeding an aquatic animal present in an aquaculture comprising the step of feeding the aquatic animal with the composition now disclosed.
In an embodiment, the step of feeding the aquatic animal may be carried out during the life span of the aquatic animal.
In an embodiment, the aquatic animal may be selected from the following list: a shellfish, fish, amberjack, arapaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama, carp, catfish, catla, chanos, char, cichlid, cobia, cod, crappie, dorada, drum, eel, goby, goldfish, gourami, grouper, guapote, halibut, java, labeo, lai, loach, mackerel, milkfish, mojarra, mudfish, mullet, paco, pearlspot, pejerrey, perch, pike, pompano, roach, Atlantic salmon, salmon, sampa, sauger, sea bass, European sea bass, seabream, gilthead seabream, white seabream, shiner, sleeper, snakehead, snapper, snook, sole, spinefoot, sturgeon, sunfish, sweetfish, tench, terror, tilapia, trout, tuna, turbot, vendace, walleye, halibut, whitefish or shrimp.
This disclosure also relates to the use of a bacterial strain selected from ABP1 with a deposit under the accession number CECT 9675, of Aug. 6, 2018, at Colección Española de Cultivos Tipo, and/or from ABP2 with a deposit under the accession number CECT 9676, of Aug. 6, 2018, at Colección Española de Cultivos Tipo, as a probiotic.
This disclosure also relates to the use of a bacterial strain selected from ABP1 with a deposit under the accession number CECT 9675, of Aug. 6, 2018, at Colección Española de Cultivos Tipo, and/or from ABP2 with a deposit under the accession number CECT 9676, of Aug. 6, 2018, at Colección Española de Cultivos Tipo, as a supplement to feedstuff, preferably as a supplement to feedstuff for fish or shellfish.
Furthermore, the present disclosure also relates to an isolated polynucleotide or polypeptide from ABP1 with a deposit under the accession number CECT 9675, of Aug. 6, 2018, at Colección Española de Cultivos Tipo, wherein the isolated polynucleotide is selected from the following list: SEQ. ID. No. 19 (ABP10666), SEQ. ID. No. 20 (ABP10667), SEQ. ID. No. 7 (ABP10654), SEQ. ID. No. 24 (ABP10671), SEQ. ID. No. 36 (ABP10829), SEQ. ID. No. 37 (ABP10830), or combinations thereof, wherein the isolated polynucleotide encodes for a polypeptide that hydrolyses a non-starch polysaccharide, preferably non-starch polysaccharides present in plant feedstuffs, preferably wherein the non-starch polysaccharide is mannan, glucan, xylan, arabinan, and/or galactan.
The present disclosure also relates to an isolated polynucleotide or polypeptide from ABP2 with a deposit under the accession number CECT 9676, of Aug. 6, 2018, at Colección Española de Cultivos Tipo, wherein the isolated polynucleotide is selected from the following list: SEQ. ID. No. 160 (ABP24564), SEQ. ID. No. 161 (ABP24565), SEQ. ID. No. 162 (ABP24566), SEQ. ID. No. 163 (ABP24567) or combinations thereof, wherein the isolated polynucleotide encodes for a polypeptide that hydrolyses a non-starch polysaccharide, preferably non-starch polysaccharides present in plant feedstuffs, preferably wherein the non-starch polysaccharide is mannan, glucan, xylan, arabinan, and/or galactan.
The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of the present disclosure.
The present disclosure relates to the isolation, identification and characterization of novel sporeforming probiotic strain(s) with NSPase (Non-Starch Polysaccharides-active hydrolases) activity isolated from fish gut microbiota, methods and uses thereof.
In an embodiment, sporeformers were isolated from the gut of European sea bass juveniles challenged with PF diets based on SBM, RSM or SFM, which have different NSPs profiles. European sea bass was the model species chosen due to its high commercial importance in European aquaculture and its carnivorous feeding habits, thus being more challenging to cope with PF-based diets. However, other models could be equally used such as gilthead seabream (Sparus aurata) or white seabream (Diplodus sargus) or Atlantic salmon (Salmo solar).
Providing fish with self-gut bacteria capable of producing carbohydrate-active extracellular enzymes that hydrolyse NSPs emerge as a strategy with enormous potential to overcome PF-diets limitations. The bacterial strains now disclosed were isolated with this purpose and their genome sequences support their view as potential NSPs-hydrolyzers that might help aquaculture fish on using high PF-diets.
Having in mind the enhanced adaptability of gut microbial communities, a selective pressure of plant-based diets on fish gut microbiota was carried out for an enrichment of bacteria with a secretome able to mobilize the dietary NSPs. By targeting bacterial spores, remarkably resistant dormant structures with increasing applications in animal health, namely as vaccines or PRO, it was possible to isolate carbohydrate-active gut bacterial strains, from European sea bass, with PRO potential. By inferring the adaptive fitness to the fish gut and the amenability to industrial processing, the best candidates were identified to become industrially valuable PRO for improvement of fish health and utilization of dietary NSPs, contributing for sustainable and more cost-effective aquaculture practices.
