WO2016090431A1 - An antimicrobial agent and its method of production - Google Patents

Abstract

The present specification relates to an antimicrobial agent, comprising a microbial cell of the genus Mesorhizobium or an extracellular polymeric substance (EPS) or polysaccharide component of the EPS, produced by said cell, various uses thereof and its method of production.

Description

AN ANTIMICROBIAL AGENT AND ITS METHOD OF

PRODUCTION

FILING DATA

[0001] This application is associated with and claims priority from Australian Provisional Patent Application No. 2014905014, filed on 11 December 2014, entitled "An antimicrobial agent and its method of production", the entire contents of which, are incorporated herein by reference.

BACKGROUND FIELD

[0002] The present specification relates to an antimicrobial agent, various uses thereof and its method of production.

DESCRIPTION OF RELATED ART

[0003] Bibliographic details of publications referred to by author in this specification are collected alphabetically at the end of the description.

[0004] Reference in this specification to any prior publication (or information derived from it) or to any matter which is known, is not, and should not be taken as, an acknowledgement or admission or any form of suggestion that the prior publication (or information derived from it) or known matter form part of the common general knowledge in the field of endeavor to which this specification relates.

[0005] Bacteria and microalgae living in aquatic ecosystems commonly secrete an extracellular polymeric substance (EPS) as adherent capsules (capsular EPS) or release slimy mucous into the surrounding environment (excreted EPS; Decho, A. W. (1990), in Barnes, ed, Oceanogr Mar BiolAnnu Rev, Aberdeen: Aberdeen Univ Press, 73; Wotton, R. S. (2004) Scientia Marina, 68, 13). EPS typically comprises polysaccharides, proteins, nucleic acids and lipids, which form a highly hydrated matrix (Flemming, H. -C. et al. (1997), in Keevil, C. W. Biofilms in the Aquatic Environment, Cambridge, UK: The Royal Society of Chemistry, 101) and provide a layer of protection to cells against toxic compounds (Bitton, G. & Friehofer, V. (1978) Microbial Ecology, 4, 119; Jeanthon, C. & Prieur, D. (1990) Applied and Environmental Microbiology, 56(11), 3308), other organisms (Caron, D. A. (1987) Microbial Ecology, 13, 203) or physical damage (Roberson, E. B. et al. (1993) Soil Biology and Biochemistry, 25(9), 1299; Selbmann, L. et al. (2002) Research in Microbiology, 153, 585; Singh, S. & Fett, W. F. (1995) Microbiology Letters, 130(2-3), 301; Krembs, C. et al. (2002) Deep-Sea Research Part I: Oceanographic Research Papers, 49(12), 2163).

[0006] Polysaccharides form the architectural network of microbial biofilms and aggregates, which facilitate intracellular interaction and biochemical exchange between organisms (Paerl, H. W. (1976) Journal of Phycology, 12, 431; Wotton, R. S. (2004) supra). In aqueous environments, polysaccharides are essential for the attachment, survival and growth of organisms within surface-bound biofilms and aggregates. However, microbial polysaccharides are also recognized to promote biofouling as a result of the production of biologic/organic, colloidal, particulate or crystalline matter by organisms proliferating within the polysaccharide matrix of the biofilm (Kucera, J. (2010) Reverse osmosis: design, processes, and applications for engineers. 9, John Wiley and Sons). The formation of biofilms and resulting biofouling is detrimental to a wide range of industries such as shipping, food processing, water and wastewater treatment, water piping and medical devices.

[0007] Traditionally, biofouling has been controlled by modifying the properties of a surface on which a biofilm may form. Various surface modification methods have been employed, including increasing surface hydrophilicity, changing surface electrical properties or incorporating biocides to kill bacteria that are already attached to the surface. Whilst these methods can lead to a reduction in biofouling, there are nevertheless, some drawbacks including reduction in pure water flux on a coated membrane, potential for environmental contamination by the use of non-specific toxins, inadequate anti-fouling effects, the frequent need to use complex and expensive coating protocols and difficulties in coating surfaces on a large scale. Thus, there is a need for an alternative approach to modify surfaces to mitigate biofouling.

[0008] It has previously been shown that substances produced by microorganisms within biofilms can lead to the disruption of biofilm formation (Armstrong, E. (2000) Biofouling, 16, 215). These substances have been proposed as ideal candidates for the production of natural anti-fouling agents. However, microorganisms that produce polysaccharides with the capability to inhibit the attachment and growth of other organisms on surfaces are extremely rare. The polysaccharides of some species of bacteria of the Alteromonas and Pseudoalteromonas genus from deep-sea hydrothermal ecosystems are reported to have such capability. These agents are used as an anti-fouling coating on surfaces of pipelines and hospital tools such as tubes or catheters (Holmstrom, C. et al. (2002) FEMS Microbial Ecology, 41, 47; Guezennec, J. et al. (2012) International Biodeterioration & Biodegradation, 6(1), 1).

SUMMARY

[0009] The present specification teaches production of an agent which exhibits antimicrobial properties and which is useful inter alia to inhibit growth and/or maintenance of other microorganisms and/or to inhibit attachment of microorganisms to a surface. The agent may also be used to reduce microbial contamination at particular sites. In an embodiment, the agent is useful as a means to reduce biofouling. Hence, in an embodiment, the agent can be used to confer biofouling resistance to a surface.

[0010] The agent may be a microorganism capable of generating a biofilm which reduces the growth and/or maintenance of other microorganisms at a particular site or their attachment to a surface, or it may be an isolated extracellular polymeric substance (EPS) produced by a microbial cell, or a polysaccharide component thereof. In an embodiment, the EPS is capsular EPS. In another embodiment, the EPS is excreted EPS.

[0011] In an embodiment, the microbial cell is a species of Mesorhizobium or is a Mesorhizobium-li e microorganism. In an embodiment, the microbial cell is in isolated form.

[0012] Reference to an "isolated form" includes an isolated cell, purified cell culture, freeze-dried sample, frozen sample or other form of the microorganism maintained in a non-naturally occurring state. A "non-naturally occurring state" means the microorganism has undergone at least one round of purification. In an embodiment, the microorganism is maintained in a dormant or semi-dormant state until ready for use.

[0013] Reference to a "Mesorhizobium or Mesorhizobium-li e microorganism" includes a Gram negative bacterium with monotrichous flagella, which grows to a colony diameter of 1 to 4 mm after 7 days on yeast mannitol agar (YMA), exhibits a generation time of 4 to 10 hours in yeast mannitol broth (YMB), tolerates a maximum growth temperature of 36°C to 39°C, exhibits a maximum NaCl tolerance for growth of 1 to >2.5 (% w/v), tolerates a pH range for growth of 4 to 10 and has the ability to grow on melibiose. [0014] In an embodiment, the microorganism is selected from the list comprising Mesorhizobium huakuii, Mesorhizobium loti, Mesorhizobium abyssinicae, Mesorhizobium albiziae, Mesorhizobium alhagi, Mesorhizobium amorphae, Mesorhizobium australicum, Mesorhizobium camelthorni, Mesorhizobium caraganae, Mesorhizobium chacoense, Mesorhizobium cicero, Mesorhizobium gobiense, Mesorhizobium hawassense, Mesorhizobium mediterraneum, Mesorhizobium metallidurans, Mesorhizobium muleiense, Mesorhizobium opportunistum, Mesorhizobium plurifarium, Mesorhizobium qingshengii, Mesorhizobium robiniae, Mesorhizobium sangaii, Mesorhizobium septentrionale, Mesorhizobium shangrilense, Mesorhizobium shonese, Mesorhizobium silamurunense, Mesorhizobium tamadayense, Mesorhizobium tarimense, Mesorhizobium temperatum, Mesorhizobium thiogangeticum or Mesorhizobium tianshanense.

[0015] In an embodiment, the microorganism is M. huakuii or M. loti or a microorganism having biochemical, physiological or genetic properties similar to M. huakuii or M. loti. Hence, the antimicrobial agent is a Mesorhizobium or Mesorhizobium- like microorganism or is an isolated EPS produced thereby or a polysaccharide component thereof.

[0016] In an embodiment, the microorganism is designated CAM543 which was deposited at the National Measurements Institute on 29 October, 2014 under accession number V14/017216. [0017] In an embodiment, the agent is an EPS produced by the subject microorganism. In another embodiment, the agent is a polysaccharide component of the EPS produced by the subject microorganism. The EPS or a polysaccharide component thereof has an attenuated total reflectance-Fourier transform infrared spectrum with an indication of carboxyl and sulfate groups. [0018] In an embodiment, the agent comprises an isolated EPS or a polysaccharide component therefrom has the attenuated total reflectance-Fourier transform infrared spectrum of Figure 1. [0019] The agent of the present invention exhibits antimicrobial properties. These include inhibiting attachment, growth and/or maintenance of other microorganisms. In an embodiment, the agent inhibits or otherwise reduces adhesion of a microorganism to a surface. Susceptible microorganisms include bacteria, algae, fungi and combinations thereof including inhibiting the production of biofilms comprising these organisms.

[0020] In an embodiment, the agent exhibits bacteriostatic or bactericidal properties.

[0021] In another embodiment, the agent exhibits anti-adhesion properties.

[0022] In an embodiment, the antimicrobial properties inhibit attachment, growth and/or maintenance of a microorganism on a surface. Hence, the surface comprises a biofilm of the Mesorhizobium or Mesorhizobium-li e microorganism or a coating of an isolated EPS or polysaccharide component thereof produced by the Mesorhizobium or Mesorhizobium-li e microorganism, which exhibits biofouling resistance due to minimized attachment, growth and/or maintenance of other microorganisms to the surface.

[0023] A surface is generally selected from silica, silicon, semi-conductors, glass, polymers, organic compounds, inorganic compounds, metals and combinations thereof. A metal surface comprises gold, copper, stainless steel, nickel, aluminum, titanium, thermosensitive alloys and combinations thereof. In an embodiment, the surface is a membrane. In another embodiment, the surface is a semipermeable membrane.

[0024] In an embodiment, the surface is a reverse osmosis filtration membrane used for purifying water from salt or brackish water.

[0025] In an embodiment, the agent is coated on a surface. In an embodiment, the agent is formulated in a spray, mist, micro- or nano-particles, an aqueous solution, a wash, a tonic, a dispersant, an atomized formulation, sludge, powder, cream, ointment, gel, patch, impregnated bandage, liquid, formulation, paint or other suitable distribution medium including topical forms of the composition. In an embodiment, the agent coated on a surface by any suitable means. In another embodiment, the agent is coated on a surface by covalent bonding. [0026] Taught herein is a method for producing an agent comprising culturing a Mesorhizobium or Mesorhizobium-like microorganism for a time and under conditions to generate a sufficient number of cells per liter (L ¹). Where the agent is an isolated EPS or polysaccharide component thereof produced by a Mesorhizobium or Mesorhizobium-like microorganism, the microorganism is cultured for a time and under conditions sufficient for EPS to be produced, which is then isolated and purified to a suitable concentration. Optionally, a polysaccharide component is isolated from the EPS.

