Methods of isolating synergistic bacterial communities

Abstract

The invention provides a method for co-isolating two or more bacterial strains co-located on a micro-scale surface. Such co-isolated bacteria may be suitable for formation of synergistic bacterial consortia.

Description

[0001] METHODS OF ISOLATING SYNERGISTIC BACTERIAL COMMUNITIES

[0002] FIELD OF THE INVENTION

[0003] This patent application pertains to the field of microbiology, specifically to synergistic microbial consortia.

[0004] BACKGROUND

[0005] The human and plant microbiomes are complex communities of microorganisms that play a pivotal role in the health, disease, growth, and development of the host. While single species bacterial inoculation has been used extensively in agriculture, medicine and industry, many promising bacterial strains are unable to perform their function in certain and / or complex environments or are not culturable when isolated alone.

[0006] Synergistic bacterial consortia represent groups of microorganisms that interact to enhance each other's function or promote survival of the consortia. These consortia benefit from collective metabolic activities and interactions and are thus promising candidates to replace single bacterial inoculation.

[0007] Previous strategies for generating synergistic bacterial consortia have relied on combinatorial culture of two or more isolated and characterised bacterial strains. Accordingly, the identification and culture of bacterial strains is an important prerequisite for this strategy and screening a large number of possible combinations of isolated bacteria is time consuming and laborious.

[0008] There is thus an unmet need for a high throughput method of identifying suitable candidate bacterial strains which may generate synergistic bacterial consortia. Specifically, a method that does not exclusive rely on combinatorial culture of previously identified bacterial strains.

[0009] SUMMARY OF THE INVENTION

[0010] Micro-scale surfaces serve as unique environments with separate and diverse microbiomes and bacteria are often affected or dependent on other species in their immediate proximity. Without being bound by theory, the present invention is based on the realisation that stable co-localization of multiple bacteria on a micro-scale surface suggests the presence of interactions within the bacterial community, such interactions possibly being synergistic. The objective of the invention is thus to provide a method for co-isolating two or more bacterial strains co-located on a micro-scale surface, and thus identifying suitable candidate bacterial strains which may generate synergistic bacterial consortia.

[0011] Against this background, the inventors have developed a method of co-isolating two or more bacterial strains co-located on a micro-scale surface.

[0012] The present invention relates to a method for co-isolating two or more bacterial strains colocated on a micro-scale surface, the method comprising :

[0013] (i) providing a sample comprising a plurality of bacterial strains and a solid support;

[0014] (ii) filtering said sample through a first filter, and reverse filtering said sample through a second filter, to obtain micro sized particles derived from said solid support in a first volume, wherein said first filter has a pore diameter greater than the pore diameter of said second filter; and

[0015] (iii) recovering in a second volume a single micro sized particle comprising two or more bacterial strains.

[0016] DETAILED DESCRIPTION OF THE INVENTION

[0017] Definitions

[0018] As used herein the term "isolating" refers to the separation of one or more bacterial strains from a mixed bacterial population or from a sample comprising said one or more bacterial strains and other components. Accordingly, the term "co-isolation", as used herein, refers to the separation of two or more bacterial strains, where the two or more bacterial strains are separated concomitantly.

[0019] The term "to comminute" or "comminuting" is to be understood as the act of reducing the average size, length and / or diameter of one or more particles of a solid material. Said reduction may comprise separating smaller particles from a mixture of particles, for example by sieving, centrifugation, fragmenting and / or homogenizing the solid material, such as by grinding, milling, cutting, or similar.

[0020] Wherever the term "about" is employed herein in the context of amounts, for example absolute amounts, such as numbers, purities, weights, concentrations, sizes, etc., or relative amounts (e.g. percentages, equivalents or ratios), timeframes, and parameters such as temperatures, pressure, etc., it will be appreciated that such variables are approximate and as such may vary by ±10%, for example ± 5% and preferably ± 2% (e.g. ± 1%) from the actual numbers specified. This is the case even if such numbers are presented as percentages in the first place (for example 'about 10%' may mean ±10% about the number 10, which is anything between 9% and 11%.

[0021] The term "staining" is used herein to refer to any process which makes an object, such as the bacteria or the solid support, visible or measurable under certain conditions. The term "dye" should be understood as encompassing any agent which is capable of binding, intercalating, or otherwise associating with an object and which can be visualised or measured. For example, dyes may include radiolabelled or fluorescently labelled probes, as well as DNA intercalating compounds.

[0022] The term "sample" is used herein to refer to a discrete quantity of material obtained from a biological, environmental, synthetic, or composite source, which is collected, isolated, or prepared for the purpose of analysis, testing, processing, or characterization. The sample contains solid constituents, including but not limited to particulate matter or solid supports, upon which microorganisms such as bacteria may reside, adhere, or proliferate. The sample may be in solid, semi-solid, or liquid form and may include biological, chemical, or physical components of interest. It may be unprocessed, partially processed, or fully processed and may originate from natural, cultivated, industrial, or engineered environments. The sample may comprise, without limitation, soil, sludge, fecal matter, horticultural substrates, plant tissue, animal tissue, human tissue, foodstuffs, fabric, plastic, plastic derivatives, or any combination thereof.

[0023] As used herein, the term "natural microbial sample(s)" is to be understood as a sample obtained directly from a natural environment. The term thus encompasses all types and samples, which are found in nature, including samples of natural origin which have been processed or altered by human action, such as processing of foodstuffs, or production of goods.

[0024] The term "solid support" is used herein as to refer to a solid constituent within the sample and may be any substance of matter to which one or more bacterial cells may be attached to simultaneously, and which does not in itself comprise bacterial cells. The solid support can be in particulate form. By way of example, in a soil sample, the solid support includes mineral matter, organic matter and / or silica aggregates, but excludes bacterial cells attached to said support. The solid supports contained in the sample may be of various sizes, e.g. micro-sized or larger. As part of the method described herein, the solid support may be filtered to isolate micro-sized particles, or comminuted into micro-sized particles, upon which bacteria are co-localised.

[0025] The term "micro-scale surface" is to be understood as a particle of matter no more than 100 pm in length along the longest axis. As used herein the terms "micro-sized particle" or "micro-scale particle" are used interchangeably to refer to a particle of matter no more than 100 pm in length along the longest axis, which is not associated, conjugated, bound or otherwise attached to other such micro-sized particles. Micro-sized particles or microscale particles may preferably be solid. Micro-sized particles or micro-scale particles may further be derived from a solid support.

[0026] As used herein the term "synergistic bacterial consortia" or "synergistic bacterial communities", refer to a bacterial composition comprising two or more bacterial species or bacterial strains, wherein at least one biological function of at least one bacterial species or strain is improved compared to that of the bacterial species or strain in isolation.

[0027] As used herein, "volume" should be understood as any three-dimensional space. In the context of the methods of the invention, the first and / or second volume may be liquid volumes, gaseous volumes or solid volumes. As an example, the first and / or second volumes may be liquid volumes held in one or more containers or distinguishably placed on a surface, such as liquid droplets. Such containers may for example be, any such used in experimental work, such as reagent tubes, wells in a microtiter plate.

[0028] As used herein "free floating bacterial cells" or "planktonic bacterial cells" are to be understood as bacterial cells that are not attached directly or indirectly to the solid support.

[0029] As used herein, "filtration" refers to the process of separating certain solids from a fluid suspension via a filter. The filter, characterized by its pore size or pore diameter, allows the fluid to pass while retaining solids above a certain size. Unless explicitly stated otherwise, "filtration" as used herein, thus encompasses the process wherein the filtrate (also called the permeate) (i.e., the fluid passing the filter) is of interest, maintained and thus used in further steps of the method, while the remaining sample is discarded. "Reverse filtration" refers to filtration wherein the retained solids (the retentate) are of interest and therefore maintained and the filtrate is discarded.

[0030] The present specification encompasses various descriptions pertaining to the dimensions of entities such as (soil) particles, bacterial cells, aggregates, or other objects. These dimensions, unless explicitly stated otherwise, should be interpreted as the length at the point of maximum extension. This point of maximum extension is typically along the longest axis of the entity in question. Accordingly, it should be noted that the term "size", as used herein, refers to the greatest linear distance between any two points on the surface of the entity, measured along a straight line passing through the centre of the entity, and extending from one surface point to another.

