The present invention relates to the prevention of bacterial colonization by Staphylococcus aureus.

The present invention also relates to compositions (e.g. pharmaceutical compositions) comprising at least one Corynebacterium sp. and Staphylococcus sp. and related methods, said compositions and methods being particularly associated with the prevention of bacterial colonization by Staphylococcus aureus. Staphylococcus aureus is a major cause of infection in humans. It is responsible for a wide range of pathologies including skin and soft tissue infections, endocarditis, and sepsis. While S. aureus can asymptomatically colonize the skin, mucus membranes and gastrointestinal tract, asymptomatic carriage is associated with an increased risk of endogenous S. aureus infection, particularly in surgical and dialysis patients. Asymptomatic carriers also act a reservoir for the transmission of S. aureus, increasing the risk of infection in both the community and in hospital settings. Indeed, at any given time, an estimated 30% of the population carries S. aureus in the anterior nares, the principal site of S. aureus colonization.

In order to decrease the risk of transmission and infection by S. aureus, in particular in hospital settings where patients are particularly susceptible, many healthcare centers recommend routine patient screening prior to admission, coupled with the elimination of S. aureus nasal carriage, otherwise known as "nasal decolonization." Adequate nasal decolonization methods are therefore critical to limiting S. aureus transmission and infection. To date, a variety of methods have been used for nasal decolonization, including the application of local antibiotics and/or antiseptics, the use of systemic antibiotics, and artificial bacterial colonization of the nares. However, local antibiotic and antiseptic treatment methods have low efficacy and have led to the rapid emergence of antiseptic and antibiotic resistance in S. aureus strains. The use of systemic antibiotics has similarly led to the rapid emergence of antibiotic resistant strains, coupled with the presence of undesirable side effects. Artificial bacterial colonization consists in the administration of bacterial species and/or strains that compete with S. aureus. Notably, artificial colonization of the nares with S. aureus strain 502A was used to prevent colonization by more virulent S. aureus strains in neonates during outbreaks, and in patients with recurrent infection, in the 1960s. However, this method was abandoned after strain 502A itself was found to be the cause of several S. aureus infections and was even linked to a death. More recently, several studies have evaluated the effects of other bacterial species on S. aureus colonization in the nares, with varying and often contradictory results. For example, Uehara et al., 2000, has shown that S. epidermidis is unable to eliminate S. aureus from the nares. In contrast, Iwase et al., 2010, has reported that S. epidermidis can in fact eliminate S. aureus carriage. Similarly, while Uehara et al., 2000, has shown that artificial colonization with Corynebacterium can eliminate S. aureus carriage, Yan et al., 2013 has more recently shown that the presence of Corynebacterium accolens in the nares is associated with carriage of S. aureus. Yan et al., 2013 has further shown that C. accolens can facilitate growth of S. aureus.

Despite the known disadvantages of local antibiotic administration, nasal decolonization currently relies essentially on the use of a locally applied antibiotic, mupirocin. In efforts to slow the development of S. aureus resistance to mupirocin, treatment is limited to severe infections, with antibiotic administration for very short periods of time (e.g. a five-day treatment course in patients undergoing resuscitation or surgery). Attaining patient compliance can be difficult, however, as mupirocin must be applied repeatedly. Furthermore, S. aureus recolonization is frequent after treatment, indicating that mupirocin has little long-term treatment effect. Indeed, while mupirocin efficacy is significant, as many as half of the patients remain colonized with the same S. aureus strain five weeks after the start of treatment. It has furthermore been reported that mupirocin treatment of individuals exempt from S. aureus colonization can be deleterious and cause them to become carriers, making it necessary to screen for carriage prior to treatment (Wertheim et al., 2005). Despite these efforts, S. aureus antibiotic-resistant strains are now detected in up to 50% of patients in rehabilitation and long-term care facilities. Of the antibiotic-resistant strains, methicillin-resistant S. aureus (MRSA) is the most prevalent, though cases of mupirocin- resistant MRSA have been reported. Infections due to antibiotic-resistant S. aureus (e.g. MRSA) are associated with increased mortality, morbidity, length of hospitalization and cost, when compared to infections due to methicillin-sensitive S. aureus (MSSA).

As a result, there exists a need for new products and methods for the prevention of S. aureus colonization on the skin and mucus membranes. In particular, there is a need for new products and methods preventing S. aureus colonization of the nares. There is a further need for new products and methods that can be administered over a long period of time (e.g. weeks or months), in particular for the duration of the healing of an accidental or surgical wound. This need is particularly important in the context of orthopedic and cardiac surgery, in patients bearing implanted devices, and in patients residing in rehabilitation or long-term care facilities, where S. aureus colonization and/or infection are frequent. Given the propensity of S. aureus to develop antibiotic and antiseptic resistance, these new products and methods should not be based on the use of either antibiotics or antiseptics, and should function in particular in the prevention of MRSA. They should also have few side effects and be available at a reasonable cost.