In an embodiment, the PRO were isolated and purified, identified to the species level, fully characterised, namely its safety following EFSA guidelines, NSPase activity, antimicrobial activity against important fish-pathogens, adaptive fitness to the fish gut and the amenability to industrial processing. The complete genome has been sequenced.
In an embodiment, there is a need for further research namely addressing the in vivo efficacy in improving PF utilization by fish and disease resistance in bacterial infection models. A preliminary assay using challenging plant-based diets (CTR−), revealed that supplementation with ABP1, or ABP1 and ABP2 (Mix) has a positive effect on the final body weight, the weight gain, the feed efficiency and the protein efficiency ratio of European sea bass juveniles, with a tendency to get closer to a FM-based diet (CTR+) (Table 1). Future analyses including digestive enzymes activity and gut microbiota modulation, might help explain the results obtained. Furthermore, a comprehensive screening of ABP1 and ABP2 genomes will potentially allow the identification of new carbohydrases or antimicrobial molecules.
83.0 ± 1.6ab
97.0 ± 2.0b
†IBW: initial body weight.
§ABW: average body weight (initial body weight + final body weight)/2.
1Mean values and standard deviation (±SD) are presented for each parameter (n = 3).
2DGI: ((final body weight1/3 − initial body weight1/3)/time in days) × 100.
3FE: (wet weight gain/dry feed intake).
4PER: (wet weight gain/crude protein intake).
In an embodiment, the fermentation of the strains can be easily reproduced by another practitioner. Furthermore, regarding commercial applications, companies within the aquaculture and feed industry may be potentially interested in acquiring these strains for the development of new PRO with digestive added-value.
In an embodiment, diet composition was formulated. Three experimental diets were formulated to be isonitrogenous (47% crude protein), isolipidic (17% crude lipid) and to contain 30% of soy bean meal (SBM diet), 30% of rapeseed meal (RSM diet) or 30% of sunflower meal (SFM diet). A FM-based diet was used as the control diet (CTR diet). Fish oil and pregelatinized maize starch were the main lipid and carbohydrate sources, respectively. Bicalcium phosphate was added to adjust dietary phosphorus level. All diet ingredients were thoroughly mixed and dry-pelleted in a laboratory pellet mill (California Pellet Mill, CPM Crawfordsville, Ind., USA), through a 3.0 mm die. Pellets were dried in an oven at 50° C. for 24 h, and then stored at −20° C. until used. Ingredients and proximate composition of the experimental diets are presented in Table 2.
aCTR, control fishmeal-based diet; SBM, soybean meal-based diet; RSM, rapeseed meal-based diet; SFM, sunflower meal-based diet
bSteam Dried LT fish meal, Pesquera Diamante, Austral Group, S.A Perú (CP: 74.7% DM; GL: 9.8% DM)
cSorgal, S.A. Ovar, Portugal (CP: 53.7% DM; GL: 2.1% DM)
dSorgal, S.A. Ovar, Portugal (CP: 37.5% DM; GL: 4.0% DM)
eSorgal, S.A. Ovar, Portugal (CP: 30.3% DM; GL: 1.0% DM)
fC-Gel Instant-12016, Cerestar, Mechelen, Belgium
gPremix, Portugal (Calcium: 24%; Total phosphorus: 18%)
hVitamins (mg kg1 diet): retinol acetate, 18,000 (IU kg−1 diet); cholecalciferol, 2000 (IU kg−1 diet); alfa tocopherol acetate, 35; sodium menadione bisulphate, 10; thiamine-HCl, 15; riboflavin, 25; calcium pantothenate, 50; nicotinic acid, 200; pyridoxine HCl, 5; folic acid, 10; cyanocobalamin, 0.02; biotin, 1.5; ascorbic acid, 50; inositol, 400
iMinerals (mg kg−1 diet): cobalt sulphate, 1.91; copper sulphate, 19.6; iron sulphate, 200; sodium fluoride, 2.21; potassium iodide, 078; magnesium oxide, 830; manganese oxide, 26; sodium selenite, 0.66; zinc oxide, 37.5; dibasic calcium phosphate, 8.02 (g kg−1 diet); potassium chloride, 1.15 (g kg−1 diet); sodium chloride, 0.44 (g kg−1 diet)
jAquacube (guar gum, polymethyl carbamide, manioc starch blend, hydrate calcium sulphate) Agil, UK.