[0031] In the description of the invention various embodiments and / or individual components are disclosed. As will be apparent to the ordinarily skilled practitioner, all combinations of such embodiments and components taught in the disclosure are possible and can result in preferred embodiments of the present invention.

[0032] Any percentages and ratios are calculated by weight unless otherwise indicated. All percentages, parts and ratios are calculated based on the total composition unless otherwise indicated.

[0033] Detailed description of the invention

[0034] Micro-scale surfaces serve as unique environments with separate and diverse microbiomes and bacteria are often affected or dependent on other species in their immediate proximity. Without being bound by theory, it is thought that in micro-scale niches present in heterogenous environments, the community composition is governed by interspecific competition and cooperation at a scale relevant for microbial interactions. Thus, stable colocalization of multiple bacteria on a micro-scale surface suggests the presence of interactions within the bacterial community, such interactions being possibly being synergistic.

[0035] The present invention provides a method of co-isolating two or more bacterial strains colocated on a micro-scale surface (i.e., within a small spatial scale). By isolating bacteria according to the method of the invention the chance of isolating synergistically interacting bacteria is increased.

[0036] Importantly, the method of the invention allows for the micro-scale surfaces to be isolated while also removing a majority of free-floating bacterial cells which may be present in naturally occurring samples, such as soil. By removing free-floating bacterial cells it is ensured that the bacteria obtained as part of the method are exclusively bound to the micro-scale surface and thus subject to interaction with other bacteria on such surface. Previous methods of obtaining two or more bacterial strains suitable for synergistic bacterial consortium formation relied on single species or strain isolation and subsequent testing of each combination of the isolated bacterial species or strain. Such methods are thus cumbersome, time consuming and rely partially or entirely on chance.

[0037] The method of the invention thus represents a fast, high throughput workflow to co-isolate synergistic bacterial consortia compared to single species isolation and subsequent combinatorial testing.

[0038] The invention provides a method for co-isolating two or more bacterial strains co-located on a micro-scale surface, the method comprising :

[0039] (i) providing a sample comprising a plurality of bacterial strains and a solid support;

[0040] (ii) filtering said sample through a first filter, and reverse filtering said sample through a second filter, to obtain micro sized particles in a first volume, wherein said first filter has a pore diameter greater than the pore diameter of said second filter; and

[0041] (iii)recovering in a second volume a single micro sized particle comprising two or more bacterial strains.

[0042] In some embodiments, the micro sized particles obtained in step (ii) are derived from said solid support. It will be apparent to one skilled in the art that one or more bacterial cells may be attached or bound to said solid support, and such bacteria are thus also bound or attached to the micro sized particles derived from said solid support.

[0043] In some embodiments, the single micro sized particle recovered in a second volume in step (iii) is a micro sized particle obtained in step (ii) in said first volume.

[0044] In some embodiments, the first volume of step (ii) is the retentate of said reverse filtering through the second filter. In some embodiments, the first volume of step (ii) is the filtrate of said filtering through the first filter. In a preferred embodiment, the first volume of step (ii) is the filtrate of said filtering through the first filter and the retentate of said reverse filtering through the second filter.

[0045] An embodiment relates a method for co-isolating two or more bacterial strains colocated on a micro-scale surface, the method comprising :

[0046] (i) providing a sample comprising :

[0047] - a plurality of bacterial strains; and - a solid support;

[0048] (ii) filtering said sample through a first filter, and reverse filtering said sample through a second filter, to obtain micro sized particles in a first volume, wherein said first filter has a pore diameter greater than the pore diameter of said second filter; and

[0049] (iii)recovering in a second volume a single micro sized particle comprising two or more bacterial strains.

[0050] Another embodiment relates to a method for co-isolating two or more bacterial strains co-located on a micro-scale surface, the method comprising :

[0051] (i) providing a sample comprising :

[0052] - a plurality of bacterial strains; and

[0053] - a solid support;

[0054] (ii) filtering according to the following steps:

[0055] - filtering said sample through a first filter to obtain a filtrate; and

[0056] - filtering said filtrate through a second filter to obtain a retentate, wherein said retentate comprises in a first volume micro-sized particles derived from said solid support, wherein said first filter has a pore diameter greater than the pore diameter of said second filter; and

[0057] (iii)recovering in a second volume a single micro-sized particle comprising two or more bacterial strains.

[0058] Micro-scale surfaces represent a relevant environment for microbial interactions as the composition of the bacteria thereon is governed by interspecific competition and cooperation at a scale relevant for microbial interactions.

[0059] Accordingly, one skilled in the art will appreciate that the obtained micro-scale particles of the invention may preferably be of at most a certain particle diameter and / or size to increase the likeliness of the two or more bacterial strains co-isolated to interact on said micro-scale particle.

[0060] Accordingly, in one embodiment of the invention, said first filter has a pore diameter in the range of about 10 pm to about 100 pm, such as about 15 pm to about 90 pm, about 15 pm to about 80 pm, about 15 pm to about 70 pm, about 20 pm to about 60 pm, about 20 pm to about 55 pm, about 20 to about 50 pm, about 25 pm to about 45 pm, about 25 pm to about 40 pm, or about 25 to about 35. In a preferred embodiment, said first filter has a pore diameter of about 30 pm. In an alternative embodiment, said first filter has a pore diameter smaller than about 100 pm, such as smaller than about 100 pm, smaller than about 95 pm, smaller than about 90 pm, smaller than about 85 pm, smaller than about 80 pm, smaller than about 75 pm, smaller than about 70 pm, smaller than about 65 pm, smaller than about 60 pm, smaller than about 55 pm, smaller than about 50 pm, smaller than about 45 pm, smaller than about 40 pm, smaller than about 35 pm, smaller than about 30 pm, smaller than about 25 pm, smaller than about 20 pm, smaller than about 15 pm, or smaller than about 10 pm. In a preferred embodiment said first filter has a pore diameter smaller than about 30 pm.

[0061] The method of the invention relies on obtaining two or more bacterial strains co-located on a micro-scale surface (such as a micro sized particle). These bacteria may be further isolated and / or cultured to multiply their number, and / or otherwise characterize these bacteria. One skilled in the art will appreciate that, in order to obtain a first and / or second volume from which the relevant two or more bacterial strains co-located on a micro-scale surface may be further isolated and / or characterized, it may be advantageous to reduce the abundance of and / or remove free-floating bacterial cells in said second volume. By way of example, if free-floating bacterial cells are abundant in said first and / or second volume, these may compete for nutrients, supress, inhibit, or otherwise hinder the two or more bacterial strains of interest, which were co-located on the micro-scale surface. Alternatively or additionally, abundance and / or presence of free-floating bacteria in said second sample may complicate the characterization of the bacterial strains co-located on the micro-scale surface, for example by contaminating nucleic acid samples or bacterial cultures derived from said second volume.

[0062] Accordingly, in some embodiments of the invention, said first and / or second volumes are essentially free from free-floating bacterial cells. In a preferred embodiment, said first volume is essentially free from free floating bacterial cells.

[0063] Free floating bacterial cells, may be removed by reverse filtering said sample. By reverse filtering the sample using a filter through a second filter having a pore diameter equal to or greater than the diameter of the free-floating bacteria but equal to or smaller than the diameter of the micro-sized particles, the free floating bacterial cells can be discarded while the micro-sized particles are maintained.

[0064] The nature of the free-floating bacterial cells found in the sample may be dependent on the sample provided in step (i) and optionally on the methods of comminuting the sample. For example, the average diameter of spherical bacteria is 0.5-2.0 pm, while for rod- shaped or filamentous bacteria, length is 1-10 pm and diameter is 0.25-1 pm. Accordingly, in some embodiments the pore diameter of said second filter is configured to allow for passage of the free-floating bacterial cells present in the sample. In an additional embodiment, the pore diameter of said second filter is configured to allow for passage of the free-floating bacterial cells present in the sample and to block passage of the microsized particles.

[0065] In an additional or alternative embodiment, said second filter has a pore diameter in the range of about 0.22 pm to about 10 pm, such as about 0.5 pm to about 10 pm, about 0.75 pm to about 9.5 pm, about 2 pm to about 9 pm, about 2 pm to about 8.5 pm, about 3 pm to about 8 pm, about 3 pm to about 7.5 pm, about 4 pm to about 7 pm, about 4 pm to about 6.5 pm, about 4 pm to about 6 pm. In a preferred embodiment, a pore diameter of 5 pm.

