COMPOSITIONS AND USES THEREOF

Abstract

The present invention relates to a composition comprising a probiotic extract, wherein the extract comprises a protein fraction derived from a secretion or lysate and having proteins of a molecular weight of up to 100 kDa. The composition may have a number of uses, such as for use in the prevention, management or treatment of bacterial infection or the enhancement and improvement of skin health.

Claims

1. A composition comprising a probiotic extract, wherein the extract comprises a protein fraction derived from a secretion or lysate and having proteins of a molecular weight of up to 100 kDa.

2. The composition according to claim 1, wherein the extract is from one or more probiotic strains.

3. The composition according to claim 1, wherein the probiotic strain is a Lactobacilli.

4. The composition according to claim 3, wherein the Lactobacilli is L. rhamnosus.

5. The composition according to claim 1, wherein the proteins have a molecular weight of up to 90 kDa.

6. The composition according to claim 5, wherein the protein fraction comprises the Subtilin biosynthesis protein C (SpaC) and proteins having a molecular weight of up to 50 kDa, and optionally one or more exopolysaccharides.

7. The composition according to claim 6, wherein the proteins having a molecular weight of up to 50 kDa comprise one or more of the following: Glyceraldehyde-3 phosphate dehydrogenase (GAPDH); Elongation factor TU (EF-Tu); Triosephosphate isomerase (TPI); and/or Enolase.

8. A composition comprising the SpaC protein and one or more of the following proteins: Glyceraldehyde-3 phosphate dehydrogenase (GAPDH); Elongation factor TU (EF-Tu); Triosephosphate isomerase (TPI); Enolase; Acyl carrier protein; Transcription elongation factor greA; Phosphopentomutase; 505 ribosomal protein S11; Dihydroxyecetone kinase; 50s Ribosomal protein; Asparaginyl tRNA synthetase; UPF0342 protein; and/or 505 ribosomal protein L22.

9. The composition according to claim 8, wherein the one or more of the following proteins comprise: Glyceraldehyde-3 phosphate dehydrogenase (GAPDH); Elongation factor TU (EF-Tu); Triosephosphate isomerase (TPI); and/or Enolase.

10. The composition according to claim 9, wherein one or more of the proteins are recombinant and/or derived from a secretion or lysate of a probiotic.

11. The composition according to claim 10, wherein the probiotic is a Lactobacilli.

12. The composition according to claim 11, wherein the Lactobacilli is L. rhamnosus.

13. The composition according to claim 12, wherein the L. rhamnosus is L. rhamnosus GG (ATCC 53103).

14. The composition according to claim 8 wherein the composition further comprises one or more pharmaceutically or cosmetically acceptable ingredients or excipients.

15-16. (canceled)

17. The composition according to claim 1, for use in the prevention, management or treatment of a microbial infection or colonisation.

18. The composition for use according to claim 17 wherein the microbial infection or colonisation is with Staphylococcus, and optionally wherein the Staphylococcus is S. aureus.

19-21. (canceled)

22. The composition for use according to claim 17, which is formulated for use as a cream, gel, oil, or spray.

23-35. (canceled)

36. The composition according to claim 4, wherein the L. rhamnosus is L. rhamnosus GG (ATCC 53103).

37. The composition as claimed in claim 1, wherein the composition further comprises one or more pharmaceutically or cosmetically acceptable ingredients or excipients.

38. The composition according to claim 1, for use in the prevention, management or treatment of a microbial infection or colonisation.

40. The composition for use according to claim 38, wherein the microbial infection or colonisation is with Staphylococcus, and optionally wherein the Staphylococcus is S. aureus.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0076] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0077] FIG. 1: Proteins mediate the effects of LGG lysate against S. aureus.

[0078] A: A combination of S. aureus (S.a) and L. rhamnosus GG lysate (Lgg lys) resulted in a significantly higher percentage of viable keratinocytes than in monolayers infected with S. aureus alone (p=0.006). All data are compared to viability of untreated monolayers (Con). Heat (Lgg+heat) or protease treatment (Lgg+tryp) destroyed the ability of the lysate to protect keratinocytes from S. aureus.

[0079] B: When the lysate was fractionated and proteins eluted in 10-70% acetonitrile, proteins with efficacy against S. aureus were contained in fractions 30-60%. NS=Non-Significant. * denotes significant data.

