Pharmaceutical composition and methods for the prevention and/or treatment of Staphylococcus aureus using artificial bacterial colonization

Abstract

The present invention relates to a pharmaceutical composition comprising at least one Corynebacterium sp, Staphylococcus pasteuri and, optionally, Staphyloccocus epidermidis for use as a medicament, in particular for use in the prevention or treatment of S. aureus colonization.

Claims

1-17. (canceled)

18. A method for preventing and/or treating colonization by Staphylococcus aureus in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising at least one Corynebacterium sp., Staphyloccocus pasteuri, and a pharmaceutically acceptable excipient.

19. The method of claim 18, wherein said composition comprises at least one Corynebacterium sp. selected from the group consisting of C. accolens, C. propinquum, C. pseudodiphtheriticum, C. amycolatum, C. glutamicum, C. aurimucosum, C. tuberculostearicum, and C. afermentans.

20. The method of claim 18, wherein said composition comprises at least 103 CFUs of Corynebacterium sp. and at least 10.sup.3 CFUs of Staphyloccocus pasteuri.

21. The method of claim 18, wherein said composition comprises a total of at least 10.sup.3 bacterial CFUs per dose.

22. The method of claim 21, wherein said composition comprises a total of at least 310.sup.3 bacterial CFUs per dose.

23. The method of claim 18, wherein said composition further comprises Staphylococcus epidermidis.

24. The method of claim 23, wherein said composition comprises at least 10.sup.3 CFUs of S. epidermidis per dose.

25. The method of claim 23, wherein said composition comprises a ratio of Corynebacterium sp. to S. pasteuri to S. epidermidis in the range of 1:0.01:0.01 to 1:1:1.

26. The method of claim 18, wherein said pharmaceutically acceptable excipient comprises at least one lyoprotectant.

27. The method of claim 26, wherein said lyoprotectant is selected from the group consisting of peptone, glycerol, lactose, gelatin, glucose, sucrose, trehalose, dextran, maltodextrin, adonitol, and sodium glutamate.

28. The method of claim 18, wherein said composition is lyophilized or freeze-dried, and is reconstituted with at least one pharmaceutically acceptable excipient prior to administration.

29. The method of claim 18, wherein said composition further comprises at least one pharmaceutically acceptable gelling agent.

30. The method of claim 18, wherein said composition is prepared as a patch, gel, cream, lotion, ointment, film, emulsion, or salve.

31. The method of claim 18, comprising administration of the pharmaceutical composition to the anterior nares and/or the skin.

32. The method of claim 18, wherein said S. aureus is methicillin-sensitive Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

33. A pharmaceutical composition comprising at least one Corynebacterium sp., Staphyloccocus pasteuri, and a pharmaceutically acceptable excipient.

34. The composition of claim 33, wherein said bacteria are lyophilized or freeze-dried.

35. A kit comprising the pharmaceutical composition of claim 33, and: a gel, a cream, a lotion, an ointment, an emulsion, or a salve, suitable for nasal use and a means for nasal administration, or a gel, a cream, a lotion, an ointment, an emulsion, or a salve, suitable for skin use.

Description

FIGURE LEGENDS

[0101] FIG. 1: Growth of C. accolens in co-culture with S. pasteuri or S. epidermidis

[0102] Bacterial growth of C. accolens AF2345 was determined in co-culture with either (A) S. pasteuri AF2653 or (B) S. epidermidis AF2302, according to the methods described in Example 2. C. accolens colonies were observed after 72 hours (A) or 7 days (B), at the same magnification. S. pasteuri and S. epidermidis spots are indicated by an arrow. C. accolens colonies are visible in co-culture with S. pasteuri with the naked eye, but are smaller with increasing distance from the S. pasteuri spot. In contrast, C. accolens colonies grown in co-culture with S. epidermidis are much smaller than those observed even at a distance from the S. pasteuri spot in FIG. 1A, and are not visible with the naked eye.

[0103] FIG. 2: Mixed biofilms with C. accolens

[0104] Bacterial growth of C. accolens AF2345 alone or as part of a single or mixed biofilm in the presence of S. pasteuri, S. epidermidis, or both S. pasteuri and S. epidermidis was evaluated according to the methods described in Example 8. Growth was observed in the presence of either S. pasteuri alone or both S. pasteuri and S. epidermidis. No significant difference in growth occurred between these two conditions (p=0.0651)

[0105] FIG. 3: Mixed biofilms with C. propinquum

[0106] Bacterial growth of C. propinquum AF1882 alone or as part of a single or mixed biofilm in the presence of S. pasteuri, S. epidermidis, or both S. pasteuri and S. epidermidis was evaluated according to the methods described in Examples 8 and 9. Growth was observed in all conditions. No significant difference in growth of C. propinquum occurred when in the presence of either S. pasteuri alone or both S. pasteuri and S. epidermidis (p=0.0748).

[0107] FIG. 4: Production of extracellular matrix when C. accolens is co-cultured with S. pasteuri, S. epidermidis or both S. pasteuri and S. epidermidis Bacteria were grown alone or in various combinations in wells of a 96-well plate filled with TSB. Biofilm formation was estimated after 48 hours of incubation at 37 C. by measuring the optical density of solubilized crystal violet-stained extracellular matrix at 595 nm. Production of extracellular matrix was significantly increased when C. accolens was co-cultured with S. pasteuri, S. epidermidis, or both S. pasteuri and S. epidermidis. The greatest effect was obtained with the combination C. accolens/S. pasteuri/S. epidermidis (7.6-fold increase vs C. accolens alone, 12-fold increase vs S. pasteuri alone and 3.1-fold increase vs S. epidermidis alone).

[0108] FIG. 5: Bacteriotherapeutic effect of C. accolens/S. pasteuri on adherent S. aureus cells

[0109] S. aureus bacteria were inoculated in a 96-well plate and were allowed to adhere for 6 h. Bacteria were then treated with C. accolens and/or S. pasteuri (510.sup.7 CFU/well for each species). After 24 h of incubation, wells were washed, immersed in an ultrasound bath to disrupt the biofilm, and S. aureus CFUs enumerated. (A) S. aureus log.sub.10 CFU. (B) Delta S. aureus log.sub.10 CFU (each condition versus control). Although a significant bacteriotherapeutic effect was observed with S. pasteuri alone, the combination C. accolens/S. pasteuri surprisingly showed a further improved synergistic effect. Control: untreated S. aureus cells. **p<0.001 versus control.

[0110] FIG. 6: Prevention of S. aureus colonization in an in vivo model of stratified squamous epithelium

[0111] (A) Picture of the hydrocolloid patch on a healthy volunteer's forearm skin. Punched holes (corresponding to skin wells) received the bacterial suspensions to be tested covered by the polyurethane sterile incision film. (B) Inhibition of S. aureus 29213 (510.sup.3 CFU per skin well) when inoculated with C. accolens, S. pasteuri and S. epidermidis alone or combined, each at a dose of 210.sup.6 CFU per well. S. aureus alone was inoculated as positive control. The [(ThresholdCT)/Threshold] ratios are plotted for each condition tested.