[0066] In a further additional or alternative embodiment, said second filter has a pore diameter greater than about 0.22 pm, such as greater than about 0.5 pm, greater than about 0.75 pm, greater than about 1 pm, greater than about 1.25 pm, greater than about 1.5 pm, greater than about 1.75 pm, greater than about 2 pm, greater than about 3 pm, greater than about 4 pm, greater than about 5 pm, greater than about 6 pm, greater than about 7 pm, greater than about 8 pm, greater than about 9 pm, greater than about 10 pm. In a preferred embodiment, said second filter has a pore diameter greater than 2 pm.

[0067] In a preferred embodiment of the invention, said first filter has a pore diameter of about 30 pm and said second filter has a pore diameter of about 5 pm.

[0068] It will be appreciated by one skilled in the art, that filtration and reverse filtration in step (ii) may be carried out in any order. Accordingly, in some embodiments the sample is first filtered and then reverse filtered. In an alternative embodiment, the sample is first reverse filtered and then filtered.

[0069] It will be appreciated that the size and / or diameter of said micro-sized particles will be determined by the pore diameters of said first and second filters. One skilled in the art will appreciate that the obtained micro-scale particles of the invention may preferably be of at most a certain particle diameter and / or size to increase the likeliness of the two or more bacterial strains co-isolated to interact on said micro-scale particle.

[0070] Accordingly, in some embodiments, the diameter of the micro-sized particles is in the range of about 100 pm to about 0.22 pm, for example about 100 pm to about 0.25 pm, about 95 m to about 0.75 pm, about 90 pm to about 1 pm, about 85 pm to about 1.5 pm, about 80 pm to about 2 pm, about 60 pm to about 3 pm, about 50 pm to about 4 pm, or about 30 pm to about 5 pm. In a preferred embodiment, the diameter of the micro-sized particles is in the range of about 5 pm to about 30 pm.

[0071] One skilled in the art will appreciate that it may be advantageous to reduce the average size, length and / or diameter of certain samples that may be provided as part of the invention and which may harbour two or more bacterial strains co-located in close proximity and may therefore be of interest. For example, in order to improve the efficacy or efficiency of filtration and / or reverse filtration of such samples. By way of example, samples comprising solid objects of a certain size or stabilized through internal structures, such as tissue (animal or plant) or soil, may be easier and / or more efficiently filtered or reverse filtered is their average size is reduced, for example by homogenising and / or fractionation. Accordingly, in some embodiments of the method of the invention, the method further comprises a step of comminuting said sample prior to step (ii).

[0072] In some embodiments, said comminuting comprises comminuting the sample into fragments of a size less than about 200 pm, such as less than less than about 200 pm, less than about 180 pm, less than about 160 pm, less than about 140 pm, less than about 120 pm, less than about 100 pm, less than about 80 pm, less than about 60 pm, less than about 40 pm, less than about 20 pm. In a preferred embodiment, the sample is comminuted into fragments of a size less than 100 pm.

[0073] Comminuting the sample may be achieved through a variety of methods which are known in the art. In some embodiments, said comminuting comprises homogenising the sample. In particular embodiments, homogenising the sample is performed by a technique selected from the group consisting of grinding, milling, crushing, sonication, cryo-milling, shearing, and extrusion.

[0074] In an additional or alternative embodiment, said comminuting comprises fractionating the sample. In particular embodiments, fractionating the sample is performed by a technique selected from the group consisting of sieving, centrifugation or size-exclusion chromatography.

[0075] In order to co-isolate two or more bacterial strains co-located on a micro-scale surface, the micro-sized particles obtained in said first volume can be recovered individually in one or more second volumes comprising a single micro-sized particle. Accordingly, it will be appreciated that recovering a single micro-sized particle in said second volume, allows for the co-isolation of only the two or more bacterial strains co-located on said micro-sized particle.

[0076] Without being bound by theory, it is thought that the two or more bacterial strains on micro-scale surfaces, such as the micro-sized particles, have increased probability of interacting, such as by interacting synergistically. As such and by way of example, if bacterial strains from more than a single micro-sized particle are co-isolated and / or characterized, each bacterial strain will not necessarily have been in close proximity on a micro-scale surface to all of the other bacterial strains co-isolated and / or characterized, and as such, no expectation of interaction and / or synergy can be assumed for such bacterial strains. As such, the method of the invention provides a step (iii) of recovering in a second volume a single micro sized particle comprising two or more bacterial strains.

[0077] One skilled in the art will appreciate that, as described throughout, a single micro sized particle should be recovered in said second volume. Accordingly, in some embodiments, step (iii) of the method does not comprise recovering more than one micro sized particle in said second volume. In an additional or alternative embodiment, step (iii) comprises recovering no more than one micro sized particle in said second volume.

[0078] One skilled in the art will appreciate that recovering in a second a second volume a single micro sized particle may be carried out through a variety of methods, some of which are known in the art.

[0079] In some embodiments, said second volume is obtained by serially diluting said first volume until said second volume only contains a single micro sized particle. In some embodiments, diluting said first volume comprises diluting said volume to extinction. It will be appreciated that, diluting a volume to extinction comprises diluting a volume, until the volume only comprises one micro-sized particle or no micro-sized particles.

[0080] In some embodiments the number of micro-sized particles in a volume is determined probabilistically, for example by diluting a known or estimated number of micro-sized particles enough times, that the probability of the final volume containing two or more particles less than 5%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.005%.

[0081] Alternatively, in some embodiments, recovering a single micro-sized particle in step (iii) may be carried out by actively separating an individual micro-sized particle from said micro sized particles of step (ii). For example, by physically moving a micro-sized particle into a second volume, such as by using a microcapillary needle. In an additional or alternative embodiment, recovering in a second volume a single microsized particle in step (iii) is carried out automatically by a machine, for example based on morphological parameters. It will be appreciated by one skilled in the art that techniques and equipment to separate a microscopic object based on certain morphological parameters are known in the art. By way, of example, many such techniques have been developed to sort specific cells from complex cell mixtures, such as flow cytometry, fluorescence associated cell sorting, immunomagnetic cell sorting, and a variety of microfluidics approaches. Such techniques can readily be implemented or adapted by one skilled in the art to separate a micro-sized particle from a mixture of micro-sized particles, for example based on morphological characteristics. Accordingly, in some embodiments, recovering in a second volume a single micro-sized particle in step (iii) is carried out by an automated particle sorter. In a preferred embodiment, said automated particle sorter is a Fluorescence Activated Cell Sorter (FACS).

[0082] It will be appreciated that, in order to increase the throughput of the method of the invention it may be advantageous to recover several micro-sized particles each in an individual second volume. Each micro-sized particle in an individual second volume may then be further used to isolate and / or characterize bacterial strains co-located on said micro-sized particle. Accordingly, in one embodiment, step (iii) is repeated to obtain in a plurality of different second volumes single micro-sized particles.

[0083] Certain embodiments of the invention may recover a single micro-sized particle in a second volume, while also producing alternative second volumes which do not comprise such single micro-sized particle. By way of example, if said single micro-sized particle in said second volume is obtained by dilution to extinction, certain second volumes of the dilution series may also be produced, which do not comprise such single second volume, and certain other second volumes may be produced which comprise two or more said microsized particles, as a result of the stochastic nature of dilutions. In such embodiments, it may be advantageous but not necessary, to verify that a certain second volume only comprises a single micro-sized particle. Accordingly, in some embodiments, the method further comprises a step (iv) of verifying that said second volume comprises only a single micro-sized particle. It will be appreciated by one skilled in the art that detecting the presence of one or more micro-sized particles in said second volume may be carried out for example, by staining the solid support of said micro-sized particle (i.e., the particle matter not consisting of bacterial cells) and detecting the presence of said stain using, for example, microscopical approaches. Accordingly, in some embodiments, said micro-sized particle recovered in step (iii) is stained prior to step (iv). In a preferred embodiment, said micro-sized particle is stained by one or more dyes capable of staining said solid support.

