CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit of, and priority from, US provisional application No. 61/987,824, filed on May 2, 2014, the contents of which are hereby incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to honey glycoproteins for use as antimicrobial agents, including for topical applications and wound dressings.

BACKGROUND OF THE INVENTION

[0003] Antibacterial properties of honey have been well documented; several compounds are found in honey that considerably contribute to its antibacterial activity, such as hydrogen peroxide [1, 2], methylglyoxal [3, 4], leptosin [5], melanoidins [6], oxidative stress and hydroxyl radicals [7-9]. The sheer number of these compounds might suggest that honey works through a multimodal mechanism of action and that it is quite possibly due to this multimodality that honey remains effective in inhibiting growth of a broad spectrum of bacterial species. In the art, there is no single compound in honey known to exhibit antibacterial efficacy exceeding the effect of other contributing antibacterial compounds that are present, or that demonstrates a direct correlation with the total honey antibacterial activity.

SUMMARY OF THE INVENTION

[0004] This invention relates to an isolated glycoprotein, isolated from honey. The glycoprotein comprises a protein moiety and at least two carbohydrate moieties. The protein moiety comprises the amino acid sequence of Major Royal Jelly Protein 1 (MRJP1), and two or more of the at least two carbohydrate moieties each comprise a mannosylated glycan. The MRJP1 sequence includes, in the C-terminus region, amino acid sequence TPFKISIHL (SEQ ID N0:3) that is cleavable to yield three antimicrobial peptides known as Jelleins, particularly Jellein 1 , Jellein 2 and Jellein 4.

[0005] As described herein, the isolated glycoprotein has surprisingly been found to have antimicrobial activity, likely due to both the lectin-like binding properties of the isolated glycoprotein and cell wall degrading activities. That is, as described herein, the isolated glycoprotein has been shown to agglutinate bacteria and to degrade bacterial cell walls or membranes.

[0006] Thus, the isolated glycoprotein provides a natural source for an antimicrobial agent that may be used to treat surfaces, including surfaces such as skin or wounds.

[0007] In one aspect, the present invention provides a composition, the composition comprising: an isolated glycoprotein comprising a protein moiety that comprises the amino acid sequence of Major Royal Jelly Protein 1 (MRJP1), the MRJP1 sequence comprising the amino acid sequence TPFKISIHL in the C-terminal region, and at least two carbohydrate moieties, wherein two or more of the at least two carbohydrate moieties each comprise a mannosylated glycan.

[0008] The MRJP1 may comprise, for example, the sequence of SEQ ID NO: 1 or SEQ ID NO:2.

[0009] In some embodiments, one or more of the mannosylated glycans may be a mannosylated N-glycan. Alternatively or additionally, one or more of the mannosylated glycans may be a mannosylated O-glycan. One or more of the mannosylated glycans may each comprises 4 to 9 mannose residues.

[0010] In the composition, the isolated glycoprotein may be present at a

concentration equal to or greater than a minimum inhibitory concentration or a minimum bactericidal concentration. For example, in some embodiments, the isolated glycoprotein is present in the composition at a concentration of from about 0.1 μg/ml to about 1 mg/ml, or any subrange or value therein.

[0011] The composition may further comprise a pharmaceutically acceptable carrier, or may comprise other components to formulate the composition for topical

administration.

[0012] The composition may further comprise an additional antimicrobial agent.

[0013] The composition may be used to treat a wound, including reduction or prevention of contamination or infection with a multidrug resistant bacteria.

[0014] In another aspect, the present invention provides a wound dressing having a surface for contacting a wound, the dressing comprising a composition applied on or impregnated in said surface, the composition comprising an isolated glycoprotein comprising a protein moiety that comprises the amino acid sequence of Major Royal Jelly Protein 1 (MRJP1), the MRJP1 sequence comprising the amino acid sequence

TPFKISIHL in the C-terminal region, and at least two carbohydrate moieties, wherein two or more of the at least two carbohydrate moieties each comprise a mannosylated glycan.

[0015] In another aspect, the present invention provides a method of treating a surface, the method comprising contacting the surface with a composition comprising an isolated glycoprotein comprising a protein moiety that comprises the amino acid sequence of Major Royal Jelly Protein 1 (MRJP1), the MRJP1 sequence comprising the amino acid sequence TPFKISIHL in the C-terminal region, and at least two carbohydrate moieties, wherein two or more of the at least two carbohydrate moieties each comprise a mannosylated glycan.

[0016] The surface may be, for example, skin or a wound of a human or non-human animal.

[0017] Contacting may comprise applying the wound dressing of the invention.

[0018] Treating may comprise reducing or preventing contamination or infection with a multidrug resistant bacteria.

[0019] In another aspect, the present invention provides use of a composition comprising an isolated glycoprotein for treating a wound, the isolated glycoprotein comprising a protein moiety that comprises the amino acid sequence of Major Royal Jelly Protein 1 (MRJPl), the MRJPl sequence comprising the amino acid sequence

TPFKISIHL in the C-terminal region, and at least two carbohydrate moieties, wherein two or more of the at least two carbohydrate moieties each comprise a mannosylated glycan.

[0020] In another aspect, the present invention provides use an isolated glycoprotein in the manufacture of a composition of the invention for treating a wound, the isolated glycoprotein comprising a protein moiety that comprises the amino acid sequence of Major Royal Jelly Protein 1 (MRJPl), the MRJPl sequence comprising the amino acid sequence TPFKISIHL in the C-terminal region, and at least two carbohydrate moieties, wherein two or more of the at least two carbohydrate moieties each comprise a mannosylated glycan.

[0021] The uses may include treating the wound to reduce or prevent contamination or infection with a multidrug resistant bacteria.

[0022] The composition used with the wound dressing, method or uses may be any composition of the invention as described herein.

[0023] Other aspects and features of the present invention will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In the figures, which illustrate, by way of example only, embodiments of the present invention are as follows.

[0025] Figure 1 is a series of SDS-PAGE profiles of proteins and glycoproteins found in various honeys.

[0026] Figure 2 is a series of micrographs of rat red blood cells treated with glycoproteins isolated from honeys, or with phytohemagglutinin (PHA). [0027] Figure 3 is a series of micrographs showing agglutination of bacterial cells by isolated honey glycoproteins.

[0028] Figure 4 is a series of micrographs showing bacterial cell wall lysis by isolated honey glycoproteins: (A) E. coli; and (B) B. subtilis.

[0029] Figure 5 is a series of micrographs depicting cell morphological changes of bacterial cultures induced by isolated honey glycoproteins.

[0030] Figure 6 is a series of electron micrographs depicting cell morphological changes of bacterial cultures induced by isolated honey glycoprotein G208.

[0031] Figure 7 shows the effect of various concentrations of isolated honey glycoproteins on bacterial growth rates.

[0032] Figure 8 shows the effect of various concentrations of isolated honey glycoproteins on bacterial survival rates.

[0033] Figure 9 shows the effect of various concentrations of non-glycosylated honey fractions on bacterial growth rates.

[0034] Figures 10 and 11 show the amino acid sequence of the protein moiety of isolated honey glycoproteins G208 and G210.

[0035] Figures 12 and 13 are series of micrographs of E. coli and B. subtilis, either untreated control or treated with glycoproteins isolated from honeys.

[0036] Figure 14 shows the antibacterial effect of glycosylated and deglycosylated glycoproteins isolated from honeys.

[0037] Figure 15 shows the amino acid sequence of the protein moiety of isolated honey glycoproteins 61 kDa band and 29 kDa band from glycoproteins isolated from honeys.

[0038] Figure 16 is a series of photographs of ID and 2D gels of proteins and glycoproteins found in various honeys. [0039] Figure 17 shows the inhibitory effect of ampicillin (positive control) and various concentrations of isolated glycoproteins from honey on E. coli and B. subtilis.

[0040] Figure 18 shows the inhibitory effect of ampicillin and isolated glycoproteins from honey on multidrug resistant bacterial strains.

[0041] Figures 19 and 20 show the inhibitory effect of isolated glycoproteins from honey on multidrug resistant bacterial strains.

DETAILED DESCRIPTION

[0042] The compositions, products, methods, and uses described herein relate to the identification of a glycoprotein isolated from honey, which has been found to have antimicrobial properties, including agglutination and bactericidal properties. The bactericidal properties may include activity against multidrug resistant bacteria, such as various clinical isolates of multidrug resistant bacterial strains.

[0043] A glycoprotein has been identified in honey that is now shown to exhibit lectin-like and bacteriolytic properties. The glycoprotein has the ability to recognize and agglutinate bacterial cells, and to inhibit bacterial growth and survival. Thus, the isolated glycoprotein may be useful for treating a surface, including a wound surface or skin, to reduce microbial load on the surface. The isolated glycoprotein may thus also be included in a wound dressing for application to a wound or skin surface.

[0044] Surprisingly, the protein sequence of the isolated glycoprotein was identified as the full-length Major Royal Jelly Protein 1 (MRJP1) precursor protein. The C- terminal portion of the MRJP1 contains three known antimicrobial peptide sequences referred to as Jelleins (specifically Jelleins 1, 2 and 4. MRJP1 is a protein found in royal jelly that is cleaved to release antibacterial Jellein peptides), which are generated by differential cleavage of the C-terminus of the protein, which contains the sequence TPFKISIHL (SEQ ID NO:3). However, as described herein, the glycosylated, full-length protein is now surprisingly shown to also exhibit antimicrobial properties in honey, including antibacterial properties, even without cleaving to release the Jellein peptides. . [0045] Thus, there is presently provided a composition that comprises a glycoprotein isolated from honey. The term "isolated" means purified, or at least partially purified, in order to remove the glycoprotein from the honey, or to remove or capture a honey fraction containing the glycoprotein but having at least some of the other honey components removed from the fraction. Thus, prior to inclusion in the composition, the isolated glycoprotein is in purified or partially purified form. Alternatively to purification from whole honey, the isolated glycoprotein may be synthesized as a recombinant protein in an expression system that results in glycosylation of the expressed protein, and may be purified or partially purified from that expression system. Thus, the isolated glycoprotein is free from, substantially free from, or at least partially free from, whole honey, regardless of whether it is obtained by fractionating whole honey or by recombinant methods.

[0046] As will be appreciated, the molecular weight of the isolated glycoprotein will vary depending on the degree of glycosylation and the precise carbohydrate groups present in any particular embodiment. In some embodiments, the isolated glycoprotein has an apparent molecular weight of about 56 kDa to about 61 kDa, or about 56 kDa, about 59 kDa or about 61 kDa, as determined by SDS-PAGE. The unglycosylated protein moiety of the isolated glycoprotein may have a molecular mass of about 48.9 kDa.