[0027] In an embodiment, the Mesorhizobium or Mesorhizobium-like microorganism is cultured in the presence of a sugar such as, but not limited to, glucose, mannitol or glycerol.

[0028] In an embodiment, the culture is a continuous culture maintained at 20°C and wherein the microorganism is suspended in a liquid broth.

[0029] In an embodiment, the agent is Mesorhizobium or Mesorhizobium-like microorganism is cultured from a freeze-dried, frozen or other form of suitable long-term storage inoculum sample.

[0030] In an embodiment, the agent is an isolated EPS or polysaccharide component thereof in the supernatant fluid of the culture. In another embodiment, the agent is an isolated EPS or polysaccharide component thereof encapsulated on the surface of the subject microorganism. [0031] Taught herein is a method of inhibiting the attachment, growth and/or maintenance of an unwanted microorganism on a surface of a selected site comprising generating a biofilm comprising Mesorhizobium or Mesorhizobium-like microorganisms or forming a coating of the isolated EPS or polysaccharide component thereof produced by a Mesorhizobium or a Mesorhizobium-like microorganism on the surface of the selected site. [0032] The present specification teaches an antimicrobial agent comprising a Mesorhizobium or Mesorhizobium-like microorganism or an EPS produced thereby or a polysaccharide component thereof. Whilst the agent may be used as a general antimicrobial agent, it has particular use in inhibiting the formation and maintenance of biofilms. Hence, the agent is biofouling resistant.

[0033] Abbreviations used herein are defined in Table 1.

- Si -

Table 1. Abbreviations

Abbreviation Description

°C Degrees centigrade

ATR Attenuated total reflectance

ATR-FTIR Attenuated total reflectance-Fourier transform infrared

BCA Bicinchoninic acid

cm, mm, μιη, nm Centimeter, millimeter, micrometer, nanometer cP Centipoise

EPS Extracellular polymeric matrix

FA Fatty acid

FAME Fatty acid ethyl ester

FID Flame ionization detector

g, mg, μg, ng Gram, milligram, microgram, nanogram

GC Gas chromatography

GC-MS Gas chromatography mass spectrometry

HEPES 4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid hr, min, sec Hour, minute, second

ITS Internally transcribed spacer

L, mL, μL· Liter, milliliter, microliter

M, mM, μΜ Molar, millimolar, micromolar

MWCO Molecular weight cut-off

OD Optical density

PCR Polymerase chain reaction

RO Reverse osmosis

rpm Revolutions per minute

SB Simple broth (peptone, yeast extract)

SB+Glu Simple broth with glucose

SM Simple media (peptone, yeast extract, agar)

YMA Yeast mannitol agar

YMB Yeast mannitol broth BRIEF DESCRIPTION OF THE FIGURES

[0034] Figure 1 is a graphical representation of EPS spectra recorded on a Thermo Scientific Nicolet 6700 spectrometer equipped with diamond ATR accessories with depth of penetration of approximately 2 μιη or less. Thirty-two scans were accumulated with a resolution of 4 cm^"1 for each spectrum. Peaks labeled with [top] wave numbers (cm ¹) and [bottom] suggested functional groups.

[0035] Figure 2 is a graphical representation of rheological characteristics of aqueous solutions of CAM543 EPS (10 mg ml^"1) measured on a Brookfield micro viscometer. Sheer stress (dynes cm^" ) versus shear rate (sec^" ) [top] and viscosity (cP) versus shear rate (sec ¹) [bottom].

[0036] Figure 3 is a graphical representation of optical density (at 600 nm) versus time for 24 cultures (3 mL) grown in 3 g L-1 peptone and 1 g L-1 yeast extract with no added carbon or with glucose (10 g L-1), mannitol (10 g L-1) or glycerol (10 gL-1) added as a carbon source. For each carbon source treatment a culture was incubated at each of six different temperatures (20°C, 22°C, 24°C, 26°C, 28°C and 30°C).

[0037] Figure 4 is a graphical representation of optical density (at 600 nm) versus time for 24 cultures (3 mL) grown in 3 g L-1 peptone and 1 g L-1 yeast extract at 30°C. Cultures were grown in triplicate with no added carbon or with acetate (10 g L-1), succinate (10 g L-1), glycine (10 g L-1), sorbitol (10 g L-1), or glycerol (10 g L-1). [0038] Figure 5 is a photographic representation of CAM543 grown in yeast mannitol broth (YMB) [top] and yeast mannitol agar (YMA) [bottom], 4 days, 30°C, magnification: 1600X; [left to right] bright field, phase contrast, differential interference contrast. DETAILED DESCRIPTION

[0039] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not to the exclusion of any other integer or step or group or integers or steps.

[0040] As used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a microbial cell" includes a single cell, as well as two or more cells; reference to "an agent" includes a single agent, as well as two or more agents; reference to "the disclosure" includes a single and multiple aspects taught by the disclosure; and so forth. Aspects taught and enabled herein are encompassed by the term "invention". All aspects are enabled within the width of the present invention.

[0041] The present specification teaches an agent having antimicrobial properties. The agent comprises a microbial cell of the genus Mesorhizobium or a Mesorhizobium-li e microorganism; or an isolated extracellular polymeric substance (EPS) produced by a microbial cell of the genus Mesorhizobium or is a Mesorhizobium-li e microorganism; or is a polysaccharide component of the EPS produced by a microbial cell of the genus Mesorhizobium or is a Mesorhizobium-li e microorganism. [0042] Reference to an "extracellular polymeric substance" or "EPS" includes both capsular and excreted EPS. Capsular EPS is excreted by microorganisms and retained as a capsule on the surface of the cell, forming the outermost layer of the cell. Excreted EPS is a distinct substance that is excreted by microorganisms into the surrounding environment. Both capsular and excreted EPS comprise high-molecular weight compounds including, but not limited to, polysaccharides, proteins, DNA, lipids and acids.

[0043] The EPS may comprise one or more types of polysaccharide. Reference to a "polysaccharide" means polymeric carbohydrate molecules comprising repeating units or monomers, such as monosaccharides, disaccharides or oligiosacharides, joined by glycosidic bonds. It is further understood that the terms "polysaccharide" and "glycan" may be used interchangeably.

[0044] In an aspect, the EPS comprises a single polysaccharide or polysaccharide derivative. In another aspect, the EPS comprises combinations of two or more polysaccharides, or polysaccharide derivatives. Hence, the agent may comprise either homogenous (homopolysaccharide) or heterogeneous (heteropolysaccharide) polysaccharide components of the EPS. In another aspect, the polysaccharide in accordance with the present invention is a combination of homopolysaccharides and heteropolysaccharides . [0045] In an embodiment, the polysaccharides are linear, and may optionally comprise degrees of branching. In another embodiment, the polysaccharide is soluble or is a soluble derivative, in particular a water-soluble polysaccharide or a water-soluble derivative of a polysaccharide. In another embodiment, the polysaccharide is insoluble or is only partially soluble. [0046] In an embodiment, the polysaccharide is an exopolysaccharide. An exopolysaccharide may additionally comprise other non-carbohydrate substituents. For example, other non-carbohydrate substituents may include acetate, pyruvate, succinate, sulfate and phosphate substituents.

[0047] In another aspect, the polysaccharide including an exopolysaccharide is a naturally-occurring polysaccharide. In still another aspect, the polysaccharide is a polysaccharide derivative. Derivatives include, for example, modified monosaccharides or monosaccharide derivatives. Examples of monosaccharide derivatives include aminosugars, sulfosugars and sugar alcohols. Furthermore, polysaccharide derivatives may comprise one or more monosaccharides modified by chemical methods known in the art.

[0048] Reference to "antimicrobial properties" means an ability to inhibit or reduce microbial attachment, growth, maintenance, replication or survival. The reduction in microbial attachment includes a reduction in or inhibition of adhesion of a microorganism to a surface. The term "microbial" includes bacteria, algae and fungi. It is proposed that the agent can, in an aspect, confer biofouling resistance to surfaces.

[0049] The terms "microbial cell" and "microorganism" may be used interchangeably throughout this specification. [0050] Reference to a "Mesorhizobium or Mesorhizobium-li e microorganism" includes a microorganism which is Gram negative, comprises a monotrichous flagella, grows to a colony diameter of 1 to 4 mm after 7 days on yeast mannitol agar (YMA), exhibits a generation time of 4 to 10 hours in yeast mannitol broth (YMB), tolerates a maximum growth temperature of 36°C to 39°C, exhibits a maximum NaCl tolerance for growth of 1 to >2.5 (% w/v), tolerates a pH range for growth of 4 to 10 and has the ability to grow on melibiose.

[0051] In an embodiment, the Mesorhizobium is selected from the list comprising Mesorhizobium huakuii, Mesorhizobium loti, Mesorhizobium abyssinicae, Mesorhizobium albiziae, Mesorhizobium alhagi, Mesorhizobium amorphae, Mesorhizobium australicum, Mesorhizobium camelthorni, Mesorhizobium caraganae, Mesorhizobium chacoense, Mesorhizobium cicero, Mesorhizobium gobiense, Mesorhizobium hawassense, Mesorhizobium mediterraneum, Mesorhizobium metallidurans, Mesorhizobium muleiense, Mesorhizobium opportunistum, Mesorhizobium plurifarium, Mesorhizobium qingshengii, Mesorhizobium robiniae, Mesorhizobium sangaii, Mesorhizobium septentrionale, Mesorhizobium shangrilense, Mesorhizobium shonese, Mesorhizobium silamurunense, Mesorhizobium tamadayense, Mesorhizobium tarimense, Mesorhizobium temperatum, Mesorhizobium thiogangeticum or Mesorhizobium tianshanense.

[0052] In an embodiment, the Mesorhizobium is M. huakuii or M. loti.

[0053] In an embodiment, the Mesorhizobium or Mesorhizobium-li e microorganism is the microbial cell designated CAM543 deposited at the National Measurements Institute on 29 October, 2014 under accession number V14/017216.

[0054] Hence, the present specification teaches an agent comprising an isolated microbial cell of the genus Mesorhizobium or a Mesorhizobium-li e microorganism, or an EPS produced thereby, or a polysaccharide component thereof wherein the microbial cell and/or EPS and/or polysaccharide exhibits antimicrobial properties towards other microorganisms.