[0084] It will be appreciated that in any of the embodiments of the invention, it may be advantageous but not necessary, to verify that the recovered micro-sized particle comprises one or more bacteria and / or to determine the number of bacterial cells and / or bacterial colonies, which are present on the single micro-sized particle. By way of example, it may be advantageous to discard micro-sized particles comprising no bacterial cells. It will be appreciated by one skilled in the art that detecting the presence of bacterial cells on such micro-sized particles may be carried out, for example, by staining the micro-sized particles by one or more dyes capable of staining the bacterial cells. It will further be appreciated, that staining said micro-sized particles with dyes capable of differentiating living from dead bacteria may be advantageous, for example, to discard micro-sized particles only comprising dead bacteria. Accordingly, in some embodiments, said microsized particle recovered in step (iii) is stained prior to step (iv) by one or more dyes capable of staining living bacterial cells and / or one or more dyes capable of staining bacterial cells having a porous membrane. In a preferred embodiment, the micro-sized particle is stained by Propidium Iodide, SYTO9, and / or SYBR. Green.

[0085] It will be appreciated that staining the solid support of the said micro-sized particles and the bacterial cells of said micro-sized particles may be carried out simultaneously. It will further be appreciated that certain dyes, which are capable of staining living and / or dead bacterial may also be able to stain the solid support of said micro-sized particle. Such stains and / or dyes may be detected using a variety of techniques known in the art. In some embodiments, step (iv) is carried out by flow cytometry and / or microscopy. In a preferred embodiment, wherein step (iv) is carried out by microscopy. In one embodiment, said microscopy is fluorescence microscopy.

[0086] It will be appreciated that any sample which comprises a plurality of bacterial strains and a solid support may be provided in step (i) of the invention. Without being bound by theory, it is thought that natural microbial samples comprise variety and abundance of bacterial strains and solid supports, and thus are appropriate samples for use in the method of the invention. Accordingly, in some embodiments, the sample is a natural microbial sample. In some embodiments, the sample is selected from the group consisting of a soil sample, a sediments sample, a plant tissue sample, an animal tissue sample, a human tissue sample, and a foodstuff sample. One skilled in the art will appreciate that the nature and abundance of bacteria in a sample comprising a solid support will be dependent on the nature of said solid support. Samples comprising certain solid supports may thus be more likely to comprise two or more specific bacterial strains and / or bacteria with specific properties, which may be of interest to coisolate as a result of the method of the invention. Accordingly, in some embodiments, the solid support comprises a material selected from the group consisting of soil, sludge, faecal matter, horticultural substrates, plant tissue, animal tissue, human tissue, foodstuffs, fabric, plastic and plastic derivatives.

[0087] In some embodiments, the sample comprises a solid support which is attached to two or more bacterial strains in nature. By "in nature" we mean without direct human intervention. It will be appreciated that such samples may exclude, by way of example only, solid supports (such as plates or beads) on which two or more bacterial strains are inoculated, or solid supports which are synthetically produced to comprise one or more binding moieties specific for two or more bacterial strains which are bound to said bacterial strains.

[0088] Soils contain a tangle of minerals, water, nutrients, gases, plant roots, decaying organic matter, and microorganisms which work together to cycle nutrients and support terrestrial plant growth. Most soil microorganisms live in periodically interconnected communities closely associated with soil aggregates. At the spatial scales most relevant for microbial biogeochemistry, soils are primarily composed of microaggregates (>200 pm), which bind soil organic carbon and protect it from removal by erosion. This scale is particularly important in shaping microbial interactions since microbial residents occupy specialized niches within the aggregate structure.

[0089] The methods of the invention are therefore suited for co-isolating soil bacteria on microscale surfaces such as soil aggregates, such bacteria possible having a synergistic interaction associated with their close proximity on the micro-scale surface. Only <5% of bacteria in soil are culturable. It is hypothesized that one of the main attributes to unculturability is co-dependency between bacteria. By co-culturing bacteria that are / were present in the same micro-niche (i.e., on the micro-scale surface), the chance of preserving metabolite exchange is increased and thus otherwise unculturable bacteria may be cultured as part of a synergistic bacterial consortium.

[0090] Accordingly, in a preferred embodiment, the sample is a soil sample. In an additional embodiment, the solid support is soil. Following step (ill) of recovering in a second volume a single micro-sized particle, it may be advantageous to multiply and / or culture the bacterial strains co-located said microsized particle. Such bacterial culture derived from the bacterial strains co-located on said micro-sized particle may for example, be suitable for further characterization, maintenance and / or other use of said bacterial strains. Accordingly, in some embodiments, the method of the invention further comprises a step (v) of inoculating bacteria on said micro-sized particle in bacterial culture media to obtain a bacterial culture. In some embodiments, step (v) comprises placing said micro-sized particle in bacterial culture media. In an alternative embodiment, step (v) comprises adding bacterial culture media to said second volume comprising said micro-sized particle. In some embodiments, said bacterial culture is selected from the group consisting of Root extract, Leaf extract, Tuber extract, Soil extract, Tryptone soy broth, R2 broth, Terrific Broth (TB), Lysogeny Broth (LB), Super Optima broth (SOC) and combinations thereof.

[0091] In one embodiment, the method of the invention further comprises a step (vi) of isolating one or more bacterial strains from the micro-sized particles of step (iii) or from the bacterial culture of step (v). In some embodiments, the one or more bacterial strains are isolated from the bacterial culture of step (v) by diluting said bacterial culture and inoculating said dilution in bacterial culture media.

[0092] It will be appreciated that the one or more of the bacteria co-located on said-micro sized particle, the bacteria of said bacterial culture or said isolated bacterial strains may be characterised using a variety of methods known in the art and commonly used in the field of microbiology. Characterising bacteria may, for example, be suitable to identify taxonomical information of such bacteria. Such bacteria may be characterized morphologically, based on the size, shape and colour of a bacterial colony thereof, biochemically, based on the nature of the metabolites produced by such bacteria, or by sequencing, based on the nature and sequence of the nucleic acid component of the bacteria. Sequencing is generally considered the optimal method of identifying the nature and taxonomical information of a bacteria.

[0093] Accordingly, in some embodiments, the method of the invention further comprises a step (vii) of isolating nucleic acid from the micro-sized particles of step (iii), from the bacterial culture of step (v) or from the isolated bacterial strains of step (vi) and sequencing said nucleic acids. One skilled in the art will appreciate that different sequencing methods are known in the art and may be used interchangeably dependent on which information is required. By way of example, sequencing of the 16S ribosomal RNA (rRNA) allows for genus level identification, while metagenome sequencing allows for strain level identification. In some embodiments, sequencing the nucleic acid comprises sequencing the 16S ribosomal RIMA, shotgun metagenome sequencing or whole genome sequencing.

[0094] It will be appreciated by one skilled in the art that step (iv), step (v), step (vi) and step (vii) may each be carried out as part of the invention or not carried out. Similarly, each of the abovementioned steps may be carried out in any order, unless dictated otherwise by context. By way of example, step (vii) of isolating and sequencing nucleic acids from the micro-sized particles, the bacterial culture or the isolated bacterial strains may be carried out regardless of whether all of steps (iv), (v), and (vi) were carried out, provided at least one of said steps was carried out. Similarly, step (vii) of isolating and sequencing nucleic acid may be carried out before step (v) of inoculating bacteria on said micro-sized particle, for example by isolating and sequencing nucleic acid directly from said micro-sized particle.

[0095] As previously described and without wishing to be bound by theory, it is thought that the two or more bacterial strains co-located on micro-scale surfaces, such as the micro-sized particles, have increased probability of interacting, such as by interacting synergistically. Accordingly, the two or more bacterial strains co-located on a micro-scale surface represent are candidates for forming a synergistic bacterial consortium. It will therefore be appreciated that synergistic bacterial consortia may be obtained by selecting from the each two or more bacterial strains co-located on micro-scale surfaces, those which interact synergistically.

[0096] Additionally or alternatively, isolated bacterial strains isolated from said micro-sized particle or from said bacterial culture may be co-cultured and screened for synergism and synergistic bacterial consortia may be selected from said co-cultures. For example, such co-cultures may comprise only two or more, three or more, four or more, five or more or six or more bacterial strains from said micro-sized particle or from said bacterial cultures. Co-cultures comprising less than the total number of bacterial strains co-localized on said micro-sized particles or cultured on said bacterial culture may have improved synergistic effect compared to all bacterial strains co-localized on said micro-sized particles or cultured on said bacterial culture. Accordingly, combinatorial co-culture of each such two or more isolated bacterial strains may be screened to select synergistic bacterial consortia.

