WO2009140715A1

WO2009140715A1 - Diagnostic method

Info

Publication number: WO2009140715A1
Application number: PCT/AU2009/000391
Authority: WO
Inventors: Lor Wai Tan
Original assignee: Central Northern Adelaide Health Service; The Queen Elizabeth Hospital Research Foundation Inc.
Priority date: 2008-05-21
Filing date: 2009-03-31
Publication date: 2009-11-26

Abstract

The present invention relates to the diagnosis and/or characterization of markers associated with biofilm formation a surface of a subject. In particular, the present invention provides methods for determining a presence of a biofilm, or a risk of formation of a biofilm, on a surface of a subject, the method comprising determining a sialic acid profile associated with one or more glycoproteins expressed by a cell of the subject.

Description

DIAGNOSTIC METHOD

PRIORITY CLAIM

The present application claims priority to Australian provisional patent application 2008902510, the contents of which are hereby incorporated by reference.

FIELD

The present invention relates to methods for determining a presence of a biofilm, or a risk of formation of a biofilm, on a surface of a subject.

BACKGROUND

Biofilms are a structured population or community of microbial cells enclosed in a self- produced polymeric matrix and adherent to an inert or living surface. Biofilm formation may occur in a series of sequential steps. Firstly microorganisms bind to a surface. Once a foothold is established, the microorganisms may rapidly multiply whilst simultaneously secreting a protective polysaccharide matrix. The colonization process is completed when the microflora switch to a sessile phenotype that cannot be easily dislodged.

Biofilms may form on a range of different surfaces. Typically, the surfaces are solid surfaces exposed to or submerged in an aqueous solution. Biofilms are particularly important in mammalian health, including human health.

Biofilms that are commonly associated with human health include biofilms that form on mucosal, urogenital and alimentary surfaces. A common example of a human disease that is often associated with the presence or formation of a biofilm on a mucosal surface is chronic rhinosinusitis (CRS). CRS is a disease which results in chronic inflammation of the paranasal sinuses, and may further include inflammation of part of the nose.

In Australia chronic rhinosinusitis (CRS) is classified as a prevalent condition within the major chronic respiratory diseases group. Between 2000-01 the collective cost of treatment for CRS and asthma exceeded $693 million, 50% of which was spent on pharmaceuticals. In the US, CRS results in 11.5 million physician visits per year with an estimated US$2 billion spent annually on medications. There clearly is a significant economic cost in terms of reduced productivity, work days lost, and increased expenditure on elective surgery that taxes an already overburdened public health system.

Recent advances in microscopy have improved the ability to detect bacterial biofilms in situ. Using scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM) techniques, clear evidence now exists for bacterial biofilms on sinus mucosa of CRS patients. Increasingly, bacterial biofilms have been implicated as aetiological agents in the development of CRS. As bacterial communities in biofilms adopt a phenotype that do not require functional growth and cell wall synthesis gene expression, conventional systemic antibiotic treatments that specifically target metabolically active bacteria undergoing replication or cell wall synthesis are of reduced efficacy.

Surgery is often performed to remove infected mucosa on a mucosal surface. However, incomplete removal of the infected mucosa often leads to the regeneration of the biofilm and reestablishment of disease.

Biofilms can also be resistant to conventional antibiotic treatments and their propensity for regeneration following incomplete surgical removal may explain the refractory and often recalcitrant nature of chronic rhinosinusitis (CRS). In light of the above, a method for determining a presence of a biofilm, or a risk of formation of a biofilm, on a surface of a subject would be desirable. On the basis of information obtained from such a determination, it would also be possible, for example, to select appropriate therapies, select preventative measures and/or provide a surgical prognosis for subjects.

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

SUMMARY

The present invention relates to the diagnosis and/or characterization of markers associated with biofilm formation on surfaces of subjects.

In particular, the present invention provides a method for determining a presence of a biofilm, or a risk of formation of a biofilm, on a surface of a subject, the method comprising determining a sialic acid profile associated with one or more glycoproteins expressed by a cell of the subject.

In some embodiments, determining a sialic acid profile associated with one or more glycoproteins may involve determining one or more of: i) an amount of sialic acid linked to a glycoprotein; ii) a molecular weight of a sialic acid linked glycoprotein; iii) a type of sialic acid-glycoprotein linkage; iv) the amount of a sialic acid-linked glycoprotein having a molecular weight of 70-

80 kDa, 80-90 kDa or 90-100 kDa; and/or v) the level of expression of a protein associated with the sialic acid profile.

In some embodiments, the presence of a biofilm, or risk of formation of a biofilm is indicative of a subject with a disease or with an increased risk of a disease.

In some embodiments, the presence of a biofilm, or risk of formation of a biofilm is indicative of a subject with a poor surgical prognosis.

In some embodiments, the presence of a biofilm, or risk of formation of a biofilm is indicative of a subject having a reduced responsiveness to antibiotic therapies for a biofilm.

In some embodiments, the presence of a biofilm, or risk of formation of a biofilm is indicative of a subject having an increased risk of recurrent colonization by biofilm- forming bacteria and/or fungi.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the above description.

In a first aspect, the present invention provides a method for determining a presence of a biofilm, or a risk of formation of a biofilm, on a surface of a subject, the method comprising determining a sialic acid profile associated with one or more glycoproteins expressed by a cell of the subject.

The term "biofilm" as used herein may be understood as a structured population or community of microbial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface. Biofilm formation may occur in a series of sequential steps. Firstly microorganisms recognise and bind to target receptors, which may include terminal sugars of glycoproteins that are expressed on a host mucosa. Once a foothold is established, the microorganisms may rapidly multiply whilst simultaneously secreting a protective polysaccharide matrix. The colonization process is completed when the microflora switch to a sessile phenotype that cannot be easily dislodged from the mucosa.