[0055] In an embodiment, enabled herein is an agent comprising an isolated microbial cell of the genus Mesorhizobium selected from the list comprising M. huakuii, M. loti, M. abyssinicae, M. albiziae, M. alhagi, M. amorphae, M. australicum, M. camelthorni, M. caraganae, M. chacoense, M. cicero, M. gobiense, M. hawassense, M. mediterraneum, M. metallidurans, M. muleiense, M. opportunistum, M. plurifarium, M. qingshengii, M. robiniae, M. sangaii, M. septentrionale, M. shangrilense, M. shonese, M. silamurunense, M. tamadayense, M. tarimense, M. temperatum, M. thiogangeticum or M. tianshanense, or an EPS produced thereby, or a polysaccharide component thereof wherein the microbial cell and/or EPS and/or polysaccharide exhibits antimicrobial properties.

[0056] Taught herein is an agent comprising an isolated microbial cell of the genus Mesorhizobium selected from M. huakuii and M. loti, or an EPS produced thereby, or a polysaccharide component thereof wherein the microbial cell and/or EPS and/or polysaccharide exhibits antimicrobial properties.

[0057] Further enabled herein is an agent comprising an isolated microbial cell designated CAM543 deposited at the National Measurements Institute on 29 October, 2014 under accession number V14/017216, or an EPS produced thereby, or a polysaccharide component thereof wherein the microbial cell and/or EPS and/or polysaccharide exhibits antimicrobial properties.

[0058] The present specification further teaches an isolated EPS or polysaccharide component thereof produced by a microbial cell of the genus Mesorhizobium or a Mesorhizobium-like microorganism wherein the EPS and/or polysaccharide exhibits antimicrobial properties.

[0059] Reference to an "isolated EPS" includes an EPS or polysaccharide component thereof which exhibits an attenuated total reflectance-Fourier transform infrared spectrum (ATR-FTIS) with an indication of carboxyl and sulfate groups. An example of an ATR- FTIS of an EPS or polysaccharide contemplated in the present invention is set forth in Figure 1.

[0060] In an embodiment, enabled herein is an isolated EPS or polysaccharide component thereof produced by a microbial cell of the genus Mesorhizobium selected from the list comprising M. huakuii, M. loti, M. abyssinicae, M. albiziae, M. alhagi, M. amorphae, M. australicum, M. camelthorni, M. caraganae, M. chacoense, M. cicero, M. gobiense, M. hawassense, M. mediterraneum, M. metallidurans, M. muleiense, M. opportunistum, M. plurifarium, M. qingshengii, M. robiniae, M. sangaii, M. septentrionale, M. shangrilense, M. shonese, M. silamurunense, M. tamadayense, M. tarimense, M. temperatum, M. thiogangeticum or M. tianshanense wherein the EPS and/or polysaccharide exhibits antimicrobial properties.

[0061] Taught herein is an isolated EPS or polysaccharide component thereof produced by a microbial cell selected from M. huakuii and M. loti wherein the EPS and/or polysaccharide exhibits antimicrobial properties. [0062] Further enabled herein is an isolated EPS or polysaccharide component thereof produced by a microbial cell designated CAM543 deposited at the National Measurements Institute on 29 October, 2014 under accession number V14/017216 wherein the EPS and/or polysaccharide exhibits antimicrobial properties.

[0063] The present specification further teaches an agent having antimicrobial properties that inhibits the attachment, growth and/or maintenance of other microorganisms on a surface.

[0064] Reference to a "surface" includes silica, silicon, semi-conductors, glass, polymers, organic compounds, inorganic compounds, metals and combinations thereof. Where a surface is a metal, reference to a "metal" includes gold, copper, stainless steel, nickel, aluminum, titanium, thermosensitive alloys and combinations thereof. In an embodiment, the surface is a membrane. In another embodiment, the surface is a semipermeable membrane. [0065] Enabled herein is a membrane with a coating of Mesorhizobium or Mesorhizobium-li e microorganisms, or an isolated EPS produced thereby, or a polysaccharide component thereof that exhibits biofouling resistance.

[0066] The agent may be applied to a surface as an inoculum, spray, paint, aerosol, solution, drops, nebulizer and the like. Alternatively, the agent is formed as a coating on a surface.

[0067] In an embodiment, a surface is chemically odified to facilitate the formation of a coating of Mesorhizobium or a Mesorhizobium-li e microorganisms, or an isolated EPS produced thereby, or a polysaccharide component thereof. [0068] The formation of a coating of Mesorhizobium or Mesorhizobium-li e microorganisms, or an isolated EPS produced thereby, or a polysaccharide component thereof may be achieved by any suitable means. In an embodiment, the coating is an isolated EPS or a polysaccharide component thereof produced by a Mesorhizobium or a Mesorhizobium-li e microorganism which is formed by covalent bonding. [0069] Methods for the covalent bonding of substances onto surfaces are well known to those skilled in the art. Interfacial reactions leading to the formation of covalent interfacial bonds are derived from well-known organic-synthetic reactions. The choice of bonding reaction depends on both the nature of the substrate material and the chemical composition of the compound of the present invention. [0070] In an embodiment, the coating of an isolated EPS or a polysaccharide component thereof produced by a Mesorhizobium or a Mesorhizobium-li e microorganism is formed on a surface by covalent bonding using a linker.

[0071] In an embodiment, the coating of an isolated EPS or a polysaccharide component thereof produced by a Mesorhizobium or a Mesorhizobium-li e microorganism is formed on a surface by covalent bonding using an inorganic linker.

[0072] In an embodiment, the coating of an isolated EPS or a polysaccharide component thereof produced by a Mesorhizobium or a Mesorhizobium-li e microorganism is formed on a surface by covalent bonding using any suitable metal alkoxide or alkoxysilane.

[0073] Reference to an "alkoxy" means a group having the formula -OR, where R is an alkyl group. Examples of linear alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentoxy, and hexoxy. Examples of branched alkoxy groups include, but are not limited to, isopropoxy, sec -butoxy, tert-butoxy, isopentoxy, and isohexoxy. Examples of cycloalkoxy groups include, but are not limited to, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy.

[0074] Reference to an "alkyl" includes straight chain and branched alkyl groups having from 1 to about 20 carbon atoms or, in some embodiments, from 1 to 8, 1 to 6, or 1 to 4 carbon atoms. In an embodiment, the alkyl group may be a cycloalkyl group.

Examples of straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups.

Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. Examples of cycloalkyl groups include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0075] One or more metal alkoxides or alkoxysilanes within the scope of the present application may react with suitable functional groups on the isolated EPS or a polysaccharide component thereof produced by a Mesorhizobium or a Mesorhizobium-li e microorganism and the surface to link the EPS or polysaccharide and the surface. In particular, the metal alkoxide or alkoxysilane may link the EPS or polysaccharide to the surface by transesterification or alcoholysis. In an embodiment, the suitable functional groups on the EPS or polysaccharide and the surface may include hydroxyl groups, carbonyl groups, carboxyl groups and combinations thereof. In another embodiment, the linker may comprise two or more metal alkoxides.

[0076] Taught herein is a metal which forms part of the metal alkoxide linker which is any suitable metal. Examples of suitable metals include, but are not limited to, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium, silver, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, and lanthanides, such as cesium, samarium, gadolinium, dysprosium, erbium and neodymium. Further taught herein is a metal comprising one or more of the metals described above. The metal of the present specification may further comprise a bivalent, trivalent, tetravalent, pentavalent, or hexavalent metals. In an embodiment, the linker may be a crosslinker derived from an alkoxysilane.

[0077] In an embodiment, the coating of an isolated EPS or a polysaccharide component thereof produced by a Mesorhizobium or a Mesorhizobium-li e microorganism is formed on a surface by covalent bonding using a titanium alkoxide, a zirconium alkoxide, an aluminum alkoxide or an alkoxysilane linker.

[0078] In another embodiment, the coating of an isolated EPS or a polysaccharide component thereof produced by a Mesorhizobium or a Mesorhizobium-li e microorganism is formed on a surface by covalent bonding using an alkoxide linker of the following formula Si(OR)₄, Ti(OR)₄, Zr(OR)₄ and Al(OR)₄. In an embodiment, the linker may be titanium isopropoxide.

[0079] Hence, the present specification teaches an immobilization strategy that uses specific crosslinking molecules to act as a bridge between the EPS or polysaccharide and a surface. In an aspect, the carboxyl (-COOH) and hydroxyl (-OH) functional groups on the surface and the hydroxyl (-OH), carbonyl (-C=0) and carboxyl (-COOH) functional groups on the EPS or polysaccharide, are reacted with the metal alkoxide to allow covalent bonding to occur.

[0080] In another aspect, titanium alkoxides are known to react with compounds containing hydroxyl groups via the protolytic loss of one or more alkoxide ligands (Figure 2; Uekawa, N., et al. (2006) Journal of the Ceramic Society of Japan, 114(10), 807; Yi, Y., et al. (2010) US Patent No. 2010/0239493; Kariduraganavar, M.Y., et al. (2009) Industrial and Engineering Chemistry Research, 48, 4002. In particular, titanium isopropoxide reacts with carboxyl groups to form carboxylate completes through chelating bidentate, bridging bidentate or monobidentate mechanisms (Figure 3; see also Hojjati, B. and Charpentier, P.A. (2001) Journal of Polymer Science Part A: Polymer Chemistry, 46, 3926; Nakamoto, K. (1997) Infrared and Raman Spectra of Inorganic and Coordination Compounds. Wiley- Interscience: New York, 59; Deacon J.B. and Phillips, R.J. (1980) Coordination Chemistry Reviews, 33, 227). [0081] The use of metal alkoxides for the formation of a coating on a surface taught by the present specification requires that the metal alkoxides are dissolved in alcohol prior to being combined with the isolated EPS or a polysaccharide component thereof produced by a Mesorhizobium or a Mesorhizobium-like microorganism and water.

[0082] In an embodiment, the alcohol is ethanol or 1-propanol. [0083] In a further embodiment, the volume ratio of titanium isopropoxide to alcohol is between 1: 10 and 1:20. Reference to "between 1: 10 and 1:20" means 1: 10, 1: 11, 1: 12, 1: 13, 1: 14, 1: 15, 1: 16, 1:17, 1: 18, 1: 19 or 1:20.

[0084] The weight/weight (w/w) ratio of the isolated EPS or a polysaccharide component thereof produced by a Mesorhizobium or a Mesorhizobium-like microorganism and the linker is proposed to be between 0.15 and 1.25. Reference to the w/w ratio of "between 0.15 and 1.5" means 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.05, 1.10, 1.15, 1.20, 1.25, 1.30, 1.35, 1.40, 1.45 or 1.50.