[0112] FIG. 7: S. aureus decolonization by bacteriotherapy in an in vivo murine model of stratified squamous epithelium.

[0113] Murine stratified squamous epithelium skin wells previously colonized with S. aureus were treated with C. accolens, S. pasteuri and, optionally, S. epidermidis in various combinations, at a dose of 10.sup.6 CFU per species. Twenty-four hours later, mice were euthanized, and the area of each skin well was swabbed to quantify S. aureus CFUs. A. S. aureus log.sub.10 CFU. B. Delta S. aureus log.sub.10 CFU (each condition versus control). A significantly lower level of S. aureus was observed when wells colonized with S. aureus were subjected to bacteriotherapy with the combination C. accolens/S. pasteuri or C. accolens/S. pasteuri/S. epidermidis. The S. aureus burden was 5.50 log.sub.10 CFU per well with the control. Control: untreated S. aureus cells. *p<0.05 versus control. **p<0.005 versus control.

EXAMPLES

[0114] The following examples are included to demonstrate preferred embodiments of the invention. All subject-matter set forth or shown in the following examples and accompanying drawings is to be interpreted as illustrative and not in a limiting sense. The following examples include any alternatives, equivalents, and modifications that may be determined by a person skilled in the art.

[0115] The representative bacterial strains used in these examples are detailed in Table I, below. The strains selected for use in the examples (listed in Table 1) include reference strains, which possess the characteristics representative of all strains of a given species.

TABLE-US-00001 TABLE I Strains used. Strain Source/origin/collection number Staphylococcus aureus USA300 ATCC MRSA, Wrist abscess BAA-1556 Staphylococcus aureus AF3147 MSSA, nasal swab, our collection Staphylococcus aureus AF2419 MSSA, nasal swab, our collection Staphylococcus aureus ATCC 29213 MSSA, S. aureus subsp. aureus Rosenbach (strain Wichita) Corynebacterium accolens AF2345 Nasal swab, our collection, CNCM I-5395 Corynebacterium accolens AF3612 Nasal swab, our collection Corynebacterium accolens CIP 104783T Collection of the Pasteur Institute, Paris, France Corynebacterium propinquum AF1882 Nasal swab, our collection, CNCM I-5393 Staphylococcus epidermidis AF2302 Nasal swab, our collection, CNCM I-5394 Staphylococcus pasteuri AF2653 Nasal swab, our collection, CNCM I-5396 Staphylococcus pasteuri AF2062 Nasal swab, our collection Staphylococcus pasteuri CIP 103540T Collection of the Pasteur Institute, Paris, France Staphylococcus pasteuri CIP 103830 Collection of the Pasteur Institute, Paris, France

[0116] Abbreviations: MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-susceptible Staphylococcus aureus. Strains with a collection number were deposited with the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, F75724 Paris CEDEX 15, France on Jan. 25, 2019 by the UFR des sciences de la sant Simone Veil, 2 avenue de la source de la Bievre, 78180 Montigny le Bretonneaux, France, represented by Prof. Jean-Louis Gaillard.

Example 1: In Vitro Inhibition of the Growth of S. aureus by C. accolens

[0117] In view of the prior art describing contradictory effects of C. accolens on S. aureus colonization in vivo, the effect of C. accolens on S. aureus growth was first evaluated here in vitro.

Materials and Methods

[0118] 250 l of a 1.0 McFarland (McF) suspension of C. accolens, prepared from a 48 h-culture on Columbia 5% sheep blood agar at 35+/2 C., was inoculated by swabbing on a 90-mm diameter blood agar plate in order to obtain 510.sup.4 CFU/cm.sup.2. 250 l of saline was inoculated as negative control. One 50 mm 0.2 m track-etched filter was placed on top of the C. accolens suspension. S. aureus suspensions prepared from a 24 h-culture on Columbia 5% sheep blood agar at 35+/2 C. and containing a target number of 100 CFUs, 10 CFUs, or 1 CFU in 10 l were spotted on the 50-mm diameter filter.

[0119] After incubation for 48 h at 35 C., S. aureus colonies on filter were harvested and bacteria resuspended in saline (2.5 to 10 mL). The number of S. aureus CFUs present in each colony was then determined by measuring optical density (OD) at 600 nm. To quantify the growth inhibition of S. aureus in the presence of C. accolens, the OD/colony with or without C. accolens was determined. Each set of experiments was repeated at least twice.

Results

[0120] C. accolens was surprisingly responsible for a reduction of 27.78% to 51.93% of S. aureus growth depending on the C. accolens and S. aureus strain tested (cf. Table II). Also, surprisingly, the growth of both MRSA (S. aureus USA300) and MSSA (S. aureus AF3147 and AF2419) strains was significantly inhibited. The percent growth inhibition appears to depend on the C. accolens strain used, as no significant differences in growth inhibition were observed between different S. aureus strains grown in the presence of a same C. accolens strain.

TABLE-US-00002 TABLE II S. aureus growth inhibition (mean (SD), %) P-value.sup.a P-value.sup.a P-value.sup.a C. accolens S. aureus strain USA300 vs USA300 vs AF3147 vs strain USA300 AF3147 AF2419 AF3147 AF2419 AF2419 AF2345 51.75 51.86 51.93 0.969 0.946 0.980 (2.93) (3.71) (3.33) CIP104783T 30.01 27.78 32.70 0.437 0.361 0.115 (3.33) (3.21) (3.33) AF3612 41.67 38.90 42.32 0.308 0.823 0.281 (2.89) (3.21) (3.85) .sup.aStudent's t-test (p values <0.01 were considered as significant), SD: standard deviation.

Example 2: Growth Promotion of C. accolens in Co-Culture with S. pasteuri

[0121] We next studied the interaction of C. accolens and S. pasteuri in vitro. The effect of S. pasteuri on growth by C. accolens was studied using plate count agar (PCA), which allows the growth of S. pasteuri, but not that of C. accolens. S. epidermidis was used as a control species.

Materials and Methods

[0122] 500 l of a 10.sup.2 dilution of a 0.5 McFarland suspension of C. accolens (strain AF2345), prepared as described in Example 1, was inoculated onto PCA medium. After complete drying, 10 l of a 1.0 McFarland suspension of S. pasteuri (strain AF2653) or S. epidermidis (strain AF2302), prepared from a 24 h-culture on Columbia 5% sheep blood agar at 35+/2 C., was spotted at the center of the plate. Ten l of sterile saline was spotted as a negative control. Growth of C. accolens was determined after 72 hours of incubation at 35 C. Growth was determined by the presence of colonies visible with the naked eye.

Results

[0123] Growth of C. accolens was detected on the periphery of the S. pasteuri spot after 72 hours of incubation (FIG. 1A) while abortive colonies were detected around the S. epidermis spot (FIG. 1B) or the control spot (not shown). The C. accolens colonies detected on the periphery of the S. pasteuri spot could be detected with the naked eye, while the abortive colonies around the S. epidermis spot could not be detected without magnification. Incubation of C. accolens in co-culture with S. epidermis for longer periods of time did not further improve C. accolens growth, even when the plates were incubated for up to a total of 7 days. Thus, in contrast to S. epidermidis, S. pasteuri promotes growth of C. accolens.