[0080] FIG. 2: Specific Fractions inhibit Staphylococcal adhesion to keratinocytes

[0081] A: Cells pre-treated with L. rhamnosus GG lysate (LGG lys) had significantly fewer adherent Staphylococci compared to cells infected with S. aureus (SA) alone. The adhesion of the pathogen to keratinocytes was significantly lower in cultures treated with fractions eluting in 30%, 40%, 50% and 60% acetonitrile (P=0.01, P=0.016 P=0.012, P=0.015 respectively, n=3).

[0082] B: The same fractions were also efficatious when added to keratinocytes 2 hours after incubation with pathogen (P=0.034, P=0.035, P=0.01, P=0.033 for 0%, 40%, 50% and 60% respectively, n=3). There was no difference between the numbers of staphylococci adherent to cells exposed to 20% acetonitrile fractions in either assay (P=0.06, n=3). However, there was a significant difference in the number of adherent staphylococci adherent to cells exposed to 50% acetonitrile fraction compared with other fractions in both assays (*P=0.02). Results are expressed as the mean?SEM, *P<0.05. N.S=Non-Significant.

[0083] FIG. 3: The pilus protein SpaC is involved in the anti-adhesive function of the lysate

[0084] A: Immunoblotting with specific anti-SpaC serum demonstrated the presence of the SpaC protein in the 50% fraction.

[0085] B: recombinant SpaC (rSpaC) inhibited staphylococcal adhesion to keratinocytes in a dose dependent manner with 25 and 50 ug/ml rSpaC providing significant protection (0.03 and 0.013 respectively).

[0086] C: 50 ?g/ml of BSA, did not inhibit staphylococcal adhesion.

[0087] FIG. 4: SpaC protects keratinocytes from the toxic effects of S. aureus.

[0088] A: Recombinant SpaC (rSpaC) at 50 ug/ml afforded significant protection to keratinocyte monolayer viability in the presence of S. aureus (S.a) (p=0.013). However, this was significantly less than the protection afforded by the whole lysate (Lgg lys, p=0.008).

[0089] B: By contrast, a SpaC deficient strain of LGG (KO SpaC) was still able to protect keratinocytes.

[0090] All data were compared to the viability of the untreated monolayer (Con).

EXAMPLES

Example 1: Methods

Bacterial Cell Culture

[0091] Lactobacillus rhamnosus GG (ATCC 53103) was cultured anaerobically in Wilkins-Chalgren broth at 37? C., and Staphylococcus aureus was cultured aerobically in nutrient broth (Oxoid) as described in Mohammedsaeed et. al. (2014) and Prince et. al. (2011) (Mohammedsaeed M., et. al., (2014). Appl. Environ. Microbiol. 80(18):5773 and Prince T. et. al., (2011) Appl Environ. Microbiol. 78(15):5119-26). The LGG SpaC knock out was produced as described in Lebeer et. al. (2012) (Lebeer S., et. al., (2012). Appl Environ. Microbiol. 78: 185-193). LGG lysate was produced according to the protocol published in Mohammedsaeed et. al., (2014). In some experiments investigating the involvement of proteins, the lysate was placed in a boiling water bath for 5 min, or treated with tryspin (0.2% w/v) in Phosphate buffered saline for 1 h at 37? C. to denature proteins.

Fractionation of the L. rhamnosus GG lysate

[0092] A 30 ml preparation of LGG lysate was adjusted to pH 5.8 using 0.1% Triflouroacetic acid and applied to a Strata XL column (pore size 100 ?m, Phenomenex Ltd, Cheshire, UK). Bound proteins were eluted from the column in 60 ml of 90% methanol at pH 2. The sample was spun in a centrifugal evaporation system for 3 h (Biotek, Bedfordshire, UK) and the resulting sample (5 ml) was applied to a 5 ml Sep-Pak C18 cartridge (pore size 37-55 ?m, Fischer Scientific, Loughborough, UK). Proteins were eluted using 5 ml aliquots of increasing concentrations of 10-70% (v/v) acetonitrile containing 0.1% (v/v) TFA solution. Each 5 ml fraction was collected into a separate tube and the eluted fractions were evaporated to remove the acetonitrile for 3 h in centrifugal evaporation system (Biotek, Bedfordshire, UK). The resulting 1 ml of each fraction was subjected to SDS-page analysis and stained with Instant Blue (Harston, Cambridgeshire, UK) to visualise the protein bands. The fractions were maintained at 4? C. for further analysis in adhesion and viability assays. For increased concentration and purification, the most efficacious fractions were further separated by HPLC using a Jupiter 90A column (Phemonenex, Cheshire, UK) with a gradient of 10-99% acetonitrile applied over 50 min.