Biofilms may be produced by and/or colonised by diverse microflora. For example, in one embodiment, the biofilm comprises a bacterial cell. The bacterial cell may be any bacterial cell that is able to make up part of biofilm. For example, the bacterial cell may include a bacterial cells from a Helicobacter species including Helicobacter pylori, Helicobacter hepaticus, Helicobacter raγγini, Helicobacter muridarum, Helicobacter bills; Streptococcus species including Streptococcus mutatis, Streptococcus pyogenes, Streptococcus pnuemoniae; Enterococci species including Enterococcus faecalis; Bacteroides species; Bifidobacterium species; Peptococcus species; Peptostreptococcus species; Pseudomonas species including Pseudomonas aeruginosa; Ruminococcus species; Clostridia species including Clostridium difficile; Lactobacillus species including Lactobacillus acidophilus; Neisseria species including Neisseria gonorrhea, Neisseria meningitides; Escherichia coll; Vibrio cholerae; Shigella species including Shigella dysenteriae, Shigella flexneri, and Shigella Sonnei, Yersinia species including Yersinia enterocolitica; Pseudomonas aeruginosa; Bordetella pertussis; Campylobacter species including Campylobacter jejuni; Haemophilus influenzae; and/or Staphyloccus species including Staphyloccus epidermis, Staphyloccus aureus.

In some embodiments, the bacterial cell is a Streptococcus species, including, for example, Streptococcus mutans, Streptococcus gordonii or Streptococcus pneumoniae.

In some embodiments the bacterial cell is a Staphylococcus species, including, for example, Staphylococcus aureus.

In addition to bacteria, the biofilms contemplated in the present invention include biofilms comprising other types of microflora such as actinobacteria, fungi, protists and the like. In one embodiment, the biofilm comprises a fungal cell. The fungal cell may include a fungal cell from Alternaria spp., Aspergillus spp. including Aspergillus fumigatus, Blastomyces dermatitidis, Candida spp., Cladophialophora bantiana, Coccidioides immiϊis, Cryptococcus spp., including Cryptococcus neoformans (Var. neoformans, var. gattii), Emmonsia parva var. parva, Emmonsia parva var. crescens, Epidermophyton floccosum, Fonsecaea compacta, Fonsecaea pedrosoi, Histoplasma capsulatum var. capsulatum, Histoplasma spp. including Histoplasma capsulatum var. duboisii, Histoplasma capsulatum var. farcinimosum, Madurella grisea, Madurella mycetomatis, Microsporum spp., Neotestudina rosatii, Paracoccidioides brasiliensis, Penicillium marneffei, Phycomycetes including Mucor, Phizopus and Absidia spp., Scedosporium apiospermum (Pseudallescheria boydii), Scedosporium proliferans (inflatum), Sporothrix schenckii and/or Trichophyton spp.

The method of the present invention may be used to determine a presence of a biofilm, or a risk of formation of a biofilm, on a surface of any suitable subject. Typically, the subject is an animal subject. Suitable animal subjects include, for example, mammalian subjects such as humans, companion animals such as dogs or cats, livestock animals such as cattle, sheep, goats and the like. In addition, the present invention may also be applied to determining a presence of a biofilm, or a risk of formation of a biofilm, on a surface of non-mammalian animal subjects such as birds including poultry birds such as chickens, reptilian subjects including companion reptiles such as turtles, tortoises and snakes; fish including wild-caught fish and fish in aquaculture.

The surface of the subject which has a presence of a biofilm, or a risk of formation of a biofilm, may be any surface of the subject on which a biofilm may establish. For example, the surface may be a mucosal surface, urinary tract surface, reproductive surface, alimentary surface, skin (in particular follicle) surface, tooth surface, ear canal surface, a corneal surface and the like

In some embodiments the surface is a mucosal respiratory surface. Mucosal respiratory surfaces include surfaces of the lungs, nostrils, nasal cavity, oropharynx, laryngopharynx, larynx, glottis, trachea, mouth and sinuses.

In some embodiments, the surface is a paranasal sinus surface. Paranasal sinuses include the maxillary sinuses the frontal sinuses the ethmoid sinuses and the sphenoid sinuses.

In some embodiments, the surface of the subject is an artificial surface. Such artificial surfaces are may be present in the subject as a result of a surgical procedure. Examples of artificial surfaces include tubing and other medical devices, such as catheters, pacemakers, prosthetic heart valves, prosthetic joints, voice prostheses, contact lenses and/or intrauterine devices. Medical devices include disposable or permanent catheters, (e.g. central venous catheters, dialysis catheters, long-term tunneled central venous catheters, short-term central venous catheters, peripherally inserted central catheters, peripheral venous catheters, pulmonary artery Swan-Ganz catheters, urinary catheters, and peritoneal catheters), long-term urinary devices, tissue bonding urinary devices, vascular grafts, vascular catheter ports, wound drain tubes, ventricular catheters, hydrocephalus shunts, heart valves, heart assist devices (e.g. left ventricular assist devices), pacemaker capsules, incontinence devices, penile implants, small or temporary joint replacements, urinary dilator, cannulas, elastomers, hydrogels, surgical instruments, dental instruments, tubing, such as intravenous tubes, breathing tubes, dental water lines, dental drain tubes, and feeding tubes, fabrics, paper, indicator strips (e.g. paper indicator strips or plastic indicator strips), adhesives (e.g. hydrogel adhesives, hot-melt adhesives, or solvent-based adhesives), bandages, orthopedic implants, and any other device used in the medical field.

As indicated, in some embodiments, the artificial surface is a prosthetic surface. The prosthetic surface may include, for example, a catheter, pacemaker, prosthetic heart valve, prosthetic joint, voice prostheses, contact lens, intrauterine device, urinary catheter, a peritoneal catheter and the like. In some embodiments, the prosthetic surface is an implant, catheter or stent. The catheter may for example be a central venous catheter, a peripheral intravenous catheter, an arterial catheter, a haemodialysis catheter, an umbilical catheter, precutaneous nontunneled silicone catheter, a cuffed tunneled central venous catheter or a subcutaneous central venous port.

In some embodiments, the presence of a biofilm on a surface of the subject may be associated with a disease state in the subject. For example, as set out previously, several human disease states are associated with biofilm formation on a surface of the subject. Such disease states include, for example: biofilm-mediated sub-types of CRS, cystic fibrosis, dental caries and related gum diseases, otitis media with effusion, sepsis and complications arising from bacterial biofilm colonization of prosthetic implants or cardiac stents and chronic eye infections from continuous use of contact lenses.