Example 3: Synergistic Inhibition of the Growth of S. aureus by C. accolens in Combination with S. pasteuri In Vitro

[0124] As S. pasteuri promoted C. accolens growth, we studied the anti-S. aureus activity of S. pasteuri and C. accolens, alone or in combination. Anti-S. aureus activity of S. epidermidis, was also tested alone or in combination with C. accolens.

Materials and Methods

[0125] For each strain alone or in combination, 250 l of a 1.0 McFarland suspension prepared from cultures on Columbia 5% sheep blood agar at 35+/2 C. (C. accolens: 48 h of incubation; S. pasteuri, S. epidermidis: 24 h of incubation) was inoculated by swabbing on a 90 mm diameter blood agar plate (510.sup.4 CFU/cm.sup.2). 250 l of saline was inoculated as negative control. One 50 mm 0.2 m track-etched filter was placed on top of the spot. S. aureus USA300 suspensions containing a target number of 100 CFUs, 10 CFUs, and 1 CFU in 10 l were spotted on the 50 mm diameter filter. Materials and methods were otherwise as described in Example 1.

Results

[0126] Table III presents the results obtained with C. accolens AF2345, S. pasteuri AF2543 and S. epidermidis AF2302. As shown, the combination of C. accolens and S. pasteuri was significantly more inhibitory than each species alone and inhibited S. aureus growth by more than 95%. In contrast, the combination of C. accolens and S. epidermidis was not synergistic and even tended to be antagonistic (mean S. aureus growth inhibition of 68.92% versus 79.28% with S. epidermidis AF2302 alone, p=0.0133).

TABLE-US-00003 TABLE III S. aureus growth Strain/combination inhibition (mean (SD), %) P-value.sup.a C. accolens AF2345 55.96 (2.59) S. pasteuri AF2653 81.87 (2.59) S. epidermidis AF2302 79.28 (3.66) C. accolens AF2345 + 96.12 (1.30) vs AF2653 alone: S. pasteuri AF2653 3.4216 10.sup.7 C. accolens AF2345 + 68.92 (3.66) vs AF2302 alone: S. epidermidis AF2302 0.0133 .sup.aStudent's t-test (p values < 0.01 were considered as significant).

[0127] We tested other combinations of C. accolens and S. pasteuri strains in the same conditions. As shown in Table IV, all combinations tested inhibited S. aureus inhibition by more than 95%.

TABLE-US-00004 TABLE IV S. aureus Combination growth inhibition (%).sup.a C. accolens AF2345 + S. pasteuri AF2062 99.43 C. accolens AF2345 + S. pasteuri 95.38 CIP103540T C. accolens AF2345 + S. pasteuri CIP103830 99.61 C. accolens CIP104783T + S. pasteuri 95.38 AF2653 C. accolens CA3612 + S. pasteuri AF2653 95.38 .sup.aOD was measured from a pool of ten colonies suspended in saline.

Example 4: Dose-Effect Relationship of the Combination of C. accolens and S. pasteuri

Materials and Methods

[0128] Serial 1/5 dilutions were prepared from a 1.0 McFarland suspension of each strain and 250 l of each bacterial suspension was inoculated by swabbing on a 90 mm diameter blood agar plate, to obtain each strain alone or in combination at a plated density of 510.sup.4, 10.sup.4, 210.sup.3, 410.sup.2, and 0.810.sup.2 CFU/cm.sup.2. Materials and methods were otherwise as described in Example 3.

Results

[0129] As shown in Table V, the combination of C. accolens and S. pasteuri was surprisingly significantly synergistic at all bacterial densities tested, with the strongest synergistic effect obtained at 0.810.sup.2 CFU/cm.sup.2. However, only the densities superior or equal to 210.sup.3 CFU/cm.sup.2 (e.g. 510.sup.4 CFU/cm.sup.2, 10.sup.4 CFU/cm.sup.2, and 210.sup.3 CFU/cm.sup.2) showed anti-S. aureus activity that inhibited bacterial growth by more than 95%.

TABLE-US-00005 TABLE V S. aureus growth inhibition P-value.sup.a (mean AF2653 + AF2345 vs Strain/combination (SD), %) AF2653 alone 5 10.sup.4 CFU/cm.sup.2 C. accolens AF2345 50.00 (5.89) S. pasteuri AF2653 86.46 (1.80) C. accolens AF2345 + S. pasteuri 97.71 (0.36) 4.1433 10.sup.5 AF2653 10.sup.4 CFU/cm.sup.2 C. accolens AF2345 41.69 (5.87) S. pasteuri AF2653 80.21 (3.46) C. accolens AF2345 + S. pasteuri 96.64 (0.47) 0.0001828 AF2653 2 10.sup.3 CFU/cm.sup.2 C. accolens AF2345 25.02 (5.91) S. pasteuri AF2653 71.88 (3.45) C. accolens AF2345 + S. pasteuri 95.06 (0.45) 2.5551 10.sup.5 AF2653 4 10.sup.2 CFU/cm.sup.2 C. accolens AF2345 14.59 (9.08) S. pasteuri AF2653 54.17 (4.17) C. accolens AF2345 + S. pasteuri 80.21 (1.81) 6.0412 10.sup.5 AF2653 0.8 10.sup.2 CFU/cm.sup.2 C. accolens AF2345 0.00 (0.00) S. pasteuri AF2653 16.66 (5.90) C. accolens AF2345 + S. pasteuri 73.96 (1.80) 3.6798 10.sup.6 AF2653 .sup.aStudent's t-test p values < 0.01 were considered as significant).

Example 5: Anti-S. aureus Activity of the Combination of C. accolens, S. Pasteuri and S. epidermidis

[0130] In view of our previous results presented in Example 3, we evaluated whether or not the addition of S. epidermidis had a negative impact on the inhibitory activity of the combination of C. accolens with S. pasteuri against S. aureus.

Materials and Methods

[0131] For each strain in combination, 250 l of a 1.0 McFarland suspension prepared from cultures on Columbia 5% sheep blood agar at 35+/2 C. (C. accolens: 48 h of incubation; S. pasteuri, S. epidermidis: 24 h of incubation) was inoculated by swabbing on a 90 mm diameter blood agar plate (510.sup.4 CFU/cm.sup.2); 250 l of saline was inoculated as negative control. One 50 mm 0.2 m track-etched filter was placed on top of the spot. A S. aureus USA300 suspension containing a target number of 100 CFUs, 10 CFUs, and 1 CFU in 10 l were spotted on the 50 mm diameter filter. Materials and methods were otherwise as described in Example 1.

Results

[0132] As shown on table VI, the same level of S. aureus growth inhibition was obtained with the combination C. accolens AF2345/S. pasteuri AF2653 versus the combination C. accolens AF2345/S. pasteuri AF2653/S. epidermidis AF2302.