Tandem Mass Spectrophotometric Analysis of Protein Fractions

[0093] Tandem Mass spectrometry (MS/MS) identification of proteins was conducted using the gel top method. Proteins were separated electrophoretically for 10 minutes at 150V by SDS-PAGE and then stained using Instant Blue. Bands of interest were excised from the gel and dehydrated using acetonitrile followed by vacuum centrifugation. Dried gel pieces were reduced with 10 mM dithiothreitol and alkylated with 55 mM iodoacetamide. Gel pieces were then washed alternately with 25 mM ammonium bicarbonate followed by acetonitrile. This was repeated, and the gel pieces dried by vacuum centrifugation. Samples were digested with trypsin overnight at 37? C. Digested samples were analysed by LC-MS/MS using an UltiMate? 3000 Rapid Separation LC (RSLC, Dionex Corporation, Sunnyvale, Calif.) coupled to a LTQ Velos Pro (Thermo Fisher Scientific, Waltham, Mass.) mass spectrometer. Peptide mixtures were separated using a gradient from 92% A (0.1% FA in water) and 8% B (0.1% FA in acetonitrile) to 33% B, in 44 min at 300 nL min.sup.?1, using a 75 mm?250 ?m i.d. 1.7 ?M BEH C18, analytical column (Waters). Peptides were selected for fragmentation automatically by data dependant analysis. Data produced was searched using Mascot data base search engine (Matrix Science UK). Data was validated using Scaffold (Proteome Software, Portland, Oreg.).

Growth of Primary Human Keratinocytes, Viability and Adhesion Assays

[0094] Primary human keratinocytes and associated adhesion and viability assays were performed exactly as described in Mohammedsaeed et. al. (2014).

Production of Recombinant SpaC

[0095] An expression plasmid construct (pKTH5319) consisting of the L. rhamnosus GG (ATCC 53103) SpaC gene encoding the backbone (residues 31-302) but lacking the N-terminal signal peptide and C-terminal sorting motif regions was produced in accordance with the protocol mentioned in Kankainen et. al. (2009) (Kankainen M., et. al, (2009) Proc Natl Acad Sci USA 106(40): 17193-8) was created. Recombinant SpaC pilin was hexa-histidine-tagged at the C-terminus. Recombinant protein expression in Escherichia coli BL21 cells was then induced by addition of 1 mM isopropyl-d-1-thiogalactopyranoside after which the culture was grown overnight at 18? C. Cells were harvested by centrifugation, resuspended in a lysis buffer consisting of 40 mM NaH2PO4 pH 7.4, 150 mM NaCl and EDTA-free protease-inhibitor cocktail tablets (Roche Ltd, Sussex UK). The cell lysate was then centrifuged at 48, 400?g for 20 min. and the cell-free lysate was loaded onto a 5 ml NiCl.sub.2-charged HiTrap chelating HP column (GE Healthcare, Amersham, UK) that had previously been equilibrated with a buffer containing 40 mM NaH.sub.2PO.sub.4 pH 7.4, 150 mM NaCl. Resin-bound SpaA protein was then eluted with buffer containing 40 mM NaH.sub.2PO.sub.4 pH 7.4, 150 mM NaCl, 250 mM imidazole using a linear gradient equivalent to ten column volumes. SpaC containing fractions were determined by SDS-PAGE, pooled and dialyzed against 20 mM HEPES pH 7.0, 150 mM NaCl, 1 mM EDTA. The dialyzed protein solution was then concentrated using an Amicon ultrafiltration device fitted with a 10 kDa molecular-weight cut-off (Amicon technologies Ltd, Kent, UK) and subsequently purified further by a Sephacryl S-200 26/60 gel-filtration column (GE Healthcare) equilibrated with dialysis buffer.

SDS-PAGE and Immunostaining

[0096] SDS-PAGE and immunostaining was performed as described in Sultanna et. al. (2013) (Sultanna R. et. al., (2013) Appl. Environ, Microbiol. 79(16) 4887-4894) using SpaC antiserum as described in Kankainen et. al. (2009) (Kankainen M., et. al., (2009) Proc Natl Acad Sci USA 106(40): 17193-8).