[0047] Thus, as will be appreciated, the isolated glycoprotein comprises a protein moiety and at least two carbohydrate moieties.

[0048] The protein moiety of the isolated honey glycoprotein comprises the amino acid sequence of MRJP1. The MRJP1 may by any variant of MRJP1 from any honey or from any bee species. The MRJP1 sequence includes the amino acid sequence

TPFKISIHL (SEQ ID NO: 3) in the C-terminal region of the full-length protein, meaning that the sequence occurs towards the C-terminal end of the protein, but that there may be one, two, five, ten or more amino acids downstream of the TPFKISIHL sequence. In some embodiments, the sequence TPFKISIHL is at the C-terminal end of the protein moiety, meaning the TPFKISIHL is the final sequence of the MRJP1 protein sequence and there are no additional amino acids downstream of the TPFKISIHL sequence in the MRJP1 protein moiety.

[0049] In some embodiments, the protein moiety may comprise, consist of, or consist essentially of the following amino acid sequence: MTRLFMLVCL GIVCQGTTGN ILRGESLNKS LPILHEWKFF DYDFGSDERR QDAILSGEYD YKNNYPSDID QWHDKIFVTM LRYNGVPSSL NVI SKKVGDG GPLLQPYPDW SFAKYDDCSG IVSASKLAID KCDRLWVLDS GLVNNTQPMC SPKLLTFDLT TSQLLKQVEI PHDVAVNATT GKGRLSSLAV QSLDCNTNSD TMVYIADEKG EGLIVYHNSD DSFHRLTSNT FDYD-PKFTK MTIDGESYTA QDGISGMALS PMTNNLYYSP

VASTSLYYVN TEQFRTSDYQ QNDIHYEGVQ NILDTQSSAK VVSKSGVLFF GLVGDSALGC WNEHRTLERH NIRTVAQSDE TLQMIASMKI KEALPHVPIF DRYINREYIL VLSNKMQKMV NNDFNFDDVN FRIMNANVNE LILNTRCENP DNDRTPFKIS IHL (SEQ ID NO: 1).

[0050] In SEQ ID NO: 1, the "-" represents an unknown in the sequence, and thus may be represented by X, which may be any amino acid or which may be absent.

[0051] In some embodiments, the protein moiety may comprise, consist of, or consist essentially of the following amino acid sequence: MTRLFMLVCL GIVCQGTTGN ILRGESLNKS LPILHEWKFF DYDFGSDERR QDAILSGEYD YKNNYPSDID QWHDKIFVTM LRYNGVPSSL NVISKKVGDG GPLLQPYPDW SFAKYDDCSG IVSASKLAID KCDRLWVLDS GLVNNTQPMC SPKLLTFDLT TSQLLKQVEI PHDVAVNATT GKGRLSSLAV QSLDCNTNSD TMVYIADEKG EGLIVYHNSD DSFHRLTSNT FDYDPKFTKM TIDGESYTAQ DGISGMALSP MTNNLYYSPV ASTSLYYVNT EQFRTSDYQQ NDIHYEGVQN ILDTQSSAKV VSKSGVLFFG LVGDSALGCW NEHRTLERHN IRTVAQSDET LQMIASMKIK EALPHVPIFD RYINREYILV LSNKMQKMVN NDFNFDDVNF RIMNANVNEL ILNTRCENPD NDRTPFKISI HL (SEQ ID NO: 2).

[0052] The term "consists essentially of or "consisting essentially of as used herein means that a polypeptide may have additional features or elements beyond those described provided that such additional features or elements do not materially affect the ability of the polypeptide to function as the protein moiety of the isolated glycoprotein as an antimicrobial agent. That is, the polypeptide may have additional features or elements that do not interfere with the agglutination functionality or the cell wall/membrane degradation functionality of the glycoprotein. For example, a polypeptide consisting essentially of a specified sequence may contain one, two, three, four, five or more additional amino acids, at one or both ends of the sequence provided that the additional amino acids do not interfere with, inhibit, block or interrupt the antimicrobial activity of the glycoprotein. Similarly, a polypeptide molecule may be chemically modified with one or more functional groups provided that such chemical groups do not inhibit, block or interrupt the antimicrobial activity of the glycoprotein.

[0053] The isolated honey glycoprotein also comprises at least two carbohydrate moieties. For example, the glycoprotein may comprise two, three, four, five, ten, fifteen, twenty, twenty-five, twenty-eight or thirty carbohydrate moieties. For example, the glycoprotein may comprise two or more, three or more, four or more, five or more, ten or more, fifteen or more, twenty or more, twenty-five or more, twenty-six or more, twenty- seven or more, twenty-eight or more, twenty-nine or more, or thirty or more carbohydrate moieties.

[0054] At least two of the carbohydrate moieties of the isolated honey glycoprotein each comprise a mannosylated glycan, which is capable of binding to Concanavalin A. In some embodiments, more than two of the carbohydrate moieties of the isolated glycoprotein is each a mannosylated glycan. In some embodiments, each of the carbohydrate moieties of the isolated glycoprotein is a mannosylated glycan.

[0055] A mannosylated glycan is any glycan comprising one or more mannose residues. For each mannosylated glycan present in the isolated glycoprotein, the mannosylated glycan may be a glycan containing multiple mannose residues, for example 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more mannose residues. In some embodiments, the mannosylated glycan comprises from 4 to 9 mannose residues. The mannosylated glycan may be, for example, a mannosylated N-glycan or a mannsoylated O-glycan, and may comprise glucosaminyl residues. For example, the mannosylated glycan may be a mannosylated N-glycan and may comprise (Manal-3[Manal-6]Man) linked to an N-acetyl β-glycosaminyl group. For example, the mannosylated glycan may comprise Man4GlcNAc2, Man5GlcNAc2, Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2, or Man9GlcNAc2. In some embodiments, the mannosylated glycan may be, for example, a mannosylated O-glycan.

[0056] In some embodiments, in addition to the two or more mannosylated glycans, the isolated glyocoprotein may comprise other types of carbohydrate moieties, including other N-glycans or O-glycans, for example sialylated glycans or fucosylated glycans.

[0057] Thus, the isolated glycoprotein is capable of binding Concanavalin A via two or more carbohydrate moieties.

[0058] The isolated glycoprotein may be readily isolated from honey, using routine laboratory protein chemistry and purification methods. For example, Concanavalin A affinity chromatography may be used, including in a manner as described in the

Examples below.

[0059] The isolated glycoprotein may also be prepared using recombinant methods. For example, a nucleic acid sequence encoding the MRJP1 protein sequence may be included in an expression vector for expression in a suitable expression system that will allow for glycosylation of the expressed protein moiety, including with at least two mannosylated glycan groups. Suitable expression systems for expressing recombinant glycoproteins are known, including for example expression systems that use baculovirus vectors in insect cells or that use mammalian vectors in CHO cells. To assist with purification of the recombinant isolated glycoprotein, the glycoprotein may be engineered to have a fused N-terminal or C-terminal affinity tag, for example a six-histidine tag, a FLAG sequence tag or a glutathione-S-transferase tag. The engineered glycoprotein with a fused affinity tag may be then be purified using conventional techniques, including the appropriate affinity chromatography column such as a nickel chelating column, an anti- FLAG antibody column or a glutathione column. The affinity tag may be subsequently cleaved from the isolated glycoprotein.

[0060] Recombinant methods may allow for convenient production of readily attainable and significant amounts of the isolated glycoprotien. Using recombinant methods to produce the glycoprotein may also allow for a consistent glycosylation pattern on the recombinant protein, as the expression conditions such as host cell type, growth medium and growth conditions can be more readily controlled.

[0061] The isolated glycoprotein, which comprises full-length, glycosylated MRJP1 , has now been shown to exhibit antimicrobial properties, meaning that it may inhibit growth, and in some cases may kill, microbes, including for example, bacteria, and fungi including yeast. The isolated glycoprotein may inhibit the growth of bacteria or kill bacteria, including a bacterial strain that exhibits multidrug resistance (MDR).

[0062] Antibiotic resistance among Gram-negative bacteria to beta-lactam antibiotics is on a rise due to emergence of extended- spectrum β-lactamase (ESBL). Emergence of MDR bacterial pathogens poses a significant threat to the ability to control and treat bacterial infections. In particular, antibiotic resistance among Gram-negative bacteria to β-lactam antibiotics is dramatically increasing. Exposure of bacteria to new β-lactam drugs intensifies frequency of mutations in β-lactamase gene leading to emergence of new, extended-spectrum β-lactamase (ESBL), including metallo^-lactamase such as the novel New Delhi metallo^-lactamase 1 (NDM-1) (Bradford, 2001). ESBL are able to hydrolyze almost all clinically-available β-lactams limiting therapeutic options (Paterson and Yu, 1999). ESBL are often encoded by genes located on large plasmids together with genes conferring resistance to other antibiotics such as aminoglycosides or tetracyclines (Paterson and Bonomo, 2005), and can be transferred to other bacterial species by horizontal gene transfer via mobile elements such as plasmids and transposons (Warnes et al, 2012). These mechanisms of transmission effectively accelerate the spread of MDR bacteria causing nosocomial outbreaks worldwide.

[0063] As use herein, reference to multidrug resistance or to multidrug resistant (MDR) bacteria is reference to bacteria that are simultaneously resistant to at least one antimicrobial agent from each of two or more different classes of antimicrobial agents, and thus may be resistant to at least two different antimicrobial mechanisms of action. MDR bacteria may be gram-negative or gram-positive (including mycobacteria) bacteria, and may include different species or strains of Klebsiella, Escherichia, Pseudomonas, Staphylococcus including methicillin resistant (MRSA) strains, Enterococcus including vancomycin resistant (VRE) strains, Proteus, Enter obacteriaceae, Acinetobacter, Streptococcus, Salmonella, and Mycobacterium. MDR bacteria include bacterial species and strains that express an extended spectrum beta lactamase (ESBL) or a New Delhi metallo beta lactamase (NDM). MDR bacteria may include strains of Escherichia coli (including ESBL or NDM strains), Staphylococcus aureus (including MRSA strains), Klebsiella pneumoniae (including ESBL strains), Pseudomonas aeruginosa, Proteus mirabilis, and Enterococcus faecium (including VRE strains).