TABLE-US-00006 TABLE VI S. aureus growth P-value.sup.a AF2345 + AF2653 inhibition (mean vs Combination (SD), %) AF2345 + AF2653 + AF2302 C. accolens AF2345 + 98.91 (0.16) 1.00 S. pasteuri AF2653 C. accolens AF2345 + 98.91 (0.16) S. pasteuri AF2653 + S. epidermidis AF2302 .sup.aStudent's t-test (p values < 0.01 were considered as significant).

Example 6: C. propinquum May Replace C. accolens in the Combinations C. accolens/S. pasteuri and C. accolens/S. pasteuri/S. epidermidis

[0133] We then studied whether or not other species of Corynebacterium, such as C. propinquum, may also have anti-S. aureus activity alone and/or in combination with S. pasteuri or with S. pasteuri and S. epidermidis.

Materials and Methods

[0134] Materials and methods were as described in Example 3 except that C. propinquum AF1882 replaced C. accolens AF2345.

Results

[0135] As shown in Table VII, the combinations C. propinquum/S. pasteuri and C. propinquum/S. pasteuri/S. epidermidis were both synergistic and had significant anti-S. aureus activity, inhibiting S. aureus growth by more than 95%.

TABLE-US-00007 TABLE VII S. aureus growth P-value.sup.a vs inhibition (mean % AF1882 Strain/combination (SD)) alone C. propinquum AF1882 81.25 (4.42) C. propinquum AF1882 + S. pasteuri 98.33 (0.30) 0.002415.sup.b AF2653 C. propinquum AF1882 + S. pasteuri 98.59 (0.27) 0.0005017.sup.b AF2653 + S. epidermidis AF2302 .sup.aStudent's t-test (p values < 0.01 were considered as significant). .sup.bAF1882 + AF2653 versus AF1882 + AF2653 + AF2302, P-value = 0.3524.

Example 7: Anti-S. aureus Activity of Bacterial Combinations at Various Corynebacterium sp.:Staphylococcus sp. ratios

[0136] The anti-S. aureus activity of various ratios of Corynebacterium sp.:Staphylococcus sp. was also evaluated. In particular, we evaluated various ratios of the combination C. accolens:S. pasteuri:S. epidermidis or the combination of C. propinquum:S. pasteuri:S. epidermidis.

Materials and Methods

[0137] Appropriate dilutions of C. accolens AF2345, C. propinquum AF1882, S. pasteuri AF2653 and S. epidermidis AF2302 were prepared from a 2.0 McFarland suspension of each strain and 25 to 250 l of each bacterial suspension were co-inoculated by swabbing on a 90 mm diameter blood agar plate, thereby obtaining 10, 510.sup.4, 10.sup.4, 510, and 10.sup.3 CFU/cm.sup.2 of C. accolens AF2345 or C. propinquum AF1882 combined with S. pasteuri AF2653 and S. epidermidis AF2302 at the following ratios (Corynebacterium:S. pasteuri/S. epidermidis): 1:1:1, 1:0.2:0.2, 1:0.1:0.1, 1:0.02:0.02, 1:0.01:0.01. Materials and methods were otherwise as described in Example 3.

Results

[0138] Tables VIII and IX show the results obtained with C. accolens- and C. propinquum-based combinations, respectively.

[0139] As shown in Table VIII, the combination C. accolens:S. pasteuri:S. epidermidis surprisingly has an anti-S. aureus activity exceeding 95% at all densities and ratios tested, except for ratios 1:0.01:0.01 at densities 10.sup.4 CFU/cm.sup.2, for ratios 1:0.02:0.02 at densities 510.sup.3 CFU/cm.sup.2, and for ratio 1:0.1:0.1 at a density of 10.sup.3 CFU/cm.sup.2. However, anti-S. aureus activities remained significant at all concentrations and densities tested. Indeed, even at the lowest tested concentration and density (e.g. a ratio of 1:0.01:0.01 at a density of 10.sup.3 CFU/cm.sup.2) S. aureus growth remained inhibited by at least 74%. Increasing either the ratio (e.g. to 1:0.02:0.02) or the density (e.g. to 510.sup.3) increased anti-S. aureus activity, inhibiting S. aureus growth by approximately 83% and 85%, respectively.

TABLE-US-00008 TABLE VIII C. accolens/S. pasteuri/S. epidermidis combination at various ratios and densities Density/ratio S. aureus growth inhibition (mean % (SD)) C. accolens at 10.sup.5 CFU/cm.sup.2 Ratio.sup.a 1:1:1 99.70 (0.10) Ratio 1:0.2:0.2 99.07 (0.19) Ratio 1:0.1:0.1 99.27 (0.41) Ratio 1:0.02:0.02 97.87 (0.19) Ratio 1:0.01:0.01 97.33 (0.19) C. accolens at 5 10.sup.4 CFU/cm.sup.2 Ratio 1:1:1 99.80 (0.00) Ratio 1:0.2:0.2 99.33 (0.19) Ratio 1:0.1:0.1 98.67 (019) Ratio 1:0.02:0.02 97.87 (0.19) Ratio 1:0.01:0.01 97.33 (0.38) C. accolens at 10.sup.4 CFU/cm.sup.2 Ratio 1:1:1 99.60 (0.00) Ratio 1:0.2:0.2 98.53 (0.19) Ratio 1:0.1:0.1 96.40 (1.18) Ratio 1:0.02:0.02 96.53 (0.38) Ratio 1:0.01:0.01 93.87 (0.38) C. accolens at 5 10.sup.3 CFU/cm.sup.2 Ratio 1:1:1 99.50 (0.10) Ratio 1:0.2:0.2 98.53 (0.19) Ratio 1:0.1:0.1 96.53 (0.38 Ratio 1:0.02:0.02 93.07 (0.38) Ratio 1:0.01:0.01 85.07 (0.75) C. accolens at 10.sup.3 CFU/cm.sup.2 Ratio 1:1:1 98.13 (0.19) Ratio 1:0.2:0.2 97.73 (0.19) Ratio 1:0.1:0.1 92.27 (0.38) Ratio 1:0.02:0.02 83.47 (0.75) Ratio 1:0.01:0.01 74.93 (0.75) .sup.aRatio is expressed as the quantity of C. accolens:S. pasteuri:S. epidermidis

[0140] As shown in Table IX, the results obtained with C. propinquum were similar to those obtained with C. accolens. Indeed, anti-S. aureus activities for bacterial combinations including C. propinquum also surprisingly exceeded 95% at all densities and ratios tested, except for ratios 1:0.01:0.01 at densities 510.sup.3 CFU/cm.sup.2, and for ratios 1:0.1:0.1 and 1:0.2:0.2 at a density of 10.sup.3 CFU/cm.sup.2. As seen for combinations with C. accolens, anti-S. aureus activities of combinations with C. propinquum remained significant at all concentrations and densities tested, and were in fact generally higher than those seen for C. accolens. Indeed, S. aureus growth was inhibited by at least 81% at the lowest tested concentration and density (e.g. a ratio of 1:0.01:0.01 at a density of 10.sup.3 CFU/cm.sup.2). Increasing either the ratio (e.g. to 1:0.02:0.02) or the density (e.g. to 510.sup.3) increased anti-S. aureus activity, inhibiting S. aureus growth by approximately 89% and 93%, respectively.