[0097] Biofilm formation is a known marker of synergy within a multi-strain community. Synergistic effects within a bacterial community have been shown to promote biofilm biomass, resistance of the biofilm to antimicrobial agents and bacterial invasion in multispecies biofilms. High prevalence of synergy in biofilm formation in multispecies consortia isolated from a natural bacterial habitat have been strongly suggested to represent interspecific cooperation in soil bacterial communities. As such, co-cultures may be screened by their biofilm formation capabilities to select for synergistic bacterial consortia.

[0098] Accordingly, in a particular embodiment, the method of the invention further comprises a step of selecting synergistic bacterial consortia, the step comprising : culturing the bacterial culture of step (v) or co-culturing two or more isolated bacterial strains of step (vi); measuring the biofilm formation capability of said culture or said co-culture; and selecting cultures or co-cultures having a greater biofilm formation capability compared to a reference.

[0099] It will be appreciated that the reference may be any biofilm formation capability value, which has been determined to be an effective cut-off value for determining bacterial synergism of two or more bacterial strains based on the biofilm formation capabilities. In some embodiments, the reference is determined as part of the method of the invention. In an alternative embodiment, the reference may be a known or calculated value. In some embodiments, the reference is one or more of the individual bacterial strains in said culture or said co-culture. In an alternative embodiment, the reference is the average biofilm formation capability of the one or more individual bacteria in said culture or said co-culture, when each bacterial strain is cultured separately.

[0100] Specific bacterial cultures comprising a known composition of bacterial strains are known to have specific biofilm formation capabilities. For example, a bacterial culture comprising Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans, and Paenibaciiius amyioiyticus has previously been shown to produce high levels of biofilm (Yang, N., Nesme, J., Roder, H.L. et al. Emergent bacterial community properties induce enhanced drought tolerance in Arabidopsis. npj Biofilms Microbiomes 7, 82 (2021)). Accordingly, in some embodiments the reference is one or more bacterial culture with known biofilm formation capabilities. In a preferred embodiment, the reference bacterial culture with known biofilm formation capability comprises Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans, and Paenibaciiius amyioiyticus.

[0101] Methods of measuring biofilm formation of a bacterial culture are known in the art. For example, a list of suitable methods is disclosed in Wilson C et al. Quantitative and Qualitative Assessment Methods for Biofilm Growth: A Mini-review. Res Rev J Eng TechnoL 2017. In some embodiments, said biofilm formation is determined using a method selected from the group consisting of viable cell counting, flow cytometry based counting, light and / or fluorescence microscopy, dry mass assessment, total organic carbon assessment, crystal violet assay, an ATP bioluminescence assay, and total protein determination. In a preferred embodiment, said biofilm formation capability is determined using a Crystal Violet assay.

[0102] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "having", "including" and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any nonclaimed element as essential to the practice of the invention.

[0103] The listing or discussion of an apparently prior published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. All patents, patent applications, journal articles, and / or other publications cited herein are incorporated by reference in their entirety.

[0104] Preferences, options, and embodiments for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences, options and embodiments for all other aspects, features and parameters of the invention.

[0105] Description of the Figures

[0106] Figure 1. - Representative fluorescence microscope images of Propidium Iodide and SYBR Green soil samples: (A) Undiluted control soil sample. (B) Fractionated and filtered soil sample. (C) 25X diluted control soil sample. (D) 25X diluted fractionated and filtered soil sample. (E) 15625X diluted fractionated and filtered soil sample fractionation and dilution. Soil particles can be seen as larger objects (having a maximum emission spectrum of about 636nm), whereas live bacteria stained with SYBR can be seen as foci (having a maximum emission spectrum of about 520 nm).

[0107] Figure 2. - Bar diagram shows normalized OD590 (Crystal Violet assay) of a representative subset of co-cultures, negative control and SPMX.

[0108] Figure 3. - Bar diagram shows normalized OD590 (Crystal Violet assay) of co-culture C259 compared to the individual bacterial strains in the co-culture (C259.1 and C259.2), and SPMX.

[0109] Figure 4. - (A) Violin plots show mean tomato yield of bacterial culture C78, C259 and C410 treated tomato plants infected with P. infestans, compared to control. (B) Representative phenotype of bacterial culture C78 treated tomato plants infected with P. infestans, compared to control.

[0110] Figure 5. - Bar diagram shows mean infection score of co-culture C53 treated potato plants infected with A. alternate.

[0111] Figure 6. - Representative images of rice plants treated with or without co-culture C306 and drought stressed.

[0112] Figure 7. - Bar graphs show In-planta effect of C259 co-culture and its individual components, Pp (C259.1) and Pk (C259.2) against drought stress in tomato plants.

[0113] Figure 8. - Non-limiting schematic representation of an exemplary embodiment of the method of the invention. A sample is provided in step (i) comprising a solid support and bacteria, where some bacteria are attached to the solid support. In step (ii) the sample is first filtered to remove particles above a certain size and the filtrate is then reverse filtered to remove non-attached bacteria and obtain micro sized particles in the retentate. In step (iii) a single micro-sized particle is recovered.

[0114] Certain embodiments and features of the present invention are also outlined in the following numbered items:

[0115] 1. A method for co-isolating two or more bacterial strains co-located on a micro-scale surface, the method comprising :

[0116] (i) providing a sample comprising a plurality of bacterial strains and a solid support; (ii) filtering said sample through a first filter, and reverse filtering said sample through a second filter, to obtain micro sized particles in a first volume, wherein said first filter has a pore diameter greater than the pore diameter of said second filter; and

[0117] (iii) recovering in a second volume a single micro sized particle comprising two or more bacterial strains.

[0118] 2. The method of item 1, wherein said first filter has a pore diameter in the range of about 10 pm to about 100 pm, such as about 15 pm to about 90 pm, about 15 pm to about 80 pm, about 15 pm to about 70 pm, about 20 pm to about 60 pm, about 20 pm to about 55 pm, about 20 to about 50 pm, about 25 pm to about 45 pm, about 25 pm to about 40 pm, or about 25 to about 35, preferably a pore diameter of 30 pm.

[0119] 3. The method of item 1, wherein said first filter has a pore diameter smaller than about 100 pm, such as smaller than about 100 pm, smaller than about 95 pm, smaller than about 90 pm, smaller than about 85 pm, smaller than about 80 pm, smaller than about 75 pm, smaller than about 70 pm, smaller than about 65 pm, smaller than about 60 pm, smaller than about 55 pm, smaller than about 50 pm, smaller than about 45 pm, smaller than about 40 pm, smaller than about 35 pm, smaller than about 30 pm, smaller than about 25 pm, smaller than about 20 pm, smaller than about 15 pm, or smaller than about 10 pm.

[0120] 4. The method of any of the preceding items, wherein said second filter has a pore diameter in the range of about 0.25 pm to about 10 pm, such as about 0.5 pm to about 10 pm, about 0.75 pm to about 9.5 pm, about 2 pm to about 9 pm, about 2 pm to about 8.5 pm, about 3 pm to about 8 pm, about 3 pm to about 7.5 pm, about 4 pm to about 7 pm, about 4 pm to about 6.5 pm, about 4 pm to about 6 pm, preferably a pore diameter of 5 pm.

[0121] 5. The method of any of items 1-3, wherein said second filter has a pore diameter greater than about 0.25 pm, such as greater than about 0.5 pm, greater than about 0.75 pm, greater than about 1 pm, greater than about 1.25 pm, greater than about 1.5 pm, greater than about 1.75 pm, greater than about 2 pm, greater than about 3 pm, greater than about 4 pm, greater than about 5 pm, greater than about 6 pm, greater than about 7 pm, greater than about 8 pm, greater than about 9 pm, greater than about 10 pm.

[0122] 6. The method of item 1, wherein said first filter has a pore size of about 30 pm and said second filter has a pore size of about 5 pm. 7. The method of any of the preceding items, wherein the diameter of the micro-sized particles is in the range of about 100 pm to about 0.25 pm, for example about 100 pm to about 0.25 pm, about 95 pm to about 0.75 pm, about 90 pm to about 1 pm, about 85 pm to about 1.5 pm, about 80 pm to about 2 pm, about 60 pm to about 3 pm, about 50 pm to about 4 pm, or about 30 pm to about 5 pm.

[0123] 8. The method of item 7, wherein the diameter of the micro-sized particles is in the range of about 5 pm to about 30 pm.

[0124] 9. The method of any of the preceding items, wherein the first volume is essentially free from free floating bacterial cells.

[0125] 10. The method of any of the preceding items, further comprising a step of comminuting said sample prior to step (ii).