Statistical Analysis

[0097] All data was presented as the mean?SEM of three independent experiments with triplicate samples within each independent experiment. Data generated was analyzed by one-way analysis of variance (ANOVA) and post hoc Tukey test using SPSS (IBM SPSS Statistics version 16.0) program. Data was considered significant if the P value was <0.05.

Example 2: Results

[0098] Heat, or Protease Treated Lysate does not Protect Keratinocytes from the Effects of S. aureus

[0099] The nature of the efficacious molecules within the LGG lysate mediating its anti-adhesive effects on S. aureus was first detected. To this end, the lysate was treated with heat or protease and then its ability to protect keratinocyte monolayer viability in the presence of S. aureus investigated. Only c. 30% of the keratinocyte monolayer was viable following 24 h incubation with S. aureus. However, in the presence of LGG lysate, monolayer viability increased to c. 65%. Heat or protease treated lysate did not protect monolayer viability (see FIG. 1a), suggesting that proteins within the lysate are the efficacious molecules.

[0100] The LGG lysate was subjected to partial fractionation using a hydrophobic interaction column and the proteins were eluted in a gradient of 10-70% acetonitrile. The ability of proteins eluting in each fraction to protect keratinocyte viability was investigated. Proteins eluting in 30-60% acetonitrile were able to protect keratinocyte from the effects of S. aureus, however, proteins eluting in other fractions did not (see FIG. 1b).

The 50% Acetonitrile Fraction Both Excludes and Displaces S. aureus from Keratinocytes

[0101] The ability of the 30-60% fractions of the lysate to exclude or displace S. aureus from keratinocyte binding sites was investigated. The data in FIG. 2 show that the 50% fraction is the most efficacious at both exclusion and displacement of S. aureus. Importantly, when tested in a spot on the lawn assay, this fraction had minimal activity against S. aureus growth compared with other fractions that conferred protection to keratinocytes as shown in Table 1 below.

TABLE-US-00001 TABLE 1 Diameter of zone of Treatment inhibition (mm, n = 3) Whole LGG lysate 14 +/? 1.6 20% fraction 0 30% fraction 5 +/? 0.8 40% fraction 0 50% fraction 0 60% fraction 10 +/? 1.7 99% acetonitrile 0

The 50% Acetonitrile Fraction Contains the Pilus Protein SpaC

[0102] To understand the involvement of SpaC in anti-adhesive processes, the 50% fraction was subjected to immunoblotting using an anti-SpaC antibody. This produced a single band, at the correct molecular weight for SpaC, suggesting that the 50% fraction of the lysate contains this protein (see FIG. 3a). To confirm the involvement of SpaC as an anti-S. aureus adhesion mechanism, recombinant SpaC was produced and its anti-adhesive activity compared against that of the crude lysate. Recombinant SpaC inhibited adhesion of S. aureus to keratinocytes in a dose dependent manner with 50 ?g/ml being the most efficacious (FIG. 3b). By contrast, 50 ?g/ml of a control protein, Bovine serum albumin did not inhibit staphylococcal adhesion to keratinocytes (see FIG. 3c). However, SpaC was only able to inhibit S. aureus adhesion to keratinocytes when added to keratinocytes before, but not after the pathogen.

SpaC Partially Preserves Keratinocyte Monolayer Viability in the Presence of S. aureus

[0103] The recombinant SpaC was tested to establish whether it could protect keratinocyte monolayers from the toxic effects of S. aureus. The effect of 50 ?g/ml crude lysate against 50 ?g/ml recombinant SpaC was compared. Although the recombinant SpaC could indeed protect keratinocytes, it was significantly less efficacious than the crude lysate (see FIG. 4a). Furthermore, a LGG strain deficient in SpaC production, was still able to protect keratinocyte viability (see FIG. 4b) and did not show significantly reduced ability to inhibit S. aureus adhesion.

The 50% Acetonitrile Fraction Contains Additional Potential Anti-Adhesive Proteins

[0104] Since deletion of SpaC in a LGG mutant strain did not negate the effects of the lysate on keratinocyte viability in response to S. aureus, the possibility that other proteins in the LGG lysate may also impact upon adhesion of staphylococci to keratinocytes was assessed. A tandem mass spectrometry (MS/MS) analysis of the proteins contained within the 50% fraction of the LGG lysate was produced. The data is summarised in Table 2 below.