TABLE-US-00009 TABLE IX C. propinquum/S. pasteuri/S. epidermidis combination at various ratios and densities Density/ratio S. aureus growth inhibition (mean (SD), %) C. propinquum at 10.sup.5 CFU/cm.sup.2 Ratio.sup.a 1:1:1 99.80 (0.00) Ratio 1:0.2:0.2 99.60 (0.00) Ratio 1:0.1:0.1 99.33 (0.19) Ratio 1:0.02:0.02 98.13 (0.19) Ratio 1:0.01:0.01 97.47 (0.19) C. propinquum at 5 10.sup.4 CFU/cm.sup.2 Ratio 1:1:1 99.50 (0.10) Ratio 1:0.2:0.2 99.50 (0.10) Ratio 1:0.1:0.1 99.07 (0.19) Ratio 1:0.02:0.02 98.67 (0.19) Ratio 1:0.01:0.01 97.73 (0.19) C. propinquum at 10.sup.4 CFU/cm.sup.2 Ratio 1:1:1 99.70 (0.10) Ratio 1:0.2:0.2 99.50 (0.10) Ratio 1:0.1:0.1 98.53 (0.19) Ratio 1:0.02:0.02 97.33 (0.19) Ratio 1:0.01:0.01 95.87 (0.19) C. propinquum at 5 10.sup.3 CFU/cm.sup.2 Ratio 1:1:1 99.60 (0.00) Ratio 1:0.2:0.2 98.93 (0.19) Ratio 1:0.1:0.1 97.33 (0.19) Ratio 1:0.02:0.02 95.87 (0.19) Ratio 1:0.01:0.01 93.73 (0.19) C. propinquum at 10.sup.3 CFU/cm.sup.2 Ratio 1:1:1 98.53 (0.19) Ratio 1:0.2:0.2 97.07 (0.19) Ratio 1:0.1:0.1 91.73 (0.38) Ratio 1:0.02:0.02 89.87 (0.75) Ratio 1:0.01:0.01 81.33 (0.75) .sup.aRatio is expressed as the quantity of C. accolens:S. pasteuri:S. epidermidis

Example 8: C. accolens Forms a Biofilm in the Presence of S. pasteuri

[0141] C. accolens requires a complex medium and tryptic soy broth (TSB) does not support its growth. Here we show that C. accolens and S. pasteuri can form a mixed biofilm supporting the growth of C. accolens in conditions where it is normally unable to grow.

Materials and Methods

[0142] Bacterial suspensions of a turbidity of McFarland 0.5 were prepared from cultures on Columbia 5% sheep blood agar plates as described in Example 3. 50 l of the suspension (approx. 210 CFU) of C. accolens AF2345 was distributed in 12 wells (row A); 50 l of a 1:100 dilution (approx. 210.sup.4 CFU) was distributed in 12 wells (row B); 50 l of the suspension of S. epidermidis AF2302 (approx. 210.sup.6 CFU) was distributed in columns 1, 2, 3, 7, 8, and 9; 50 l of the suspension of S. pasteuri AF2653 (approx. 210.sup.6 CFU) was distributed in columns 4, 5, 6, 7, 8, and 9. Columns 10, 11, and 12 contain C. accolens AF2345 suspended in 100 l of culture medium.

[0143] The plates were centrifuged, the suspension saline was removed and 100 l of TSB were added to each well. After 24 hours of incubation, 50 l of the medium was removed and replaced with fresh TSB. The plates were examined for growth. After 48 hours the plates were observed, the medium removed and the wells gently washed twice with saline. 200 l of saline was added to each well and the plate immersed in an ultrasound bath to disperse and resuspend the biofilm. For each well, 5 l ( 1/40th) was transferred to 95 l of saline ( 1/800.sup.th). 5 l of this suspension was then transferred to a fresh plate containing 95 l of saline ( 1/16000.sup.th). 10 l of each suspension was then applied to a Mueller Hinton 2 agar plate and to a Mueller Hinton 5% blood NAD enriched defibrinated agar plate containing mupirocin (128 g/ml).

[0144] The bacteria grown after 24 h were suspended in 2.2 ml ampules and the turbidity measured using a McFarland reader.

Results

[0145] The results obtained with the high and the low inocula of C. accolens (row A and row B) are qualitatively similar. However, the bacterial density obtained after 24 hours of incubation with the lower inoculum was too low to allow quantification using the McFarland turbidimeter. Data reported hereunder are obtained from the higher inoculum tested (row A).

[0146] As expected, C. accolens does not grow in TSB and does not form a biofilm. In contrast, both S. epidermidis and S. pasteuri form a biofilm in TSB (data not shown). Surprisingly, C. accolens is capable of growing within a S. pasteuri biofilm or a mixed S. pasteuri and S. epidermidis biofilm, although it does not grow within a biofilm of S. epidermidis alone (cf. FIG. 2). As expected, no growth is observed in the control wells containing C. accolens in TSB, since it is known that TSB does not support the growth of C. accolens. The difference between the growth of C. accolens in a S. pasteuri biofilm and a S. epidermidis biofilm is significant (p=0.0065). The difference between the growth of C. accolens in a S. pasteuri biofilm and a mixed S. pasteuri and S. epidermidis biofilm does not reach statistical significance (p=0.0651).

Example 9: Growth of C. propinquum in Mixed Biofilms

[0147] The same experiment presented in Example 8 was performed by replacing C. accolens with C. propinquum.

Materials and Methods

[0148] Experiments were performed as described in Example 8 except that C. propinquum AF1882 were tested instead of C. accolens AF2345.

Results

[0149] As shown in FIG. 3, C. propinquum is capable of forming a robust biofilm in TSB. It can also form mixed biofilms in combination with S. epidermidis and S. pasteuri. The growth within S. epidermidis is significantly lower than growth in biofilms containing S. pasteuri (p=0.019). However, as was the case with C. accolens, the presence of S. epidermidis does not adversely affect the growth within C. accolens containing biofilms (C. propinquum with S. pasteuri versus C. propinquum with S. pasteuri and S. epidermidis, p=0.0748).

Example 10: Synergistic Effect of Combining C. accolens with S. pasteuri and/or S. Epidermidis on Biofilm Formation

[0150] In addition to the quantification of bacterial growth (as illustrated above in Examples 8 and 9), biofilm formation of C. accolens grown alone or in combination with S. pasteuri, S. epidermidis, or both S. pasteuri and S. epidermidis was evaluated here by staining the extra-cellular matrix with crystal violet, which allows the relative quantification of biofilm formation in vitro.