The present invention is directed to a composition comprising at least one Corynebacterium sp. and at least one Staphylococcus sp., preferably comprising at least one Corynebacterium sp. and at least S. pasteuri. The composition according to the invention may comprise any of the preferred characteristics described below relative to the pharmaceutical composition, which is itself a preferred embodiment of the composition. In particular, the composition comprises at least one Corynebacterium sp. and Staphyloccocus pasteuri, wherein said at least one Corynebacterium sp. is selected from C. accolens, C. propinquum, C. pseudodiphtheriticum, C. striatum, C. amycolatum, C. glutamicum, C. aurimucosum, C. tuberculostearicum, and C. afermentans, preferably C. accolens and/or C. propinquum. Preferably, the composition further comprises Staphylococcus epidermidis. Preferably, the composition comprises at least 10 ³ CFUs of Corynebacterium sp., at least 10 ³ CFUs of Staphyloccocus pasteuri, and, optionally, at least 10 ³ CFUs of Staphylococcus epidermidis per dose. Preferably, the composition comprises a total of at least 10 ³ bacterial CFUs per dose, preferably at least 1.2x10 ³ , more preferably at least 1.4x10 ³ , even more preferably at least 3x10 ³ bacterial CFUs per dose. Preferably, the ratio of Corynebacterium sp. to Staphylococcus pasteuri to Staphyloccocus epidermidis is comprised in the range of 1 : 0.01 : 0.01 to 1 : 1 : 1 . Preferably, the composition further comprises at least one excipient, preferably a pharmaceutically acceptable excipient. Preferably said at least one excipient comprises at least one lyoprotectant, preferably selected from peptone, glycerol, lactose, gelatin, glucose, sucrose, trehalose, dextran, maltodextrin, adonitol, and sodium glutamate. Preferably, the composition is lyophilized or freeze-dried. Preferably, the composition further comprises at least one gelling agent, preferably a pharmaceutically acceptable gelling agent.

Preferably, the present invention is directed to a pharmaceutical composition comprising at least one Corynebacterium sp. and at least one Staphylococcus sp., preferably comprising at least one Corynebacterium sp. and at least S. pasteuri. Said at least one Corynebacterium sp. is preferably selected from C. accolens and C. propmquum, or comprises both C. accolens and C. propinquum. The present invention is further directed to a pharmaceutical composition comprising at least one Corynebacterium sp., S. pasteuri, and at least one additional Staphylococcus sp., preferably S. epidermidis. Thus, according to a particularly preferred embodiment, the present invention is further directed to a pharmaceutical composition comprising C. accolens and/or C. propinquum, S. pasteuri and S. epidermidis. The invention is further directed to said composition for use as a medicament, in particular in the prevention of S. aureus colonization. Indeed, the inventors have surprisingly found that a composition comprising a combination of at least one Corynebacterium sp. and S. pasteuri or a combination of at least one Corynebacterium sp., S. pasteuri and S. epidermidis can prevent S. aureus colonization.

The combination of these bacterial species is particularly advantageous as the inventors have surprisingly shown that the three bacterial species C. accolens, S. pasteuri and S. epidermidis, individually inhibit S. aureus colonization, with C. accolens and S. pasteuri having a strong synergistic effect, inhibiting S. aureus growth by more than 95%. The inventors have also surprisingly shown that other Corynebacterium sp., such as C. propinquum similarly inhibit S. aureus colonization, with C. propinquum and S. pasteuri having a particularly strong synergistic effect, inhibiting S. aureus growth by more than 98%. Moreover, growth of C. accolens is potentiated by S. pasteuri, as C. accolens does not grow in the absence of S. pasteuri in certain conditions. The presence of S. pasteuri, or of both S. pasteuri and S. epidermidis, improves persistence of Corynebacterium sp., such as C. accolens or C. propinquum by enabling the establishment of a biofilm. Furthermore, although a combination of C. accolens and S. epidermidis has an antagonistic tendency, reducing the inhibitory effect on S. aureus growth seen with S. epidermidis alone, the combination of all three bacterial species also has surprisingly high anti-S. aureus activity, inhibiting S. aureus growth by more than 95%. As S. epidermidis is an essential colonizer of the nasal flora, it is preferably included in the pharmaceutical combination. S. epidermidis may notably provide a protective bacterial layer in vivo, contributing to the prevention of S. aureus re-establishment. Thus, a pharmaceutical composition comprising at least one Corynebacterium sp., S. pasteuri and S. epidermidis prevents S. aureus colonization, and is particularly advantageous.

The pharmaceutical composition of the present invention is advantageous as the composition is not based on the administration of antibiotics or antiseptics, but rather on the administration of at least two, preferably three bacterial species, thereby minimizing the risk of the emergence of further S. aureus resistance. Advantageously, said composition can be administered for longer periods of time than current methods. Advantageously, the present composition can prevent colonization by antibiotic resistant S. aureus, such as MRSA, in a patient. The composition of the invention is also advantageous as none of the selected bacterial species possess virulence traits. Even more advantageously, all selected bacterial species are commonly detected in the nasal microflora and/or on the skin of healthy subjects.

Prevention of colonization via the pharmaceutical composition of the present invention is particularly advantageous as colonization is associated with both an increased risk of endogenous infection and transmission. Thus, preventing S. aureus colonization represents the most effective way of reducing the total number of S. aureus infections. Furthermore, current methods for treating S. aureus are not recommended for prophylactic use due to the possibility of increasing antibiotic resistance. In contrast, the pharmaceutical composition according to the invention can be administered prophylactically to subjects.

The term "patient" or "subject" is defined herein as any human individual, regardless of their age. In a preferred embodiment, the subject is hospitalized for any reason. In a more preferred embodiment, the subject is hospitalized for cardiac or orthopedic surgery, bares an implanted device, or resides in a rehabilitation or long-term care facility. In cases where the subject is not colonized by S. aureus at the time of hospital admission, the pharmaceutical composition of the invention may be used prophylactically, to prevent later colonization by S. aureus.

The subject may be an adult or child. The term "adult" refers herein to an individual of at least 16 years of age. The term "child" comprises infants from 0-1 years of age and children from 1 -8 years of age, 8-12 years of age, and 12-16 years of age. The term "child" further comprises neonatal infants from birth to 28 days of age and post-neonatal infants from 28 to 364 days of age. The composition may be administered to an adult or a child, including neonatal infants. The pharmaceutical composition of the invention is particularly advantageous as existing treatment methods using antibiotics are limited in children due to unwanted side effects.