In an embodiment, the animal experiment was performed at the Marine Zoology Station, Porto University, Portugal, with European sea bass, juveniles obtained from a commercial fish farm (Maresa S. A., Ayamonte, Huelva, Spain). After transportation to the experimental facilities fish were first submitted to a quarantine period of 30 days before transfer to the experimental system where they were allowed to adapt for 15 days. Before the experimental period, fish were fed a commercial diet (48% protein, 11% lipids, 5% starch). The trial was performed in a recirculating water system equipped with 12 cylindrical fiberglass tanks of 100 l water capacity and thermo-regulated to 22.0±1.0° C. Tanks were supplied with continuous flow of filtered seawater (2.5-3.5 l min−1) of 34.0±1.0 g l−1 salinity and dissolved oxygen was kept near saturation (7 mg l−1). Thereafter, 20 European sea bass with an initial mean body weight of 34.4 g were distributed to each tank and the experimental diets randomly assigned to triplicate groups. The trial lasted 45 days and fish were fed by hand, twice daily, 6 days a week, until apparent visual satiation. The experiment was performed by accredited scientists (following FELASA category C recommendations) and was conducted according to the EU directive 2010/63/EU on the protection of animals for scientific purposes.
In an embodiment, sampling was carried out as follows. Fish in each tank were bulk-weighed at the beginning and at the end of the trial, after 1 day of feed deprivation. For that purpose, fish were slightly anaesthetized with 0.3 ml l−1 ethylene glycol monophenyl ether (Sigma-Aldrich, Steinheim, Germany). On the sampling days (at day 15 after the beginning of the trial and at the end of the trial or day 45), fish were fed several times over the day to guarantee that gut was full at sampling time. At 4 h after the first meal, 3 fish per tank were randomly sacrificed with an overdose of ethylene glycol monophenyl ether, for collection of biological samples under aseptic conditions. To overcome inter-fish variation, the resulting material was pooled into one sample per tank to assess differences between dietary groups. Whole-gut (without pyloric caeca) were aseptically excised and squeezed to collect the digesta contents.
In an embodiment, the isolation of sporeforming bacteria was performed as follows. Each sample of digesta (1 g) obtained from fish fed the different dietary treatments was homogenized in 9 ml of buffered saline solution (0.9%). Serial dilutions were prepared in Bott & Wilson (B&W) salts and 100 μl aliquots spread on the surface of LB agar medium, after 20 min heat treatment at 65° C., for sporeformers selection. Plates were incubated at 30° C. in aerobic conditions for up to 5 days. Following selection, sporeformers were isolated and characterized for morphology in DSM, to confirm spore production by phase-contrast microscopy. Colonies representing different morphologies were picked at random and purified by restreaking on agar plates of the same media, before storage at −80° C. in LB broth with 30% glycerol. Sporeformers isolates were routinely grown aerobically at 37° C. in LB or DSM. The laboratory strain B. subtilis 168 [14] was used as a control in most of the experiments described in the present disclosure.
In an embodiment, screening sporeforming bacteria for carbohydrates metabolization was carried out as follows. Each sporeformer isolate was cultured on solid M9 minimal medium [15] supplemented with 0.2% (w/v) of each of the following carbohydrates: D-glucose (G7528), D-fructose (F3510), D-xylose (X3877), L-arabinose (A3256), D-galactose (G0750), D-mannose (63580), all purchased from Sigma-Aldrich, Steinheim, Germany-Aldrich Co. LLC. The Xylooligosaccharides (XOS) and Galactooligosaccharides (GOS) are commercially available prebiotics from Qingdao FTZ United International Inc. (Quingdao, China) that were added at the same concentration (0.2%). Growth after 24 h at 37° C. was recorded by photographing colonies in a Gel Doc XR System (Bio-Rad) using the Image Lab software v.4.0.1 (Bio-Rad). Growth quantification was assessed by measuring the colony volume on fixed areas with local background subtraction (adjusted volume=[CNT*mm2] data counts/mm2) using the Quantity One software v.4.6.9 (Bio-Rad). Quantification of carbohydrates utilization in liquid M9 was performed after an overnight enrichment in liquid LB at 37° C. with agitation. Each isolate was diluted to an initial optical density (OD600; absorbance measured at 600 nm) of 0.1 in liquid M9 minimal medium alone or supplemented with 0.2% of the different carbohydrates previously tested. Bacterial growth was followed during 48 h and quantified by measuring the OD600. In both solid and liquid medium assays, results presented were corrected by subtracting the colony volume/OD600 measured in M9 alone.
In an embodiment, the taxonomic identification of PRO isolates was performed as follows. Identification was carried out for all the isolates with promising extracellular carbohydrolytic activities. Total genomic DNA extraction was performed from overnight LB cultures, using the EZNA bacterial DNA purification kit (Omega Bio-Tek, USA), according to the manufacturer's instructions and quantified with the Qubit 2.0 Fluorometer (Invitrogen, Oregon, USA). PCR amplification of the small-subunit rRNA (16S rRNA) was carried at an annealing temperature of 55° C. using primers 27F and 1492R. Each 20 μl reaction contained 1×DreamTaq Buffer (Thermo Scientific, Vilnius, Lithuania), 0.2 mM of each dNTP (Thermo Scientific, Vilnius, Lithuania), 0.2 μM of each primer (STAB Vida, Lisboa, Portugal), 1 U of DreamTaq DNA Polymerase (Thermo Scientific, Vilnius, Lithuania) and 25 ng of DNA template. The Bioinformatics Resources Sequence Match package of the Ribosomal Database Project 11 (http://rdp.cme.msu.edu) and BLAST of the GenBank nonredundant (nr) nucleotide database (http://www.ncbi.nlm.nih.gov) were used to analyse the sequencing data.