[0126] 11. The method of item 10, wherein said comminuting comprises comminuting the sample into fragments of a size less than about 200 pm, such as less than less than about 200 pm, less than about 180 pm, less than about 160 pm, less than about 140 pm, less than about 120 pm, less than about 100 pm, less than about 80 pm, less than about 60 pm, less than about 40 pm, less than about 20 pm.

[0127] 12. The method of any of items 10 or 11, wherein said comminuting comprises homogenising the sample.

[0128] 13. The method of item 12, wherein homogenising the sample is performed by a technique selected from the group consisting of grinding, milling, crushing, sonication, cryo-milling, shearing, and extrusion.

[0129] 14. The method of any of items 10-13, wherein said comminuting comprises fractionating the sample.

[0130] 15. The method of item 14, wherein fractionating the sample is performed by a technique selected from the group consisting of sieving, centrifugation or size-exclusion chromatography.

[0131] 16. The method of any of the preceding items, wherein said second volume is obtained by serially diluting said first volume. 17. The method of item 16, wherein said second volume is obtained by serially diluting said first volume until said second volume only contains a single micro sized particle.

[0132] 18. The method of any of the preceding items, wherein recovering a single micro-sized particle in step (iii) is carried out by an automated particle sorter.

[0133] 19. The method of item 18, wherein said automated particle sorter is a Fluorescence Activated Cell Sorter (FACS).

[0134] 20. The method of any of the preceding claims, wherein step (iii) is repeated to obtain in a plurality of different second volumes each containing a single micro-sized particle.

[0135] 21. The method of any of the preceding claims, further comprising a step (iv) of verifying that said second volume comprises only a single micro-sized particle.

[0136] 22. The method of claim 21, wherein said micro-sized particle recovered in step (iii) is stained prior to step (iv).

[0137] 23. The method of claim 22, wherein said micro-sized particle is stained by one or more dyes capable of staining living bacterial cells and / or one or more dyes capable of staining bacterial cells having a porous membrane.

[0138] 24. The method of item 22 or 23, wherein said micro-sized particle is stained by one or more dyes capable of staining said solid support.

[0139] 25. The method of any of items 22-24, wherein the micro-sized particle is stained by Propidium Iodide, SYTO9, and / or SYBR. Green.

[0140] 26. The method of any of items 21-25, wherein step (iv) is carried out by microscopy.

[0141] 27. The method of any of the preceding items, wherein the sample is a natural microbial sample.

[0142] 28. The method of item 27, wherein the sample is selected from the group consisting of a soil sample, a sediments sample, a plant tissue sample, an animal tissue sample, a human tissue sample, and a foodstuff sample. 29. The method of any of the preceding items, wherein the solid support comprises a material selected from the group consisting of soil, sludge, fecal matter, horticultural substrates, plant tissue, animal tissue, human tissue, foodstuffs, fabric, plastic and plastic derivatives.

[0143] 30. The method of any of the preceding items, further comprising a step (v) of inoculating bacteria on said micro sized particle in bacterial culture media to obtain a bacterial culture.

[0144] 31. The method of any of the preceding items, further comprising a step (vi) of isolating one or more bacterial strains from the micro-sized particles of step (iii) or from the bacterial culture of step (v).

[0145] 32. The method of any of the preceding items, further comprising a step (vii) of isolating nucleic acid from the micro-sized particles of step (iii), from the bacterial culture of step (v) or from the isolated bacterial strains of step (vi) and sequencing said nucleic acids.

[0146] 33. The method of item 32, wherein sequencing the nucleic acid comprises sequencing the 16S ribosomal RIMA, shotgun metagenome sequencing or whole genome sequencing.

[0147] 34. The method any of items 30-31, further comprising a step of selecting synergistic bacterial consortia, the step comprising : culturing the bacterial culture of step (v) or co-culturing two or more isolated bacterial strains of step (vi); measuring the biofilm formation capability of said culture or said co-culture; and selecting cultures or co-cultures having a greater biofilm formation capability compared to a reference.

[0148] 35. The method of item 34, wherein the reference is one or more of the individual bacteria in said culture or said co-culture.

[0149] 36. The method of item 34, wherein the reference is one or more bacterial communities with known biofilm formation capabilities.

[0150] 37. The method of item 36, wherein the bacterial community with known biofilm formation capability comprises Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans, and Paenibacillus amylolyticus. 38. The method of any of items 34-38, wherein said biofilm formation capability is determined using a Crystal Violet assay.

[0151] 39. The method of any of the preceding items, wherein the micro sized particles obtained in step (ii) are derived from said solid support.

[0152] 40. The method of any of the preceding items, wherein the single micro sized particle recovered in a second volume in step (iii) is a micro sized particle obtained in step (ii) in said first volume.

[0153] 41. The method of any of the preceding items, wherein, the first volume of step (ii) is the filtrate of said filtering through the first filter and the retentate of said reverse filtering through the second filter.

[0154] 42. A composition comprising two or more bacterial strains obtained by culturing two or more bacterial strains comprised in the second volume recovered in step (iii) according to the method of any of the preceding claims.

[0155] 43. A method for co-isolating two or more bacterial strains co-located on a microscale surface, the method comprising :

[0156] (i) providing a sample comprising :

[0157] - a plurality of bacterial strains; and

[0158] - a solid support;

[0159] (ii) filtering said sample through a first filter, and reverse filtering said sample through a second filter, to obtain micro sized particles in a first volume, wherein said first filter has a pore diameter greater than the pore diameter of said second filter; and

[0160] (iii)recovering in a second volume a single micro sized particle comprising two or more bacterial strains.

[0161] 44. A method for co-isolating two or more bacterial strains co-located on a microscale surface, the method comprising :

[0162] (i) providing a sample comprising :

[0163] - a plurality of bacterial strains; and

[0164] - a solid support;

[0165] (ii) filtering according to the following steps:

[0166] - filtering said sample through a first filter to obtain a filtrate; and

[0167] - filtering said filtrate through a second filter to obtain a retentate, wherein said retentate comprises in a first volume micro-sized particles derived from said solid support, wherein said first filter has a pore diameter greater than the pore diameter of said second filter; and (iii)recovering in a second volume a single micro-sized particle comprising two or more bacterial strains.

[0168] EXAMPLES

[0169] Certain embodiments and features of the present invention are presented in the following non-limiting examples.

[0170] EXAMPLE 1 - co-isolation of bacterial strains located on micro scale surface

[0171] Background and aim

[0172] The method of the invention provides an efficient method of co-isolating bacterial strains co-located on a micro-scale surface. The present example demonstrates the usefulness of one embodiment of the invention to obtain single micro-sized soil particles harbouring bacteria attached thereto in a volume which is free from free-floating bacteria.

[0173] Methods

[0174] Sample preparation

[0175] 10 mL of rhizosphere soil slurry in a sterile 0.9% NaCI solution was wet sieved through a sterilized 250 pm mesh, a 75pm mesh and finally a 35pm mesh, to remove larger soil aggregates and then reverse filtered through a 5pm filter, to remove free floating cells (processed sample). A separate rhizosphere sample, which was not wet sieved or filtered was used as a control (control sample).

[0176] Dilution

[0177] The processed or the control sample was then diluted 5X 10 times, resulting in 5mL per dilution.

[0178] Staining and visualization

[0179] A ImL aliquot of each dilution was used for visualization of the diluted samples. An aliquot of the undiluted processed and control samples was also used for visualization. Each sample was dual-stained with the BacLight™ (Live / Dead Bacterial Viability Kit, L-7007, Molecular Probes) and Propidium Iodide (60pM) and SYBR Green (IX) were added to the aliquots. Propidium Iodide stains the soil particles, while living bacterial cells were stained by SYBR Green. The stained samples were incubated in the dark for 20 minutes and then visualized on a Zeiss CLSM confocal microscope, to visualize and / or count the number of soil particles and the number of planktonic cells. Results

[0180] Following sample preparation and dilution of the rhizosphere soil slurry sample, an aliquot of each dilution of the processed and control samples was stained and visualized (Figure 1). For comparison, the undiluted samples were likewise stained and visualized.

[0181] Soil particles could clearly be distinguished as larger particles stained by PI, while bacterial cells were observed as smaller foci stained by SYBR. Green. Bacterial cells attached to soil particles could be observed as green / yellow foci on the larger soil particles (see arrows - Figure 1).