The pharmaceutically acceptable composition comprises a sufficient quantity of each of the bacterial strains to be therapeutically effective. As a non-limiting example, the pharmaceutical composition may comprise at least 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , or 10 ¹² colony forming units (CFUs) of each of the bacterial strains, or a total of at least 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , or 10 ¹² CFUs of all bacterial strains taken together. The quantities of each bacterial strain need not be identical. The ratio of the amount of each of the bacterial strains to the other one or more strains is also such that the pharmaceutically acceptable composition is therapeutically effective. As a non- limiting example, when two strains are comprised in the pharmaceutical composition, the ratio of one strain to the other may range from 1 : 0.01 to 1 : 2, including for example, ratios of 1 : 0.02, 1 : 0.04, 1 : 0.2, 1 : 0.4, or 1 : 1 . As a non-limiting example, when three strains are comprised in the pharmaceutical composition, the ratio of one strain to each of the others may range from 1 : 0.01 : 0.01 to 1 : 1 : 1 , including for example, ratios of 1 : 0.02 : 0.02, 1 : 0.1 : 0.1 , or 1 : 0.2 : 0.2. In a preferred embodiment, the pharmaceutical composition of the present invention comprises from 10 ³ to 10 ¹² CFUs of the at least one Corynebacterium sp., from 10 ³ to 10 ¹² CFUs of S. pasteuri, and, optionally, from 10 ³ to 10 ¹² CFUs of S. epidermidis per dose. More preferably, the composition comprises from 10 ⁴ to 10 ¹⁰ CFUs of the at least one Corynebacterium sp., from 10 ⁴ to 10 ¹⁰ CFUs S. pasteuri and, optionally, from 10 ⁴ to 10 ¹⁰ CFUs of S. epidermidis per dose. Even more preferably, the composition comprises 10 ⁵ to 10 ⁹ CFUs of the at least one Corynebacterium sp., 10 ⁵ to 10 ⁹ CFUs S. pasteuri and, optionally, from 10 ⁵ to 10 ⁹ CFUs of S. epidermidis per dose.

Preferably, the composition of the present invention comprises a total bacterial concentration of at least 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , or 10 ¹² CFUs, preferably at least 1 .2, 1 .4, 2 or 3 x10 ³ CFUs, at least 5, 5.02, 5.04, 5.2, or 5.4 x10 ³ CFUs, at least 1 .02, 1 .04, 1 .2, 1 .4, 1.5, 2 or 3x10 ⁴ CFUs, at least 5, 5.02, 5.04, 5.2, or 5.4 x10 ⁴ CFUs, at least 1 .02, 1 .04, 1 .2, 1 .4, 1 .5, 2, or 3 x10 ⁵ CFUs, at least 1 .02, 1 .04, 1.2, 1.4, 2, or 3 x10 ⁶ CFUs, at least 1 .02, 1 .04, 1 .2, 1.4, 2, or 3 x10 ⁷ CFUs, at least 1 .02, 1 .04, 1 .2, 1 .4, 2, or 3 x10 ⁸ CFUs, at least 1 .02, 1 .04, 1 .2, 1 .4, 2 or 3 x10 ⁹ CFUs, at least 10 ¹⁰ CFUs, at least 1 .02, 1 .04, 1 .2, 1.4, 2 or 3 x10 ¹⁰ CFUs, at least 1 .02, 1 .04, 1 .2, 1.4, 2 or 3 x10 ¹¹ CFUs, or at least 1 .02, 1.04, 1.2, 1.4, 2 or 3 x10 ¹² CFUs per dose.

The term "dose" is defined herein as amount of the pharmaceutical composition that is administered at a given time. Preferably, the dose administered is therapeutically effective. The dose may be expressed herein in terms of weight, volume, or CFUs of the pharmaceutical composition.

The term "colony forming unit" or "CFU" is defined herein as a unit used to indicate the number of viable bacteria in a sample, wherein one colony forming unit corresponds to one viable bacterial cell. In the pharmaceutical composition of the present invention, the ratio in CFUs of the at least one Corynebacterium sp. to the at least one Staphyloccocus sp. (e.g. the ratio of the at least one Corynebacterium sp., preferably C. accolens, : S. pasteuri) is preferably comprised in the range from 1 : 0.01 to 1 : 2, more preferably 1 : 0.01 to 1 : 1 . In the pharmaceutical composition of the present invention, the ratio in CFUs of the at least one Corynebacterium sp. to S. pasteuri to S. epidermidis is preferably comprised in the range from 1 : 0.01 : 0.01 to 1 : 1 : 1 . Advantageously, the ratio of the at least one Corynebacterium sp., preferably C. accolens, to S. pasteuri to S. epidermidis is comprised in the range from 1 : 0.02 : 0.02 to 1 : 1 : 1 , preferably from 1 :0.1 : 0.1 to 1 : 1 : 1 , even more preferably from 1 : 0.2 : 0.2 to 1 : 1 : 1 .

As a non-limiting example, the at least one Corynebacterium sp. may be selected from C. accolens, C. propinquum, C. pseudodiphtheriticum, C. striatum, C. amycolatum, C. glutamicum, C. aurimucosum, C. tuberculostearicum, or C. afermentans in the pharmaceutical composition. The above-mentioned species have notably all been isolated from the nares and/or the skin. The similar effects of different Corynebacterium spp. are notably illustrated in the examples. In some cases, two or more Corynebacterium spp. may be present in the pharmaceutical composition. For example, C. accolens may be combined with at least one other Corynebacterium sp., for example selected from one or more of those listed above, such as C. propinquum. According to a preferred embodiment, the at least one Corynebacterium sp. is selected from C. accolens, C. propinquum, C. pseudodiphtheriticum, C. striatum, C. amycolatum, C. glutamicum, C. aurimucosum, C. tuberculostearicum, and C. afermentans. According to a more preferred embodiment, the at least one Corynebacterium sp. comprises C. accolens or C. propinquum, or a combination of both C. accolens and C. propinquum, according to any of the embodiments described herein. The similar effects of different C. accolens strains are notably illustrated in the examples.