In an embodiment, the screening of PRO isolates for NSPases was performed as follows: to tentatively obtain a set of primers specific for the genes encoding NSPs degrading enzymes (NSPases), an initial search was conducted at the Protein Knowledgebase—UniProtKB with terms “family:hydrolase AND annotation:(type:location AND secreted) AND taxonomy:“Bacteria”. A file containing bacterial secreted glycosyl hydrolases (GH) was then created and the ones involved in the utilization of NSPs of interest were chosen for further analysis. Enzymes chosen included mannanases, mannosidases, arabinofuranosidases, arabinanases, glucosidases, glucanases, fructosidases (fructanases), fructafuranosidases, galactorunases, xylosidases, and xylanases. The protein sequence of each individual enzyme was used to search for similar proteins in the translated nucleotide database (tblastn) (http://www.ncbi.nlm.nih.gov) and to make nucleotide alignments between the sequences obtained with ClustalW algorithm using Geneious R7 v7.1.7 (Biomatters, Auckland, New Zealand). Regions of sequence conservation were chosen to design primer pairs (Table 4) with the Vector NTI 10 software (Invitrogen, Carlsbad, Calif.), with a calculated annealing temperature of approximately 55° C. and an amplicon size of 200 to 250 base pairs (bp). PCR amplification was done essentially as described for the 16S rRNA (previous section), adjusting the annealing temperature to 55° C. and the extension time to 30 s.
In an embodiment, biosafety issues in particular antibiotics susceptibility and hemolytic activity were also evaluated. Antimicrobial resistance was studied by testing susceptibility of sporeforming isolates to different classes of antibiotics, namely Macrolides (Erythromycin, EM), Aminoglycosides (Kanamycin, KM, Streptomycin, SM, and Gentamycin, GM), Tetracyclines (Tetracycline, TC), Glycopeptides (Vancomycin, VA) and Cloramphenicol (CL), following the recommendations of the EFSA Panel on Additives and Products or Substances used in Animal Feed [16]. Minimal inhibitory concentrations (MIC) were determined using Etest® (bioMérieux, Inc.). Hemolysis was determined on Columbia 5% sheep blood agar plates streaked with colonies from fresh LB plates, after incubation at 37° C. for 24, 48 and 72 h.
In an embodiment, antimicrobial activity screening assays were performed as follows. The antimicrobial activity of selected sporeforming isolates was assessed by a colony overlay assay using as targets different fish pathogens. Zones of growth inhibition around the producer strains spots after 24 h incubation at 25° C. (for Photobacterium damselae, Vibrio harveyi, Tenacibaculum maritimum and Aeromonas bivalvium) or 37° C. (for Staphylococcus aureus) were considered as positives and the corresponding growth-inhibition halos diameter measured (mm). A cell-free supernatant screening assay was performed by inoculating BHI or Marine Agar (for T. maritimum) plates with overnight cultures of indicator strains, assuring a uniform and complete coverage of the agar plate. After 15 min rest to allow plates to dry, 1 cm holes where done in the agar and consequently filled with 200 μl of cell-free supernatant of each producer strain, previously centrifuged and filtered through a 0.2 μm cellulose filter, from stationary phase LB cultures (grown overnight at 37° C.). Zones of growth inhibition around the producer strains supernatant holes obtained after 24 h incubation at 25° C. or 37° C. (as before) were considered as positive. All observations were recorded by photographing in a Gel Doc XR System (Bio-Rad) using the Image Lab Software (Bio-Rad).
In an embodiment, sporulation, germination and resistance to gut environment were also carried out as follows. The kinetics of spore formation and germination was quantified using adaptations of well-established methods [15, 17]. Sporulation occurred in DSM for 24 h at 37° C. in an orbital shaker at 200 rpm, and its efficiency was determined by plating serial dilutions made in B&W isotonic buffer (Bott and Wilson salts: 1.24% K2HPO4, 0.76% H2PO4, 0.1% trisodium citrate, 0.6% [NH4]2SO4, pH 6.7) on LB agar, before and after a 20 min heat treatment at 80° C. to eliminate vegetative cells. Following 24 h incubation at 37° C., visible colonies were counted, and sporulation efficiency calculated as the titre of colony forming units (CFU ml−1) before and after the heat treatment.
Preparation of highly purified spores was done as follows: in brief, 48 h spores preparations (in liquid DSM) of each isolate were centrifuged for 10 min at 10000 g and 4° C. Cell pellets were suspended in 50 mM Tris-HCl (pH 7.2) containing 50 μg ml−1 of lysozyme, and incubated for 1 h at 37° C. After a single wash with 1 volume of distilled water (10 min at 10000 g, 4° C.), cell pellets were suspended in 0.05% SDS, followed by three washes with distilled water and finally suspended in 1 volume of distilled water. Spores purity and recovery yields were determined by plating serial dilutions on LB agar, before and after a 20 min heat treatment at 80° C.