TABLE-US-00002 TABLE 2 Proteins identified by Tandem Mass Spectrometry in the 50% acetonitrile fraction of the LGG lysate. Molecular Protein weight (kDa) UDP-glucose 4 epimerase 90 B-galactosidase chain D 33 30S Ribosomal protein S7 95 Acyl carrier protein 9 Glyceraldehyde-3 phosphatedehydrogenase 36 ElongationfactorTU 44 Triosephosphateisomerase 27 50S ribosomal protein S11 15 Dehydroxyacetone kinase 21 50S ribosomal protein L22 13 Asparaginyl tRNA synthetase 50 Enolase 47 GMP synthase 58 UPF0342 protein LRH 13 30S ribosomal protein S5 32 Glucose 1 phosphate thymidylyltransferase 75 B-galactosidase chain D 33 DNA-directed RNA polymerase alpha subunit 38 Phosphoribosylpyrophosphate synthetase 25 Phosphoglycerate mutase 46 Aspartyl tRNA synthetase 64 M29 family amonopeptidase 21 Glycine cleavage system H 11 50S ribosomal protein 13 UPF0342 protein 13 Note: These proteins were consistently found in n = 3 column fractionations. The bold text highlights proteins with molecular weights corresponding to abundant proteins in the sample as judged by SDS-PAGE.

[0105] To further concentrate and identify proteins of interest a further round of purification of the 50% fraction using Reverse-Phase HPLC and proteins were eluted from a C 18 reverse phase column using a gradient of 0-100% acetonitrile. The concentrated fractions were collected based on ultraviolet absorption at 215 nm and 4 specific peaks containing proteins were collected at between 21-32 minutes of elution. These peaks, named F1-4, (to differentiate their retention times on the column) were used in both staphylococcal adhesion assays and keratinocyte viability assays. The proteins contained within F4 were the most efficacious in both assays (data not shown). Hence, F4 was subjected to analysis both by gel electrophoresis and MS/MS analysis. The proteins contained within F4 are shown in Table 3 below.

TABLE-US-00003 TABLE 3 Proteins identified by Tandem mass Spectrometry in the F4 fraction of the lysate Footnote. Molecular Protein weight (kDa) Acyl carrier protein 9 Glyceraldehyde-3phosphatedehydrogenase 36 ElongationfactorTU 43 Transcription elongation factor greA 23 Phosphopentomutase 43 Triosephosphateisomerase 27 50S ribosomal protein S11 15 Dihydroxyecetone kinase 21 50s Ribosomal protein 15 Asparaginyl tRNA synthetase 50 Enolase 47 UPF0342 protein 13 50S ribosomal protein L22 13 The bold text indicates proteins with molecular weights corresponding to abundant proteins in the fraction as judged by SDS-PAGE.

[0106] The proteins highlighted in bold in Table 2, elongation factor Tu, (EF-Tu) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase and triosephosphate isomerase (TPI), are common to both the 50% acetonitrile fraction and the F4 fraction. These proteins are likely to be major constituents of F4 because the most abundant proteins in the F4 fraction (as judged by electrophoresis) correspond to the known molecular weights of these proteins. Of significance, EF-Tu, GAPDH, enolase and TPI are known adhesion molecules in other lactobacilli.

Discussion

[0107] The inventors have been investigating the potential of LGG cell-free lysates as a topical therapy targeted at prevention/treatment of S. aureus infection. For topical applications, a cell-free lysate has many advantages over the use of viable bacteria. Importantly, the safety concerns surrounding use of live bacteria on the skin are negated as are the potential problems associated with formulating live bacteria. However, if probiotics like LGG are to fulfil their potential as topical therapeutic agents, an understanding of the bacterial molecules mediating their effects is a pre-requisite.

[0108] In the current experiments, the inventors investigated the molecules mediating the anti-adhesive effects of LGG against S. aureus. The efficacious molecules appear to be proteins because heat denaturation or protease treatment completely destroyed the activity of the lysate against S. aureus. However, the possibility that other molecules such as sugars on the surface of LGG may also be important to its adhesive action cannot be completely excluded. Indeed, exopolysaccharides have been shown to be important for LGG binding to the host in the intestine. Although exopolysaccharides and other molecules may be involved in the activity of the LGG lysate, since glycosidase treatment of the LGG lysate resulted in no significant loss of efficacy against S. aureus it was concluded that in the main, the inhibitory action of the lysate against S. aureus adhesion is mediated by proteins.