In the context of the pharmaceutical composition, any strain of S. epidermidis may be used. As a non-limiting example, a strain of S. epidermidis may be selected from the strains isolated from the nares.

In the context of the pharmaceutical composition, any strain of the S. pasteuri species may be used. The similar effects of different S. pasteuri strains are notably illustrated in the examples.

The pharmaceutical composition may be administered prophylactically, in the absence of detection of S. aureus, preferably to the skin and/or nares, more preferably to the anterior nares. Alternatively, the pharmaceutical composition may be administered following detection and elimination of S. aureus on the skin or in the nares. Alternatively, the pharmaceutical composition may be administered in the absence of testing.

The composition may be administered to a subject one or more times daily (e.g. one, two, three, or four times) and can be administered for several consecutive days (e.g. for one, two, three, four, five, six, seven, eight, nine, ten days or more), weeks (e.g. for one, two, three, four, five, six, seven, eight, nine, ten weeks or more), or months (e.g. for one, two, three, four, five, six months or more). In a preferred embodiment, the composition is administered 1 to 3 times per day for 8 to 12 days, preferably in subjects hospitalized for cardiac or orthopedic surgery. In an alternative preferred embodiment, the composition is administered for weeks or months in subjects bearing an implanted device or residing in a rehabilitation or long-term care facility, or for the duration of the healing of an accidental or surgical wound. In a particularly preferred embodiment, the composition is administered 1 to 3 times per day for one to four weeks, or for one to six months, in subjects bearing an implanted device, residing in a rehabilitation or long-term care facility, or healing from an accidental or surgical wound. In an even more preferred embodiment, the composition is administered for the duration of a hospital stay, the duration of the presence of an implanted device, the duration of residence in a rehabilitation or long-term care facility, or for the duration of the healing of an accidental or surgical wound, preferably 1 to 3 times per day.

Alternatively, the composition may be administered to a subject intermittently (e.g. every other day, every two days, or as necessary) or cyclically (e.g. administration for several days in a row, followed by an absence of administration, or "withdrawal," for several days, at the end of which the cycle is repeated). In a preferred embodiment, the composition is administered for 8-12 days followed by a period of withdrawal.

Alternatively, the composition may be administered on demand.

If a protective effect is observed following administration, administration may be repeated as appropriate to maintain said protective effect. If S. aureus colonization is detected following administration of the composition, the administration regimen should be repeated.

The duration and frequency of administration of the composition can be adapted by the skilled person, based on their general knowledge. In particular, the adaptation of these parameters can depend on the length of time during which it is desired that S. aureus colonization is absent in the nares and/or on the skin of a subject. For example, it may be desired that S. aureus colonization is absent for the duration of a hospitalization.

In a preferred embodiment of the invention, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient. The term "pharmaceutically acceptable excipient" is defined herein as a component, or combination of components, that is compatible with the pharmaceutical composition, does not generate unwanted side-effects in the patient, and that is generally considered to be non-toxic. A pharmaceutically acceptable excipient is most commonly implicated in facilitating administration of the composition, increasing product shelf-life or efficacy, or improving the solubility or stability of the composition. The pharmaceutically acceptable excipient is generally considered to be pharmacologically inactive. However, in some cases, the excipient itself may also have a therapeutic effect, for example, by making it more difficult for S. aureus to colonize the treated site (i.e. site of administration).

Pharmaceutically acceptable excipients or carriers are well-known in the prior art and can easily be adapted by the skilled person based on the desired galenic formulation of the composition. The galenic formulation, method of administration, and dosage can further be determined based on widely-accepted criteria for adapting patient treatments, including their general health, age, weight, tolerance to treatment, etc., as necessary.

The pharmaceutical composition of the present invention may be administered topically to the epithelium (e.g. the skin) and/or mucus membranes. Preferably, the pharmaceutical composition of the present invention may be administered to the nares, sinuses, or on the axilla, groin, inguinal and perirectal regions, or elsewhere on the skin or mucus membranes. According to a preferred embodiment, the composition is administered to the nares. The term "nares" as defined herein comprises both the anterior nares or nostrils and the nasal cavity. Preferably, the pharmaceutical composition of the invention is administered to the anterior nares, or nostrils.

While S. aureus may colonize various sites on the human body, it is recognized that its primary ecological niche is the anterior nares, from which it can spread to other tissues or organs (Kluytmans et al., 1997). Much like the skin (e.g. forearm skin), the anterior nares are lined by skin-like, fully keratinized, squamous epithelium with hairs, sebaceous glands and sweat glands. Genetic studies have moreover demonstrated that microbial communities from these two physically distinct niches share identical compositional patterns (Yan et al., 2013). Thus, S. aureus colonization is similar in the skin and anterior nares, with the skin furthermore representing an excellent and easily accessible model for both of these sites. Advantageously, the composition of the present invention comprises any suitable pharmaceutical preparation for administration to the skin and/or mucus membranes, such as a patch, gel, cream, lotion, ointment, film or salve. Advantageously, the viscosity or texture of the composition is modulated to improve application or efficacy (e.g. contact time). Advantageously, the composition is such that it does not cause adverse effects to the nasal epithelium. Even more advantageously, the pH and/or the osmolarity of the composition is similar to that of the nares.