Spore germination in response to the addition of 100 mM L-alanine or to a mixture of 100 mM KCl, 56 mM glucose, 56 mM fructose and 33 mM L-asparagine (AGFK), was performed at 37° C. in 50 mM Tris-HCl, pH 7.5.
Potential resistance to gut transit was evaluated by determining the acid and bile tolerance of each selected isolate. For that purpose, 48 h DSM spores preparations were heat-treated for 20 min at 80° C. to eliminate vegetative cells and harvested by centrifugation. After a double wash with Phosphate-buffered saline (PBS), serial dilutions made in B&W salts were plated onto LB agar plates to determine the initial bacterial counts. Spores were then diluted in 1 volume of 0.85% NaCl, pH 2, containing 3 mg ml−1 pepsin (Sigma-Aldrich, Steinheim, Germany), to mimic stomach conditions. Following 4 h incubation at 37° C. with agitation, serial dilutions made in B&W were again plated onto LB agar plates to determine bacterial counts, and, after a single wash with PBS, spores were resuspended in LB, pH 8 containing 1 mg ml−1 pancreatin (Sigma-Aldrich, Steinheim, Germany) and 0.3% bile salts (Sigma-Aldrich, Steinheim, Germany). Bacterial incubation continued for 24 h at 37° C. with agitation to mimic passage through the gut. Finally, serial dilutions made in B&W were again plated onto LB agar plates to determine the final bacterial counts. All plates were incubated at 37° C. during 24 h prior to colonies count.
In an embodiment, shotgun genome sequencing was carried out at the Research and Testing Laboratory (Lubbock, Tex., USA) using the PacBio RSII sequencer (Pacific Biosciences, CA, USA). A total of 78,219 and 96,855 reads (with a mean read length of 13,383 and 15,478 base pairs) were obtained for ABP1 and ABP2, respectively, using as reference the Bacillus subtilis subsp. subtilis str. 168 (AL009126.3) [14]. The raw sequences were assembled using Pacific Biosciences SMRT Analysis v2.3.0. The total size of the assembly was around 4,068 Mb (2 final contigs) for ABP1 and 4,308 Mb (3 final contigs) for ABP2. A BLAST analysis against the RefSeq_genome database (NCBI) revealed that the best match for ABP1 is the Bacillus subtilis subsp. subtilis str. BSP1 (CP003695.1; (11)) while for ABP2 a best match is Bacillus sp. LM 4-2 (CP011101.1; (12)). The BLAST version used was the 2.7.1.
Both assemblies were analysed by using the Rapid Annotation Subsystem Technology (RAST) server. The amino acid sequences of each gene identified in RAST were processed using BLASTP+ against the RefSeq_Protein (NCBI), RefSeq_RNA (NCBI) and All-tRNA [4] (http://gtrnadb.ucsc.edu/) databases and then passed along the DAVID web service to determine other crucial annotation data such as GO Terms, PFAMs, TIGRFAMS, EC numbers or KEGG Pathways.
In an embodiment, statistical analysis was conducted by one-way ANOVA using the SPSS 21 software package for Windows (IBM® SPSS® Statistics, New York, USA). Data were tested for normality and homogeneity of variances by the Shapiro-Wilk and Levene's test, respectively. When normality was not verified, data were transformed prior to ANOVA. Significant differences among groups were determined by the Tukey's multiple range test. The probability level of 0.05 was used for rejection of the null hypothesis.
More than 200 bacterial isolates were obtained from the heat-treated gut contents of European sea bass fed each dietary situation (CTR, SBM, RSM and SFM). Following purification, 160 isolates representing different samples and colony morphologies (illustrated in
In an embodiment, the carbohydrolytic activity of gut sporeformes was evaluated. The entire collection of 160 isolates was screened for their carbohydrolytic potential by substrate specific culture-based methods, and different profiles of carbohydrate utilization could be assigned to different isolates, as illustrated in
In an embodiment, the carbohydrate-active gut sporeformes were testes as PRO for aquaculture as follows. The selected 43 isolates were checked for minimal biosafety requirements to be considered as PRO, following the guidelines from the EFSA and the World Health Organization (WHO) [8, 17]. The majority (33) of the isolates exhibited some degree of hemolytic activity when cultivated on 5% sheep blood agar plates, with 14 isolates showing strong or β hemolysis (Table 3). Half of the isolates revealed to be resistant to at least 1 antimicrobial, and 10 isolates were resistant to 2 or more antimicrobials, defined as MR in Table 3 and detailed in Table 5. These tests allowed selecting a strict group of 11 isolates as good candidates to become a PRO for European sea bass (Table 3, highlighted in bold lettering), as isolates showing strong hemolytic activity or any antimicrobial resistance to the different classes of antibiotics tested were not further studied.
ABP3
B. thuringiensis; B. cereus
ABP4
Bacillus sp.