[0182] The undiluted control sample consisted of a heterogeneous mixture of soil particle sizes and had an abundance of free-floating cells (Figure 1A). Dilution of the control sample unsurprisingly resulted in fewer soil particles per diluted sample. These particles were heterogeneous and free-floating bacterial cells were still clearly present in the 25X dilution (Figure IB).

[0183] The soil particles of the processed sample that had been wet sieved and filtered displayed a much more homogeneous size range. Additionally, almost no free-floating bacteria were observed (Figure 1C). When diluted 25X the processed samples showed only few soil particles and no observable free-floating bacteria (Figure ID). Finally, samples diluted 15625X were verified as containing only one or zero soil particle(s) per 5 pl volume and no visible free-floating bacterial cells (Figure IE).

[0184] One or more bacterial cells could be detected for most soil particles observed in all of the samples. Wet-sieving, filtration and / or dilution thus did not seem to reduce bacterial presence on the soil particles.

[0185] EXAMPLE 2 - culturing bacterial strains co-isolated from micro-scale surface

[0186] Background and aim

[0187] Example 1 demonstrated that the methods of the invention can be used to obtain single micro-sized soil particles in a volume which is free from free-floating bacteria. Staining and visualization of the soil particles further showed that bacteria were attached to these particles. To further study and / or utilise the bacterial strains present on the soil particles, the bacteria can be further cultured, for example in bacterial growth media. As different bacterial strains may require a unique nutrient composition, not all bacterial strains present on the soil particle are likely to multiply and by culturable in a single bacterial growth medium. To mitigate this limitation, an aliquot of each dilution obtained in Example 1 was culture in a variety of bacterial growth media.

[0188] Methods

[0189] 5 pl aliquots of each 300X dilution as obtained in Example 1 (i.e., a volume containing only 1 or 0 soil particles), were inoculated in different culture media (Root extract, Leaf extract, Tuber extract, Soil extract, Tryptone soy broth, R2 broth), with and without fungal antibiotic Cycloheximide, and incubated for 14 days. Each media was also incubated without such aliquot, as a negative control. After 14 days each culture was measured for bacterial growth by Optical density (OD600).

[0190] Results

[0191] 14 days post inoculation, growth was observed in 1137 out of 1235 inoculated wells based on optical density measurements (OD600), compared to the negative control. Each coculture was then transferred to fresh culture media. Each co-culture was given a unique identification code consisting of the letter C and a number (i.e., Cl, C2, C3...).

[0192] EXAMPLE 3 - Biofilm formation of co-cultures

[0193] Aim and Background

[0194] Biofilm formation has been extensively used as a proxy for synergism in bacterial multistrain communities and synergistic effects within a bacterial community have been shown to promote biofilm biomass, resistance of the biofilm to antimicrobial agents and bacterial invasion in multispecies biofilms. Accordingly, the presence of synergistic interactions within each co-culture may be tested by determining each co-cultures biofilm formation capabilities.

[0195] Methods

[0196] Co-cultures obtained from Example 2 were normalized based on OD600 to setup a Crystal Violet assay. Overnight cultures of the bacterial communities inoculated in Tryptone soy broth were OD normalized to the same cell density. 2pl of normalized culture was inoculated in 198pl of TSB in a 96-well plate and incubated for 24h at 25°C on agitation (200rpm). After 24h, the wells are washed with lx Phosphate buffer saline (PBS), and then stained with 160 pL of 0,5% CV stained. The stained wells were further washed three times with PBS, and biofilm production was measured spectrophotometrically at OD590 against a positive control (known 4 strain community SPMX that shows high synergy (Yang, N., Nesme, J., Roder, H.L. et al. Emergent bacterial community properties induce enhanced drought tolerance in Arabidopsis. npj Biofilms Microbiomes 7, 82 (2021)). Biofilm production was compared between co-cultures and their individual bacteria using the same assay.

[0197] Results

[0198] Of the 1137 co-cultures assayed, 24.3% (277) displayed equal or enhanced biofilm production capabilities compared to the positive control, SPMX, and were thus classified as high-performers. Biofilm formation data of representative co-cultures is detailed in the Table 1 and shown in Figure 2.

[0199] Table 1 - Quantitative summary of biofilm formation of select co-cultures:

[0200] EXAMPLE 4 - Co-culture biofilm formation compared to constituent isolated bacterial strains

[0201] Background and aim

[0202] To verify that the biofilm formation capabilities of high performing co-cultures was related to synergism, the constituent bacterial strains of a representative high performing coculture were isolated and tested for their individual biofilm formation capabilities.

[0203] Methods

[0204] A 10X dilution series was made from a representative high performing co-culture of Example 3 for up to 10 dilutions. At the 108dilution, two distinct colony morphologies were visible and two representative single colonies were picked to obtain the constituent bacterial strains (C259.1 (also called Pp) and C259.2 (also called Pk)). Biofilm formation capability of each bacterial strain and the co-cultured were assessed as detailed in Example 3.

[0205] Results

[0206] Co-culture C259 showed significantly increased biofilm formation compared to the constituent bacterial strains (n=12, p < 0.0001), strongly suggesting that the co-culture results in synergistic interaction of the bacterial strains.

[0207] EXAMPLE 5 - Co-culture effect on P.infestans infection in tomato plants

[0208] Background and aim

[0209] Select co-cultures which displayed increased biofilm formation capabilities compared to SPMX in Example 3, were then tested for their ability to suppress Phytophthora infestans in greenhouse trials. P. infestans is an agronomically important plant pathogen infecting crops from the Solanaceae family (potato and tomato). Most notably it causes late blight disease in potato. There is a long felt need to develop commercial biological alternatives to pesticides for this pathogen. Select co-cultures showed antagonist activity against P. infestans in-vitro. Hence, they their efficacy as a multi-strain biopesticide was tested in greenhouse trials.

[0210] Methods

[0211] Each co-culture at an OD600= 0.1 was first coated on surface sterilized tomato seeds. P. infestans spores (105spores / mL) were then added as a secondary coat on the seeds before transplanting them to the soil. The positive control included chemical pesticide, Revus, which was applied according to the manufacturers instructions.

[0212] Results

[0213] Treatment with specific (15 out of 22) co-cultures were found to protect tomato plants against late blight disease caused by P. infestans. (n=6, p< 0.01). Plants treated with cocultures C78, C259 and C410 showed a 29.4%, 29.5% and 46.4% increase in tomato yield harvest, respectively. Mean harvest weight and mean dry biomass for tomato plants treated with the specific co-cultures and with and without P. infestans infection are summarised in Figure 4 and Table 2 below.

[0214] Table 2 - Mean harvest and mean dry biomass for tomato plants treated with the specific co-cultures and with and without P. infestans infection.

[0215] EXAMPLE 6 - Co-culture effect on A. alternata infection (leaf blight disease-) in potato plants.

[0216] Background and aim

[0217] Co-culture C53 which displayed increased biofilm formation capabilities compared to SPMX in Example 3, was also tested for the ability to suppress Alternaria alternata in greenhouse trials. A. alternata is an agronomically important plant pathogen of potatoes for which commercial biological alternatives to pesticides do not exist. It is the causative agent of the early blight disease. Hence, the efficacy of the co-cultures as biopesticide against early blight disease was tested in greenhouse trials.

[0218] Methods

[0219] C53 at an OD600= 0.1 was coated on the roots of young potato seedlings and leaves were sprayed with A. alternata spore suspension (105spores / mL). Adult plants were scored for early blight (Alternaria leaf infection) based on disease symptoms, the status of infection on potato leaves was categorized into 5 levels, where: 0= non-infected; l=green leaves with less than 20 lesions; 2= green leaves with more than 20 lesions; 3= partially yellow leaves with lesions; 4 = whole yellow leaves with lesions; 5=dead leaves. Summarized plant infection score was decided by the average of the infection scores of each expanded leaf on the plant.

[0220] Results

[0221] Treatment with C53 was found to protect potato plants strongly against blight caused by A. alternata (n=5, p< 0.01). With co-culture treated plants showing only 50% of the infection symptoms compared to untreated plants (Figure 5 and Table 3).