[0085] In an embodiment, the coating is formed on a surface by treating the surface with one or more metal alkoxides and the isolated EPS or a polysaccharide component thereof produced by a Mesorhizobium or a Mesorhizobium-like microorganism.

[0086] Application of the Mesorhizobium or a Mesorhizobium-like microorganism to a surface is proposed to be within the range 10 cells cm -^"2 to 1010 cells cm -^"2. Reference to " 10 cells cm^"2 to 10¹⁰ cells cm^"2" means 10, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹ or 10¹⁰ cells cm^" . Alternatively, a surface is seeded with a low number of cells and the cells are permitted to expand into a biofilm. In terms of the EPS or its polysaccharide component thereof, the amount of the agent applied to a surface is in the order of 0.5 μg cm^" to 500 μg cm^"2 Reference to "0.5 μg cm^"2 to 500 μg cm^"2" means 0.5, 1, 10, 50, 100, 200, 300, 400 or 500 μg cm^"2.

[0087] Hence, the present specification teaches a coating formed on a surface comprising a Mesorhizobium or Mesorhizobium-li e microorganism, or an isolated EPS produced thereby, or a polysaccharide component thereof.

[0088] Enabled herein is a surface comprising a microbial cell of the genus Mesorhizobium selected from the list comprising M. huakuii, M. loti, M. abyssinicae, M. albiziae, M. alhagi, M. amorphae, M. australicum, M. camelthorni, M. caraganae, M. chacoense, M. cicero, M. gobiense, M. hawassense, M. mediterraneum, M. metallidurans, M. muleiense, M. opportunistum, M. plurifarium, M. qingshengii, M. robiniae, M. sangaii, M. septentrionale, M. shangrilense, M. shonese, M. silamurunense, M. tamadayense, M. tarimense, M. temperatum, M. thiogangeticum or M. tianshanense, or an isolated EPS produced thereby, or a polysaccharide component thereof.

[0089] Further taught herein is a surface comprising a microbial cell selected from M. huakuii and M. loti, or an isolated EPS produced thereby, or a polysaccharide component thereof.

[0090] In a particular embodiment, the present specification enables a surface comprising a microbial cell designated CAM543 deposited at the National Measurements Institute on 29 October, 2014 under accession number V14/017216, or an isolated EPS produced thereby, or a polysaccharide component thereof.

[0091] The present specification teaches a method for inhibiting the growth of an unwanted microorganism at a selected site by generating a biofilm comprising Mesorhizobium or Mesorhizobium-li e microorganisms. In an embodiment, the biofilm or an EPS produced by Mesorhizobium or Mesorhizobium-li e microorganisms within the biofilm inhibit or otherwise reduce adhesion of other microorganisms to the surface.

[0092] Reference to a "biofilm" includes an aggregate of microorganisms wherein cells are embedded within a self -produced matrix of EPS, or polysaccharide component thereof, to adhere to each other and/or a surface. The biofilm comprising the Mesorhizobium or Mesorhizobium-li e microorganisms promotes biofouling resistance.

[0093] In an embodiment, a method is provided for inhibiting the growth of an unwanted microorganism at a selected site by generating a biofilm comprising microbial cells of the genus Mesorhizobium selected from the list comprising M. huakuii, M. loti, M. abyssinicae, M. albiziae, M. alhagi, M. amorphae, M. australicum, M. camelthorni, M. caraganae, M. chacoense, M. cicero, M. gobiense, M. hawassense, M. mediterraneum, M. metallidurans, M. muleiense, M. opportunistum, M. plurifarium, M. qingshengii, M. robiniae, M. sangaii, M. septentrionale, M. shangrilense, M. shonese, M. silamurunense, M. tamadayense, M. tarimense, M. temperatum, M. thiogangeticum or M. tianshanense.

[0094] Enabled herein is a method for inhibiting the growth of an unwanted microorganism at a selected site by generating a biofilm comprising microbial cells of the genus Mesorhizobium selected from M. huakuii and M. loti.

[0095] Taught herein is a method for inhibiting the growth of an unwanted microorganism at a selected site by generating a biofilm comprising microbial cells designated CAM543 deposited at the National Measurements Institute on 29 October, 2014 under accession number V14/017216.

[0096] In another aspect, the agent is formulated into a composition comprising Mesorhizobium or a Mesorhizobuim-li e microorganisms, or an isolated EPS produced thereby, or a polysaccharide component thereof with one or more carriers, diluents or excipients for use in inhibiting the growth of an unwanted microorganism at a selected site.

[0097] The terms "formulation" and "composition" may be used interchangeably throughout this specification.

[0098] In an embodiment, the composition is in the form of a spray, mist, micro- or nano-particles, an aqueous solution, a wash, a tonic, a dispersant, an atomized formulation, sludge, powder, cream, ointment, gel, patch, impregnated bandage, liquid, formulation, paint or other suitable distribution medium including topical forms of the composition. [0099] Useful applications of the agent include formulations of sprays, dispersants, aerosols, solutions, drops and paints to inhibit the growth of an unwanted microorganism at a selected site. Where the selected site is a surface, treatment of the surface with the formulation confers biofouling resistance. [00100] A composition comprising Mesorhizobium or Mesorhizobuim-li e microorganisms, or an isolated EPS produced thereby, or a polysaccharide component thereof described herein generally includes a carrier, excipient, diluent, preservative, stabilizer and/or a solid or liquid additive. Compositions comprising the microorganism of the present specification, or an isolated EPS produced thereby, or a polysaccharide component thereof may also be used in a formulation such as a hand wash, body wash or shampoo.

[0101] The composition may take a wide variety of forms depending on the intended method of administration. In preparing the compositions, usual media may be employed such as, for example, water, glycols, oils, alcohols, preservatives and/or coloring agents. The compositions may take the form of a liquid preparation such as, for example, suspensions, elixirs and solutions. Carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like may be used. The composition may also be in the form of sprays, dispersants and paints. [0102] The Mesorhizobium or Mesorhizobuim-li e microorganism, or an isolated EPS produced thereby, or a polysaccharide component thereof is administered directly to the site, generally for a time and under conditions sufficient to inhibit the growth of an unwanted microorganism at a selected site. This includes the inhibition of the growth of an unwanted microorganism on filtration membranes used for desalination or decontamination of aqueous fluid. Hence, the treated membranes are biofouling resistant.

[0103] When administered by aerosol or spray, the compositions are prepared according to techniques well-known in the art of chemical formulation and may be prepared in solutions of saline, employing benzyl alcohol or other suitable preservatives, fluorocarbons and/or other solubilizing or dispersing agents known in the art. This, of course, is dependent on whether the composition comprises a live microorganism. In an embodiment, a composition comprising the microorganism of the present specification is prepared as an aerosol or spray to inoculate surfaces with the microorganism to allow a biofilm to form. [0104] In an embodiment, the present specification teaches a method for inhibiting the growth of an unwanted microorganism at a selected site, the method comprising coating the site with Mesorhizobium or Mesorhizobium-li e microorganisms, or an EPS produced thereby or a polysaccharide component thereof.

[0105] Taught herein is a method for inhibiting the growth of an unwanted microorganism at a selected site, the method comprising coating the site with microbial cells of the genus Mesorhizobium selected from the list comprising M. huakuii, M. loti, M. abyssinicae, M. albiziae, M. alhagi, M. amorphae, M. australicum, M. camelthorni, M. caraganae, M. chacoense, M. cicero, M. gobiense, M. hawassense, M. mediterraneum, M. metallidurans, M. muleiense, M. opportunistum, M. plurifarium, M. qingshengii, M. robiniae, M. sangaii, M. septentrionale, M. shangrilense, M. shonese, M. silamurunense,

M. tamadayense, M. tarimense, M. temperatum, M. thiogangeticum or M. tianshanense, or an EPS produced thereby, or a polysaccharide component thereof.

[0106] Enabled herein is a method for inhibiting the growth of an unwanted microorganism at a selected site, the method comprising forming a coating of microbial cells of the genus Mesorhizobium selected from M. huakuii and M. loti, or an EPS produced thereby, or a polysaccharide component thereof at the selected site.

[0107] Taught herein is a method for inhibiting the growth of an unwanted microorganism at a selected site, the method comprising forming a coating of microbial cells designated CAM543 deposited at the National Measurements Institute on 29 October, 2014 under accession number V14/017216, or an EPS produced thereby, or a polysaccharide component thereof at the selected site. [0108] In an embodiment, the isolated EPS or a polysaccharide component produced by a Mesorhizobium or Mesorhizobuim-li e microorganism exhibits an ATR-FTIS with an indication of carboxyl and sulfate groups.

[0109] In an embodiment, the isolated EPS or a polysaccharide component produced by a Mesorhizobium or Mesorhizobuim-li e microorganism exhibits an ATR-FTIS of Figure 1.

[0110] In an embodiment, the Mesorhizobium or Mesorhizobuim-li e microorganism, or an EPS produced thereby, or a polysaccharide component thereof exhibits antimicrobial properties selected from inhibiting the attachment, growth and/or maintenance of bacteria, algae or fungi.

[0111] In an embodiment, the Mesorhizobium or Mesorhizobuim-li e microorganism, or an EPS produced thereby, or a polysaccharide component thereof exhibit bacteriostatic or bactericidal properties.

[0112] The present specification teaches a method for producing an agent having antimicrobial properties. The method comprises the culturing of a Mesorhizobium or a Mesorhizobium-li e microorganism for a time and under conditions to generate a sufficient number of cells per liter (L ¹).

[0113] Reference to a "culture" includes a culture of a Mesorhizobium or a Mesorhizobium-like microorganism which is stored as a freeze-dried, frozen or otherwise maintained in a dormant or semi-dormant state and which can be grown into a suitable inoculum for large scale culturing.

[0114] Reference to an "inoculum" includes the source material for the transfer of microorganisms into the culture system.

[0115] In an embodiment, the agent is an isolated EPS obtained from the supernatant fluid of a culture of a Mesorhizobium or a Mesorhizobium-li e microorganism.

[0116] In an embodiment, the agent is an isolated EPS obtained from the surface of microbial cells in a culture of Mesorhizobium or Mesorhizobium-li e microorganisms. [0117] In an embodiment, the culture is a continuous culture maintained at 20°C and wherein the microorganism is suspended in liquid broth comprising a sugar.