[0064] The isolated glycoprotein exhibits lectin-like properties. That is, the isolated glycoprotein is able to agglutinate bacteria, with specificity similar to that of

Concanavalin A, but may not be able to hemagglutinate red blood cells. Without being limited by theory, the isolated glycoprotein appears to be able to recognise certain cell wall or membrane components of microbes, including bacteria, such as certain carbohydrate structures, which may be present on microbial cell surfaces but not on the surface of red blood cells. Recognition and binding of the microbial cell wall/membrane component leads to agglutination of the microbes by the isolated glycoprotein, causing clumping and aggregation. The isolated glycoprotein also appears to be involved in cell wall or membrane degradation, including bacterial cell wall or membrane degradation, which in some cases may lead to changes in morphology of the microbe, and/or death of the microbe. The cell wall/membrane degradation may occur via permeabilization, for example by pore formation, by the isolated glycoprotein acting on the microbial cell wall or membrane. Thus, the isolated glycoprotein may have dual functionality: (i) agglutination activity, mediated at least in part by the carbohydrate moiety; and (ii) wall/membrane permeabilization, likely mediated at least in part by the protein moiety.

[0065] The isolated glycoprotein may be included in the composition in an effective amount, including antimicrobially effective. The term "effective amount" as used herein means an amount effective, at dosages and for periods of time necessary to achieve the desired result, for example, to render a surface including a wound surface, antimicrobial, or to have an antimicrobial effect. Thus, the glycoprotein may be included in the composition at a concentration sufficient for the glycoprotein to have an antimicrobial effect, i.e. an effective amount, including an antimicrobially effective amount.

[0066] For example, the isolated glycoprotein may be present at a concentration equal to or greater than the minimum inhibitory concentration (MIC) or the the minimum bactericidal concentration (MBC). The MIC and the MBC for an. isolated glycoprotein prepared from a specific honey can be readily determined using routine laboratory methods. Assays to determine MIC and MBC are known in the art, including for example as described in the Examples below. For example, the MIC or MBC may be determined using a broth microdilution assay in compliance with the National Committee on Clinical Laboratory Standards (NCCLS) at a bacterial concentration of 10 ⁶ CFU/ml and the standard plate count, respectively. The amount of isolated glycoprotein required to have an antimicrobial effect may depend on bacterial load (concentrations) under the particular circumstances for which the composition is to be used. For example, for higher concentrations of bacteria, 2xMBC or a greater multiplicity of MBC of the isolated glycoprotein may be required to have a bactericidal effect, and 2xMIC or a greater multiplicity of MIC of the isolated glycoprotein may be required to have a growth inhibition effect. As well, the multiplicity of MBC/MIC required may vary depending on the growth phase of the bacteria: exponential growth (dividing) versus stationary growth (nondividing).

[0067] In some embodiments, the isolated glycoprotein may be present in the composition at a concentration from about 0.1 μg/ml to about 1 mg/ml, from about 0.1 μg/ml to about 100 μg/ml, from about 1 μg/ml to about 1 mg/ml, from about 10 μg/ml to about 1 mg/ml, from about 25 μg/ml to about 1 mg/ml, from about 50 μg/ml to about 1 mg/ml, from about 75 μg/ml to about 1 mg/ml, from about 100 μg/ml to about 1 mg/ml, from about 250 μg/ml to about 1 mg/ml, from about 500 μg/ml to about 1 mg/ml, from about 750 μg/ml to about 1 mg/ml, or from about 1 μg/ml to about 100 μg/ml. The isolated glycoprotein may also be present in the composition at any intermediate concentration or sub-ranges falling within these recited ranges. The concentration of the isolated glycoprotein in the composition may exceed the concentration of the

glycoprotein that naturally occurs in honey. [0068] As indicated above, the isolated glycoprotein may weaken the cell wall or membrane of the microbe. In order to increase the antimicrobial efficacy of the composition, it may be desirable to include an additional antimicrobial agent in the composition. For example, the additional antimicrobial agent may be an antibiotic, a fungicide, a viricide, a sulphonamide, a silver salt, a copper salt, a zinc salt, or a plant essential oil. In some embodiments, the composition may include hydrogen peroxide as an additional antimicrobial agent.

[0069] The composition may further include other additional components depending on the particular application for which the composition is intended to be used. For example, the composition may include pharmaceutically acceptable carriers, buffers, antioxidants, protease inhibitors or stabilizers, or any additional components required to stabilize the isolated glycoprotein, and/or provide the composition with desired consistency and cohesion for the relevant application.

[0070] The composition may be formulated as a solution, a spray, a gel, a cream, a foam, a lotion, a hydrogel, or other form that allows for application to a surface, including a surface such as skin or a wound.

[0071] Thus, in some cases, the surface may be human or animal skin, or a human or animal wound, or a surface intended to contact human or animal skin or wound.

Therefore, the composition may be formulated to be pharmaceutically acceptable. The compositions may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives and various compatible diluents and carriers. The proportion and identity of any pharmaceutically acceptable diluent or carrier is determined by chosen route of administration, compatibility with live cells, and standard pharmaceutical practice. Generally, the pharmaceutical composition will be formulated with components that will not destroy or significantly impair the biological properties of the isolated glycoprotein.

[0072] The pharmaceutical composition can be prepared by known methods for the preparation of pharmaceutically acceptable compositions suitable for administration to subjects, such that an effective quantity of the isolated glycoprotein, and any additional active substance or substances, is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999. On this basis, the pharmaceutical compositions include, albeit not exclusively, solutions of isolated glycoprotein, in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffer solutions with a suitable pH and iso-osmotic with physiological fluids.

[0073] The composition may be useful for applying to a surface in order to provide the surface with antimicrobial activity and/or to reduce the microbial load on the surface. For example, the composition may be used topically to sanitize skin or a wound, including skin or a wound that may be susceptible to contamination of infection with, or that may be contaminated or infected with, MDR bacteria. Thus, the composition may be formulated with suitable vehicles, diluents, carriers, etc. for topical administration to a non-human or human animal subject.

[0074] Thus, the composition may be applied directly to skin or to a wound surface in order to provide the skin or wound with antimicrobial activity. For example, the composition may be applied by spreading or spraying over a region of skin or over a surface of a wound.

[0075] The composition may also be applied to a material or product that is to be placed over the skin or wound, including for example a wound dressing. To this end, there is also provided a wound dressing comprising the composition.

[0076] The wound dressing has a surface intended for contact with a wound to be dressed, such that in use, the surface is placed in contact with or adjacent to the wound. The surface for contacting a wound has the above-described composition applied thereon or impregnated therein, and thus carries or includes the composition.

[0077] The wound dressing may be any material that is to be applied to a wound in order to prevent infection and/or to assist in healing of the wound. The wound dressing may be occlusive, semi-permeable or permeable, depending on the type of dressing and the type of wound to be dressed. The dressing may be designed to absorb exudate or to allow exudate to pass through to a secondary layer of dressing, to stop bleeding, to protect the wound from infection or contamination, and/or to promote healing of the wound.

[0078] The wound dressing may comprise any material used to dress a wound, including for example, fabric, gauze, polymeric material, or silicone. The wound dressing may comprise a low adherent gauze or fabric, or may comprise a polymeric or silicone film.

[0079] The wound dressing may be further impregnated or coated with other agents useful in wound healing, for example biological molecules involved in wound repair, and/or additional antimicrobial agents.

[0080] The compositions and wound dressings as described herein can be used to treat a surface, which surface is intended to be provided with antimicrobial activity. For example, human or non-human animal skin or a wound of a human or non-human animal.

[0081] Thus, there is also provided a method of treating a surface. As indicated above, the surface may be any surface that is to be treated to inhibit or kill microbes or to reduce microbial load on the surface. In some embodiments, the surface is skin of a human or non-human animal. In some embodiments, the surface is a wound of a human or non-human animal, for example a surface of a wound.

[0082] In the method, the composition is applied to the surface.

[0083] The composition may be reapplied as necessary, including at regular intervals, over a period of time during which the surface is desired to be provided with

antimicrobial activity.

[0084] The composition may be applied in amount sufficient to provide a layer of the composition to the surface. The composition may be spread on or smoothed over the surface, if desired.

[0085] Additionally or alternatively, a wound dressing may also be applied to the surface, particularly in embodiments where the surface is a wound surface. Thus, in some embodiments, the method is a method of dressing or treating a wound, comprising applying the composition as described herein and/or the wound dressing as described herein to a surface of a wound.

[0086] If the wound dressing is to be applied to the wound, the wound dressing is applied so that the surface of the dressing that carries or includes the composition is in contact with the wound.

[0087] The wound dressing may be removed and a fresh wound dressing applied as necessary or desired, including at regular intervals, for example over a period of time during which the surface is desired to be provided with antimicrobial activity or during which the wound is dressed to assist with healing.

[0088] Also contemplated are uses of the composition and wound dressing, included uses relating to treating a surface, or uses related to dressing or treating a wound.

[0089] The present compositions, products, methods and uses are further exemplified by way of the following non-limiting examples.

EXAMPLES

[0090] EXAMPLE 1

[0091] Summary

[0092] Concavalin A chromatography was used to isolate glycoproteins from honey. The glycoprotein fractions (glps) from the affinity chromatography, but not the flow- through fractions, were found to exhibit strong growth inhibitory and bactericidal properties. The MIC/MBC values of glps isolated from different honeys ranged from 13^g/ml to 2^g/ml against Escherichia coli to the MICs 13.8-1.24μg/ml against Bacillus subtilis. The glps agglutinated both bacterial species but did not agglutinate red blood cells. Light and scanning electron microscopy demonstrated that glps caused membrane permeabilization and shrinkage of red blood cells, while in bacteria they caused cell wall degradation, formation of spheroplast/protoplasts and led to a complete cell lysis. Glps thus appear to operate by two distinct functionalities; via binding selectivity to bacterial targets and agglutination, likely due to the high-mannose structure, and a non-specific pore-formation/ membrane permeabilization activity observed in both bacterial cells and erythrocytes. Time-kill kinetics in combination with microscopic observations revealed that the fate of cell wall-deficient forms dependent of glps concentration, and bacterial growth phase. At concentration of lxMBC, glps irreversibly led to cell lysis of exponentially growing cells, reducing bacterial counts by >5-log ₁₀ within 15 min incubation. In contrast, sub-optimal glps concentrations allowed some cell wall- deficient forms to revert to mature cells. MALDI-TOF and electrospray quadrupole time of flight mass spectrometry, (ESI-Q-TOF-MS/MS) analysis of the main 61kDa band shared by all isolated glps showed sequence identity with the Major Royal Jelly Protein 1 (MRJP1) precursor that harbors three antimicrobial peptides: Jelleins 1, 2 and 4.