[0222] Table 3 - Mean A. alternata infection score of potato plants treated with and without C53 co-culture

[0223] EXAMPLE 7 - Co-culture effect on root rot caused by R. solani infection

[0224] Background and aim

[0225] Co-cultures C1038 and C1095 which displayed increased biofilm formation capabilities compared to SPMX in Example 3, were also tested for the ability to suppress Rhizoctonia solani in greenhouse trials. R. solani is a major global plant pathogen with a broad host range. It causes root rot, stem canker, black scurf, damping off and wire stem diseases on various crops, including potatoes and tomatoes. The two co-cultures showed the ability to inhibit spore germination and mycelial growth of / ?, solani in-vitro. Hence, their efficacy as a multi-strain biopesticide against early blight disease was tested in greenhouse trials.

[0226] Methods

[0227] C53 at an OD600= 0.1 was coated on the roots of young potato seedlings. Agar plugs of R. solani mycelia were placed near the plant roots in the soil about 3 weeks after transplanting the seedlings into the pots. The positive control included chemical pesticide Rizolex, applied according to manufactures instructions. The tuber and root-shoot junction were assessed for presence of necrotic lesions 1 week after infection. A qualitative infection scale was developed based on the size and frequency of lesions.

[0228] Results

[0229] C1038 and C1095 were found to strongly protect potato plants against root rot caused by R. solani. With co-culture treated plants showing only 25% of the infection symptoms compared to untreated plants (n = 12, p< 0.01). Mean infection sites per plant for plants treated with and without co-cultures C1038 and C1095 and infected with R. solani are shown in Table 4.

[0230] Table 4 - Mean infection sites per plant for plants treated with and without co-cultures C1038 and C1095 and infected with R. solani.

[0231] EXAMPLE 8 - Co-culture effect on drought stress in rice plants

[0232] Background and aim

[0233] Elevated biofilm formation has been shown to induce drought tolerance in several plant hosts, for both single strains and multi-strain communities. Co-culture C306, which displayed increased biofilm formation capabilities compared to SPMX in Example 3, was therefore also tested for the ability to protect rice plants against drought stress.

[0234] Methods

[0235] Plants treated with and without co-culture C306 were watered normally for 14 days after transplantation in soil. Then they underwent a 23-day drought treatment where they received no water, followed by a 8-day rewatering phase post drought. The plant survivability was measured as ratio of the dead plants to healthy plants.

[0236] Results

[0237] C306 was found to protect rice plants against drought stress (n=3, p< 0.01). Mean survival rate is summarized in Table 5. Representative images of rice plants treated with or without C306 and drought stressed are shown in Figure 6.

[0238] Table 5 - Mean survival rate of co-culture C306 treated rice plants subjected to drought stress.

[0239] EXAMPLE 9 - Co-culture in nlanta effect on drought stress in tomato plants compared to constituent isolated bacterial strains

[0240] Background and aim

[0241] To confirm that high performing co-cultures as obtained in Example 3 showed synergistic effect on a target plant treated with the co-culture, the constituent bacterial strains of a representative high performing co-culture were isolated and tested for their individual effect on tomato plants undergoing drought stress.

[0242] Methods

[0243] The constituent bacterial strains of co-culture C259 (C259.1 (also called Pp) and C259.2 (also called Pk)) were obtained as described in Example 4. Constituent strains or the coculture at OD600= 0.1 were coated on surface sterilized tomato seeds, and adult plantes were then subjected to drought conditions during two 7-day drought periods separated by a 5-day recovery period.

[0244] Results

[0245] Plants treated with Pp and Pk individually, showed increased root elongation and higher length of the overall root-shoot axis compared to the un-inoculated controls (p<0.05). Plants treated with Pp and Pk as a two-strain co-culture outperformed plants treated with single strains in the development of lateral roots, overall root length (p<0.05), and drought recovery score (p<0.05). A Quantitative summary is shown in Table 6 below and Figure 7 shows corresponding bar graphs.

[0246] REFERENCES

[0247] 1. Wilson C etal. Quantitative and Qualitative Assessment Methods for Biofilm Growth: A Mini-review. Res Rev J Eng TechnoL 2017.

[0248] 2. Yang, N., Nesme, J., Roder, H.L. et al. Emergent bacterial community properties induce enhanced drought tolerance in Arabidopsis. npj Biofilms Microbiomes 7, 82 (2021)

Claims

CLAIMS1. A method for co-isolating two or more bacterial strains co-located on a micro-scale surface, the method comprising :(i) providing a sample comprising a plurality of bacterial strains and a solid support;(ii) filtering said sample through a first filter, and reverse filtering said sample through a second filter, to obtain micro sized particles derived from said solid support in a first volume, wherein said first filter has a pore diameter greater than the pore diameter of said second filter; and(iii) recovering in a second volume a single micro-sized particle comprising two or more bacterial strains.

2. The method of claim 1, wherein the sample is a natural microbial sample.

3. The method of claim 1 or 2, wherein the sample is selected from the group consisting of a soil sample, a sediments sample, a plant tissue sample, an animal tissue sample, a human tissue sample, and a foodstuff sample.

4. The method of claim 3, wherein the sample is a soil sample.

5. The method of any of the preceding claims, wherein said first filter has a pore diameter in the range of about 20 to about 50 pm.

6. The method of any of the preceding claims, wherein said second filter has a pore diameter in the range of about 2 pm to about 10 pm.

7. The method of any of the preceding claims, wherein said first filter has a pore diameter of about 30 pm and said second filter has a pore diameter of about 5 pm.

8. The method of any of the preceding claims, wherein the first volume is essentially free from free-floating bacterial cells.

9. The method of any of the preceding claims, further comprising a step of comminuting said sample prior to step (ii).

10. The method of claim 9 wherein said comminuting comprises comminuting the sample into fragments of an average size less than about 100 pm.

11. The method of any of the preceding claims, wherein said second volume is obtained by serially diluting said first volume.

12. The method of any of the preceding claims, wherein recovering a single micro-sized particle in step (iii) is carried out by an automated particle sorter.

13. The method of any of the preceding claims, further comprising a step (iv) of verifying that said second volume comprises only a single micro-sized particle.

14. The method of claim 13, wherein said micro-sized particle recovered in step (iii) is stained prior to step (iv).

15. The method of claim 11, wherein said micro-sized particle is stained by one or more dyes capable of staining living bacterial cells, one or more dyes capable of staining bacterial cells having a porous membrane, and / or one or more dyes capable of staining said solid support.

16. The method of any of the preceding claims, further comprising a step (v) of inoculating bacteria on said micro sized particle in bacterial culture media to obtain a bacterial culture.

17. The method of any of the preceding claims, further comprising a step (vi) of isolating one or more bacterial strains from the micro-sized particles of step (iii) or from the bacterial culture of step (v).

18. The method any of claims 13 or 14, further comprising a step of selecting synergistic bacterial consortia, the step comprising : culturing the bacterial culture of step (v) or co-culturing two or more isolated bacterial strains of step (vi); measuring the biofilm formation capability of said culture or said co-culture; and selecting cultures or co-cultures having a greater biofilm formation capability compared to a reference.

19. The method of claim 18, wherein reference is one or more bacterial culture with known biofilm formation capabilities, optionally the reference bacterial culture comprises Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans, and Paenibaciiius amyioiyticus.

20. The method of claim 18, wherein the reference is one or more of the individual bacteria in said culture or said co-culture.

21. The method of any one of claims 18-20, wherein said biofilm formation capability is determined using a Crystal Violet assay.

22. A composition comprising two or more bacterial strains obtained by culturing two or more bacterial strains comprised in the second volume recovered in step (iii) according to the method of any of the preceding claims.

23. A method for co-isolating two or more bacterial strains co-located on a microscale surface, the method comprising :(i) providing a sample comprising :- a plurality of bacterial strains; and- a solid support;(ii) filtering said sample through a first filter, and reverse filtering said sample through a second filter, to obtain micro sized particles in a first volume, wherein said first filter has a pore diameter greater than the pore diameter of said second filter; and(iii)recovering in a second volume a single micro sized particle comprising two or more bacterial strains.

24. A method for co-isolating two or more bacterial strains co-located on a microscale surface, the method comprising :(i) providing a sample comprising :- a plurality of bacterial strains; and- a solid support;(ii) filtering according to the following steps:- filtering said sample through a first filter to obtain a filtrate; and- filtering said filtrate through a second filter to obtain a retentate, wherein said retentate comprises in a first volume micro-sized particles derived from said solid support,wherein said first filter has a pore diameter greater than the pore diameter of said second filter; and(iii)recovering in a second volume a single micro-sized particle comprising two or more bacterial strains.

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