Materials and Methods

[0151] 1.0 McFarland suspensions of C. accolens AF2345, S. pasteuri AF2653 and S. epidermidis AF2302 were prepared in tryptic soy broth (TSB) as described in Example 1. Fifty L of the undiluted C. accolens suspension and 50 L of a 10.sup.2 dilution of the S. pasteuri and S. epidermidis suspensions were seeded in the wells of a 96-well plate either alone or combined as follows: C. accolens/S. pasteuri, C. accolens/S. epidermidis and C. accolens/S. pasteuri/S. epidermidis. The volume was completed to 200 L per well with TSB and the plate was incubated at 37 C. After incubation for 48 hours, the wells were rinsed with distilled water, air-dried and the biofilm was stained with 200 L of a 0.2% crystal violet solution prepared in distilled water. The wells were then rinsed with distilled water, air-dried and the fixed crystal violet solubilized in 200 L of a 30% acetic acid solution prepared in distilled water. 100 L of the solubilized crystal violet solution was transferred to a new 96-well plate and the biofilm extra-cellular matrix quantified by measuring the optical density of the suspension at 595 nm.

Results

[0152] As shown in FIG. 4, significantly more extracellular matrix was produced when C. accolens was grown in combination with S. pasteuri, S. epidermidis or both S. pasteuri and S. epidermidis than with each bacterial species alone. Enhancement of extracellular matrix production was the highest with the combination C. accolens/S. pasteuri/S. epidermidis.

[0153] Furthermore, the quantity of extracellular matrix produced by the combined bacteria was greater than the expected additive effect expected in view of the quantity produced by each species alone. Thus, a synergistic effect is observed when C. accolens is co-cultured with S. pasteuri and/or S. epidermidis. The effect is maximal when C. accolens is co-cultured with both S. pasteuri and S. epidermidis.

Example 11: Treatment of Adherent S. aureus Cells with the Combination C. accolens/S. pasteuri Inhibits S. aureus Development and Biofilm Formation

[0154] We further studied whether treatment with C. accolens alone, S. pasteuri alone or the combination C. accolens/S. pasteuri had a bacteriotherapeutic effect on S. aureus, in particular inhibiting the development and biofilm formation of already adherent S. aureus.

Materials and Methods

[0155] A 1.0 McF suspension of S. aureus (strain USA300) was prepared from a 24 h-culture on Columbia 5% sheep blood agar at 35+/2 C., in sterile water. 200 l of this suspension diluted 1:100,000 in Trypto-Casein-Soy (TCS)+5% glucose was inoculated into wells (10.sup.3 CFU/well) of a 96-well plate and incubated for 6 h at 35+/2 C.

[0156] 50 McF, 5 McF and 3 McF suspensions of C. accolens (strain AF2345) and S. pasteuri (strain AF2653), respectively, were prepared in TCS supplemented with 0.05% polysorbate 80 (also referred to as Tween 80 or T80) from a 48 h-culture (C. accolens) or a 24 h-culture (S. pasteuri) on Columbia 5% sheep blood agar at 35+/2 C. These suspensions were further diluted in TCS+T80 0.05% in order to obtain a bacterial concentration of 10.sup.8 CFU/mL

[0157] After 6 h of incubation at 35+/2 C., 100 l of the S. aureus suspension was eliminated per well in the 96-well plate, and replaced by 100 l of bacterial suspension (C. accolens alone, S. pasteuri alone, or a combination of C. accolens/S. pasteuri), thus at a concentration of 10.sup.7 CFU/well for each species. 100 l of TCS+T80 0.05% also was added in place of the bacterial suspension in control wells (untreated S. aureus).

[0158] After further incubation for 24 h at 35+/2 C., wells were washed three times, filled with 200 l of sterile water, and immersed in an ultrasound bath to disperse and resuspend the biofilm. S. aureus CFUs were enumerated on MRSA agar plates (Biomrieux, Marcy-I'Etoile, France). Results were measured in triplicate and expressed as the mean log.sub.10 CFU and as the mean percent of CFU reduction versus control.

Results

[0159] As shown in FIG. 5, while both C. accolens and S. pasteuri alone had a bacteriotherapeutic effect, inhibiting S. aureus development and biofilm formation, the combination C. accolens/S. pasteuri showed an even stronger bacteriotherapeutic effect. S. aureus levels were notably reduced from 8.90 log.sub.10 CFU/mL to 7.53 log.sub.10 CFU/mL (FIG. 5A), corresponding to a mean CFU reduction of 95.0%.

[0160] Surprisingly, the bacteriotherapeutic effect observed with the combination of C. accolens/S. pasteuri was synergistic as it was greater than with C. accolens alone, S. pasteuri alone, or the expected effect of the combination of C. accolens/S. pasteuri (FIG. 5B).

[0161] Thus, the development of S. aureus cells adhering on a surface is the most efficiently combatted by the synergistic combination of at least C. accolens and S. pasteuri.

Example 12: Treatment of Adherent S. aureus Cells with the Combination C. propinquum/S. pasteuri Inhibits S. aureus Development and Biofilm Formation

[0162] We further determined if other species of Corynebacterium, such as C. propinquum, may also inhibit S. aureus development and biofilm formation.

Materials and Methods

[0163] Materials and methods were as described in Example 11 except that C. propinquum AF1882 (bacterial suspension: 2 McF) replaced C. accolens AF2345 (bacterial suspension: 50 McF). Reference strain S. pasteuri CIP 103830 was used in a similar manner to S. pasteuri AF2653 as described in Example 11.

Results

[0164] The mean log.sub.10 CFU of S. aureus was significantly lower when adherent S. aureus cells were treated with the combination C. propinquum/S. pasteuri (8.450.24 versus 9.610.12 for untreated S. aureus cells, p=0.0008), with a mean CFU reduction of 92.76%. Thus, the combination of C. propinquum/S. pasteuri may also inhibit S. aureus development and biofilm formation.

Example 13: Reconstituted Lyophilized Bacteria Prevent S. aureus Colonization

[0165] As the final formulation may include freeze-drying the bacteria, we verified that this process does not impair C. accolens ability, in combination with S. pasteuri and S. epidermidis, to prevent growth of S. aureus in vitro. We therefore compared the anti-S. aureus activity of fresh versus reconstituted freeze-dried bacteria.

Materials and Methods

[0166] Bacterial suspensions of C. accolens AF2345, S. pasteuri AF2652 and S. epidermidis AF2302 were combined at a 1:1:1 or a 1:0.1:0.1 ratio, respectively, in freeze-drying buffer. One part of these suspensions, hereafter named fresh bacteria, was used to inoculate 90 mm petri dishes at final densities of 10.sup.3 CFU/cm.sup.2, 10.sup.2 CFU/cm.sup.2 or 10.sup.1 CFU/cm.sup.2 by swabbing 300 L of the suspension on the surface of the agar plate. 300 l of sterile water was inoculated as negative control. One 50 mm 0.2 m track-etched filter was placed on top of the inoculated surface and S. aureus USA300 suspensions containing a target number of 10 and 100 CFU in 10 l were spotted in triplicate on the filter surface. Materials and methods were otherwise as described in Example 3. The other part of the prepared suspensions was lyophilized in a shelf freeze dryer, using a two-stage process at negative pressure (primary drying: sub-zero temperatures; secondary drying: 15 C.). At the end of the process the vials were sealed under vacuum and stored at 4 C. in the dark until use. On the day of the assay, the lyophilized cake was resuspended in 400 L of sterile water and the reconstituted suspensions were inoculated according to the same conditions and dilutions as were used for the fresh bacterial suspensions.