Together, the presence of Jellein sequences within the glps protein moiety could reconcile and explained membrane damaging effects caused by MRJP while the high-mannose moiety in the MRJP 1 could explain the lectin-like activity.

[0093] Materials and Methods

[0094] Honeys: Honeys were donated by Canadian beekeepers and included unprocessed, raw samples (Table 1).

Table 1. Honey Types and Sources

Year of

Honey Botanical origin Melissopalynology Donated by:

harvest

Parker-Bee

HI 77 buckwheat 2010 fagopyrum

Apiaries

Podolski Honey

H207 rapeseed/salix 2012 brassica/ salix

Farm

goldenrod/rapeseed/ solidago/brassica/ Honey& Q H208 2013

buckwheat fagopyrum Corporation

rhamnaceae/ligustrum/

H210 wildflower 2012 Bees41ife

trifolium [0095] Isolation of Honey Glycoproteins: Glycoproteins were directly isolated from 25% aqueous honey solutions using the agarose-immobilized Concavalin A spin columns (Glycoprotein Isolation Kit, ConA, Thermo Scientific, Rockford IL). The isolation was conducted according to the manufacturer's manual. The isolation process was monitored by SDS-PAGE followed by Coomassie blue staining, PAS-staining and functional assays. In the functional assay, the flow-through fractions, wash-out fractions and fractions retained by the ConA columns were tested for their growth inhibitory activity in broth microdilution susceptibility tests.

[0096] Determination of Protein Concentration: Protein quantification was performed by addition of 200 of Bradford dye reagent (brilliant blue G, 0.1 mg/niL; ethanol, 5% (v/v); phosphoric acid, 10% (v/v) and water) to 40 μΐ, of samples, pre- diluted 100 times [36]. The photometrical measure was performed at 595 nm. Bovine serum albumin was used to generate a standard curve. Measurements were done in triplicate.

[0097] SDS-PAGE Electrophoresis: SDS-PAGE was performed according to the method described by Laemmli, 1970 [37]. Honey proteins were analyzed on 7.5% gel separation gel with attached 5% stacking gel, while glycoproteins were analyzed on 12% gels. 30 μΐ of a honey solution (50% v/v) or 15μ1 of a fraction from ConA

chromatography were mixed with the sample buffer, denatured at 100°C for 5 min and loaded on a gel. The electrophoresis was carried out in duplicate at a constant current of 100 Volts using a Mini Protean III electrophoresis cell (Bio-Rad Laboratories, Hercules, CA). After electrophoresis, the gel was either stained with Brilliant Blue R-250 (Bio- Rad) for honey proteins or stained with periodic acid Schiff base (PAS-staining) to visualize glycoproteins. The molecular weight of the proteins was determined using molecular weight standards (Fermentas Life Sciences, PageRuler Prestained Protein Ladder #SM0671).

[0098] Periodic Acid Schiff (PAS) Staining: Following SDS-PAGE, glycosylated proteins were detected using the Pierce Glycoproteins staning kit (Thermo Scientific, Rockford IL), based on the standard periodic acid Schiff base that employs acidic fuchsin dye. The kit included PAS-positive (horseradish peroxidase) and PAS-negative controls (soybean trypsin inhibitor).

[0099] Preparation of rat red blood cells suspension: Rat blood was collected in tubes containing EDTA and centrifuged at 3,000 x g for 5 min at 4°C. Red blood cells were washed three times in Tris- buffered saline (TBS) containing 10 mM EDTA, followed by two other washes with 20 volumes of TBS (pH 7.2) containing 75 mM NaCl. Erythrocytes were then resuspended in TBS as a 10% (vol/vol) suspension and immediately used in hemagglutination assay.

[00100] Rapid Hemagglutination Assays: The rapid determination of

hemagglutination activity of glycoprotein fractions was performed on microscope slides using freshly prepared rat red blood cells (RBCs). Ten microliters of glycoprotein fractions was mixed with an equal volume of a 2% RBC suspension and after gentle shaking for 1 min, the agglutination was observed in a light microscope at 400x magnification and photographed. TBS and phytohemagglutinin were used as a negative and positive control, respectively.

[00101] Bacterial Strains and Growth Cultures and Experimental Design:

Standard strains of Bacillus subtilis (ATCC 6633) and Escherichia coli (ATCC 14948) purchased from Thermo Fisher Scientific Remel Products (Lenexa, KS 66215) were grown in Mueller-Hinton Broth (MHB) (Difco Laboratories) overnight in a shaking water bath at 37°C. Overnight cultures were diluted with broth to the equivalent of the 0.5 McFarland standard.

[00102] Broth Microdilution Assay and Determination of the MIC: The susceptibility of E. coli and B. subtilis (10 ⁶ CFU/ml) to honey glycoproteins was analyzed spectrophotometrically using broth microdilution assay in a 96 well microtitre plate format. Each well contained 1 ΙΟμΙ of inoculum and 15μ1 of two-fold serial dilutions of glycoproteins (starting from 40 μg/ml), as described previously for honey [2]. Bacterial growth was measured at A595 nm using the Synergy HT multidetection microplate reader (Synergy HT, Bio-Tek Instruments, Winooski, VT, USA). Growth inhibition and the MIC was evaluated by recording the concentration of glycoproteins that reduced bacterial growth by 99% in comparison to a control, untreated culture, after 18 h incubation with shaking at 37°C. Statistical analysis and dose response curves were obtained using K4 software provided by Synergy HT (Bio-Tek Instruments, Winooski, VT, USA).

[00103] Determination of bactericidal activity of glycoproteins and the MBC:

Bacterial survival, after 18 hr incubation with glycoproteins, was assessed using a standard plate count. The entire contents (ΙΟΟμΙ) of experimental wells (in triplicate) from the broth microdilution assays were directly streaked into the Mueller-Hinton agar (MHA) plates. The viable cells were enumerated after overnight incubation of agar plates at 37°C and compared to the viable cell counts of control cultures. To verify the final cell density of bacteria not exposed to glycoproteins, ΙΟμΙ samples were taken from wells containing inoculum only (assay control) and serially 10-fold diluted with sterile water to obtain approximate cell density of 10 ⁴ and 10 ² CFU/ml. Then ΙΟμΙ and ΙΟΟμΙ aliquot from each dilution was streaked, in duplicate, onto agar plates. The MBC endpoint was determined as the minimum concentration of glycoproteins at which 99.9% of the initial inoculum was eradicated and at which only one or no colonies could be seen on MHA.

[00104] Determination of Time-Kill Kinetics and Morphological Changes: E. coli and B. subtilis culture (10 ⁶ CFU/ml in 1 10 μΐ) were grown in 96-well plates until they reach early log phase (A59 ₅ nm 0.2-0.3) at which point each of glycoproteins (G177, G208 and G210) was added (15 μΐ) in triplicate to separate wells at lxMIC and 0.5x MIC concentrations. The plates were incubated at 37° C for 18 hr. The plates served as a starting material to examine (a) the time-kill kinetics and (b) morphological changes.

[00105] Time-Kill Kinetics: The time-kill assay was used to determine both the rate of killing by glycoporteins and the concentrations of glycoproteins required for cell death. After bacterial cultures reached exponential growth, the inhibitory action and killing rate were measured simultaneously at 0, 15 min, 30 min, 45 min, 60 min, 120 min and 18 hr by the absorbance readings at A ₅ 9 ₅ nm and by viable cell counts using a standard plate count. The killing curves were constructed by withdrawing ΙΟμΙ aliquots from wells containing inoculum (assay control) and experimental wells. The ΙΟμΙ aliquots were serially 10-fold diluted with sterile water to obtain cell density ranging from 10 ⁴ to 10 ² CFU/ml and then a 10 μΐ and 100 μΐ aliquot from each dilution were streaked onto Mueller-Hinton agar (MHA) plates. After 18 hr incubation at 37°C, the viable cells were enumerated.

[00106] Cell Morphology Examination by Light Microscopy: To examine morphological changes in bacterial cells induced by glycoproteins at different growth phases, 10 μΐ samples were removed from the experimental and control wells at the exponential phase of growth (after 3 hr incubation) and at stationary phase (18 hr incubation) from the 96 well plates. The samples were examined on glass slides at 400 x magnifications under light microscope (Zeiss, Axiolab, Germany). Images were viewed and photographed using the digital camera and built-in software (Singer Instruments MSM 400, Somerset, UK).

[00107] Bacterial Agglutination Assay: For qualitative determination of agglutinating activity, the 10 μΐ of undiluted overnight cultures of B. subtilis and E. coli were mixed with 10 μΐ glycoproteins on glass slides, while ΙΟμΙ of Concavalin A (100 μg/ml) and TBS were used as a positive and negative control, respectively. The mixtures were incubated for 15 min with gentle rotation and the agglutination was viewed under light at the magnification of 400 ^χ under light microscope (Zeiss, Axiolab, Germany). Images were viewed and photographed using the digital camera and built-in software (Singer Instruments MSM 400, Somerset, UK).

[00108] Scanning Electron Microscope (SEM): For SEM analysis, overnight cultures of B. subtilis and E. coli were incubated with glycoproteins at IxMBC for 1 hr at 37°C. Cells were fixed in 2.5% glutaraldehyde (EM grade, Sigma-Aldrich, St. Louis, MO, USA) in 0.1 M Tris-buffered saline (TBS), pH 7.3 for 15 min at room temperature. The fixed samples were dehydrated with graded ethanol series, and re-suspended in pure ethanol. One drop of the solution was put on the membrane (0.22 μπι, Corning, NY, USA), and then air dried. The samples were sputter coated with gold before SEM observation. The microscopy was performed with a Hitachi S-3000 (Hitachi, Japan) and operated at 10 kV. [00109] Enzymatic Deglycosylation: Lyophilized glycoproteins (100 μg) were dissolved in 30 μΐ deionized water, heated to 100°C for 5 min, cooled to room

temperature and deglycosylated using the Enzymatic Protein Deglycosylation Kit (Sigma- Aldrich, St. Louis, MO, USA), according to the manufacturer's instruction. The extent of deglycosylation was assessed based on the mobility shifts on SDS-PAGE gels as compared to undigested original sample.