ABP5
B. subtilis
ABP6
B. cereus
ABP7
B. pumilus; B. safensis
ABP8
B. licheniformis
ABP9
B. cereus
ABP10
B. simplex; B. macroides
ABP1
B. subtilis
ABP11
B. sp
ABP12
B. safensis
ABP13
B. licheniformis
ABP14
B. pumilus
ABP15
B. cereus; B. subtilis
ABP16
B. safensis; B. pumilus
ABP17
B. subtilis; B. mojavensis
ABP18
B. licheniformis; B. aerius
ABP19
B. subtilis
ABP20
B. subtilis; B. amyloliquefaciens
ABP21
B. licheniformis
ABP22
B. licheniformis; B. aerius
ABP23
B. licheniformis
ABP24
B. licheniformis
ABP25
B. pumilus
ABP26
B. licheniformis
ABP27
B. subtilis; B. amyloliquefaciens
ABP28
B. cereus
ABP29
B. licheniformis; B. aerius
ABP30
B. cereus
ABP31
B. cytotoxicus
APB32
B. licheniformis
ABP33
B. safensis
ABP34
Bacillus sp.
ABP35
B. licheniformis
ABP2
B. subtilis
ABP36
B. simplex; B. macroides
ABP37
B. subtilis
ABP38
Bacillus sp.
ABP39
Bacillus sp.
ABP40
Bacillus sp.
ABP41
B. licheniformis
ABP42
B. licheniformis
ABP43
B. thuringiensis; B. cereus
aIn underlined lettering are the isolates showing strong hemolytic activity or any antimicrobial resistance, discarded from the rest of the disclosure and in bold the 11 isolates used in subsequent tests.
bCTR, control fishmeal-based diet; SBM, soybean meal-based diet; RSM, rapeseed meal-based diet; SFM, sunflower meal-based diet.
cSpores detected by phase-contrast microscopy of 24 h cultures in DSM agar.
dCatalase activity tested by resuspending a colony in a 3% solution of hydrogen peroxide (Sigma).
eHemolysis determined on Columbia 5% sheep blood agar plates after incubation at 37° C. for 24, 48 and 72 h (shown is the final reading at 72 h incubation). β-hemolysis, the bacterial hemolytic enzymes completely break down the blood cells; α-hemolysis, the bacterial hemolytic enzymes only partially break down the blood cells; γ-hemolysis corresponds to essentially no hemolytic activity detected.
fAbR-Antimicrobial resistance determined by the E-test method against several antibiotics (Table 5). R—resistance to one antimicrobial; MR—resistance to 2 or more antimicrobials; — no resistance phenotype detected.
gClosest known species found using RDP based on partial sequences (600 to 800 nt) of the 16S rRNA gene.
The selected 11 isolates were then simultaneously cultured in M9 liquid medium to quantify bacteria growth after 24 h in liquid M9 supplemented with the different carbohydrates (
The presence of specific carbohydrases coding genes in these 11 isolates was investigated by using oligonucleotide primers specifically designed to target the genes coding for β-glucanase (bglS), levanase or β-D-fructofuranosidase (sacC), mannan endo-1,4-β-mannosidase (gmuG), endo-1,5-α-L-arabinanase (abnA), and arabinoxylan arabinofuranohydrolase (xynD) (Table 4). Their broad carbohydrolytic phenotype could not be correlated with the presence of the target genes, since no PCR amplification was obtained for the most promising isolates (ABP38 and ABP40) while all target genes seem to be present in the worst fish isolates ABP4 and ABP5 (
aThe enzyme Glycoside Hydrolase Family (GH) number and the EC number are providing in brackets
bgene name in B. subtilis strain 168 genome, whose sequence was used to design the oligonucleotide primers
Table 5. Susceptibility of selected isolates to various antimicrobial agents
In an embodiment, sporeforming isolates ABP7, ABP1, ABP20, ABP27, ABP34, ABP2, ABP36, ABP38, and ABP40, that simultaneously met the minimal safety requirements to be eligible as PRO and were the most efficient isolates in metabolizing the carbohydrates tested, were further characterized to determine their sporulation efficiency, an important characteristic for future industrial production and feed incorporation.
In an embodiment and by comparison with the well-studied standard strain B. subtilis 168 [14], isolates ABP36, ABP38, and ABP40 did not reach a minimum titer of 107 ml−1 heat-resistant cells, after 24 h sporulation induction by nutrient exhaustion in DSM liquid medium (
In an embodiment, the potential to survive passage through the gastrointestinal tract, important for in vivo efficacy, was determined by exposure to sequential simulated stomach and gut conditions. Purified spores of isolates ABP7, ABP1, ABP20, ABP27, ABP34 and ABP2 were first subjected during 4 h to acidified NaCl containing pepsin, to mimic stomach conditions, followed by 24 h exposure to alkalinized LB medium containing pancreatin and bile salts. While 4 h in simulated stomach conditions had nearly no effect on the isolates survival, the subsequent 24 h exposure to simulated gut conditions lead to a reduction in each bacterial population (
In an embodiment, the remaining four isolates, namely ABP1, ABP20, ABP27, ABP2, were characterized for their antimicrobial activity against several fish pathogenic strains, namely P. damselae, V. harveyi, T. maritimum, A. bivalvium, and S. aureus. As illustrated in
In an embodiment, and in an attempt to infer the germination capacity of these strains inside the animal gut, spores of the same four isolates, ABP1, ABP20, ABP27 and ABP2, were subject to different germinants, namely L-alanine and a mixture of KCl, glucose, fructose and L-asparagine (AGFK). For the conditions tested, isolates ABP20 and ABP27 were unable to germinate (
In an embodiment, the 4,068,058 bp genome of ABP1 was found to consist on 4,304 open reading frames (ORFs) with 4,184 genes identified (30 rRNAs genes and 86 tRNA genes).