As the pH of the nasal epithelium is generally within the range of 5.5 to 7.5, the pharmaceutical composition of the present invention advantageously has a pH in said range. The one or more pharmaceutically acceptable excipients included in the composition of the present invention may further comprise one or more antioxidants, buffers, bulking substances, suspending agents, solubilizing agents, lyoprotectants, matrix forming additives, film formers, humectants, diluents, solvents, plasticizers, oily/emulsifying/ aqueous bases, gelling agents, preservatives, tonicity adjusting agents, vehicles, and stabilizers.

As a non-limiting example, the composition of the present invention may comprise at least one pharmaceutically acceptable excipient or carrier well known to the skilled person. Said at least one excipient may be selected from water, glycerin, mineral oils (e.g. vaseline, paraffin), animal oils or fats (e.g. beeswax or wool fat), semi-solid hydrocarbons (oleaginous), or vegetable oils (e.g. coconut oil, mango butter, palm oil, soybean oil, olive oil, shea butter and cacao butter), glyceride, xanthan gum, lactose, dextran, mannitol, methylcellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, sodium carboxy methylcellulose, carboxypropyl methylcellulose, polyethelyne glycol, polypropylene glycol, hyaluronic acid, starch-based biodegradeable polymers, tragacanth, pectin, chitin and chitosan derivatives, carrageenan, guar gum, agar, alginate, gelatin, fibrin, albumin, phosphate buffered saline, Polycarbophil, hydrogenated palm oil, glyceride, Carbomer 934P, macrogol, methyl paraben, glyceryl polymethacrylate, cyanoacrylate, triethanolamine, sorbic acid, NaOH, Carbopols (e.g. Carbopol 974P), diazolidinyl urea, Poloxamer 184, dimethicone, cellulose gum, lactic acid, methyl paraben, propylparaben, polyvinyl pyrrolidone, polyvinyl pyrrolidone-vinyl acetate, polyvinyl alcohol, polyacrylic acid, polyacrylamide, homo- and copolymers of acrylic acid crosslinked with a polyalkenyl polyether, sodium hydroxide, sodium chloride, and potassium chloride, and the like.

In a preferred embodiment, the pharmaceutically acceptable composition is a gel, preferably a bioadhesive or mucoadhesive gel. The term "gel" is defined herein as a dispersion of particles interpenetrated with a liquid to generate a semisolid material. The term "bioadhesive gel" is defined herein as a gel which adheres to a biological surface (e.g. stratified squamous epithelium such as the skin or anterior nares). The term "mucoadhesive gel" is defined herein as a gel which adheres to a mucus coat (e.g. mucus membrane).

In a preferred embodiment, the at least one pharmaceutically acceptable excipient is a gelling agent selected from hydroxypropyl methylcellulose, hydroxyethyl cellulose, chitosan and derivatives thereof, for example chitin, carrageenan and derivatives thereof, alginate and derivatives thereof, pectin and derivatives thereof, homo- and copolymers of acrylic acid crosslinked with a polyalkenyl polyether, or mixtures thereof.

If a tonicity adjusting agent is present, said tonicity adjusting agent is preferably selected from potassium chloride, mannitol, dextrose, glycerin, or sodium chloride. In a preferred embodiment, the pharmaceutical composition comprises at least one gelling agent and at least one tonicity adjusting agent. In a preferred embodiment, sodium chloride is present in the pharmaceutical composition at an isotonic concentration.

If a vehicle is present, said vehicle is preferably selected from water, a water miscible solvent (e.g. glycerin) or a water immiscible solvent (e.g. vegetable oil). If a preservative is present, said preservative is preferably selected from benzyl alcohol, cresols, benzoic acid, phenol, parabens and sorbic acid.

If a stabilizer is present, said stabilizer is preferably selected from surfactants, polymers, polyols, a poloxamer, albumin, gelatin, trehalose, proteins, sugars, polyvinylpyrrolidone, N-acetyl-tryptophan ("NAT")), caprylate (i.e. sodium caprylate), a polysorbate (i.e. P80), amino acids, and divalent metal cations such as zinc. According to a further aspect of the invention, bacteria are lyophilized or freeze-dried. "Lyophilization" is a process well known to the skilled person for the removal of water from frozen bacterial cultures by sublimation under reduced pressure. General methods are described in "Lyophilization: Introduction and Basic Principles" (Jennings, 1999). Said process includes the following steps: providing an aqueous formulation comprising viable bacteria, freezing, primary drying (sublimation), and secondary drying (desorption). Lyophilized bacteria are obtained from said process.

According to a preferred mode, bacteria are lyophilized in the presence of a lyoprotectant. The lyoprotectant may be intracellular or extracellular or a combination of both. Preferably, said lyoprotectant is selected from peptone, glycerol, lactose, gelatin, glucose, sucrose, trehalose, dextran, maltodextrin, adonitol, and sodium glutamate.

According to a preferred mode, bacteria are lyophilized in presence of a matrix forming additive. Preferably, said matrix forming additive is mannitol or skim milk.

Preferably, lyophilized bacteria are stored at refrigeration temperatures (e.g. +3 to +6°C) or at room temperature (e.g. between 20 and 25°C).

Preferably, each bacterial species is lyophilized separately. According to a first preferred embodiment, each lyophilized bacterial species is reconstituted separately with at least one pharmaceutically acceptable excipient just before use. Reconstituted bacteria may then be administered individually or admixed prior to administration. According to a second preferred embodiment, all bacterial species are admixed together following lyophilization. In this preferred embodiment, lyophilized bacteria are reconstituted together with at least one pharmaceutically acceptable excipient just before use. According to a third preferred embodiment, all bacterial species are admixed together prior to lyophilization. In this preferred embodiment, the mixture is reconstituted with at least one pharmaceutically acceptable excipient just before use.