In some embodiments, the present invention specifically contemplates a method of determining a presence of a biofilm, or a risk of formation of a biofilm, on a surface of a subject, wherein a respiratory disease is associated with the biofilm. In some embodiments, the present invention specifically contemplates a method of determining a presence of a biofilm, or a risk of formation of a biofilm, on a surface of a subject, wherein chronic rhinosinusitis is associated with the biofilm.

As set out above, the method of the present invention contemplates determining a presence of a biofilm, or a risk of formation of a biofilm, on a surface of a subject, by determining a sialic acid profile associated with one or more glycoproteins expressed by a cell of the subject.

In some embodiments, the present invention contemplates determining a sialic acid profile associated with one or more mucin glycoproteins expressed by a cell of the subject. Airway mucous consists of mucin glycoproteins, water, ions, lung secretions and serum protein transudates. Mucin glycoproteins are part of the body's innate immune system against ingested or inhaled aeroallergens. They comprise a protein backbone to which N-linked and/or O-linked glycoside residues are attached. The MUC genes code for the apoprotein backbone whilst glycosyltransferase genes determine the types of oligosaccharides attached. Mucin glycoproteins are typically membrane bound, glycosylated phosphoproteins.

Examples of mucin glycoproteins include, for example, glycoproteins comprising a MUCl, MUC5AC, MUC5B, or MUC7 apoprotein.

Examples of the MUCl protein are described under gene card accession number GC01M153424. The MUCl protein may also be referred to as CD227 antigen.

Examples of the MUC5AC protein are described under gene card accession number GC11P001169.

Examples of the MUC5B protein are described under gene card accession number GC11P001200.

Examples of the MUC7 protein are described under gene card accession number GC04P071372.

Oligosaccharides can make up to 80% of the molecular weight of a mucin glycoprotein. The most common sugar groups associated with mucin glycoproteins are sialic (neuraminic) acid (Sia), N-Acetyl galactosamine (GaINAc), N-Acetyl glucosamine (GIcNAc), galactose (Gal) and fructose. Sialic acids with α2-3Gal and α2-6Gal linkages are present in all ciliated and non-ciliated, goblet and basal cells of the respiratory epithelium. As set out above, the present invention is predicated, in part, on determining a sialic acid profile associated with one or more glycoproteins expressed by a cell of the subject.

The present invention contemplates any way in which a sialic acid profile associated with one or more glycoproteins may be determined.

In some embodiments, glycoproteins are isolated from a cell or serum. Serum from which glycoproteins may be isolated include mucus, saliva, blood and lymph. Cell lysates may be prepared by methods known in the art including freeze-thaw lysis, sonication, cell membrane shearing, osmotic lysis and detergent treatment. Membrane bound glycoproteins may be separated from intracellular glycoproteins by centrifugation. Glycoproteins may be separated from other proteins using glycoprotein specific antibodies or lectins, including, for example wheat germ agglutinin (WGA). Numerous glycoprotein isolation kits are also available including, for example, Qproteome Total Glycoprotein kit (Qiagen, USA).

In some embodiments, the determination of the sialic acid profile associated with the one or more glycoproteins may involve the analysis of the glycoprotein on an intact cell. For example, the sialic acid profile associated with the one or more membrane bound glycoproteins may be determined by flow cytometry. In these embodiments, the glycoprotein does not need to be isolated.

In some embodiments, determining the sialic acid profile associated with the one or more glycoproteins comprises determining an amount of a sialic acid linked to the one or more glycoproteins.

The present invention contemplates any suitable method for determining the amount of a sialic acid linked to the one or more glycoproteins. Such methods may include an analysis of the glycoprotein before and after treatment with a desialylation agent, including, for example, treatment with neuraminidase.

For example, methods for determining the amount of a sialic acid linked to the one or more glycoproteins may include HPLC analysis, for instance, as described in Helander et al. (Clinical Chemistry 50: 954-958, 2004). As another example, the method may involve mass spectrometry analysis including, for instance, including the method described in Hiki et al. (Kidney International 59: 1077-1085, 2001). Alternatively, the amount of sialic acid linked to the one or more glycoproteins may be determined by ELISA. The ELISA will typically include the use of a detectably labelled sialic acid binding lectin or antibody. Quantitative measurements may be made using an appropriately matched detection device. For example, if the detectable label is a fluorescent tag, an appropriate detection device may be a flow cytometer. The glycoprotein may optionally be bound to a substrate. An example of sialic acid quantitation by ELISA is provided by Leung et al. (Nephron Experimental Nephrology 107(3): 107-118, 2007).

In another embodiment, determining the sialic acid profile associated with the one or more glycoproteins comprises determining a molecular weight of a sialic acid linked glycoprotein. It will often be the case that the molecular weights of a number of glycoproteins will be determined from the subject. All methods of determining the molecular weight of one or more sialic linked glycoproteins are contemplated. These methods will typically involve, for example, separation of glycoproteins based on their molecular weight and/or identification of sialic linked glycoproteins. For instance, the glycoproteins may be run on SDS-PAGE gel to separate glycoproteins of different molecular weights, blotted onto a membrane by western blotting and then bands containing sialic linked glycoproteins identified using labelled sialic acid-specific lectins or antibodies. The molecular weight is determined by comparing the position of the bands with control bands representing proteins of known molecular weight. Alternatively, sialic linked glycoproteins may be separated from non-sialic linked glycoproteins using sialic acid specific lectins or antibodies by methods such as chromatography. Methods of separating proteins by weight, charge, size or composition are known in the art and include methods provided in Kenny and Fowell, Practical Protein Chromatography (Humana Press Inc, Totowa, New Jersey, 1992). The molecular weight of the separated sialic acid linked glycoproteins may then be determined by methods known in the art.

Methods for determining the molecular weight of proteins include mass spectrometry, SDS page, flow cytometry, HPLC and methods detailed in Van Eyk and Dunn, Clinical Proteomics: From Diagnosis to Therapy (Wiley-VCH Verlag GmbH & Co, Weinheim, Germany, 2008).