[0118] In an embodiment, where the agent is an isolated EPS, cellular material is removed by centrifugation and the EPS isolated by filtration. [0119] In terms of in vitro culture technology, the Mesorhizobium or Mesorhizobium- like microorganism, or the EPS produced thereby, or the polysaccharide component thereof can be produced on either a small scale or on a larger scale. In terms of small scale production, this may be effected in, for example, batch culture flasks, or under continuous culture conditions which can be scaled up for producing populations of cells for a given application. One means of achieving large scale production is via the use of a bioreactor.

[0120] Bioreactors are designed to provide a culture process that can deliver medium and at controlled concentrations and rates. Bioreactors are available commercially and employ a variety of types of culture technologies. Of the different bioreactors used for microbial cell culture, most have been designed to allow for the production of high density cultures of a single cell type and as such, find use in the present invention.

[0121] Hence, the method for producing the agent comprises the steps of culturing a Mesorhizobium or a Mesorhizobium-li e microorganism for a time and under conditions to generate a sufficient number of cells L^"1.

[0122] In an embodiment, the method for producing the agent comprises the steps of culturing a microbial cell of the genus Mesorhizobium selected from the list comprising M. huakuii, M. loti, M. abyssinicae, M. albiziae, M. alhagi, M. amorphae, M. australicum, M. camelthorni, M. caraganae, M. chacoense, M. cicero, M. gobiense, M. hawassense, M. mediterraneum, M. metallidurans, M. muleiense, M. opportunistum, M. plurifarium, M. qingshengii, M. robiniae, M. sangaii, M. septentrionale, M. shangrilense, M. shonese, M. silamurunense, M. tamadayense, M. tarimense, M. temperatum, M. thiogangeticum or M. tianshanense for a time and under conditions to generate a sufficient number of cells L^"1. [0123] Taught herein is a method for producing the agent comprises the steps of culturing a microbial cell of the genus Mesorhizobium selected from M. huakuii and M. loti for a time and under conditions to generate a sufficient number of cells L^"1.

[0124] Enabled herein is a method for producing the agent comprises the steps of culturing a microbial cell designated CAM543 deposited at the National Measurements Institute on 29 October, 2014 under accession number V14/017216 for a time and under conditions to generate a sufficient number of cells L^"1.

[0125] The sufficient number of cells L^"1 depends on whether the aim is to generate a population of cells for a particular use (e.g. application to a surface) or whether the aim is to generate EPS. Sufficient levels of cells include, for example, 10 cells L^"1 to 10¹⁰ cells L^" Reference to " 10 cells L^"1 to 10¹⁰ cells L^"1" includes 10, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹ or 10¹⁰ cells L^"1 and levels in between.

[0126] Further contemplated here is the genetic modification of the Mesorhizobium or Mesorhizobium-like microorganism such as to introduce particular traits. Examples of genetic modification include generation of auxotrophic mutants and mutants which have the capacity to metabolize and grown on an expanded spectrum of carbon sources. The mutants may also be useful as proprietary markers.

[0127] Kits are also encompassed by the present invention. The kits include multi- compartmental containers wherein a first compartment may contain an inoculum of the Mesorhizobium or Mesorhizobium-like, microorganism; other compartments may comprise growth medium or various other nutrients or supplements and/or excipients.

[0128] The kits may be packaged for use at industrial sites. Antimicrobial kits comprising the isolated EPS, or a polysaccharide component thereof and other integers to be used to generate antimicrobial formulations and/or compositions. EXAMPLES

[0129] Aspects of certain embodiments of the present invention are further described by reference to the following non-limiting Examples.

Protocols

Isolation of CAM543

[0130] Simple Media (SM) was used to isolate bacteria from membrane samples and included 1000 ml of ultrapure (type 1) water, such as Milli-Q (trade mark) water: 3 g peptone, 1 g yeast extract, 15 g agar. SM with added glucose (SM+Glu) included 30 g L^"1 glucose, which was autoclaved separately and added prior to pouring plates. Simple Broth (SB) included all media components of SM except agar. Media was adjusted to pH 7 prior to autoclaving.

[0131] Spiral wound reverse osmosis (RO) membrane was used for 12 months in a brackish water treatment plant. Strips of RO membrane were sampled aseptically and place in 50 ml plastic test tubes. A 1 ml aliquot of sterile ultrapure (type 1) water, such as Millies (trade mark) water was added to the tubes, which were then mixed and 100 ml aliquots were removed to tubes containing 900 ml sterile water and diluted serially. Aliquots (200 μΐ) of 10^"3, 10^"4 and 10^"5 dilutions samples were spread on to SM plates. After air drying in a laminar flow cabinet, plates were incubated at 20°C until growth was visible. Individual colonies were transferred to new plates, streaked for isolation and incubated at 20°C. Isolates were sub cultured onto the same media on which they were originally isolated. This procedure was continued until one type of colony morphology was present on each plate.

Preliminary characterization of bacteria

[0132] Gram staining was used for initial characterization of bacterial isolates. Bacteria were examined at 1000X magnification with a Zeiss Axioplan microscope using phase contrast optics. The image was analyzed using Axio Vision 3.1 software.

[0133] Colony morphology of isolates was determined after growth for 3 weeks on SM. Growth of isolates sub cultured onto SM with added glucose was compared to that of the same isolate grown without glucose for the same period of time. Growth and presence of mucoid morphology were qualitatively assessed. For long term storage, isolates were suspended in liquid broth prepared with 30% (v/v) glycerol and transferred to 1.5 plastic cryovials and stored at -80°C. Growth of CAM543 for EPS production

[0134] Bacteria were revived from cryogenic storage by streaking the culture onto solid SM. For EPS production, SB contained HEPES (20 mM, pH range 6.8-8.2) as a biological buffer. SB with added glucose (SB+Glu) was prepared as described above. All media was adjusted to pH 7 prior to autoclaving. [0135] A 15 ml bottle of the SB+Glu was inoculated with individual colonies of strain CAM543 grown on SM and incubated at 20°C and shaken at 200 rpm on an orbital shaking table. After seven days, this liquid culture (primary) was used to inoculate 250 mL of SB+Glu in polycarbonate baffled flask, capped with a lid incorporating a 0.2 μιη filter.

[0136] After seven days, this secondary culture was transferred to a 2L flask containing 500 mL sterile SB+Glu. One week later, flasks were then inoculated with an inoculum volume equivalent to 10% (v/v) of the total. Culture media volumes, glucose concentrations and length of incubation varied as laid out in Table 2. Culture were incubated at 20°C and shaken at 200 rpm on an orbital shaking table.

Table 2. Description of treatments for shake flask cultures of CAM543

A SM+lO g/L GIc 500 2 7

C SM+30 g/L Glc 500 2 7

D SM+30 g/L Glc 250 1 7

E SM+40 g/L Glc 500 2 7

F SM+40 g/L Glc 250 1 7

J SM+30 g/L Glc 250 1 14

O SM+30 g/L Glc 500 2 21

[0137] The eight flasks were incubated at 20°C and shaken at 200 rpm until harvested for polysaccharides. After one week, a 10 mL aliquot was aseptically removed from each flask to a sterile test tube for pH, viscosity, glucose and optical density measurements. Isolation of polysaccharides

[0138] Cultures were centrifuged at 10, 000 X g and 20°C for 2 hr to remove cellular material. Supernatants were decanted and cell pellets were frozen, freeze-dried and weighed. Sodium azide (0.2 g L^"1) was added to supernatants to prevent bacterial growth. The liquids were filtered through a muffled (450°C, 18 hr) glass fiber filter and then through a cellulose nitrate filter (0.45 μιη). The filtrates were subjected to ultrafiltration to remove small proteins and media components and concentrate the EPS (100,000 Dalton MWCO). The concentrated retentates were frozen, freeze-dried and weighed. Characterization of polysaccharide

Crude chemical characterization

[0139] Solutions were prepared from EPS harvested from eight different flasks. The EPS were dissolved in ultrapure (type 1) water, such as Milli-Q (trade mark) water (1 mg mL^"1) and used for subsequent colorimetric assays. Uronic acid content of the EPS was determined by the meta-hydroxydiphenyl method (Blumenkrantz, N. & Asboe-Hansen, G. (1973) Analytical Biochemistry, 54 , 484; Filisetti-Cozzi, T. M. C. C. & Carpita, N. C. (1991) Analytical Biochemistry, 197, 157), with D-glucuronic acid as a standard. Protein content was determined by the bicinchoninic acid (BCA) protein assay (Smith, P. K., et al. (1985) Analytical Biochemistry, 150, Issue 1 , 76) with bovine serum albumin as the standard. The total neutral carbohydrate content was determined by the orcinol-sulfuric acid method (modified by Rimington, C. (1931) Biochemical Journal, 25, 1062), using D- glucose as a standard.

ATR-FTIR analysis of polysaccharide

[0140] Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectra were recorded on a Thermo Scientific Nicolet 6700 spectrometer equipped with diamond ATR accessories. For the diamond ATR crystal, the depth of penetration was approximately 2 μιη or less (depending on the wavenumber of the vibration band). Thirty- two scans were accumulated with a resolution of 4 cm^"1 for each spectrum. Rheometry

[0141] A 1 niL aliquot of a solution (10 mg^'ml^"1) of the polysaccharide was placed in the sample cup of a Brookfield micro viscometer connected to a recycling water bath, which maintained the temperature of the samples at 25°C during viscosity measurements. Sample temperature was allowed to equilibrate for 2 minutes, and then the shear rate was varied from 12 to 100 rpm. After 2 min at each shear rate, the viscosity was recorded.

Characterization of bacterial growth parameters

Growth of CAM543 in Pall microreactor

[0142] Two studies were performed using the Micro-24 micro reactor system. In the first experiment, SB media was supplemented with no additional carbon source (control) or with 1 % (w/v) of one of three alternatives: mannitol, glycerol or glucose. The pH was set to 7 and maintained with NH₃ (flow rate 5 cc min^"1). Dissolved oxygen was set to 50% (v/v) saturation. Temperature was varied (20, 22, 24, 26, 29 and 30°C). Cell number was determined by optical density (600 nm) as an indication of growth. Uninoculated media were used as blanks. Seed cultures of CAM543 were revived from cryostorage in SB. Cultures were incubated overnight at 30°C before being used as inoculum for this study.

[0143] In the second experiment, SB media was supplemented with no carbon source (control) or with 1 % (w/v) of one of five carbon sources: acetate, succinate, glycine, sorbitol, glycerol. Three micro-cultures (3 mL) were grown with no carbon source or with each of the five carbon source alternatives. The pH was set to 7 and maintained with NH₃ (flow rate 5 cc/min). Dissolved oxygen set to 50% (v/v) saturation. The temperature was set to 30°C. Inocula for this experiment were prepared as described above.