[00110] Protein Identification by MALDI-ToF Mass Spectrometry (MS): The deglycosylated 61 kDa protein bands were excised from Coomassie blue-stained gels, cut into 1-mm cubes, and washed in distilled water followed by acetonitrile: 100 mM

NH ₄ HC0 ₃ solutions, as described in Protocols, the Biological Mass Spectrometry Laboratory, UWO (www.bmsl.uwo.ca/in-gel_digestion.html). Proteins were reduced with dithiothreitol, followed by alkylation with iodoacetamide to prevent free thiol groups from cross-linking. Gel bands were dried in acetonitrile and rehydrated in ammonium bicarbonate. Proteins in the gel were digested by incubation with nonself cleaving trypsin (Promega, Madison, WI) for 16 h at 37°C, extracted with a acetonitrile/sodium bicarbonate solution and dried in a speed vacuum. Prior to mass spectrometric analysis, dried peptide samples were re-dissolved in a 0.2% formic acid and 3 μΐ of volumes were injected into mass spectrometer.

[00111] ESI-Q-TOF-MS MS Analysis: ESI-Q-TOF- MS/MS analyses

(electrospray quadrupole time of flight mass spectrometry) were performed using a Q- TOF Global mass spectrometer (Waters) and run in positive mode using electrospray interface. MS acquisition was obtained with MassLynx 4.1 software (Waters). The columns consisted of Waters nanoAcquity UPLC column (BEH130 CI 8) with a trap column (Symmetry CI 8). Peptides were resolved at 300 nl/min over a 75 min in a gradient of water (A) and 0.2% formic acid (B): methanol as shown in Table 2. Table 2. MS Solvent Gradient

%B %A Time (min)

5 95 Initial

60 40 40.0

95 5 42.5

[00112] All MS/MS spectra were analyzed using the following databases:

Mascot® using the NCBI database (NCBI 2010) with all entries and PEAKS.

[00113] Protein identification was performed by the MASCOT search engine

(www.matrixscience.com) against the NCBInr protein and Swiss-Prot/TrEMBL databases using peptide mass fingerprinting (PMF). The following parameters were used for database search: 1) taxonomy group 2) mass tolerance of 0.2 Da, 3) one missed tryptic cleavage allowed, 4) carboamidomethylation of cysteine (as a fixed modification) and 5) oxidation of methionine (as a variable modification). Proteins were identified by

MASCOT using the probability-based MOWSE score, equal to -lOXLog(P), where P is the probability that the observed match is a random event. Protein scores of >53 were considered statistically significant (P<0.05) under the selected variables.

[00114] PEAKS has also integrated PTM and mutation characterization through automatic peptide sequence tag based searching (SPIDER) and PTM identification.

[00115] Statistical Analysis: Analyses were performed using the statistical program GraphPad Instat version 3.05. (GraphPad Software Inc.). Data were analyzed using a one-way ANOVA with subsequent Tukey-Kramer Multiple Comparison test or an unpaired t-test. Differences between means were considered to be significant at p<0.05.

[00116] Figure Legends

[00117] Figure 1. Comparison of protein and glycoprotein profiles in different honeys by SDS-PAGE. Panel I, (A) and (B): Differences in polypeptide profiles in honeys of different botanical origin. Panel II. Positive identification of glycoproteins separated by the ConA affinity chromatograph: (A) Coomassie Blue- stained proteins, (B) PAS-stained glycoproteins, including PAS-positive (horseradish peroxidase) and PAS-negative controls (soybean trypsin inhibitor). Panel III (A) Comparison of protein and glycoprotein profiles before and after ConA chromatography in different honeys. (B) Mobility shift of deglycosylated glycoproteins (the arrows).

[00118] Figure 2. Membrane effects of honey glycoproteins on rat RBCs. A. Microscopic visualization of morphological changes in RBCs treated with glycoproteins or phytohemagglutinin (PHA) as compared to untreated cells, a. Control, b. G207, c. G210, d. PHA, e. G177 and f, 208. Insets illustrate a transition of RBCs morphology from biconcave (a) to echinocyte form (b and f) after treatment with glycoproteins.

[00119] Figure 3. Agglutination of bacterial cells by honey glycoproteins; (a)E. coli agglutination by soybean lectin (b) E. coli agglutination by glycoprotein G208, (c)B. subtilis agglutination by Concavalin A, and (d) B. subtilis agglutination by glycoprotein G177.

[00120] Figure 4. A. E. coli cell wall lysis caused by glycoproteins, a. Control culture of E. coli at logarithmic phase of growth, b. E. coli incubated with G177, c. G208 and d. G210. B. B. subtilis cell wall lysis caused by glycoproteins, a. Control cultures of B. subtilis at logarithmic phase of growth, b. Cultures of B. subtilis incubated with G177, c. G208 and d. G210.

[00121] Figure 5. The transition of bacterial cell shape from rod-like to coccoidal forms at stationary phase of growth, (a) B. subtilis- control, (b) B. subtilis incubated with glycoprotein G208, (c) E. coli -control, (d) E. coli incubated with glycoprotein G208.

[00122] Figure 6. Scanning electron microscopy of E. coli transition from rod-like form to coccoidal form during incubation with glycoprotein G208. a. Control culture, b. and c. shape-changes in E. coli cells treated with G208, (d) cell lysis after G208- treatemnet (scale bar, 5 μιη). Backgrounds are formed by filter.

[00123] Figure 7. Effect of glycoprotein concentrations (lxMIC and 0.5xMIC) on growth rates of E. coli and B. subtilis. [00124] Figure 8. Effect of glycoprotein concentrations (lxMIC and O.^xMIC) on survival of E. coli and B. subtilis.

[00125] Figure 9. Growth inhibition of E. coli and B. subtilis by flow-through fractions from ConA chromatography.

[00 26] Figures 10 and 11. Summary of fully annotated matched peptides between the MRJP1 and 61 kDa, deglycosylated protein (Figure 10: G208, Figure 11 : G210). Blue bars indicate sequence coverage and confirmed identifications.

[00127] Results

[00128] Isolation Of Lectin-Like Glycoproteins From Honey

[00129] Examination of the most dominant proteins in honeys of different botanical origin using SDS-PAGE revealed that all honeys contained prominent bands grouped in the range of 60-70 kDa, while proteins of 25 and 35 kDa seemed to be uniquely present in honeys HI 77, 206, 207, 208 that showed significant amounts of buckwheat pollen (Figure 1, panel I- A, B and Table 1).

[00130] Since many disease resistance proteins are postranslationally glycosylated [20], PAS-staining, a method that is specific for detecting glycans, was used to visualize honey glycoproteins on SDS gels (Figure 1, panel II- A, B). As seen in Figure 1 (panel III A), the SDS-PAGE profiles of glycoproteins (glps) and honey proteins from which they originated were surprisingly similar, suggesting that glycosylation might have here a functional significance. While glycosylation increases structural stability of proteins and their resistance to proteolysis, carbohydrates in glps may also function as recognition determinants of other glycoconjugates and are often responsible for cell adhesion and agglutination. Therefore, there was a high potential that honey glps could have ability to react with surface-exposed carbohydrates on bacterial membranes, causing their agglutination, thereby contributing to the overall antibacterial activity.

[00131] Honey glps were selectively separated using Concavalin-A (ConA) affinity chromatography. ConA is a lectin that specifically binds high-mannose-type N- glycans, a type of glycans often present in the cell wall of Gram- positive and Gram- negative bacteria [21]. Among ConA-captured proteins were two abundant polypeptides; the 31 kDa protein, present in G177 and G208, and the 61 kDa protein present in all tested honeys (with exception of honey H207) (Figure 1. Panel IIIA).

[00132] Agglutinating activity of the isolated glps was monitored using a rapid, direct hemagglutination assay on microscope slides using freshly isolated rat red blood cells (RBCs). The effects of the interaction between glps G177, G208, G207 and G210 with RBCs were examined under light microscope using phytoheamagglutinin (PHA) as a positive control. PHA, another high-mannose-type lectin, clearly formed clumps of rat RBCs on microscopic slides during 30 min incubation at room temperature (Figure 2d). In contrast, honey glps were unable to bind and agglutinate RBS under the same conditions (Figure 2). However, the exposure of RBCs to glps G177 and G208 resulted in a rapid transition from biconcave shape of normal erythrocytes into crenated, shrank echinocytes (Figure 2, e and f). The transition was accompanied by the formation of holes and membranes blebs (Figure 2, insets) indicating that the integrity of erythrocyte membranes became compromised. Although glps G207 and G210 did not produce such extensive membrane defects, they too influenced membrane permeability, causing changes that resembled the early phases of echinocyte development (Figure 2, b).

Differences in the glps profiles, as seen in the SDS-PAGE (Figure 1, panel III A), seemed to be a factor that distinguished glps actions based on severity of the membrane effects they caused to the rat RBC (Figure 2). It could be concluded that some of these lower molecular weight polypeptides, such as those present in glps G177 and G208, contributed more to the membrane disruptions than others (G207 and G210).

[00133] Agglutinating And Cell Wall Depleting/ Lytic Activities Of Honey Glycoproteins

[00134] Exposure of overnight bacterial cultures to glps resulted in visible agglutination of both Gram- positive Bacillus subtilis (ATCC-6633) and Gram-negative Escherichia coli (ATCC-14948) (Figure 3). All four glycoproteins clumped bacterial cells in a similar way to ConA-lectin (Figure 3). [00135] To determine whether glps exert similar membrane effect on bacterial cells, the bacterial susceptibility to glps was analyzed using broth microdilution assay in 96-well format. Overnight cultures of B. subtilis and E. coli diluted to 10 ⁶ CFU/ml were exposed to serially two-fold diluted glps. At two time-points of bacterial growth; at the beginning of exponential growth and at the stationary phase, 10 μΐ aliquots were withdrawn from incubation wells for microscopic observations. Results revealed an extensive disruption of the bacterial cell walls leading to the appearance of small, rounded forms (Figure 4 A and B). This lytic activity of glycoproteins was specifically intensive in dividing bacteria during their exponential growth and occurred

indiscriminately in both Gram-positive and Gram-negative bacteria (Figure 4 A and B).

[00136] At the entry to a stationary phase, the majority of E. coli and B. subtilis cells had their cell-shape changed from the rod-like forms to coccoidal forms of different sizes (Figure 5). In contrast to the rod-like cells of untreated culture, the coccoidal forms, at least those which ^' originated from B. subtilis, did not retain Gram stain that specifically binds cell wall peptidoglycans. Thus, the coccoidal forms of both E. coli and B. subtilis resembled spheroplasts/protoplasts depleted of cell wall.

[00137] The cell-shape changes were clearly depicted in scanning electron micrographs (Figure 6). The treatment of E. coli cells with the suboptimal concentration of glp G208 (0.5xMIC) caused several distinct morphological changes: cells became shorter, rounded and prone to lysis (Figure 6).