In some embodiments, determining the sialic acid profile associated with the one or more glycoproteins comprises determining:

(i) the amount of a sialic acid linked glycoprotein having a molecular weight of 70-80 kDa; (ii) the amount of a sialic acid linked glycoprotein having a molecular weight of 80-90 kDa; and/or (iii) the amount of a sialic acid linked glycoprotein having a molecular weight of 90-100 kDa.

In the above embodiments, and specifically including those embodiments where the subject is a human subject, the amount of a sialic acid linked glycoprotein having a molecular weight of 70-80 kDa, 80-90 kDa or 90-100 kDa is indicative of the presence or absence of a biofilm on a surface of the subject or is indicative of the risk of formation of a biofilm on a surface of the subject.

Furthermore, in the above embodiments an increase in: (i) the amount of a sialic acid linked glycoprotein having a molecular weight of 70-80 kDa; (ii) the amount of a sialic acid linked glycoprotein having a molecular weight of 80-90 kDa; and/or

(iii) the amount of a sialic acid linked glycoprotein having a molecular weight of 90-100 kDa is indicative of the presence of a biofilm on a surface of the subject and/or an increased risk of formation of a biofilm on a surface of the subject.

An "increase" in the amount of the subject glycoproteins should be understood to include an elevated amount of the glycoprotein relative to a healthy normal subject. Similarly, an "increased risk" of biofilm formation as referred to herein, refers to a risk elevated relative to healthy, normal subjects.

In some embodiments, determining the sialic acid profile associated with the one or more glycoproteins comprises determining a type of sialic acid-glycoprotein linkage. In one specific embodiment, the determined type of sialic acid-glycoprotein linkage is an α2-6 linkage. In another specific embodiment, the determined type of sialic acid- glycoprotein linkage is an α2-3 linkage.

In some embodiments, the present invention exploits the binding properties of plant lectins to sialic groups based on their linkage patterns. Three exemplary lectins suitable for use in accordance with the present invention include (i) Sambucus nigra (SNA) that bind to sialic residues attached to either Gal or GaINAc via α2-6 linkages; (ii) Maackia amurensis (MAA) that recognises α2-3 linked sialic groups and (iii) wheat germ agglutinin (WGA) that binds sialic groups.

Thus, in some embodiments, determining the sialic acid profile associated with one or more glycoproteins comprises using a wheat germ agglutinin which binds to the sialic acid. In some embodiments, determining an α2-6 linkage comprises using an extract of Sambucus nigra which binds sialic acid linked glycoproteins comprising an α2-6 linkage.

In some embodiments, determining an α2-3 linkage comprises using an extract of Maackia amurensis which binds sialic acid linked glycoproteins comprising an α2-3 linkage.

In further embodiments, the bound sialic acids are separated from unbound sialic acids.

A commercially available lectin-based affinity column chromatography technique (Sialic Glycoprotein Column Fractionation Kit, Qiagen Germany) can be used to enrich for sialic linked glycoproteins harvested from very small volumes of crude exudates from cells including, for example, cells from sinus mucosal tissue explants. Together with an automated protein electrophoresis system that utilises microfluidic separation techniques and fluorescent sample detection (Experion Pro260 Electrophoresis Chips, Biorad USA), various species of terminal sialic acid glycoproteins can be discriminated precisely depending on their linkage patterns and molecular weights.

RNA/DNA microarray profiling technology may also be adapted to analyse glycoproteins (e.g. Qproteome Glycoarray Kit, Qiagen Germany). Here, the expression of terminal sugars for any particular glycoprotein can be determined provided the appropriate monoclonal antibody to that glycoprotein is available. An exemplary method for glycoprotein array analysis is described by Rosenfeld et al. (J Biochem Biophys Methods 70: 415-426, 2007)

Accordingly, in some embodiments, the sialic acid profile is determined by using a glycoprotein microarray. In some embodiments, determining a sialic acid profile associated with one or more glycoproteins expressed by a cell of the subject comprises determining the level of expression of a protein associated with the sialic acid profile by one or more cells of the subject.

A "protein associated with the sialic acid profile" of a subject includes any protein which forms part of a sialic acid linked glycoprotein and/or is involved in the synthesis or degradation of such proteins.

Thus, in some embodiments, a protein associated with a sialic acid profile may be an apoprotein of a glycoprotein. Examples of such apoproteins include, for example, the apoprotein backbones of mucin glycoproteins as hereinbefore described. In some embodiments, as described later, a protein associated with the sialic acid profile of a subject may also be a sialyl transferase.

Determining the level of expression of the protein associated with the sialic acid profile may include determining the level of protein expression and/or the level of expression of a nucleic acid which encodes the protein.

Methods for determining the level and/or pattern of expression of a nucleic acid or protein are known in the art.

Exemplary methods of the detection of RNA expression include methods such as quantitative or semi-quantitative reverse-transcriptase PCR (eg. see Zaheer et al, Neurochem. Res. 20:1457-63, 1995), in-situ hybridization, including fluorescent in situ hybridisation (FISH) (eg. see Jin and Lloyd, / Clin Lab Anal. 11(1): 2-9, 1997; Calmels and Mazurais, Methods MoI Biol. 366: 159-80, 2007; and Pauletti et al, Journal of Clinical Oncology 18(21): 3651-3664, 2000); northern blotting (eg. see McMaster et al, Proc. Natl. Acad. Sci. USA 74: 4835-38, 1977); and the like. Exemplary methods for analysing the expression of a polypeptide include, for example, Western blotting (eg. see Fido et al, Methods MoI Biol. 49: 423-37, 1995); ELISA (eg. see Fujiwara et al, J Immunol Methods. 112(1): 77-83, 1998); or immunohistochemistry.

In some embodiments, the expression level of a protein associated with the sialic acid profile may be determined by determining the copy number of a nucleic acid encoding a protein present in the genomic DNA of one or more cells of the subject. In this embodiment, multiple copies of a nucleic acid encoding a protein in the genome are predictive of a relatively high expression level of the nucleic acid sequence and/or the protein in the cell. In some embodiments, a relatively high level of the nucleic acid sequence and/or the protein expression is indicated by the presence of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 copies of the nucleic acid in the genome of one or more cells of the organism.