Growth of CAM543 on YMB and YMA

[0144] Further characterization of CAM543 was carried out on cultures grown on yeast mannitol broth (YMB), which consisted of mannitol (10 g^'L^"1), KH₂P0₄ (0.5 g^'L^"1), MgS0₄7H₂0 (0.9 g L^"1), NaCl (0.1 g L^"1), CaC0₃ (1.0 g L^"1), YE (0.4 g L^"1). Yeast mannitol agar (YMA) was prepared as above with the addition of agar (15 g L^"1) prior to autoclaving. The pH adjustments were made with 1 M NaOH. Morphological characteristics of cells and colonies were noted after 7 days of growth on YMA at 30°C. Colony diameter, color, surface appearance, opacity, texture, form, elevation and margin were recorded according to descriptive criteria set out by Smibert and Krieg (in Gerhardt, P., et al. (1994) Methods for General and Molecular Bacteriology, Washington D. C: American Society for Microbiology, 611).

[0145] CAM543 was tested for the ability to use melibiose as an alternative carbon sources to mannitol. YMA was prepared as above with the following changes. Either mannitol or melibiose was added at 1 g L^"1. YE was omitted and replaced with filter- sterilized trace metal supplement (10 mL L ¹ of a lOOx stock solution) and a filter- sterilized vitamin solution (10 mL L^"1 of a lOOx stock solution).

Flagella stain

[0146] Cultures of CAM543 were prepared for flagella staining (Heimbrook, M. E. et al. (1989) Journal of Clinical Microbiology, 27, 2612) by incubation in liquid YMB and on YMA for four days at 30°C. Bacteria were examined at 1600X magnification, (combination of xlOO Plan-Neofluar NA1.4 objective and Optovar 1.6) under a Zeiss Axioplan microscope using phase contrast, bright field and differential interference contrast optics. The image was captured using a Zeiss Axiocam model HRc and analyzed using Axio Vision 4.8.2 software.

Taxonomic characterization

16S rRNA gene sequence analysis

[0147] Colonies of CAM543 grown on YMA for 4 days were suspended in sterile distilled water and heated to 100°C for 10 min. After centrifugation, the supernatant was used as a template in polymerase chain reactions (PCR). PCR was carried out with a 'BAK' primer pair that targeted the 16S rRNA gene (Dasen, G. et al. (1998) Systematic and Applied Microbiology, 21, Issue 2, 251). PCR was also carried out with a primer pair targeting the internally transcribed spacer (ITS) region (Kwon, S. -W. et al. (2005) International Journal of Systematic and Evolutionary Microbiology, 55, Issue 1, 263). The PCR reaction solution included I μL· each of the appropriate primer solution (10 μΜ), 2 μL· mixed nucleotide solution (5 mM), 1 μL· Taq polymerase, 5 μΐ_^ PCR buffer (10X concentration), 5μί DNA template, 35 μΐ_^ distilled water. The PCR cycling reaction consisted of the following steps (1) 5 min at 94°C, (2) 1 min at 94 °C, (3) 1 min at 58°C, (4) 2 min at 72°C, (5) 10 min at 72°C, (6) 4°C, indefinitely. Steps 2 through 4 were repeated 35 times. The PCR product was purified with QIAquick kit and treated with BigDye terminator labeling reaction before being sequenced. Sequences were aligned using Vector NTI software then compared to online databases (National Centre for Biotechnology Information, U.S. National Institutes of Health).

Fatty acid analysis

[0148] Biomass of CAM543 was prepared for fatty acid (FA) analysis by inoculating YMA plates with cryostock cultures. Plated were incubated at 30°C for one week. Cell material was scraped from the agar with a sterile loop and transferred to CHCl₃-washed glass test tubes fitted with a Teflon-lined screw cap. Cell material was directly trans- methylated to produce FA methyl esters (FAME) using methanol-chloroform-conc. hydrochloric acid (3 ml, 10: 1: 1, 80°C, 2 hr, White, L. O. (1972) Journal of General Microbiology, 72, Issue 3, 565). FAME were extracted into hexane-chloroform (4: 1, 1.8 ml). The FAME samples were dried on a heat block (40°C) under a stream of nitrogen gas and re-dissolved in chloroform for the instrumental analysis.

[0149] FAME samples were analyzed by gas chromatography (GC) and filtered with a fused silica capillary column (15 m x 0.1 mm i.d., Ο. ΐμιη film thickness), an FID, a split/split less injector and an auto sampler and injector. Helium was the carrier gas. Samples were injected in split less mode at an oven temperature of 120°C. After injection, the oven temperature was raised to 270°C at 10°C min ¹ and finally to 300°C at 5°C min ¹. GC results are subject to an error of +5% of individual component area.

[0150] GC-mass spectrometric (GC-MS) analyses were performed on a Finnigan Thermo Electron Corporation Trace GC Ultra DSQ GC-MS; the system was fitted with an on-column injector and Thermoquest Xcalibur software (TX, USA). The GC was fitted with a capillary column of similar polarity to that described above. Individual components were identified using mass spectral data and by comparing retention time data with those obtained for authentic and laboratory standards. A full procedural blank analysis was performed concurrent to the sample batch, with FA below detection.

Microorganism deposit

[0151] Microorganism CAM543 was deposited at the National Measurements Institute on 29 October, 2014 under accession number V14/017216.

Example 1

Isolation of CAM543 and preliminary characterization

[0152] CAM543 was isolated from ultrapure (type 1) water, such as Milli-Q (trade mark) water that had been mixed with a small strip cut from an RO membrane. The cell suspension had been diluted (1: 1000) and spread on agar plates (SM, pH 9).

[0153] When grown on SM+Glu at pH 9 for 10 days at 20°C, the culture showed enhanced mucoid morphology relative to growth on SM under the same conditions.

Example 2

Growth of CAM543 for EPS production

[0154] CAM543 was grown in liquid shake flask cultures for EPS production. Final optical density of culture broths, measured at the time of harvesting, ranged from approximately 1.20 to 1.35 absorbance units, irrespective of the amount of glucose used or the length of incubation. The culture pH dropped from the initial pH of 7 to between 4.4 and 4.7 for all cultures. Final culture viscosity ranged from 1.1 to 1.8 cP (Table 3).

[0155] Biomass yield, as measured by the dry weight of the cell pellet after centrifugation of the culture, ranged from 600 mg L^"1 culture broth to 870 mg L^"1. EPS yield, as measured by the weight of freeze-dried EPS per liter of the culture broth, ranged from 200 mg L^"1 to 320 mg L^"1. Table 3. Results from shake flask cultures of CAM543 grown for extracellular polymeric substance production.

A SM+10 g L^_1 Glu 7 1.212 4.72 1.13 605 208

C SM+30 g L^_1 Glu 7 1.220 4.55 1.39 663 237

D SM+30 g L^_1 Glu 7 1.354 4.64 1.82 805 326

E SM+40 g L^_1 Glu 7 1.302 4.57 1.60 773 267

F SM+40 g L^_1 Glu 7 1.362 4.61 1.75 869 326

J SM+30 gl^_1 Glu 14 1.190 4.49 1.69 722 320

0 SM+30 g L^_1 Glu 21 1.190 4.43 1.50 639 220 Example 3

Characterization of polysaccharides

Crude chemical characterization

[0156] The results of analysis of crude chemical composition of seven EPS from Flasks A, C, D, E, F, J and O are presented in Table 4. Neutral sugars (63-85%) were the most abundant component on a percent of total dry weight EPS. Uronic acids comprised from 12-23% of the total dry weight. Proteins comprised 6-16% of the total dry weight of the EPS.

Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) analysis of polysaccharide

[0157] ATR-FTIR analysis yielded spectra with absorbance's centered around 1031, 1065, 1236, 1366, 1730, 2880, 2920, 3310 cm^"1 (Figure 1, top). The bonds indicated by these absorbance's are presented in Figure 1 (bottom).

Rheometry

[0158] The viscosity of small volume (1 mL) samples from a concentrated (10 mg mL^{" l}) aqueous EPS solution was measured in a cup and cone micro viscometer. The liquid seemed to display non-Newtonian behavior as shear stress did not vary at the same proportion as shear rate (Figure 2, top). In the viscosity versus shear rate plot (Figure 2, bottom), typical pseudo plastic characteristic was observed, as the liquid demonstrated 'shear thinning' behavior.

Table 4. Crude chemical composition of EPS derived from sever shake flask batch cultures of CAM543.

Neutral Uronic

Flask sugars acids Protein

designation (% total (% total (% total

dry wt) dry wt) dry wt)

A 63 20 16

C 74 20 8

D 72 20 13

E 81 23 6

F 76 21 9

J 67 12 10

0 85 17 6

Example 4

Characterization of bacteria

Growth of strains in Pall microreactor

[0159] In the first of two experiments in the micro reactor, CAM543 reached the maximum optical density (OD) after approximately 70 h in all cases. Glycerol produced the highest ODs of all four treatments. For cultures grown with no additional carbon source or with mannitol or glycerol, the highest OD was observed in cultures incubated at 30°C. For cultures grown with glucose added, the maximum OD was observed in cultures incubated at 28 °C (Figure 3).

[0160] In the second experiment, the maximum OD was observed after approximately 45 h in all cultures with the exception of the cultures grown in acetate or with glycine (Figure 4). In the acetate cultures, the maximum OD was reached after 70 h. Growth curves from this experiment were used to calculate a generation time of 10 h (Table 5). Glycine inhibited the growth of CAM543 in this experiment. Growth of CAM543 on YMB and YMA

[0161] CAM543 grown on YMA at 30°C for 7 days produced semi-translucent, off- white colonies that were 0.5 mm in diameter. Colonies had a smooth, convex surface, butyrus texture, circular shape and entire margin. Cells were Gram negative rods 2.5 μιη wide (Figure 5, bottom). Maximum temperature for growth on this media was 36°C. Growth was observed on YMA between pH 4 and 10. Growth occurred on YMA supplemented with 2.5% NaCl (w/v). Growth was also observed on melibiose as sole carbon source (Table 5).

[0162] Cultures of CAM543 growth on both YMB and YMA were observed at 1600X magnification with bright field, phase contrast, differential interference contrast (Figure 5) after flagella staining. There is a tentative indication of flagella in the phase contrast image (Figure 5, middle, top).

Table 5. Phenotypic characteristics of CAM543 and other closely related species of Mesorhizobium .