Results

[0167] As shown below in Table X, the inhibition of S. aureus growth is similar when using fresh or reconstituted lyophilized C. accolens/S. pasteuri/S. epidermidis combinations, regardless of the bacterial density or ratio tested in the assay (no significant differences are observed).

TABLE-US-00010 TABLE X Anti-S. aureus activity of fresh or reconstituted C. accolens/S. pasteuri/S. epidermidis combinations at various densities. P-value.sup.a S. aureus growth inhibition, Fresh vs mean % (SD) lyophilized Density Fresh bacteria Lyophilized bacteria bacteria 1:1:1 C. accolens/S. pasteuri/S. epidermidis ratio C. accolens at 96.82 (0.70) 96.54 (0.46) 0.60 10.sup.3 CFU/cm.sup.2 C. accolens at 90.35 (2.00) 88.95 (0.56) 0.31 10.sup.2 CFU/cm.sup.2 C. accolens at 63.40 (3.08) 66.12 (3.08) 0.34 10.sup.1 CFU/cm.sup.2 1:0.1:0.1 C. accolens/S. pasteuri/S. epidermidis ratio C. accolens at 90.95 (0.65) 91.11 (0.76) 0.80 10.sup.3 CFU/cm.sup.2 C. accolens at 66.77 (3.50) 61.32 (6.60) 0.27 10.sup.2 CFU/cm.sup.2 C. accolens at 23.95 (10.85) 20.65 (8.04) 0.69 10.sup.1 CFU/cm.sup.2 .sup.aStudent's t-test (p < 0.05 for significance).

[0168] Thus, freeze-drying a combination of bacteria (in this case C. accolens, S. pasteuri and S. epidermidis) does not impair the anti-S. aureus activity of the bacterial combination. S. aureus colonization may therefore still be successfully prevented.

Example 14: The Combination C. accolens/S. Pasteuri, and, Optionally, S. Epidermidis Inhibits S. aureus Nasal Colonization in Mice

[0169] We studied here the capacity of the combination C. accolens/S. pasteuri, and, optionally, S. epidermidis to reduce S. aureus nasal colonization in the mouse.

Materials and Methods

[0170] Six-week old BALB/c_JRj mice were obtained from Janvier Labs (Le Genest-Saint-Isle, France) and were used after one week of acclimatization (IERP, INRA, Jouy-en-Josas, France). A 10 McF suspension of S. aureus (strain USA300) in normal saline (10.sup.9 CFU/mL) was prepared from a 24 h-culture on Columbia 5% sheep blood agar at 35+/2 C. Lyophilized C. accolens (strain AF2345), S. pasteuri (strain AF2653) and S. epidermidis (strain AF2302) were reconstituted with sterile distilled water in order to obtain a suspension of 2.10.sup.10 CFU/mL for each species. Mice were inoculated intranasally with S. aureus (10.sup.7 CFU) and the combination C. accolens/S. pasteuri/S. epidermidis (0.10.sup.8 CFU for each species, thus 310.sup.8 CFU, ratio 1:1:1) at a final volume of 10 l per nostril; S. aureus alone was inoculated as a control (untreated S. aureus). At 6 h post-inoculation, mice were euthanized and the two nostrils were sampled with a single 0.6 mm interdental brush (GUM; Levallois-Perret; France) to quantify S. aureus CFUs as described in example 11. Results were expressed as the mean+/standard deviation (SD) per animal (five measurements) of the log.sub.10 CFU of S. aureus and as the mean percentage of S. aureus CFU reduction versus control.

Results

[0171] The application of the combination C. accolens/S. pasteuri/S. epidermidis significantly reduced S. aureus nasal colonization (mean (SD): 3.91 (0.46) log.sub.10 CFU vs 4.68 (0.64) log.sub.10 CFU for control; p=0.030), with a mean CFU reduction of 86.4%.

[0172] Thus, the combination C. accolens/S. pasteuri/S. epidermidis also shows anti-S. aureus activity by the intranasal route.

Example 15: Synergistic Effect of Combining C. accolens with S. pasteuri and/or S. epidermidis in Preventing of S. aureus Colonization In Vivo in a Human Skin Model

[0173] We studied the capacity of freeze-dried strains of C. accolens, S. pasteuri and S. epidermidis formulated alone or in various combinations to inhibit S. aureus growth on the forearm skin of a healthy individual using punched hydrocolloid sterile patches as templates to outline the experimental area (as illustrated in FIG. 6A). Indeed, as indicated previously, the forearm skin is an excellent model of both the skin and anterior nares, in view of the similarities in structure and microbial community composition between these sites.

Materials and Methods

[0174] Vials of lyophilized C. accolens strain AF2345, S. pasteuri strain AF2652 and S. epidermidis strain AF2302 were prepared as described in Example 13. Nine evenly spaced 4.5 mm diameter holes were punched in hydrocolloid sterile patches, each hole constituting a 30 l skin-bottom well. On the day of the assay, two patches were applied to the skin of the internal aspect of the forearm of an informed consenting healthy human volunteer. The lyophilized bacterial cakes were resuspended in sterile water and diluted to a concentration of 310.sup.8 CFU/mL. S. aureus ATCC 29213 was subcultured on blood agar and a saline suspension having a turbidity of 1 McFarland was prepared and diluted to obtain a suspension of 10.sup.8 CFU/mL.

[0175] C. accolens, S. pasteuri and S. epidermidis alone or in various combinations were added to the skin wells at a dose of 210.sup.6 CFU per species per well and S. aureus at a dose of 510.sup.3 CFU per well. The hydrocolloid patches and the surrounding skin was then covered by sterile polyurethane adhesive surgical incision film (FIG. 6A). The patches were left in place for 30 hours, after which they were peeled off and the skin area corresponding to each well sampled with a humidified swab to quantify S. aureus. The head of the swabs were cut using sterile surgical clippers and collected in sterile containers. DNA was extracted using Dneasy Blood and Tissue Kit (Qiagen), according the manufacturer's protocol. DNA extracts were amplified by quantitative real time PCR (qPCR) on the CFX96 C1000 Touch real time system (Biorad) using primers targeting the Spa gene. The reaction mixture was prepared with 1 Itaq Universal Probes supermix (Biorad), 500 nM for each primer, 250 nM probe and DNA template in a final volume of 20 l. For qPCR, an initial denaturation step (95 C., 5 min) was followed by 40 cycles of 95 C. 15 s and 60 C. for 1 min. Forty amplification cycles were performed and the CT determined. Results were expressed as the [(ThresholdCT)/Threshold] ratio. This ratio reaches one when Spa DNA is detectable without amplification, and reaches zero when there is no detection of Spa DNA at the end of the 40-cycle amplification.