[0109] A number of protein adhesins have been previously identified in lactobacilli. Of these, the involvement of the pilus protein SpaC as a mucus binding protein has been shown in a number of studies. SpaC may also be involved in the mechanism by which LGG inhibits S. aureus adhesion. This is suggested by a number of observations: Firstly, fractionation of the lysate and analysis of the fractions show the most efficacious fraction to contain SpaC. Second, recombinant SpaC inhibits S. aureus adhesion in a dose dependent manner and lastly, the toxic effects of S. aureus on keratinocyte viability are negated by SpaC, but not a control protein, BSA.

[0110] Overall, these data are consistent with a conclusion that SpaC is involved in the mechanism by which LGG inhibits the adhesion of S. aureus to keratinocytes but it is almost certainly does not act alone and other proteins are likely to be involved. Of note, the SpaC knockdown still retained an ability to protect keratinocyte viability in the presence of S. aureus. This could be explained by a previous observation by the inventors that inhibition of adhesion is not the only mechanism used by LGG to protect keratinocytes from the toxic effects of S. aureus. Previous studies showed that the LGG lysate also inhibits the growth of S. aureus. This is probably part of the explanation as to why the SpaC knockout LGG strain still retains ability to protect keratinocyte viability i.e. the knock out strain would still retain the molecules that inhibit staphylococcal growth. The inhibition of S. aureus growth is almost certainly mediated by completely different molecules to the adhesins because in separate experiments, we could show no inhibition of S. aureus growth by the 50% acetonitrile fraction. Indeed, inhibition of pathogenic growth was found to be contained in other discreet fractions of the lysate.

[0111] A second piece of evidence suggesting that SpaC is not the entire explanation for the anti-adhesive effects of the LGG lysate comes from the observation that recombinant SpaC cannot replicate the displacement activity of the lysate and the 50% fraction against S. aureus. This suggests that other proteins may be involved in the full anti adhesive activities of the lysate. Indeed, several other proteins were found in the 50% fraction and were concentrated further by HPLC into a fraction (F4) which showed the highest efficacy in both adhesion and viability assays. The most abundant proteins in this F4 fraction are likely to be elongation factor Tu, (EFTU) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase and triosephosphate isomerase (TPI) because the major components of the fraction (as judged by gel electrophoresis) were proteins of the same molecular weights as these. Interestingly, SpaC must be a low abundance protein in both the 50% and F4 fractions because Tandem Mass Spectrometry identification of proteins did not detect it. Indeed this technique is known to be reliable only for identification of abundant proteins. However, SpaC was shown to be present by western blotting suggesting it is present in the fraction and hence may be part of the anti-adhesive mechanism.

[0112] The proteins EFTU, GAPDH, enolase and TPI have been previously reported to be important for adhesive function in several species of lactobacilli. All these proteins have been previously described as so-called moonlighting proteins i.e. proteins with an ability to perform functions unrelated to the canonical function ascribed to the protein. For example, GAPDH is an intracellular enzyme central to glycolysis. However, it is found as a cell surface adhesion protein on several prokaryotes including L. plantarum and L. crispatus. TPI, another glycolytic enzyme, has been shown to be involved in competitive exclusion and displacement of Clostridium sporogenes and Enterococcus faecalis from Caco-2 cells by L. plantarum. EF-Tu is involved in protein translation but is found at the cell surface as an adhesion mediating attachment of lactobacilli to mucins. Many of these moonlighting proteins have been shown to mediate bacterial adhesion to eukaryotic cells by binding to specific eukaryotic proteins such as fibronectin. Although EFTU, GAPDH, enolase and TPI have not been shown to be adhesins specifically in LGG, very recent evidence has demonstrated three of them, (enolase, EFTU, GPDH) to be cell surface proteins of LGG as well as having their normally cytoplasmic location. Such dual localisation is usually suggestive of moonlighting function. Unfortunately, since all these proteins have canonical functions important to central bacterial metabolism, knock down generally results in lethality making their exact contributions to the anti-staphylococcal adhesive function of LGG difficult to demonstrate.

[0113] In summary, it is believed that the overall anti-adhesive function of LGG against S. aureus may be facilitated by a number of proteins including SpaC, and several moonlighting proteins expressed at the cell surface. These may include TPI, enolase, GAPDH and EFTU although others may also be involved. Observations showed that many fractions were efficacious in our assays and we analysed only the fractions showing the greatest efficacy.

[0114] The forgoing embodiments are not intended to limit the scope of the protection afforded by the claims, but rather to describe examples of how the invention may be put into practice.