The "reconstitution" of the lyophilized bacterial composition is defined herein as placing the composition in contact with a specific amount of at least one pharmaceutically acceptable excipient (e.g. a liquid or gel), said excipient hydrating the lyophilizate. Preferably, the mixture is agitated or stirred to ensure complete hydration. Preferably, reconstitution is performed within the range of 17°C to 37°C, more preferably at or above room temperature (20°C to 25°C), even more preferably at room temperature. Preferably, the composition is reconstituted immediately before use. If the reconstituted composition is not administered immediately, the reconstituted composition is preferably refrigerated until administration. If the reconstituted composition is not administered immediately and is not refrigerated, the reconstituted composition is preferably administered the same day as reconstitution. The skilled person can easily select appropriate reconstitution and storage conditions according to his general knowledge.

According to a first preferred embodiment, separately reconstituted bacteria are administered separately, one after the other. According to a second preferred embodiment, separately reconstituted bacteria are admixed prior to administration for simultaneous application. According to a third preferred embodiment, bacteria that are reconstituted together are administered simultaneously.

Alternatively, viable bacteria may be stored at temperatures comprised between about +4°C and +8°C (e.g. for several days), at temperatures comprised between about -25°C and -40°C (e.g. for several weeks), or at temperatures comprised between about -72°C and -85°C (e.g. for several years). Lyophilized bacteria may also be stored at temperatures comprised between +4°C and +8°C for several days following reconstitution. Appropriate storage conditions at each of these temperature ranges are well-known to the person skilled in the art.

The present invention further comprises the pharmaceutical composition for use in the prevention of S. aureus colonization. Said pharmaceutical composition for use in the prevention of S. aureus colonization comprises all aspects of the pharmaceutical composition of the invention as described herein. Preferably, the invention comprises the pharmaceutical composition for use in the prevention of nasal colonization by S. aureus, more preferably, comprising the administration of the composition to the anterior nares, and/or for use in the prevention of skin colonization by S. aureus. Skin colonization by S. aureus is advantageously prevented in a subject having increased susceptibility to said colonization, for example in a subject having an eczema, such as atopic dermatitis, or having diabetes mellitus.

In a preferred embodiment, the invention comprises the pharmaceutical composition for use in the prevention of colonization by antibiotic-resistant S. aureus, preferably for use in the prevention of colonization by methicillin-resistant S. aureus. More preferably, the pharmaceutical composition is used in the prevention of nasal colonization by methicillin- resistant S. aureus. The term "methicillin-resistant" indicates the lack of susceptibility of a bacterial strain to the bactericidal effects of methicillin. MRSA strains may comprise resistance to additional antibiotics (e.g. penicillin, vancomycin, mupirocin, quinolones, etc.). Alternatively, the pharmaceutical composition of the invention may be used in the prevention of nasal colonization by methicillin-sensitive S. aureus. Methicillin-sensitive strains are susceptible to the bactericidal effects of methicillin but may comprise resistance to other antibiotics.

The term "colonization by S. aureus" is defined herein as the presence of at least one strain of S. aureus on a body surface, but that does not induce a detectable immune response and/or that does not invade tissue or otherwise cause tissue damage. Alternatively, colonization may be considered to be the presence of S. aureus in the absence of infection. Colonization may be temporary, intermittent, long-term or even permanent in some cases. In particular, colonization by S. aureus may occur in the nares, sinuses, throat, gastrointestinal tract, or on the axilla, groin, inguinal and perirectal regions, on wounds, or elsewhere on the skin or mucus membranes. S. aureus colonization is a risk factor for later S. aureus infection.

The term "infection by S. aureus" is defined herein as the presence of at least one strain of S. aureus on a body surface inducing a detectable immune response and/or undergoing uncontrolled bacterial growth. The immune response may be specific or nonspecific (e.g. fever). S. aureus infection can cause skin and soft tissue infections (SSTIs), such as abscesses, furuncles, carbuncles, boils, impetigo, bacterial focculitis or cellulitis, stys, rash, staphylococcal scalded skin syndrome, necrotizing fasciitis, pneumonia, wound infection, endocarditis, gangrene, osteomyelitis, septic arthritis, septicemia, etc.

The term "nasal colonization by S. aureus" is more specifically defined herein as the presence of S. aureus bacteria in the nares. The term "nasal decolonization" is defined herein as a method for reducing or eradicating S. aureus from the nares. Complete decolonization is defined herein as the absence of detection of S. aureus in the nares for at least two consecutive weeks. Partial decolonization is defined herein by the reduction of S. aureus in the nares by at least 50% for at least two consecutive weeks as compared to the level of S. aureus in the nares prior, preferably by at least 80%, more preferably by at least 90%, more preferably by at least 95%, and even more preferably by at least 99%.

The presence of S. aureus bacteria in the nares can be detected by methods well-known in the art, comprising obtaining a sample from the anterior nares, for example via a nasal swab, and detecting S. aureus. S. aureus can be detected at the species and/or strain level. Detection methods include, for example, culture-based methods (e.g. sample plating on selective media, such as chromogenic and/or blood agar) and molecular diagnostic methods (e.g. PCR, real-time PCR, ribotyping, pulsed-field gel electrophoresis, random amplified polymorphic DNA sequencing, BOX-A1 R-based repetitive extragenic palindromic-PCR (BOX-PCR), multilocus sequence typing, whole genome sequencing, etc.).