Phenotypic characteristic CAM54 M. huakuii^b M. lotf

Monotrichous flagella +? + +

Colony Diam. on YMA 30C (incubation time) 2-4 (7 days) 2-4 (5-6 days) 1(7 days)

Generation Time (h) ~10 4-6 N D

Max growth TemptC) 36 37 39

Max NaCl tolerance for growth (% w/v) >2.5 1 2

pH range for growth 4-10 5-9.5 4-10

Gram reaction - - -

Growth on Melibiose + + - this study

bdata from Chen, W.X. et al. (1991) International Journal of Systematic Bacteriology, 41(2), 275 within Nandasena, K. G. et al. (2009)

International Journal of Systematic Bacteriology, 59(9), 2140.

c from Jarvis, B. D. W. et al. (1982) International Journal of Systematic Bacteriology, 32(3), 378; Velazquez, E. et al. (2001)

International Journal of Systematic and Evolutionary Microbiology, 51(3), 1011 within Nandasena, K. G. et al. (2009) International

Journal of Systematic Bacteriology, 59(9), 2140.

Example 5

Taxonomic characterization

16S rRNA gene sequencing

[0163] In PCR carried out with the "BAK-primer" pair targeting the 16S rRNA gene of CAM543, the readable sequence (1381 base pairs) was most similar to that of Mesorhizobium huakuii and also very similar to other Mesorhizobium species when compared to online GenBank nucleotide databases. When compared to RNA sequences in the GenBank online database, the sequence of CAM543 was most similar to Mesorhizobium loti. PCR carried out with primer pairs targeting the internally transcribed spacer region of CAM543 DNA resulted in a sequence (1134 base pairs) most similar to Mesorhizobium huakuii in the online GenBank nucleotide database.

Fatty acid analysis

[0164] Results from whole cell fatty acid analyses of CAM543 grown on YMA, incubated at 30°C for 7 days are reported. Individual fatty acids in the profile for each culture are listed as a percentage of total area (Table 6). The most abundant fatty acids in all three cultures were 16:0, z^'17:0, 18: lco7c, Unknown 1 (19: l/c l9:0-like), c l9:0 and Unknown 2. These corresponded to the six most abundant fatty acids found in previous analyses of CAM543 grown in 2 L batch fermentation (freeze dried cell pellet, SM + 30g/L Glc, pH 7, 20°C, 9 days, data not shown) for EPS production (Table 6). When compared to fatty acid profiles reported in the literature (Tighe, S. W. et al. (2000) International Journal of Systematic and Evolutionary Microbiology, 50, Issue 2, 787) for Mesorhizobium huakuii and Mesorhizobium loti. There were similarities (16:0, z^'17:0, c l9:0), but several differences were also observed (Unknown 1, 11 methyl 18: lco7c and Unknown 2). The fatty acid 18: lco7c was present in CAM543 in significant amounts (15.5% of total fatty acids). Summed feature 7 included 18: lco7c (Tighe, S. W. et al. (2000) supra), but the relative abundance of this fatty acid in the total fatty acids of Mesorhizobium huakuii and Mesorhizobium loti is unclear from this previous study.

Table 6. Percentages of whole cell fatty acid of CAM543 grown on YMA at 30°C for 7 days compared to those found in the literature for other Mesorhizobium species grown under similar conditions.

Percent of Total Fatty Acids

Fatty Acids CAM543 M. huakuii ^Ta M loti ^Ta

1 6:0 9.7 1 5.8 14.5

i1 7:0 6.9 5.9 8.2

summed feature† - 42.0 33.8

1 8:1 co7c 1 5.5 - -

Unknown 1^c 1 7.5 - - cy 1 9:0 35.4 23.0 21 .9

1 1 methyl 1 8:1co7c - 4.8 1 1 .1

Unknown 2 6.6 - -

Total 91 .7 91 .5 89.5

a Tighe ef al. (2000) supra

b 18:½7c/aj9t ro12t, 18:1_m7c/aj9c/a>12t

c 19:1/cy19:0-like

[0165] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the inventions includes all such variations and modifications which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

BIBLIOGRAPHY

Armstrong, E. (2000) Biofouling, 16: 215-224

Bitton, G. & Friehofer, V. (1978) Microbial Ecology, 4, 119-125

Blumenkrantz, N. & Asboe-Hansen, G. (1973) Analytical Biochemistry, 54: 484-489

Caron, D. A. (1987) Microbial Ecology, 13: 203-218

Chen, W. X. et al. (1991) International Journal of Systematic Bacteriology, 41(2): 275-280

Dasen, G. et al. (1998) Systematic and Applied Microbiology, 21(2): 251-259

Deacon, G.B. and Phillips, R.J. (1980), Coordination Chemistry Reviews, 33, 227

Decho, A. W. (1990). In Barnes, M. ed, Oceanogr Mar Biol Annu Rev, Aberdeen: Aberdeen Univ Press: 73-153

Filisetti-Cozzi, T. M. C. C. & Carpita, N. C. (1991) Analytical Biochemistry, 197: 157-162

Flemming, H. -C. et al. (1997). In Keevil, C. W. et al. eds. Biofilms in the Aquatic Environment, Cambridge, UK: The Royal Society of Chemistry: 1-12

Gerhardt, P., et al. (1994) Methods for General and Molecular Bacteriology, Washington D. C: American Society for Microbiology: 611

Guezennec, J. et al. (2012) International Biodeterioration & Biodegradation, 6(1): 1-7

Heimbrook, M. E. et al. (1989) Journal of Clinical Microbiology, 27: 2612-2615

Hojjati, B. and Charpentier, P.A. (2001), Journal of Polymer Science Part A: Polymer Chemistry, 46, 3926

Holmstrom, C. et al. (2002) FEMS Microbial Ecology, 41 : 47-58

Jarvis, B. D. W. et al. (1982) International Journal of Systematic Bacteriology, 32(3): 378- 380

Jeanthon, C. & Prieur, D. (1990) Applied and Environmental Microbiology, 56(11): 3308- 3314

Kariduraganavar, M.Y, et al. (2009), Industrial and Engineering Chemistry Research, 48, 4002

Krembs, C. et al. (2002) Deep-Sea Research Part I: Oceanographic Research Papers, 49(12): 2163-2181

Kucera, J. (2010) Reverse osmosis: design, processes, and applications for engineers. 9, John Wiley and Sons

Kwon, S. -W. et al. (2005) International Journal of Systematic and Evolutionary Microbiology, 55(1): 263-270

Nakamoto, K. (1997), Infrared and Raman Spectra of Inorganic and Coordination Compounds. A Wiley-Interscience Publication, New York, Part B, 59

Nandasena, K. G. et al. (2009) International Journal of Systematic and Evolutionary Microbiology, 59(9): 2140-2147

Paerl, H. W. (1976) Journal ofPhycology, 12: 431-435

Rimington, C. (1931) Biochemical Journal, 25: 1062-1071

Roberson, E. B. et al. (1993) Soil Biology and Biochemistry, 25(9): 1299-1301

Selbmann, L. et al. (2002) Research in Microbiology, 153: 585-592

Singh, S. & Fett, W. F. (1995) Microbiology Letters, 130(2-3): 301-306

Smith, P. K. et al. (1985) Analytical Biochemistry, 150(1): 76-85

Uekewa, N. et al. (2006), Journal of the Ceramic Society of Japan, 114(10), 807 Velazquez, E. et al. (2001) International Journal of Systematic and Evolutionary Microbiology, 51(3): 1011-1021

White, L. O. (1972) Journal of General Microbiology, 72(3): 565-574

Wotton, R. S. (2004) Scientia Marina, 68(suppl): 13-21

Yi, Y. et al. (2010), US Patent No. 2010/0239493

Claims

CLAIMS:

1. An agent comprising:

i. an isolated microbial cell of the genus Mesorhizobium or a Mesorhizobium-li e microorganism; or

ii. an extracellular polymeric substance (EPS) produced by a microbial cell of the genus Mesorhizobium or a Mesorhizobium-li e microorganism; or iii. a polysaccharide component of the EPS produced by a microbial cell of the genus Mesorhizobium or a Mesorhizobium-li e microorganism;

wherein the microbial cell and/or EPS and/or polysaccharide exhibits antimicrobial properties towards other microorganisms.

2. The agent of Claim 1 wherein the Mesorhizobium or Mesorhizobium-li e microorganism comprises a Gram negative bacterium with monotrichous flagella, grows to a colony diameter of 1 to 4 mm after 7 days on yeast mannitol agar (YMA), exhibits a generation time of 4 to 10 hours in yeast mannitol broth (YMB), tolerates a maximum growth temperature of 36 to 39°C, exhibits a maximum NaCl tolerance for growth of 1 to >2.5 (%w/v), tolerates a pH range for growth of 4 to 10 and exhibits an ability to grow on melibiose.

3. The agent of any one of Claims 1 or 2 wherein the Mesorhizobium is a species selected from the list comprising Mesorhizobium huakuii; Mesorhizobium loti; Mesorhizobium abyssinicae; Mesorhizobium albiziae; Mesorhizobium alhagi; Mesorhizobium amorphae; Mesorhizobium australicum; Mesorhizobium camelthorni; Mesorhizobium caraganae; Mesorhizobium chacoense; Mesorhizobium cicero; Mesorhizobium gobiense; Mesorhizobium hawassense; Mesorhizobium mediterraneum; Mesorhizobium metallidurans; Mesorhizobium muleiense; Mesorhizobium opportunistum; Mesorhizobium plurifarium; Mesorhizobium qingshengii; Mesorhizobium robiniae; Mesorhizobium sangaii; Mesorhizobium septentrionale; Mesorhizobium shangrilense; Mesorhizobium shonese; Mesorhizobium silamurunense; Mesorhizobium tamadayense; Mesorhizobium tarimense; Mesorhizobium temperatum; Mesorhizobium thiogangeticum; and Mesorhizobium tianshanense.

4. The agent of Claim 3 wherein the species of Mesorhizobium is Mesorhizobium huakuii ox Mesorhizobium loti.

5. The agent of Claim 4 wherein the microbial cell is designated CAM543 and deposited at the National Measurements Institute on 29 October, 2014 under accession number V14/017216.

6. The agent of any one of Claims 1 to 5 wherein the EPS or its polysaccharide component exhibits an attenuated total reflectance-Fourier transform infrared spectrum with an indication of carboxyl and sulfate groups.

7. The agent of any one of Claims 1 to 5 wherein the EPS or its polysaccharide component exhibits an attenuated total reflectance-Fourier transform infrared spectrum of Figure 1.

8. The agent of Claim 1 wherein the antimicrobial properties are selected from inhibiting attachment, growth or maintenance of bacteria, algae and/or fungi to a surface.