In some embodiments, determining the level of expression of a protein associated with the sialic acid profile comprises determining the expression of protein variants. The variants may be functional or non-functional variants with relation to the sialic acid profile associated with the glycoprotein.

Examples of protein variants include splice variants resulting in proteins with different isoforms or the variants may be different glycoforms, either through posttranslational or cotranslational modification. Protein variants may also be conformational variants, wherein the proteins possess the same amino acid sequence, but are folded in a different configuration which can influence the activity of the protein. Protein variants may also possess an amino acid sequence change which can affect the folding of the protein, the charge of the protein, the activity of the protein and/or the location of the protein.

In some embodiments, determining the expression of protein variants may be more accurate than, for example, merely determining the copy number of the nucleic acid sequence encoding the protein. In the case of protein variants comprising functional and non-functional proteins, an assessment on the level of expression of functional proteins may provide a more accurate assessment of the level of expression of a protein associated with a sialic acid profile.

Protein variants that result from alternate splicing may be determined at the mRNA level by methods including RT-PCR. At the protein level, variants may be determined by methods including mass spectroscopy or HPLC, or methods which utilise antibodies or lectins which are able to discriminate between the variants.

In some embodiments, the expression level of a protein associated with the sialic acid profile may be determined by determining the presence or absence of mutations or polymorphisms in promoter or regulatory regions associated with the gene encoding the protein. For example, a mutation in a promoter region may upregulate, downregulate or change the pattern of expression of a gene or a particular splice variant thereof.

In some embodiments, the protein associated with the sialic acid profile may be a sialyltransf erase.

Sialyltransferases (STs) are members of the glycosyltransferase family of enzymes that catalyse the addition of sialic acid to the terminal portion of an oligosaccharide side chain. There are approximately twenty different STs that are distinguished by (i) the acceptor structure on which they act and (ii) the type of sugar links they form. Three exemplary STs that are of particular relevance to the present invention are: ST6Gal I which catalyses α2-6 linked sialic acids (sialic α2-6Gal[31-4GlcNAc-R) and ST3Gal3 and ST3Gal4 that catalyse α2-3 linked sialic acids (sialic α2-3Gal βl-3GlcNAc-R or Gal βl-4 GIcNAc-R). As such, in some embodiments, the sialyltransferase is SToGaII, ST3Gal3 or ST3Gal4.

Examples of the SToGaII (ST6 beta-galactosamide alpha-2,6-sialyltranferase 1) are described under GeneCard accession number GC03P188132.

Examples of the ST3GAL3 (ST3 beta-galactoside alpha-2,3-sialyltransferase 3) are described under GeneCard accession number GC01P043946.

Examples of the ST3GAL4 (ST3 beta-galactoside alpha-2,3-sialyltransferase 4) are described under GeneCard accession number GC11P125732.

In some embodiments, the level of expression of SToGaII, ST3Gal3 or ST3Gal4 by a cell is indicative of a subject with the presence or absence of a biofilm on a surface or is indicative of the risk of formation of a biofilm on a surface of the subject.

In some embodiments, increased expression of SToGaII is indicative of the presence of a biofilm on a surface of the subject or indicative of a subject with an increased risk of formation of a biofilm on a surface of the subject.

An "increased" expression of SToGaII should be understood to include elevated expression relative to a healthy normal subject. Similarly, an "increased risk" of biofilm formation as referred to herein, refers to a risk elevated relative to healthy, normal subjects.

In some embodiments, the presence of the biofilm, or an increased risk of formation of a biofilm, on a surface of a subject, is indicative of a subject with a disease or with an increased risk of a disease. In this regard, determining a sialic acid profile can be used as an indicator for the presence of a disease, or an increased risk of disease development in the subject. As hereinbefore disclosed, several human disease states are associated with biofilm formation on a surface of the subject. Such disease states include, for example: biofilm- mediated sub-types of CRS, cystic fibrosis, dental caries and related gum diseases, otitis media with effusion, sepsis and complications arising from bacterial biofilm colonization of prosthetic implants or cardiac stents and chronic eye infections from continuous use of contact lenses

In some embodiments, the disease is a respiratory disease. The disease may be a chronic respiratory disease including, for example, chronic rhinosinusitis.

Rhinosinusitis is generally described as an inflammation of the nasal cavity and/or paranasal sinuses and involves the nasal mucosa. Chronic rhinosinusitis (CRS) is diagnosed when signs or symptoms of inflammation persist for 8-12 weeks or longer. Symptoms of CRS include nasal obstruction, loss of sense of smell, nasal or postnasal discharge, nasal congestion, and facial pain/pressure (typically over the affected sinus area).

In some embodiments, the presence of the biofilm, or an increased risk of formation of a biofilm, on a surface of a subject, is indicative of a subject with a poor surgical prognosis. In particular, determining a sialic acid profile may be used to identify a subject with a poor surgical prognosis, for a surgery associated with the presence of a biofilm on a surface of the subject.

"Poor surgical prognosis" should be understood to include recurrence of biofilm formation after surgery and/or associated complications including, for example, reestablishment of infection, a biofilm or associated disease.

As such, in some embodiments the poor surgical prognosis is associated with a sinus surgery. Sinus surgery is common in the treatments of sinusitis including chronic rhinosinusitis. In some embodiments, the surgery may be associated with the implantation of a prosthetic implant, catheter or stent. In these embodiments, formation of a biofilm on the implant, catheter or stent may be regarded as a poor surgical outcome.

In some embodiments, the presence of a biofilm or an increased risk of formation of a biofilm on a surface of a subject is indicative of a subject having reduced responsiveness to antibiotic therapies for a biofilm. As such, determining a sialic acid profile may be indicative of a subject having reduced responsiveness to antibiotic therapies for a biofilm.

In some embodiments, the presence of a biofilm or an increased risk of formation of a biofilm, on a surface of a subject, is indicative of a subject having an increased risk of biofilm establishment and/or recurrence. As such, determining a sialic acid profile associated with one or more glycoproteins expressed by a cell of the subject, may be indicative of a subject having an increased risk of recurrent colonisation by biofilm- forming bacteria.