According to another aspect, the present invention comprises a method of preventing S. aureus colonization in a human subject in need thereof, comprising administering a therapeutically effective dose of the pharmaceutical composition according to the invention. All aspects of the pharmaceutical composition as described herein are comprised in the method of preventing S. aureus colonization.

The term "preventing" as used herein are not meant be an exclusively absolute term. Indeed, the term "prevention" as used herein may be a delay in colonization by S. aureus, a reduction in the level of colonizing S. aureus, or a reduction in the frequency of S. aureus colonization.

The term "therapeutically effective dose" as used herein refers to an amount of the pharmaceutical composition that is sufficient for preventing S. aureus colonization at least partially, preferably completely, at the treated site. In the context of prevention of S. aureus colonization, this term notably refers to an amount sufficient to delay S. aureus colonization or to reduce the frequency of S. aureus colonization. The therapeutically effective dose may be administered in one or more administrations. In some cases, the pharmaceutical composition may be used to prevent S. aureus colonization at multiple sites within a single subject (e.g. to both the skin and the anterior nares). In this case, a therapeutically effective dose is administered to each site, and according to the appropriate schema of administration.

Accordingly, the present invention encompasses the use of the pharmaceutical composition for preventing colonization by S. aureus based on the modalities described herein. According to a first aspect, the method of preventing S. aureus colonization comprises the administration of the pharmaceutical composition in which the ratio of Corynebacterium sp. to S. pasteuri is comprised in the range of 1 : 0.1 to 1 : 2, or in which the ratio of Corynebacterium sp. to S. pasteuri to S. epidermidis is comprised in the range of 1 : 0.01 : 0.01 to 1 : 1 : 1. According to a second aspect, the method of preventing S. aureus colonization comprises the administration of the pharmaceutical composition having at least 10 ³ CFUs of Corynebacterium sp., at least 10 ³ CFUs of S. pasteuri, and, optionally, at least 10 ³ CFUs of S. epidermidis per dose. Preferably, the method of preventing S. aureus colonization comprises the administration of the pharmaceutical composition having from 10 ³ to 10 ¹² CFUs of Corynebacterium sp., from 10 ³ to 10 ¹² CFUs of S. pasteuri, and, optionally, from 10 ³ to 10 ¹² CFUs of S. epidermidis per dose. According to a preferred aspect, the method of preventing S. aureus colonization comprises the administration of the pharmaceutical composition comprises a total of at least 10 ³ , preferably at least 1.2, 1.4, 2 or 3x10 ³ CFUs per dose. Preferably, said pharmaceutical composition is lyophilized or freeze-dried.

Preferably, the method of preventing S. aureus colonization comprises the administration of the pharmaceutical composition further comprising at least one pharmaceutically acceptable excipient. According to a first aspect, said at least one pharmaceutically acceptable excipient is a lyoprotectant, preferably selected from peptone, glycerol, lactose, gelatin, glucose, sucrose, trehalose, dextran, maltodextrin, adonitol, and sodium glutamate. According to a second aspect, said at least one pharmaceutically acceptable excipient is a gelling agent. Said at least one pharmaceutically acceptable excipient may be selected from those listed above, or from excipients that are well known in the art. Preferably, the method of preventing S. aureus colonization comprises the administration of the pharmaceutical composition to the anterior nares and/or the skin.

Preferably, the method of preventing S. aureus colonization of the present invention prevents S. aureus nasal colonization. Preferably, the method of preventing S. aureus colonization of the present invention prevents MRSA or MSSA nasal or skin colonization. A further object of the present invention is method for preventing nasal or skin colonization by S. aureus in a subject comprising: a) determining if S. aureus is present in the nares or on the skin; and

b) administering the pharmaceutical composition of the invention to said subject if S. aureus is not detected.

The present invention also comprises the use of the composition according to the invention for the manufacture of a medicament for the prevention of colonization by S. aureus. The present invention also comprises the use of the composition according to any of the embodiments described herein in the prevention of colonization by S. aureus, preferably in the nares and/or on the skin.

The present invention also comprises a method for preventing colonization by S. aureus in a subject in need thereof, preferably in the nares and/or skin, comprising administering the composition described herein.

The subject of any of the methods provided herein may be any subject previously identified in the context of the use of said composition. In particular, said subject may be a subject hospitalized for cardiac or orthopedic surgery, a subject bearing an implanted device, a subject residing in a rehabilitation or long-term care facility, a subject having an accidental or surgical wound, a subject having an eczema, such as atopic dermatitis, or a subject having diabetes mellitus.

The present invention has for further object a kit comprising the composition, preferably the pharmaceutical composition, described herein, a gel suitable for nasal use and a means for nasal administration.

Preferably, the kit comprises: a) the pharmaceutical composition comprising C. accolens, S. pasteuri, and, optionally, S. epidermidis, and at least one pharmaceutically acceptable excipient, said composition having a lyophilizate formulation,

b) at least one pharmaceutically acceptable component,

c) a means for nasal administration of said composition, and

d) optionally, a notice of use.

Preferably, the at least one pharmaceutically acceptable component of b) is an aqueous solution for the reconstitution of said lyophilizate or a gelling agent. More preferably, the component of b) comprises both an aqueous solution for the reconstitution and a pharmaceutically acceptable gelling agent.