Results

[0176] FIG. 6B shows the [(ThresholdCT)/Threshold] ratios obtained with S. aureus qPCR after co-incubation of C. accolens, S. pasteuri and S. epidermidis alone and in various combinations. When taken alone, none of the species inhibited S. aureus growth. In contrast, a synergistic inhibition of S. aureus growth was observed for the bacterial combinations C. accolens/S. pasteuri (2.9-fold reduction S. aureus in growth when compared to growth of S. aureus alone) or the combination C. accolens/S. pasteuri/S. epidermidis (12.6-fold reduction), in accordance with the present invention.

[0177] No irritation, erythema or alteration of the skin of the volunteer was observed.

Example 16: The Combination C. accolens/S. Pasteuri Shows a Synergistic S. aureus Decolonization Effect in a Murine Model of Stratified Squamous Epithelium

[0178] Our previous results obtained in vitro showed a synergistic bacteriotherapeutic activity of the combination C. accolens/S. pasteuri against S. aureus cells in vitro (see Example 11). We studied here the capacity of this combination to reduce S. aureus colonization (S. aureus decolonization) in an in vivo model of stratified squamous epithelium relevant for both the skin and anterior nares, using Nude mice. We further evaluated whether S. epidermidis had an antagonistic effect towards the combination C. accolens/S. pasteuri.

Materials and Methods

[0179] Six-week old BALB/cAnNRj-Fox1 nu/nu mice were obtained from Janvier Labs (Le Genest-Saint-Isle, France) and were used after one week of acclimatization (IERP, INRA, Jouy-en-Josas, France). A 10 McF suspension of S. aureus (strain USA300) in physiological water (PSA) was prepared from a 24 h-culture on Columbia 5% sheep blood agar at 35+/2 C. After homogenization, 250 l was vaporized (Amber boston glass bottle, One Trillion, China) on the back of each mouse. Twenty-four hours later, a hydrocolloid sterile patch (Compeed, Paris, France) having five evenly spaced punched holes (diameter: 4.5 mm) was applied to the back of each mouse, with each hole constituting a 30 l skin-bottom well. Lyophilized suspension of C. accolens (strain AF2345), S. pasteuri (strain AF2653) and optionally S. epidermidis (strain AF2302) were reconstituted with distilled sterile water to obtain a suspension of 10.sup.10 CFU/mL for each species. Bacteria were inoculated in the skin well at a volume of 10 l (10.sup.8 CFU per skin well). The hydrocolloid patch and the surrounding skin were then covered by sterile polyurethane adhesive surgical incision film. Four conditions were tested: C. accolens alone; S. pasteuri alone; C. accolens combined with S. pasteuri; C. accolens combined S. pasteuri and S. epidermidis); 10 l of physiological water was inoculated as control (untreated S. aureus).

[0180] Twenty-four hours later, mice were euthanized, the adhesive surgical incision film was peeled, and the skin area of each well was sampled with a humidified swab (ESwab, COPAN, Brescia, Italy), and S. aureus CFUs enumerated by counting CFUs on MRSA agar (Biomrieux, Marcy-I'Etoile, France). Results were measured in duplicate and expressed as the mean+/SD log.sub.10 CFU and as the mean percentage of CFU reduction versus control.

Results

[0181] Firstly, it should be noted that the mean number of S. aureus CFUs found with the control was 5.51 per well area (15.9 mm.sup.2). Thus, S. aureus skin colonization reached a density of 210.sup.4 CFU/mm.sup.2 48 h post-inoculation, confirming the relevance of our model compared to available models of S. aureus nasal colonization in rodents. Indeed, in comparison, in an optimized model of nasal colonization using mice treated with oral streptomycin to reduce the natural nasal flora, 510.sup.3 CFUs of S. aureus/nose was found 8 hours after the intranasal instillation of 510.sup.8 CFUs of S. aureus (Park et al., 2011). Similarly, in the cotton rat model, a well-established model for S. aureus nasal colonization which also requires the oral treatment of animals with streptomycin, S. aureus colonization was 510.sup.4 CFU/nose 8 hours after inoculation (Baur et al., 2014). As S. aureus colonization occurs in the murine model used herein at levels similar to those described existing murine models, this model represents a clinically relevant model that should be considered as an appropriate equivalent to existing models.

[0182] The combination C. accolens/S. pasteuri showed a significant decolonization effect compared to the control (untreated S. aureus), with a mean CFU reduction of 72.6% (p<0.005 versus control); this effect was synergistic as it was greater than with C. accolens alone, S. pasteuri alone, or the expected effect of the combination of C. accolens/S. pasteuri (FIG. 7B).

[0183] The combination C. accolens/S. pasteuri/S. epidermidis also showed a significant effect, with a comparable CFU reduction (66.5%, p<0.005 versus control). S. epidermidis thus had no impact on the decolonization effect of the combination of C. accolens/S. pasteuri.

CONCLUSION

[0184] Taken together, these Examples illustrate the surprising synergistic anti-S. aureus activity that occurs when combining at least one Corynebacterium sp., such as C. accolens or C. propinquum, with S. pasteuri, and, optionally, S. epidermidis. Furthermore, these results indicate that S. pasteuri particularly promotes growth of Corynebacterium spp. such as C. accolens, and furthermore promotes biofilm formation of C. accolens in a synergistic manner when in co-culture as illustrated by quantification of the extracellular matrix. Biofilm formation was similarly improved in a synergistic manner when a Corynebacterium sp. was cultured with both S. pasteuri and S. epidermidis. Advantageously, a composition comprising said bacterial species can furthermore be successfully lyophilized and reconstituted with no loss of effect, as the reconstituted composition prevents S. aureus colonization at the same level as fresh bacteria. Bacteriotherapeutic effects were clearly illustrated in murine models of both S. aureus nasal colonization and more generally in decolonization of stratified squamous epithelium, which functions as model of both the skin and the anterior nares.

[0185] Furthermore, administration of the composition to human forearm skin, which represents the most common ecological niches of S. aureus (i.e. the skin and the anterior nares), yielded similar results, greatly reducing colonization by S. aureus in vivo.

[0186] These Examples provide the first evidence that a composition comprising at least one Corynebacterium sp., preferably at least C. accolens or C. propinquum, and S. pasteuri, and, preferably also S. epidermidis, represents a novel, unexpected and highly advantageous composition for use in the prevention and/or treatment of both MSSA and MRSA S. aureus colonization.

REFERENCES

[0187] Baur et al., 2014._PLoS Pathog, 10(5):e1004089. [0188] Iwase et al., 2010. Nature, 465:346-349. [0189] Jennings, 1999. Lyophilization: Introduction and Basic Principles. Interpharm/CRC Press, Denver. [0190] Kluytmans et al., 1997. Clin Microbiol Rev. 10(3):505-20. [0191] Park et al., 2011. PLoS ONE. 6(10): e25880. [0192] Uehara et al., 2000. J Hosp Infect, 44(2): 127-133. [0193] Wertheim et al., 2005. Antimicrob Agents Chemother, 49(4):1465-1467. [0194] White, 1963. Antimicrob Agents Chemother, 161: 667-70. [0195] van Belkum et al., 2009. J Infect Dis. 199: 1820-26. [0196] Yan et al., 2013. Cell Host and Microbe, 14(6): 631-640.