As set out above, the present invention is predicated on determining a sialic acid profile associated with one or more glycoproteins expressed by a cell of the subject. The cell may be any cell which expresses one or more glycoproteins.

In some embodiments, the cell of the subject is an epithelial cell. The epithelium includes skin (epidermis), the outer surface of the cornea the lining inside of the lungs, the gastrointestinal tract, the reproductive and urinary tracts, mucous membranes such as the mouth, the esophagus, and part of the rectum. The epithelium also includes the endothelium, which is the inner lining of blood vessels, the heart, and lymphatic vessels and the mesothelium which forms the walls of the pericardium, pleurae, and peritoneum.

In some embodiments the cell of the subject is a basal or goblet cell. Basal cells are epithelial cells which may be found on basement membranes of the epithelium. Basal cells are capable of rapid division to replenish shedded or damaged epithelial cells. Goblet cells are glandular epithelial cells which secrete mucus. Goblet cells are found scattered among the epithelial lining of many organs, including the intestinal and respiratory tracts. In the respiratory tract, goblet cells may be found inside the trachea, bronchus, and larger bronchioles.

In some embodiments, the cell may be a cell from a serous gland, a mucous gland or a seromucous gland.

Cells may be isolated for glycoprotein analysis by any method known in the art. Suitable methods of isolation will depend on the type of cell required for analysis and the location of the cell.

For example, in some embodiments, blood collection, swabbing and/or collection of tears, saliva, urine, fecal material, sweat or perspiration, etc may provide sufficient cells for analysis, in particular where the analysis is at the genetic level. The term "swabbing" generally denotes contacting an applicator/collector ("swab") containing or including an adsorbent material to a surface in a manner sufficient to collect surface debris and/or dead or sloughed off cells or cellular debris. Such collection may be accomplished by swabbing nasal, oral, rectal, vaginal or aural orifices, by contacting the skin or tear ducts, etc. Methods for collecting epithelial cells from urine are provided in Gasparini et al. (N. Engl. J. Med 320: 809, 1989). Commonly, buccal smears are performed to collect somatic cells from the inside of the cheek due to the simplicity and non-invasive nature of the procedure. An example of a method for conducting buccal smears is disclosed by Lench et al. (The Lancet 1: 1356-8, 1988)

In other embodiments, a biopsy may be required. The biopsy may involve a needle biopsy, a punch biopsy or an excision biopsy. The cells may then be homogenised or lysed prior to analysis. In some embodiments, the isolated cells may be cultured prior to analysis. For example explants of a biopsy sample may be generated.

In a particular embodiment, the cell of the subject is a cell from the sinus mucosa. The sinus mucosa includes cells of the paranasal sinuses. As provided above, paranasal sinuses include the maxillary sinuses the frontal sinuses the ethmoid sinuses and the sphenoid sinuses.

In one embodiment, the one or more cells of the subject on which the level of expression of the protein is determined are one or more cells from a sinonasal biopsy. Methods for performing sinonasal biopsies include, for example, methods described by Wormald et al. (Laryngoscope 113(5): 867-73, 2003).

Finally, reference is made to standard textbooks of molecular biology that contain methods for carrying out basic techniques encompassed by the present invention, including DNA restriction and ligation for the generation of the various genetic constructs described herein. See, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, 1982) and Sambrook et al. (2000, supra).

The present invention is further described by the following non-limiting examples:

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows sections of mucosa from a healthy patient (panel A) and a chronic rhinosinusitus patient (panel B) viewed by confocal scanning fluorescent microscope (2OX magnification). Panel A shows a mixture of live (for example, arrow a) and dead (for example, arrow b) nuclei of epithelial cells. Panel B shows a bacterial biofilm characterised by pinpoints of live bacteria (for example, arrow c) embedded in a polysaccharide matrix amongst nuclei (for example, arrow d) of the pseudostratified columnar epithelial cells.

Figure 2 shows a two-way table of the SNA specific sialic acid profiles of biofilm positive (Bl-8), no biofilm (NB1-2) and control (Cl-9) patient samples. The rows represent SNA specific glycoproteins separated in 10 kDa increments, while the columns represent each of the patient samples. The clustering of the patient samples, as represented on the table, was based on common glycoprotein expression as calculated using the Bray Curtis Similarity Index (Stress value = 0.1968).

Figure 3 shows a two-way table of the WGA specific sialic acid profiles of biofilm positive (Bl-8), no biofilm (NB1-2) and control (Cl-9) patient samples. The rows represent SNA specific glycoproteins separated in 10 kDa increments, while the columns represent each of the patient samples. The clustering of the patient samples, as represented on the table, was based on common glycoprotein expression as calculated using the Bray Curtis Similarity Index (Stress value = 0.1414).

Figure 4 shows a graph representing the level of expression of the ST6Gal I gene in biofilm positive CRS patients and controls. The average level of ST6Gal I gene expression (represented by the horizontal bars) was statistically significantly higher in biofilm positive CRS patients than in controls.

Figure 5 shows a two way table of MUC7 glycosylation profiles of biofilm positive CRS (Biofilm 1-13), no biofilm CRS (non 1-19) and control (Control 1-13) patient samples. The rows represent the sugar groups on the MUC7 glycoprotein, while the columns represent each of the patient samples. EXAMPLE 1 Detection of bacterial biofilm by CSLM

A Zeiss Apotome fluorescent microscope with confocal scanning ability was used to determine the biofilm status of CRS patients undergoing sinus surgery using a BacLight LIVE/DEAD kit. Healthy mucosa from a control patient shows a mixture of live and dead nuclei of epithelial cells (Figure 1 - upper panel). Patient mucosa with a bacterial biofilm is characterized by pinpoints of live bacteria (1 μm length) embedded in a polysaccharide matrix amongst the nuclei of the pseudostratified columnar epithelial cells (Figure 1 - lower panel)

EXAMPLE 2 SNA specific sialic acid profiles differ between biofilm positive patients and controls

In a study consisting of 8 biofilm positive (B), 2 non biofilm (NB) CRS patients and 10 healthy controls (C), we found 88 % (7/8) biofilm positive patients (B) expressed more α2-6 linked SNA specific sialic acids of between 80-90 kDa compared with healthy controls (C) as illustrated by Figure 2.