9. The agent of Claim 8 wherein the agent exhibits bacteriostatic or bactericidal properties.

10. The agent of Claim 1 wherein the antimicrobial properties comprise inhibition or reduction of adhesion of a microorganism to a surface.

11. An isolated EPS or its polysaccharide component produced by a microbial cell of the genus Mesorhizobium or by a Mesorhizobium-li e microorganism.

12. The EPS of Claim 11 wherein the EPS is excreted EPS.

13. The EPS of Claim 11 wherein the EPS is capsular EPS.

14. The EPS or polysaccharide of Claim 11 wherein the Mesorhizobium or Mesorhizobium-li e microorganism comprises a Gram negative bacterium with monotrichous flagella, grows to a colony diameter of 1 to 4 mm after 7 days on yeast mannitol agar (YMA), exhibits a generation time of 4 to 10 hours in yeast mannitol broth (YMB), tolerates a maximum growth temperature of 36 to 39°C, exhibits a maximum NaCl tolerance for growth of 1 to >2.5 (%w/v), tolerates a pH range for growth of 4 to 10 and exhibits an ability to grow on melibiose.

15. The EPS or polysaccharide of Claim 11 wherein the Mesorhizobium is a species selected from the list comprising Mesorhizobium huakuii; Mesorhizobium loti; Mesorhizobium abyssinicae; Mesorhizobium albiziae; Mesorhizobium alhagi; Mesorhizobium amorphae; Mesorhizobium australicum; Mesorhizobium camelthorni; Mesorhizobium caraganae; Mesorhizobium chacoense; Mesorhizobium cicero; Mesorhizobium gobiense; Mesorhizobium hawassense; Mesorhizobium mediterraneum; Mesorhizobium metallidurans; Mesorhizobium muleiense; Mesorhizobium opportunistum; Mesorhizobium plurifarium; Mesorhizobium qingshengii; Mesorhizobium robiniae; Mesorhizobium sangaii; Mesorhizobium septentrionale; Mesorhizobium shangrilense; Mesorhizobium shonese; Mesorhizobium silamurunense; Mesorhizobium tamadayense; Mesorhizobium tarimense; Mesorhizobium temperatum; Mesorhizobium thiogangeticum; and Mesorhizobium tianshanense.

16. The EPS of polysaccharide of Claim 11 wherein the species of Mesorhizobium is Mesorhizobium huakuii or Mesorhizobium loti.

17. The EPS or polysaccharide of Claim 11 wherein the microbial cell is designated CAM543 and deposited at the National Measurements Institute on 29 October, 2014 under accession number V14/017216.

18. An isolated polysaccharide derived from the EPS of Claim 11.

19. The agent of any one of Claims 1 to 6 or the EPS of Claim 11 or the polysaccharide of Claim 18 wherein the agent, EPS or polysaccharide inhibits attachment, growth and/or maintenance of a microorganism on a surface.

20. The agent of any one of Claims 1 to 6 or the EPS of Claim 11 or the polysaccharide of Claim 18 wherein the agent, EPS or polysaccharide inhibits or reduces adhesion of a microorganism to a surface.

21. The agent, EPS or polysaccharide of any one of Claims 19 or 20 wherein the surface is selected from the group consisting of silica, silicon, semi-conductors, glass, polymers, organic compounds, inorganic compounds, metals and combinations thereof.

22. The agent, EPS or polysaccharide of Claim 21 wherein the metal is selected from the group consisting of gold, copper, stainless steel, nickel, aluminium, titanium, thermosensitive alloys and combinations thereof.

23. The agent of any one of Claims 19 to 21 wherein the surface is a membrane.

24. The agent of any one of Claims 19 to 21 wherein the surface is a semipermeable membrane.

25. A method for producing the agent of Claim 1 comprising culturing a Mesorhizobium or a Mesorhizobium-li e microorganism for a time and under conditions sufficient to generate a sufficient number of cells/L.

26. The method of Claim 25 wherein the culture is a continuous culture maintained at 20°C and wherein the microorganism is suspended in liquid broth comprising a sugar.

27. The method of Claim 26 wherein EPS is separated from cellular material by centrifugation and isolated by filtration.

28. A surface comprising a Mesorhizobium or a Mesorhizobium-li e microorganism, or an EPS produced thereby, or a polysaccharide component thereof.

29. The surface of Claim 28 wherein the Mesorhizobium or Mesorhizobium-li e microorganism comprises a Gram negative bacterium with monotrichous flagella, grows to a colony diameter of 1 to 4 mm after 7 days on yeast mannitol agar (YMA), exhibits a generation time of 4 to 10 hours in yeast mannitol broth (YMB), tolerates a maximum growth temperature of 36 to 39°C, exhibits a maximum NaCl tolerance for growth of 1 to >2.5 (%w/v), tolerates a pH range for growth of 4 to 10 and exhibits an ability to grow on melibiose.

30. The surface of Claim 28 wherein the Mesorhizobium is a species selected from the list comprising Mesorhizobium huakuii; Mesorhizobium loti; Mesorhizobium abyssinicae; Mesorhizobium albiziae; Mesorhizobium alhagi; Mesorhizobium amorphae; Mesorhizobium australicum; Mesorhizobium camelthorni; Mesorhizobium caraganae; Mesorhizobium chacoense; Mesorhizobium cicero; Mesorhizobium gobiense; Mesorhizobium hawassense; Mesorhizobium mediterraneum; Mesorhizobium metallidurans; Mesorhizobium muleiense; Mesorhizobium opportunistum; Mesorhizobium plurifarium; Mesorhizobium qingshengii; Mesorhizobium robiniae; Mesorhizobium sangaii; Mesorhizobium septentrionale; Mesorhizobium shangrilense; Mesorhizobium shonese; Mesorhizobium silamurunense; Mesorhizobium tamadayense; Mesorhizobium tarimense; Mesorhizobium temperatum; Mesorhizobium thiogangeticum; and Mesorhizobium tianshanense.

31. The surface of Claim 30 wherein the species of Mesorhizobium is Mesorhizobium huakuii or Mesorhizobium loti.

32. The surface of Claim 28 wherein the Mesorhizobium or Mesorhizobium-li e microorganism is designated CAM543 and deposited at the National Measurements Institute on 29 October, 2014 under accession number V14/017216.

33. The surface of any one of Claims 28 to 32 wherein the surface is selected from the group consisting of silica, silicon, semi-conductors, glass, polymers, organic compounds, inorganic compounds, metals and combinations thereof.

34. The surface of any one of Claims 28 to 32 wherein the surface is a metal selected from the group consisting of gold, copper, stainless steel, nickel, aluminium, titanium, thermosensitive alloys and combinations thereof.

35. The surface of any one of Claims 28 to 32 wherein the surface is a membrane.

36. The surface of any one of Claims 28 to 32 wherein the surface is a semipermeable membrane.

37. A method for inhibiting growth of an unwanted microorganism at a selected site, said method comprising generating a biofilm comprising Mesorhizobium or Mesorhizobium-li e microorganisms, or forming a coating of the EPS or a polysaccharide component thereof produced by a Mesorhizobium or a Mesorhizobium-li e microorganism at the selected site.

38. The method of Claim 37 wherein the Mesorhizobium or Mesorhizobium-like microorganism comprises a Gram negative bacterium with monotrichous flagella, grows to a colony diameter of 1 to 4 mm after 7 days on yeast mannitol agar (YMA), exhibits a generation time of 4 to 10 hours in yeast mannitol broth (YMB), tolerates a maximum growth temperature of 36 to 39°C, exhibits a maximum NaCl tolerance for growth of 1 to >2.5 (%w/v), tolerates a pH range for growth of 4 to 10 and exhibits an ability to grow on melibiose.

39. The method of Claim 38 wherein the Mesorhizobium is a species selected from the list comprising Mesorhizobium huakuii; Mesorhizobium loti; Mesorhizobium abyssinicae; Mesorhizobium albiziae; Mesorhizobium alhagi; Mesorhizobium amorphae; Mesorhizobium australicum; Mesorhizobium camelthorni; Mesorhizobium caraganae; Mesorhizobium chacoense; Mesorhizobium cicero; Mesorhizobium gobiense; Mesorhizobium hawassense; Mesorhizobium mediterraneum; Mesorhizobium metallidurans; Mesorhizobium muleiense; Mesorhizobium opportunistum; Mesorhizobium plurifarium; Mesorhizobium qingshengii; Mesorhizobium robiniae; Mesorhizobium sangaii; Mesorhizobium septentrionale; Mesorhizobium shangrilense; Mesorhizobium shonese; Mesorhizobium silamurunense; Mesorhizobium tamadayense; Mesorhizobium tarimense; Mesorhizobium temperatum; Mesorhizobium thiogangeticum; and Mesorhizobium tianshanense.

40. The method of Claim 39 wherein the species of Mesorhizobium is Mesorhizobium huakuii ox Mesorhizobium loti.

41. The method of Claim 40 wherein the Mesorhizobium or Mesorhizobium-li e microorganism is designated CAM543 and deposited at the National Measurements Institute on 29 October, 2014 under accession number V14/017216.

42. The method of Claim 37 wherein the EPS or its polysaccharide component exhibits an attenuated total reflectance-Fourier transform infrared spectrum with an indication of carboxyl and sulfate groups.

43. The method of Claim 37 wherein the EPS or its polysaccharide component exhibits an attenuated total reflectance-Fourier transform infrared spectrum of Figure 1.

44. The method of Claim 37 wherein the microbial cells, EPS or polysaccharide exhibit antimicrobial properties comprising inhibiting attachment, growth and/or maintenance of bacteria, algae and/or fungi.

45. The method of Claim 37 wherein the microbial cells, EPS or polysaccharide inhibit or reduce adhesion of microorganisms to a surface.

46. The method of Claim 37 wherein the microbial cells, EPS or polysaccharide exhibit bacteriostatic or bactericidal properties.

47. A formulation comprising the agent of Claim 1 wherein the agent is an EPS or its polysaccharide component.

48. A kit comprising a storable sample of a Mesorhizobium or Mesorhizobium-li e microorganism for use in inoculating a culture flask or bioreactor or for applying to a selected site.

49. The kit of Claim 48 further comprising nutrients to facilitate the growth of the microorganism.

50. The kit of any one of Claims 48 or 49 further comprising instructions for use.

51. Use of a Mesorhizobium or Mesorhizobium-li e microorganism in the manufacture of an antimicrobial agent.

52. The use of Claim 51 wherein the antimicrobial agent is an EPS or a polysaccharide component thereof derived from a Mesorhizobium or Mesorhizobium-li e microorganism.

53. The use of any one of Claims 51 or 52 wherein the agent inhibits or reduces adhesion of a microorganism to a surface.