[00138] Although the transition from a rod-shaped cell during exponential phase to a coccoidal form upon entry into stationary phase has been observed [22, 23] this event does not involve agglutination and cell wall lysis, in contrast to the results obtained in these experiments. These latter phenomena implicate glps as immediate effector molecules responsible for the membrane effects. The glycoprotein-evoked changes in cell wall-deficient forms were found to be lethal, as described below.

[00139] Bactericidal Activity of Honey Glycoproteins

[00140] Exposure of freshly prepared, yet non-dividing E. coli and B. subtilis cultures (10 ⁶ CFU/ml) to serially two-fold diluted glps ranging from 10C^g/ml to

1.25μg/ml caused a complete growth inhibition with the MIC values varied from

13^g/ml (G208), 4^g/ml (210) and 2^g/ml (G177) against E. coli to the MICs 13.8- 1.24μg/ml against B. subtilis (Table 3). The relatively high concentration of G208 required to inhibit bacterial growths was likely related to this fraction heterogeneity where the presence of several polypeptides (in G208) fractions could "mask" a true growth inhibitory activity of an "effector" molecule.

Table 3. MIC/MBC values of glycoproteins

Protein concentration

MIC/MBC E. coli B. subtilis

G177 2.5 1.24

G208 13.8 13.8

G210 4.9 2.5

[00141] No surviving cells were found using the standard plate count by seeding undiluted 100 μΐ aliquots from the wells after 18 hr incubation (Figure 8). These results together with microscopic observations indicated that the growth-inhibitory and bactericidal activities of glps on bacteria cells at the lag phase were likely due to their damaging action on cell membranes from which cells could not recover.

[00142] There was a strong possibility that growth of actively dividing cells is equally sensitive to the lytic action of glps. With this in mind, E. coli and B. subtilis culture (10 ⁶ CFU/ml) were grown in 96-well plates until they reach early log phase (A ₅ 9 ₅ nm 0.2-0.3) at which point glps were added at IxMIC and 0.5x MIC concentrations. The inhibitory action and killing rate were measured simultaneously at 0, 15 min, 30 min, 45 min, 60 min, 120 min and 18 hr by the absorbance readings and viable cell counts. The first 15 min of incubation with glycoproteins at IxMIC resulted in a rapid reduction of bacterial growth of both E. coli and B. subtilis with a >5-logio reduction of viable bacteria (Figure 7 and 8). The longer incubation times did not change the levels of inhibition or killing rates. In contrast, the incubation of bacterial cultures with

glycoproteins at 0.5 x MICs reduced the growth by about 50% during the first 15 minutes (Figure 8). After that time, a steady decrease in growth rates was observed, finally reaching 80% after 1 hr. Glycoproteins at 0.5xMIC were also less efficient in reducing the number of viable cells at shorter incubation times, although >3-log ₁₀ decrease in CFU/ml was achieved after 1 hr incubation.

[00143] The time-kill study showed that glps caused a rapid killing of both B. subtilis and E. coli. At bactericidal concentrations, glps triggered the abrupt loss of cell membrane that was irreparable and led to cell death. It has to be noted that much higher rate of killing of bacterial cells was observed at a lag phase compared to the exponential phase (Figure 8). These results may suggest that there might be a threshold ratio of the glycoprotein molecules per the number of bacterial cells in order to efficiently conduct cell lysis. At the lag phase, the number of cells in control cultures (10 ⁶ CFU/ml) was constant for a period of first 2-3 hr, while at the exponential phase the number of cell grew tolO ⁹ CFU/ml. Moreover, glps at 0.5xMIC required time to achieve 80% growth inhibition and >3 log reduction of viable cells. Surviving cells that escaped lysis at this concentration were able to revert to mature bacterial forms and repopulated the cultures after 18 hr incubation.

[00144] Thus, the number of cells, growth rate and glps concentrations were variables that contributed to bactericidal effect.

[00145] Specificity of the Interaction of Glycoproteins with Bacterial Targets

[00146] Only glps that were captured by ConA affinity chromatography showed growth inhibitory and killing activities. The flow-through and post-wash fractions from ConA chromatography showed either greatly reduced activity or were completely inactive, respectively (Figure 9). Although the flow-through fractions showed some degree of growth inhibition, they did not reduce the number of viable cells measured by the standard plate count. It became apparent that a high-mannose subset of glps retained by ConA- column was endowed with a specific targeting of bacterial membranes and follow up destruction.

[00147] Taken together, the microscopic and time-kill results demonstrated that glps were effector molecules responsible for the specific interactions with bacterial cells that included (a) recognition of cell wall determinants, (b) agglutination, (c) cell wall degradation, and (d) reversible or irreversible changes in the cell shape that, depending on glps concentrations, led either to restoration of the mature bacterial forms or to their complete lysis, respectively.

[00148] Sequencing of 61 and 31k Da Proteins from Glycoproteins G177 and G208

[00149] The similar levels of lytic and bactericidal activities of glycoprotein fractions G177, G208 and G210 suggested that they all possess the effector molecule responsible for these cytotoxic actions. The comparison of the SDS profiles of glps pointed out to a 61 kDa protein as a putative candidate because this protein was shared by all bactericidal glps. To enable identification of the 61 kDa protein, glps were extensively deglycosylated using multi-enzyme deglycosylation system, E-DEGLY (Sigma- Aldrich). The E-DEGLY Kit contained all the enzymes required to completely remove all N-linked and O-linked carbohydrates from glycoproteins. After

deglycosylation, the 61 kDa bands from G208 and G210 with changed electrophoretic mobilities (Figure 1 , panel III-B) were excised from the Coomassie Blue stained gels, subjected to in-gel trypsin digestion followed by MALDI-TOF and electrospray quadrupole time of flight mass spectrometry, ESI-Q-TOF- MS/MS. Subsequently, the characteristic peptide mass fingerprints of the 61 kDa proteins were analyzed by databased search using Mascot and PEAKS, respectively. Both Mascot and PEAKS searches revealed identity of the 61 kDa protein with the major royal jelly protein 1 precursor (Apis mellifera, accession number: gi 58585098) with the score of 136 (greater than 95% confidence) and 298 (greater than 99.2% confidence identification), respectively (Table 4). The major royal jelly protein 1 (MRJP1) precursor harbors three known antimicrobial peptides: Jelleins (Table 5). Fully annotated matched peptides of the deglycosylated 61kDa protein from G208 and G210 are presented in Figures 10 and 1 1. Table 4. Summary of ESI-O-TOF- MS/MS statistical data

for glycoproteins G208 and G210

Protein Accession Score -lOlgP Coverage #Peptides #Unique Mass Name kDa

MRJP-1

G210 gi|58585098 99.2 142 20 14 48.88

(56kDa)

MRJP-1

G208 gi|58585098 99.2 140.23 13 48.88

56kDa)

Table 5. Summary of the Jellein sequences in the major royal jelly protein 1

precursor ami their antimicrobial activities (li niPro-Klt/Swiss-Prot)

Antimicrobial

Name Peptide span Sequence

activity

Jellein- 1 425-432 PFKISIHL-NH ₂ Yes

Jellein-2 424-432 TPFKISIHL-NH ₂ Yes

Jellein-4 424-431 TPFKISIH-NH2 No

[00150] Jelleins 1, 2 and 4 correspond to SEQ ID NOS: 4, 3 and 5, respectively.

[00151] Thus, the 61 kDa glycoprotein moiety was thus identified by mass

spectrometry as the full length MRJP 1 , which is a precursor protein for three

antimicrobial peptides known as Jelleins. The MICs of synthetic Jellein- 1 and -2 have been shown to vary between 2.5 μ^ηιΐ against E. coli (CCT 1371) to 15 μ§Λη1 against S.

saprophyticus; these values compare well with the MIC values of glps obtained in this study.

[00152] EXAMPLE 2

[00153] Summary

[00154] The glycoproteins identified in Example 1 were further characterized, using the Concavalin A chromatography and MALDI-TOF techniques. The 61 kDa band from G177, G208 and G210 was confirmed to contain full length MRJP 1, the precursor protein for Jelleins 1 , 2 and 4. The smaller glycoprotein from G177 and G208 (described as 31 kDa band in Example 1) was found to in fact be approximately 29 kDa, and was identified as a fragment of MRJP 1. In contrast, the larger band for G207, approximately 58.5 kDa, was identified as containing MRJP 2, and the smaller band for G207, approximately 27 kDa, was identified as a fragment of MRJP 2.

[00155] Materials and Methods

[00156] Samples were prepared and experiments were conducted as described above in Example 1.

[00157] Figure Legends

[00158] Figure 12. SEM images of glycoprotein-induced morphological changes in log-phase E. coli and B. subtilis. a. Control culture of E. coli, b. control cultures of B. subtilis, c. E. coli treated with bactericidal concentrations of glps, G208, d. B. subtilis treated with bactericidal concentrations of glps, G208.

[00159] Figure 13. SEM images of E. coli and B. subtilis treated with flow-through fractions from ConA chromatography. Note lack of effects of flow-through fractions on bacterial cell shape.

[00160] Figure 14. Comparison of antibacterial activities of glycosylated versus deglycosylated glycoproteins G207 and G208 analyzed using agar well diffusion assay. Inhibition zone was determined by measuring clear zone produced by test samples against E. coli and B. subtilis. Ampicillin was used as a positive control. Columns represent mean ± SEM; pO.001 (***); p<0.05 (*).

[00161] Figure 15, Summary of fully annotated matched peptides between the MRJPl and 61 kDa and 29 kDa protein. Bars indicate sequence coverage and confirmed identifications.

[00162] Results

[00163] Further Characterization of Honey Glycoproteins [00164] As seen in Example 1, only glps that were captured by ConA affinity chromatography showed any growth inhibitory or killing activities. These activities for G208 were tested on E. coli and B. substilis cultures, and the results are shown in Figure 12.

[00165] In contrast, the flow-through and post- wash fractions from the ConA columns showed either greatly reduced inhibitory activity or were completely inactive. Given the small amount of residual bacteriostatic activity, the flow-through fractions were tested for their influence on bacterial cell shape, although none was observed (Figure 13, compare with panels c and d of Figure 12).

[00166] Among the ConA-captured proteins were two abundant polypeptides; a 61 kDa protein present in all tested honeys with exception of H207 and a 29 kDa protein, present in HI 77 and H208. Honey H207 was enriched with mannosylated 58.5 kDa protein and 27 kDa peptide. The mass spectrometric analysis revealed that the 29 kDa protein of G177 and G208 was a fragment of the 61 kDa protein, while the 58.5 kDa protein and its 27 kDa polypeptide of G207 were not related to 61 kDa protein and represented a different molecule, as described below in the MALDI-TOF results.