The left vertical column represents SNA specific glycoproteins separated in 10 kDa increments; horizontal row represents patients clustered together based on common glycoprotein expression as calculated using the Bray Curtis Similarity Index (Stress value=0.1968)

Materials and Methods

Crude glycoprotein secretions were harvested and pooled from sinus biopsies cultured in collagen coated inserts at air liquid interface at 37°C, 80% relative humidity and CO2 incubator over a 2-5 day period (all dependent on how much glycoprotein is harvested which is determined by the size of the original piece of tissue collected at the time of sinus surgery). Sialic acids were extracted using a Qproteome sialic acid fractionation kit (Qiagen, Germany). Eluate was concentrated using acetone, allowed to precipitate on ice, spun down, supernatant removed and air dried in a fume hood. The dried down pellet was then resuspended in loading buffer (variation of a Laemmli buffer with beta mercaptoethanol). Using Experion loading buffer (Biorad USA), a small sample was loaded on the Pro260 Experion chip and electrophoresed as per the manufacturer's instructions. A spreadsheet of the data was generated by the Experion software from which the amount of any particular glycoprotein present in that patient's tissue may be determined. Each preparation was run in triplicate.

A presence/absence spreadsheet was generated and imported into PATN multivariate analysis programme. A Bray Curtis Similarity Index is generated which clusters patients together according to how many similar MW sialic residues are expressed.

EXAMPLE 3

WGA specific sialic acids profiles differ between biofilm positive patients and controls

Using the same patient cohort and methods as described in Example 2, Figure 3 shows that 100% (8/8) of biofilm positive patients (B) express WGA specific sialic acids of between 70-80, 80-90 and 90-100 kDa that are not present in the majority of the healthy controls (C) Stress value= 0.1414.

EXAMPLE 4

Expression of the ST6Gal I gene is statistically significant between biofilm positive CRS patients and healthy controls

As shown in Figure 4, using real time Reverse Transcriptase PCR higher expression of the ST6 Gal 1 gene in biofilm positive CRS patients was observed relative to controls.

This implies that biofilm positive patients have higher activity of the gene that catalyses the addition of α2-6 linked sialic acids onto nascent oligosaccharide chain of mucin glycoproteins.

Materials and Methods

RNA was extracted from sinus tissue harvested from patients using the QIAzol RNA extraction kit (Qiagen). RNA is reverse transcribed using Qiagen RT kit to get cDNA and a duplex real time RT-PCR run using primers specific for the target gene. The housekeeping gene used is SDHA and relative expression levels calculated using the 2- DDCt method. Prism v5 was used in determining statistical significance.

EXAMPLE 5

MUC7 glycosylation profiles differ significantly between biofilm positive CRS patients and controls

As shown in Figure 5, significant and marked differences were observed in the MUC7 glycosylation profiles between CRS patients with biofilms (Biofilm), or without biofilm (Non) and healthy controls (Control) undergoing trans sphenoidal pituitary surgery (stress value 0.088). The majority of the healthy controls not only had more of the sugar groups per se and also in larger quantities (darker blocks) compared to both biofilm and non biofilm patients (lighter blocks).

Materials and Methods

This data was generated using the Qiagen Glycoarray kit. Tissue lysate from patients are bound onto the array (glass slide) and probed with a MUC7 specific mouse monoclonal antibody and visualised with a secondary Cy3 goat anti mouse antibody. The slides were scanned using an Axon Scanner (Adel Uni Microarray Facility) and the software provided by Qiagen was used for analysis. Again, an Excel spreadsheet is generated showing which of the MUC7 terminal sugars are expressed. Data analysis done using PATN. EXAMPLE 6 Determination of biofilm status of CRS patients using CSLM

Sinus tissue from patients undergoing sinus surgery is harvested and their biofilm status determined. Suitable methods for performing these procedures are described in Psaltis et al. (Am J Rhinol 22: 1-6, 2008) and Psaltis et al. (Laryngos 117: 1302-1306, 2007).

Briefly, sinonasal biopsies are harvested in Dulbecco's Modified Eagles Media pH 7.2 without antibiotics and stained with Syto 9 and Propidium iodide (BacLight LIVE/DEAD kit, Molecular Probes, Invitrogen USA) within 2 hours of collection. Control tissue is collected from healthy patients undergoing trans sphenoidal surgery who show no overt signs of sinus disease.

EXAMPLE 7 Determination of patient MAA, SNA and WGA sialic acid linkage patterns

Mucus glycoproteins are harvested from sinus tissue explants placed in 6 well collagen coated inserts that can be cultured at an air liquid interface. This allows the collection of crude glycoprotein exudates from tissue explants that are fed basolaterally using Hams F12/DMEM media pH 7.2. Separation of MAA, SNA and WGA specific sialic acids is carried out using Sialic Glycoprotein Fractionation kits (Qiagen Hilden Germany). Their molecular weights are determined by gel electrophoresis (Experion Pro 260 protein chips, Biorad Hercules Calif USA) and glycoprotein profiles examined by PATN analysis.

EXAMPLE 8

Determination by real time RTPCR relative expression levels of ST6 Gal 1, ST3 Gal3,

ST3 Gal4

Tissue harvested is placed immediately in RNA Later (Ambion, Texas USA) to prevent degradation of RNA. Tissue is homogenized (Tissue Ruptor, Qiagen), total RNA extracted (RNeasy Lipid Tissue Extraction kit, Qiagen), cDNA reversed transcribed (Quantitect Reverse Transcription kit, Qiagen) and using Taqman probes and primers (Multiplex Power Mix, Biorad) a multiplex reaction carried out. A standard housekeeping gene SDHA has been shown to be invariant in its expression between different patients. A 2-DDCt method is used to determine relative expression levels of the target genes (as described in Livak et al. Method. Methods 25:402-408, 2001) Statistical analysis is carried out using Prism v5a.

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 invention includes all such variations and modifications. 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 the steps or features.