In an embodiment, isolate ABP2 contained a slightly bigger genome, with 4,308,180 bp. A total of 4,643 genes (from 4,759 ORFs) were identified, including 28 rRNAs genes and 82 tRNA genes.
In an embodiment, the G+C content of ABP1 and ABP2 genomes was estimated to be 43.9% and 43.4% respectively.
In an embodiment, an exhaustive comparative analysis against the reference B. subtilis str. 168 [14] using Geneious R7 v7.1.7 software (Biomatters, Auckland, New Zealand) revealed the absence of more than 200 genes from ABP1 and ABP2 genomes. These are mostly associated with prophage like regions, namely Prophage3, SPR and the Skin element, or with mobile genetic elements such as the integrative and conjugative element ICEBs1. As previously described for other gut isolates including B. subtilis str. BSP1 [17], several negative regulators of sporulation (e.g. rapE, rapK) are also absent, resulting in a higher sporulation efficiency of these isolates when compared to the B. subtilis str. 168, with the advantage that this behaviour can have at the industrial and gut levels. Interestingly, both isolates lack the sdpABCIR operon of sporulation delaying proteins, which might result in even earlier trigger of sporulation.
In an embodiment, ABP1 and ABP2 genomes also accommodate new genes, some of which coding for NSPs-active hydrolases. For instance, additional genes that might be involved in xylose and mannose metabolism are found in ABP1, while ABP2 contains one myo-inositol catabolic operon, that might contribute to the cycling of inositol phosphates in the marine environment or to their bioavailability (from PF-diets) inside the fish-gut.
In an embodiment, by sequencing the 16S rRNA gene, all isolates could be assigned to a genus, being Bacillus the most prevalent (>60%). Affiliation to a species based on a single molecular marker (16S rRNA) was limited, as expected. This was the case for isolates belonging to the B. cereus group (B. cereus, B. anthracis, B. thuringiensis, B. mycoides, B. pseudomycoides, B. weihenstephanensis, and B. cytotoxicus) or to the B. subtilis-B. licheniformis clade (B. subtilis, B. vallismortis, B. mojavensis, B. atrophaeus, B. amyloliquefaciens, B. licheniformis, B. sonorensis, and B. tequilensis), whose 16S rRNA gene sequences obtained do not differ enough to distinguish them.
Although several Bacillus spp. are quite common in the gut of different animals, including the ones with high-fiber feeding habits, such as soil invertebrates or the giant panda [19], few studies have focused on their carbohydrolytic potential. For example, predominant B. subtilis strains from the gut microbial community of the giant panda, seem to have the capacity to growth in a higher fiber environment [20], opening the possibility that also in fish, Bacillus spp. may have a decisive role in shaping their host digestive capacity towards the efficient utilization of PF-diets. In fact, two recent studies, although limited to cellulase and xylanase activities, reported the isolation of carbohydrate-active Bacillus spp. from the gut of different fish species. The 160 isolates tested in the present disclosure showed different, and in some cases potent, hydrolytic capacities when using as sole carbon source selected carbohydrates including xylose, galactose, arabinose, or mannose. This observation was further sustained by the presence of genes coding for specific extracellular CAZymes that can help fish in obtaining the otherwise unavailable energy trapped in PF. The absence of amplification for these specific genes in some isolates, despite showing broad carbohydrolytic activities, is not surprising considering that some studies suggests that new or substantially different CAZymes involved the metabolization pathways are yet to be found in the Bacilli group of organisms. Furthermore, the lack of PCR amplification of these genes, observed with some isolates, may also be caused by mismatches of the primer pairs, due to the difficulty to design gene-specific primers regarding genomic regions poorly conserved.