[00167] As indicated in Example 1, exposure of RBCs to glps G177 and G208 resulted in rapid transition from biconcave shape of normal erythrocytes into crenated, shrunken echinocytes, accompanied by formation of membrane blebs, thus demonstrating compromise of the erythrocyte membranes. Glps G207 and G210 did not produce membrane defects to the same degree as glps G177 and G208. Differences in the glps profiles seemed to be a factor that distinguished glps actions based on severity of the membrane effects they caused to the rat RBC.

[00168] Since G207 possessed only agglutinating activity, while Gl 77, G208 and G210 had both agglutinating and bactericidal activity, both G207 and G208 glps were subjected to 24-hr enzymatic deglycosylation using the multienzyme deglycosylation system, E-DEGLY (Sigma- Aldrich) that removes all N-linked and O-linked

carbohydrates from glycoproteins. [00169] The glycosylated and deglycosylated glps samples (4(^g/ml) were diluted two- and four-fold, and the samples' antibacterial activities were determined using the agar well diffusion assay (Figure 14). Ampicillin (25 μg/ml to 500 μg/ml) was used as a control in these assays. Despite the fact that the 24-hr deglycosylation reduced the molecular size of the main protein bands of G207 and G208 only by 10% on SDS-PAGE (not shown), the treatment produced significant changes in antibacterial activities of these two glycoproteins (Figure 14).

[00170] The size of the inhibition zones produced by G208 was dependent on concentrations of glps added to the well. Deglycosylation of G208 caused a reduction of the inhibition zones at all three dilutions in comparison to glycosylated G208, although these decreases were not statistically significant (Figure 14).

[00171] In contrast, G207 produced a zone of inhibition only at its highest concentration, and deglycosylation of G207 resulted in a significant decrease (B. subtilis) or a complete loss (E. coli) of the antibacterial activity of this glycoprotein.

[00172] These results indicate that for G207, glycosylation appears to be important for the agglutinating activity. In contrast, the mechanism by which G208 produces antibacterial effect does not appear to be solely glycosylation-dependent.

[00173] As seen in Example 1, the 61 kDa protein was found to contain the full- length MRJP 1 protein sequence. The most abundant 29 kDa protein co-eluting with the 61kDa MRJPl band in glps G208 and G177 was then also analyzed by MALDI-TOFMS. Mascot database search using the peptide mass fingerprints produced a statistically significant score of 281 (where scores >88 are significant p<0.05), matching major royal jelly protein 1 precuror from Apis mellifera (Accession number: gi|58585098). ESI-Q- TOF- MS/MS data showed 98.8% homology with major royal jelly protein 1 precuror from Apis mellifera (Accession number: gi|58585098) (Figure 15), therefore indicating that the 29 kDa band was a fragment of the MRJPl (Table 6). The presence of the 29 kDa protein enhanced cell wall damaging effects of the 61 kDa protein. Table 6. Summary of ESI-Q-TOF- MS/MS statistical data tor glycoproteins G177 and G208

Peptide Score - lOIgP Mass ppm tn/z RT Start End (SEQ ID NO) (%)

K.LLTFDLTTSQLLK.Q

99.6 41.83 1491.8549 41.0 746.9653 37.58 154 166 (6)

K.YDDCSGIVSASK.L

99.6 40.58 1300.5605 46.2 651.3176 24.58 1 15 126 (7)

R.EYILVLSNK.M

97.7 30.42 1077.6069 37.2 539.8308 32.17 376 384 (8)

R.YNGVPSSLNVISK.K

86.2 24.94 1376.7300 33.1 689.3951 30.05 83 95 (9)

[00174] In contrast, MALDI-TOF sequence data of glp G207 and its 27 kDa fragment excised from the SDS gels showed identity with the Major Royal Jelly Protein 2, MRJP2 of Apis mellifera (Table 7). Mascot score of 325 (>55) indicates identity or extensive homology (p<0.05). It should be noted that MRJP2 does not contain

antimicrobial Jelleins in its sequence; this could explain the result of some protein agglutinating activity but low growth inhibitory activity that was observed in the well diffusion assays.

Table 7. Summary of ESI-Q-TOF- MS/MS statistical data for 58.5 kDa glycoprotein

G207

Accession Protein Mr MASCOT Matches Sequences

ID (kDa) Score gi|58585108 MRJP2 51.441 325 6(2) 5(2)

[00175] EXAMPLE 3 [00176] Summary

[00177] The antibiotic resistance among Gram-negative bacteria to beta-lactam antibiotics is on the rise due to emergence of extended- spectrum β-lactamase (ESBL). Here, semi-quantitative radial diffusion assays and broth microdilution assays were used to evaluate susceptibility of a number of multidrug resistant clinical isolates to the MRJPl-contaning honey glycoproteins.

[00178] Bacterial strains comprised 3 MRSA, 4 Pseudomonas aeruginosa, 2 carbapenemase-producing Klebsiella pneumoniae, 2 Enterococcus faecium with high level of streptomycin and vancomycin resistance, 1 Escherichia coli NDM-1 , and 5 Extended- spectrum beta-lactamase (ESBL) identified as 1 Proteus mirabilis, 3 Escherichia coli and 1 Klebsiella pneumoniae. All MDR isolates were found to be susceptible to glps, including ESBL organisms such Klebsiella spp, E. coli NDM-1 , K. pneumoniae and P. aeruginosa. MIC ₉₀ values ranged from 3.62 μg/ml against B. subtilis to 14.37 μg/ml against P.

aeruginosa, K pneumoniae, Klebsiella spp ESBL and E. coli. Thus, glps could be effective in inhibiting growth of various MDR organisms irrespectively of their resistance mechanism.

[00179] Materials and Methods

[00180] Isolation of Honey Glycoproteins: Glycoproteins were isolated from 25% aqueous honey solutions using the agarose-immobilized Concavalin A spin columns

(Glycoprotein Isolation Kit, ConA, Thermo Scientific, Rockford, IL, USA) according to the manufacturer's instructions. The isolation process was monitored by SDS-PAGE followed by Coomassie blue staining.

[00181] Determination of Protein Concentration: Protein quantification was performed by Bradford methods in the microplate version, using the Bio-Rad Protein Assay (Bio-Rad Laboratories Inc. Hercules, CA, USA) according to the manufacturer's instructions.

[00182] SDS-PAGE Electrophoresis (ID): SDS-PAGE was performed according to the method described by Laemmli, 1970 [37]. Honey glycoproteins were analyzed by 7.5% gel separation gel with attached 5% stacking gel. 30 μΐ of a honey solution (50% v/v) or 15μ1 of a fraction from ConA chromatography were mixed with the sample buffer, denatured at 100°C for 5 min and loaded on a gel. The electrophoresis was carried out in duplicate at a constant current of 100 Volts using a Mini Protean III electrophoresis cell (Bio-Rad

Laboratories, Hercules, CA, USA). After electrophoresis, the gel was stained with Brilliant Blue R-250 (Bio-Rad). The molecular weight of the proteins was determined using molecular weight standards (Fermentas Life Sciences, Fisher Scientific-Canada, Ottawa, ON, Canada). [00183] Two-Dimensional Electrophoresis (2D): Protein samples were rehydrated in a solution of 7M urea/ 2M thiourea/ 4% CHAPS/0.5% carrier ampholytes/ 5mM

tributylphosphine. Each sample was loaded onto a 17cm pH 3-10 nonlinear strip (BioRad Cat #163-2009) and allowed to passively rehydrate for 18 hours at 20°C. IEF strips were focused on a BioRad PROTEAN IEF cell at 10,000 volts for 100,000 volt/hours.

[00184] Following the first dimension, the IEF strips were equilibrated in a solution of 50 rriM Tris-HCl pH 8.8/ 6M urea/30%glycerol/2%SDS prior to loading the second dimension. At this step the strips were also reduced and alkylated using 1% DTT followed by 2.5% iodoacetamide. Once equilibrated, the strips were loaded on a 10% homogeneous Tris Glycine gel and run overnight at 50 volts and 12°C.

[00185] Protein Digestion Using Micromass Massprep Robotic Protein Handling System: Gels were stained using nondestructive silver stain. Protein plugs were excised from the gel using a coring device with a 2mm diameter. Gel plugs were de-stained in 15mM potassium ferricyanide / 50mM sodium thiosulphate and reduced in lOmM dithiothreitol in lOOmM ammonium bicarbonate for 30 minutes. Samples were alkylated in 55mM

iodoacetamide in lOOmM ammonium bicarbonate for 20 minutes, washed in lOOmM ammonium bicarbonate and dehydrated in 100% acetonitrile. The samples were digested with 6 ng/uL trypsin (Promega Sequencing Grade Modified Trypsin) in 50mM ammonium bicarbonate (25uL) for 5 hours at 37°C. Peptides were extracted from the gel plug with a solution of 30uL 1% formic acid/ 2% acetonitrile, and then with 24uL of 50% acetonitrile follow the first extraction. Pooled extractions were evaporated down to a volume of approximately 20uL using a ThermoSavant Speedvac concentrator model SPD121P.

[00186] MALDI-TOF Mass Spectrometry: Spectra were acquired in positive-ion reflector mode and analyzed on a Voyager DePro (Applied Biosystems Corporation) using Data Explorer Version 5.1 software (Applied Biosystems Corporation). The mass range scanned was 1000 Da to 3000 Da. Raw spectra were processed under Data Explorer 5.1 software (Applied Biosystems). Peptide peaks with a signal to noise ratio of greater than 2:1 were selected for a database search. Search parameters were set at trypsin with up to one missed cleavage, fixed carbarn idomethyl modification of cysteine and variable oxidation of methionine. Database source was the Mascot search engine using the NCBInr data base at 50 ppm mass accuracy. [00187] Antibacterial Assay of Glycoproteins by Agar Well Diffusion Method:

The plates were prepared using Mueller-Hinton agar (MHA) poured in a 4mm deep layer. The 100 μΐ of bacterial inoculum (107 CFU/ml) was uniformly spread using a bent-glass rod. Wells were performed with a sterile plastic bore of 3 mm diameter. Each well was filled with 8 μΐ of sample of glycoproteins (40 μg/ml) prepared in 3 dilutions; undiluted, 2x and 4x diluted with sterile water. Each plate contained well filled with ampicillin (1 μg/10 μΐ or 5 μg/10μl) as a positive control and BSA (5 μg/10 μΐ), as a negative control. In addition, the reference MHA plates were produced, inoculated with 100 μΐ of either B. subtilis or E. coli (107 CFU/ml) that contained wells filled with series of ampicillin concentrations ranging from 2.5 to 500 μg/ml. The agar plates were incubated for 24 h at 37°C. After incubation, the zone of inhibition of the bacterial growth was measured using a digital, electronic caliper (Flower Company Inc. Newton, MA, USA). Tests were performed in duplicate.