Also, it must be noted that, as used herein, the singular forms "a", "an" and "the" include plural aspects unless the context already dictates otherwise.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method for determining a presence of a biofilm, or a risk of formation of a biofilm, on a surface of a subject, the method comprising determining a sialic acid profile associated with one or more glycoproteins expressed by a cell of the subject.

2. A method according to claim 1, wherein the biofilm comprises a bacterial cell.

3. A method according to claim 2, wherein the bacterial cell is a Streptococcus species.

4. A method according to claim 3, wherein the Streptococcus species is Streptococcus mutans, Streptococcus gordonii or Streptococcus pneumoniae.

5. A method according to claim 2, wherein the bacterial cell is a Staphylococcus species.

6. A method according to claim 5, wherein the Staphylococcus species is Staphylococcus aureus.

7. A method according to claim 1, wherein the biofilm comprises a fungal cell.

8. A method according to any one of claims 1 to 7, wherein the subject is mammalian.

9. A method according to claim 8, wherein the mammalian subject is a human.

10. A method according to any one of claims 1 to 9, wherein a disease is associated with the biofilm.

11. A method according to any one of claims 1 to 10, wherein a respiratory disease is associated with the biofilm.

12. A method according to any one of claims 1 to 11, wherein chronic rhinosinusitis is associated with the biofilm.

13. A method according to any one of claims 1 to 12, wherein the glycoprotein is a mucin glycoprotein.

14. A method according to claim 13, wherein the mucin glycoprotein is MUCl, MUC5AC, MUC5B, or MUC7.

15. A method according to any one of claims 1 to 14, wherein determining the sialic acid profile associated with the one or more glycoproteins comprises determining an amount of a sialic acid linked to the one or more glycoproteins.

16. A method according to any one of claims 1 to 15, wherein determining the sialic acid profile associated with the one or more glycoproteins comprises determining a molecular weight of a sialic acid linked glycoprotein.

17. A method according to any one of claims 1 to 16, wherein determining the sialic acid profile associated with the one or more glycoproteins comprises determining a type of sialic acid-glycoprotein linkage.

18. A method according to claim 17, wherein the type of sialic acid-glycoprotein linkage is an α2-6 linkage.

19. A method according to claim 17, wherein the type of sialic acid-glycoprotein linkage is an α2-3 linkage.

20. A method according to any one of claims 1 to 19, wherein determining the sialic acid profile associated with the one or more glycoproteins comprises determining the amount of a sialic acid linked glycoprotein having a molecular weight of 70-80 kDa.

21. A method according to any one of claims 1 to 20, wherein determining the sialic acid profile associated with the one or more glycoproteins comprises determining the amount of a sialic acid linked glycoprotein having a molecular weight of 80-90 kDa.

22. A method according to any one of claims 1 to 21, wherein determining the sialic acid profile associated with the one or more glycoproteins comprises determining the amount of a sialic acid linked glycoprotein having a molecular weight of 90-100 kDa.

23. A method according to any one of claims 19 to 22, wherein the amount of a sialic acid linked glycoprotein having a molecular weight of 70-80 kDa, 80-90 kDa or 90-100 kDa is indicative of the presence or absence of a biofilm on a surface of the subject or is indicative of the risk of formation of a biofilm on a surface of the subject.

24. A method according to any one of claims 19 to 23, wherein an increased amount of the one or more sialic acid linked glycoproteins is indicative of the presence of a biofilm on a surface of the subject or is indicative of an increased risk of formation of a biofilm on a surface of the subject.

25. A method according to any one of claims 1 to 24, wherein determining a sialic acid profile associated with one or more glycoproteins expressed by a cell of the subject comprises determining the level of expression of a protein associated with the sialic acid profile by one or more cells of the subject.

26. A method according to claim 25, wherein the protein is an apoprotein.

27. A method according to claim 25, wherein the protein is a sialyltransf erase.

28. A method according to claim 27, wherein the sialyltransferase is SToGaII, ST3Gal3 or ST3Gal4.

29. A method according to claim 33, wherein increased expression of SToGaII is indicative of the presence of a biofilm on a surface of the subject or indicative of a subject with an increased risk of formation of a biofilm on a surface of the subject.

30. A method according to any one of claims 1 to 29, wherein the presence of the biofilm, or an increased risk of formation of a biofilm, on a surface of a subject, is indicative of a subject with a disease or with an increased risk of a disease.

31. A method according to claim 30, wherein the disease is a respiratory disease.

32. A method according to claim 30 or 31, wherein the disease is a chronic respiratory disease.

33. A method according to any one of claims 30 to 32, wherein the disease is chronic rhinosinusitis.

34. A method according to any one of claims 1 to 33, wherein the presence of a biofilm or an increased risk of formation of a biofilm, on a surface of a subject, is indicative of a subject with a poor surgical prognosis.

35. A method according to claim 34, wherein the poor surgical prognosis is associated with a sinus surgery.

36. A method according to any one of claims 1 to 35, wherein the presence of a biofilm or an increased risk of formation of a biofilm on a surface of a subject is indicative of a subject having reduced responsiveness to antibiotic therapies for a biofilm.

37. A method according to any one of claims 1 to 36, wherein the presence of a biofilm or an increased risk of formation of a biofilm, on a surface of a subject, is indicative of a subject having an increased risk of recurrent formation of a biofilm.

38. A method according to any one of claims 1 to 37, wherein the surface is a mucosal surface.

39. A method according to any one of claims 1 to 38, wherein the surface is a mucosal respiratory surface.

40. A method according to any one of claims 1 to 39, wherein the surface is a paranasal sinus surface.

41. A method according to any of claims 1 to 38, wherein the surface is a alimentary, reproductive or ear canal surface.

42. A method according to any one of claims 1 to 41, wherein the surface is an artificial surface.

43. A method according to any one of claims 1 to 42, wherein the cell of the subject is an epithelial cell.

44. A method according to any one of claims 1 to 43, wherein the cell of the subject is a cell from the sinus mucosa.

45. A method according to any one of claims 1 to 44, wherein the cell is a basal or goblet cell.

46. A method according to any one of claims 1 to 44 wherein the cell is a cell from a serous gland, a mucous gland or a seromucous gland.