In an embodiment, PRO approval within EU for incorporation into animal feed, including aquafeeds, is subject to strict and exhaustive exigencies following EFSA guidelines on quality, safety, and efficacy of the candidate(s) bacterial strain(s) [21]. Besides the obligation of strain deposition in an internationally recognised culture collection, candidate PRO isolates must be tested for the presence of any acquired antibiotic resistance genes [11, 16, 22]. PRO, which are given to animals in massive amounts, should not contribute to the escalation of antimicrobial resistance by acting as vehicles of transferable genetic determinants. Unfortunately, these rules do not apply worldwide, and very recently antimicrobial resistant strains were found in PRO products used in Vietnamese shrimp culture or in Chinese human commercial products, with all the risks those findings pose to the aquaculture production sector and to public health [23]. Although EFSA guidelines only require the absence of acquired (transmittable) resistance genes, allowing the use of bacterial strains whose antibiotic resistance is chromosomally encoded, the option of eliminating all the strains showing any antimicrobial resistance to the different classes of antibiotics tested. Adding to that criterion, strains showing strong hemolytic activity, indicative of virulence potential in several pathogenic bacterial species, including sporeforming ones, were also not further tested. These tests allowed to select a group of 11 PRO candidates that qualify with the minimal biosafety issues to be approved by EFSA.
In an embodiment, to demonstrate efficacy, EFSA requires three in vivo studies showing statistically-significant effects on each target animal species [21]. To conduct such follow-up in vivo studies in European sea bass growth and digestibility trials, it was necessary, for practical reasons, to narrow the group of interesting and potential PRO candidates. These were subjected to a series of consecutive tests to analyze some desired characteristics on a future PRO product. First the sporulation yield, an important parameter in industrial and economical terms, was determined by comparison with the well-studied standard laboratory strain B. subtilis 168 [14]. Six isolates demonstrated high yield spore formation, which anticipates a good suitability for cost-effective spores' production in industrial scale. Additionally, higher sporulation levels might also act as a form of propagation inside the animal gut, maximizing these strains beneficial effect [10, 17]. Second, exposure of purified spores to sequential simulated gastric and gut conditions, revealed the four isolates best equipped to survive passage through the gastrointestinal tract, important to guarantee their in vivo efficacy. In particular, isolates ABP1 and ABP2, which also showed higher sporulation efficiency, seem to be the best suited to reach, at higher numbers, the gut where their PRO action can take place. To take advantage of these isolates as PRO, upon passage through the stomach and anterior gut, spores must germinate to originate new vegetative cells that can produce the enzymes/molecules thought to benefit their host. In nature, spore germination is believed to occur in response to specific nutrients. For example, B. subtilis spores are known to germinate in response to L-alanine, L-valine and L-asparagine but not in response to their D-enantiomers. Taken this, and although the mechanisms of germination of spores of different Bacilli (independently of their specific species) are thought to be essentially the same, it cannot be ruled out that some of the isolates might respond efficiently to other germination molecules that might be abundant in vivo (inside the animal gut), explaining the germination failure of isolates ABP20 and ABP27 under the conditions assayed. Finally, and besides their carbohydrolytic potential, these PRO might also benefit the fish host by minimizing colonization with pathogenic species, known to be especially problematic in marine aquacultures. This is the case of T. maritimum whose growth was efficiently inhibited in vitro, when exposed to both cells and cell-free culture medium of isolate ABP2.
In an embodiment, sequencing ABP1 and ABP2 genomes allowed a comprehensive screening of their genomic potential to better meet the EFSA criteria for PRO. Both genomes accommodate new genes, some of which coding for NSPases (NSP-active hydrolases). For example, additional genes that might be involved in xylose (e.g. ABP10666, ABP10667), mannose (e.g. ABP10654, ABP10671) and sucrose (e.g. ABP10829, ABP10830) metabolism are found in ABP1, while ABP2 contains one myo-inositol catabolic operon (ABP24564 to ABP24567), that might contribute to the cycling of inositol phosphates in the marine environment or to their bioavailability (from PF-diets) inside the fish-gut.
In an embodiment, dissecting these genomes permitted to detail and further document their biotechnological value as PRO and/or as sources of carbohydrases or antimicrobial molecules. For instance, determining the number and type of CAZymes present in each genome provides deeper understanding on their carbohydrolytic potential also allows identification of genomic features responsible for adaptation to life within the gut that may support the role of Bacillus spp. as PRO [10, 12-13, 17-18]. The growing applications of spores in biomedicine and biotechnology (as oral vaccines, PRO or display systems) [12-13, 18], and the fact that there are approximately 30 PRO strains approved as feed additives in EU, but only one for aquaculture (Bactocell®, which is not a sporeformer formulation), underscore the importance of this disclosure.
This disclosure relates to 2 PRO strains, ABP1 and ABP2, which genome comprises at least one polynucleotide encoding a protein which is involved in PRO behavior, and which polynucleotide is substantially identical to a polynucleotide sequence according to ABP10666, ABP10667, ABP10654, ABP10671, ABP10829, ABP10830 for ABP1 and ABP24564 to ABP24567 for ABP2.
In an embodiment, the polynucleotides as listed in Table 6 were isolated from Bacillus subtilis strain ABP1 or ABP2, and are absent or divergent in/from B. subtilis 168 [14], and at least in 5 out of 11 sequenced Bacillus isolates including:
The term “comprising” whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.
The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.
The above described embodiments are combinable.
The following claims further set out particular embodiments of the disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 115101 | Oct 2018 | PT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2019/059131 | 10/24/2019 | WO | 00 |