[00188] Broth Microdilution Assay and Determination of the MIC: The susceptibilities of clinical iolates and standard bacteria (106 CFU/ml) to honey glycoproteins were analyzed using broth microdilution assay in a 96 well microtitre plate as described previously (Brudzynski and Sjaarda, 2014). Each well contained 50μ1 of inoculum and 8μ1 of two-fold serial dilutions of glycoproteins (starting from 40μg/ml). Bacterial growth was measured at an optical density at A595 nm using the Synergy HT multidetection microplate reader (Synergy HT, Bio-Tek Instruments, Winooski, VT, USA). The MIC was evaluated by recording the concentration of glycoproteins that reduced bacterial growth by 99% in comparison to a control, untreated culture, after 18 h incubation with shaking at 37°C.

Statistical analysis and dose response curves were obtained using K4 software provided by Synergy HT (Bio-Tek Instruments, Winooski, VT, USA).

[00189] Strains Tested: Strains used were fresh clinical isolates from the Clinical

Microbiology Laboratory, LHSC, London, Ontario. List of strains and their antibiotics susceptibility is presented in Table 9. The isolates tested per species were as follows: 4 isolates of Escherichia coli, 3 isolates of MRSA, 4 isolates of Pseudomonas aeruginosa, 3 isolates of Klebsiella pneumoniae, 5 isolates of ESBL organisms including Klebsiella oxytoca, 1 isolate of Proteus mirabilis, 1 isolate of Escherichia coli NDM-1 , 2 isolates of

VRE. Control strains were E. coli ATCC 14948 and B. subtilis ATCC 6633. [00190] Figure Legends

[00191] Figure 16. 1-D and 2D gel electrophoresis of glycoprotein G208. Lane i: protein molecular weight standards; ii: buckwheat honey H208; iii: glycoprotein G208; iv: glycoprotein G217; v: 2D gel electrophoresis of G208. The arrows indicate the location of the 61 kDa and 29 kDa bands.

[00192] Figure 17. Linear relationship between ampicillin and glycoprotein concentrations and the diameter of the zone of inhibition.

[00193] Figure 18. Susceptibility of multidrug resistant clinical isolates to honey glycoproteins, (a) Susceptibility of K. pneumonia (# 1 and #19), P. aeruginosa (#34 and #35), E. 7o/ NDM (#4) and E. coli. ESBL (# 7) to G208 and G217. Ampicillin-sensitive E. coli (ATCC 14948) served as a control, (b) Susceptibility of MRSA 1 (#5) and ampicillin- sensitive B. subtilis (ATCC 6633) to G208, G207, G217 and G125 isolated from different honeys.

[00194] Figure 19. Concentration-dependent growth inhibition of multidrug resistant clinical isolates by honey glycoproteins in well diffusion assay.

[00195] Figure 20. Concentration-dependent growth inhibition of clinical isolates by honey glycoproteins evaluated using broth microdilution assay.

[00196] Results

[00197] Honey Glycoproteins

[00198] Honey glycoproteins, G208 and G217, isolated via Concavalin A-affinity chromatography showed high degree of purity when analyzed on ID and 2D gels, as seen in Figure 16.

[00199] Two-dimensional (2D) gel electrophoresis coupled with mass spectrometry was employed to identify proteins in the fraction of G208. Two selected spots from the 61 kDa and 29 kDa bands were manually excised, subjected to in-gel tryptic digestion and the peptide fragments were analyzed by MALDI TOF. The generated PMS were analyzed using the Mascot database.

[00200] The data obtained revealed that the 61 kDa and 29 kDa of G208 protein presented a mixture of the major royal jelly protein 1 and 2 precursor proteins (Apis mellifera, accession numbers: gi 58585098 (MRJP1) and gi 58585108 (MRJP2)), with scores of 281, 174 and 149 for the 61 kDa band and scores of 325 and 202 for the 29 kDa band. The individual ions scores > 55 indicate identity or extensive homology (p<0.05). The protein identification data is detailed in Table 8.

Table 8. Summary of MASCOT Search Results

[00201] Clinical Isolates

[00202] Clinical isolates used in this study included MRSA (n=3), Pseudomonas aeruginosa (n=4), Klebsiella pneumoniae (n=2), VRE (n=2) and ESBL (n=5) (Table l).The isolates were identified to genus and species and their susceptibility to antibiotics was confirmed using an automated system (Vitek®, Biomerieux®) by the Clinical Microbiology Laboratory, London Health Science Centre, London, Ontario, Canada.

[00203] As can be seen in Table 9, clinical isolates used in this study were simultaneously resistant to a number of antibiotics representing different chemical classes, therefore the isolates were classified as multidrug resistant pathogens. Table 9. Antibiogram of Clinical Isolates Used

ifjaipiG s s

[00204] Susceptibility of Clinical Isolates to Glycoproteins

[00205] In vitro evaluation of susceptibility of multidrug resistant clinical isolates was performed using a semi-quantitative radial well diffusion and broth microdilution assay. To compare the results of bacterial susceptibility to honey glycoproteins in a quantitative manner, the reference method has been designed that established the relationship between ampicillin dilutions and zone of inhibition (ZOI) in a well diffusion assay.

[00206] As shown in Figure 17, the series of two-fold diluted honey glycoproteins and ampicillin (lmg/ml stock solutions) produced concentration-dependent ZOIs with R ² =0.99 for ampicillin, and R ² = 0.96 and 0.95 for glps tested against E. coli and B. subtilis, respectively. MIC values from the well diffusion assay against E. coli and B. subtilis (1x10ό CFU/ml) were <0.025μg/well or <2^g/ml for ampicillin and <0.046 μg/well or 5.7 μg/ml for glps.

[00207] In a similar manner, each of the isolates was tested for susceptibility to glycoproteins by well diffusion assay. The agar plates were divided into three sections: the middle section was used for spread plating of the standard E. coli (ATCC 14948) sensitive to ampicillin while the right and left sections were spread plated with the MDR isolates. The susceptibility of isolates to two glycoproteins, G208 and G217, isolated from two different buckwheat honeys, was then evaluated and compared to that of ampicillin-sensitive control. Both glycoproteins have been shown to exhibit antibacterial activity against K. pneumonia 1 and 2 (Table 9) (lab number #1 and #19), P. aeruginosa 3 and 4 (lab number #34 and #35), E. coli NDM-1 4 (lab number #4) and E. coli ESBL 2 (lab number #7). These results indicate that multidrug resistance of isolates to antibiotics did not affect antibacterial action of glycoproteins (Figure 3X and Table 9). [00208] All MRSA isolates were also found to be highly sensitive to honey glycoproteins (Figure 18). Moreover, in addition to G208, glycoproteins G217, G125 and G207 isolated from different honeys, produced zones of inhibition of comparable diameters (Figures 17 and 18), indicating that antibacterial activity of glycoproteins may reside in common structural elements.

[00209] The serial two-fold dilution of glps showed a concentration-dependent inhibition of growth of isolates in the radial diffusion assay with susceptibility <0.^g/well or <12.5 μg/ml (Figure 19). Differences in the mean of inhibition zone diameters between bacterial species pointed out to some variation in the sensitivity of individual microorganism to glps. Glycoproteins were found less potent in inhibition of growth of K. pneumoniae and P. aeruginosa compared to MRSA, VRE and E. coli NDM-1 or standard, antibiotic-sensitive bacteria, as judged by the size of ZOI they produced at comparable concentrations (Figures 18 and 19).

[00210] In the broth microdilution assay, glps showed considerable antibacterial activity with MIC90 values ranging from 3.62μg/ml against B. subtilis to 14.37μg/ml against P. aeruginosa, K. pneumoniae, Klebsiella spp ESBL and E. coli. The results of broth microdilution assay correspond well with data obtained from well diffusion assay (Figures 19 and 20).

[00211] Thus, honey glps showed activity against multidrug resistant clinical isolates, irrespectively of the mechanism by which the resistance was conferred.

[00212] The effectiveness of glps against Gram-negative pathogens of ESBL type is specifically encouraging and justifies continuation of research in this field.

[00213] Thus, an antibiogram of clinical isolates used in this study indicates that in addition to being resistant to β-lactams, these isolates were also resistant to fluoroquinolones, aminoglycosides and tetracyclines, therefore presenting multidrug resistant phenotype.

Among isolates tested there were (ESBL)-producing carbapenem- resistant Klebsiella pneumoniae (KPC), P. aeruginosa (resistant to imipenem and cephalosporin, ceftazidime) and E. coli NDM-1 (New Delhi metallo-P-lactamase). Resistance to carbapenems represents a significant threat in the management of multidrug-resistant isolates, since due to their potency and broad-spectrum activity these carbapenems tend to be considered last resort antibiotics in the treatment of severe infections (Nordmann et al, 2009).

[00214] In this context, these results show that MRJP1 -containing honey

glycoproteins were able to inhibit growth of these multidrug resistant clinical isolates.

Specifically, glps showed desirable efficacy against ESBL organisms such Klebsiella spp, E. coli NDM-1, K. pneumoniae and P. aeruginosa. Both well diffusion assays and broth micodilution assays demonstrated susceptibility of clinical isolates to glps with MIC ₉₀ values ranging from 3.62μg/ml against B. subtilis to 14.37μg/ml against P. aeruginosa, K.

pneumoniae, Klebsiella spp ESBL and E. coli. Thus, in these results, glps can be seen to exhibit a consistent activity against the multidrug resistant isolates, irrespective of the isolates' resistance mechanism. Moreover, glycoproteins G217, G125, and G207 isolated from different honey varieties also efficiently inhibited growth of MDR isolates, suggesting that the activity may be due to a common glps structure.

[00215] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[00216] Concentrations given in this specification, when given in terms of percentages, include weight/weight (w/w), weight/volume (w/v) and volume/volume (v/v) percentages.

[00217] As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural reference unless the context clearly dictates otherwise. As used in this specification and the appended claims, the terms "comprise",

"comprising", "comprises" and other forms of these terms are intended in the non- limiting inclusive sense, that is, to include particular recited elements or components without excluding any other element or component. As used in this specification and the appended claims, all ranges or lists as given are intended to convey any intermediate value or range or any sublist contained therein. Unless defined otherwise all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.

[00218] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

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