Corynebacterium sp., may preferably be selected from C. accolens, C. pseudodiphtheriticum, C. striatum, C. amycolatum, C. glutamicum, C. aurimucosum, C. tuberculostearicum, C. afermentans, and/or C. propinquum in the kit. C. accolens may notably be combined with at least one other Corynebacterium spp., such as C. propinquum in the kit. According to a preferred embodiment, C. accolens is replaced by C. propinquum in the kit. A means is also provided herein for dispensing the pharmaceutical composition such that it is applied to the epithelium or mucous membranes, preferably to the nares. The term "means" is defined herein as any device for local, topical application. Preferably, the means for dispensing the pharmaceutical composition also mixes the lyophilized bacteria with the at least one pharmaceutically acceptable excipient, when lyophilized bacteria are used.

In cases where the pharmaceutical composition is present as multiple doses in a single means, a specific means for measuring a single dose may be further comprised. As an example, a single dose may be the equivalent of one or two depressions of a dispenser. FIGURE LEGENDS

Figure 1 : Growth of C. accolens in co-culture with S. pasteuri or S. epidermidis

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.

Figure 2: Mixed biofilms with C. accolens

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 )

Figure 3: Mixed biofilms with C. propinquum

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).

Figure 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 595nm. 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. pasteurilS. 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).

Figure 5: Prevention of S. aureus colonization in an in vivo model of stratified squamous epithelium (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 (5x10 ³ CFU per skin well) when inoculated with C. accolens, S. pasteuri and S. epidermidis alone or combined, each at a dose of 2x10 ⁶ CFU per well. S. aureus alone was inoculated as positive control. The [(Threshold - CT)/ Threshold] ratios are plotted for each condition tested.

EXAMPLES

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.

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 I. Strains used.

Abbreviations: MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin- susceptible Staphylococcus aureus. Example 1 : In vitro inhibition of the growth of S. aureus by C. accolens

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 250 μΙ of a 1 .0 McFarland suspension of C. accolens, prepared from a 48h-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 =5x10 ⁴ CFU/cm ² . 250 μΙ of saline was inoculated as negative control. One 50 mm 0.2 μηη track-etched filter was placed on top of the C. accolens suspension. S. aureus suspensions prepared from a 24h-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 μΙ were spotted on the 50-mm diameter filter.

After incubation for 48h 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

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 II

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

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

500 μΙ of a 10 ^"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 μΙ of a 1 .0 McFarland suspension of S. pasteuri (strain AF2653) or S. epidermidis (strain AF2302), prepared from a 24h-culture on Columbia -5% sheep blood agar at 35+/-2°C, was spotted at the center of the plate. Ten μΙ 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

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. 1 B) 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

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

For each strain alone or in combination, 250 μΙ of a 1 .0 McFarland suspension prepared from cultures on Columbia -5% sheep blood agar at 35+/-2°C (C. accolens: 48h of incubation; S. pasteuri, S. epidermidis: 24h of incubation) was inoculated by swabbing on a 90 mm diameter blood agar plate (=5x10 ⁴ CFU/cm ² ). 250 μΙ of saline was inoculated as negative control. One 50 mm 0.2 μηη 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 μΙ were spotted on the 50 mm diameter filter. Materials and methods were otherwise as described in Example 1 . Results

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 III

aStudent's t-test (p values <0.01 were considered as significant).

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 IV

OD 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

Serial 1/5 dilutions were prepared from a 1 .0 McFarland suspension of each strain and 250 μΙ 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 =5x10 ⁴ , =10 ⁴ , =2x10 ³ , =4x10 ² , and =0.8x10 ² CFU/cm ² . Materials and methods were otherwise as described in Example 3.

Results 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 =0.8x10 ² CFU/cm ² . However, only the densities superior or equal to =2x10 ³ CFU/cm ² (e.g. =5x10 ⁴ CFU/cm ² , =10 ⁴ CFU/cm ² , and =2x10 ³ CFU/cm ² ) showed anti-S. aureus activity that inhibited bacterial growth by more than 95%.

Table V

tu ent s t-test p va ues < . were cons ere as s gn cant .

Example 5: Anti-S. aureus activity of the combination of C. accolens, S. pasteuri and S. epidermidis

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

For each strain in combination, 250 μΙ of a 1.0 McFarland suspension prepared from cultures on Columbia -5% sheep blood agar at 35+/-2°C (C. accolens: 48h of incubation; S. pasteuri, S. epidermidis: 24h of incubation) was inoculated by swabbing on a 90 mm diameter blood agar plate (=5x10 ⁴ CFU/cm ² ); 250 μΙ of saline was inoculated as negative control. One 50 mm 0.2 μηη 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 μΙ were spotted on the 50 mm diameter filter. Materials and methods were otherwise as described in Example 1 .

Results

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 VI

Example 6: C. propmquum may replace C. accolens in the combinations C.

accolens/S. pasteuri and C. accolens/S. pasteuri/S. epidermidis

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

Results

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 VII

'Student's t-test (p values <0.01 were considered as significant).

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

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. pasteurr.S. epidermidis or the combination of C. propinquum:S. pasteurr.S. epidermidis.

Materials and Methods

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 μΙ of each bacterial suspension were co-inoculated by swabbing on a 90 mm diameter blood agar plate, thereby obtaining =10 ⁵ , =5x10 ⁴ , =10 ⁴ , =5x10 ³ , and =10 ³ CFU/cm 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

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

As shown in Table VIII, the combination C. accolens:S. pasteurr.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 ⁴ CFU/cm ² , for ratios 1 : 0.02 : 0.02 at densities <5x10 ³ CFU/cm ² , and for ratio 1 : 0.1 : 0.1 at a density of 10 ³ CFU/cm 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 ³ CFU/cm 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 5x10 ³ ) increased anti-S. aureus activity, inhibiting S. aureus growth by approximately 83% and 85%, respectively. 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 ⁵ CFU/cm ²

Ratio ³ 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 =5x10 ⁴ CFU/cm 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 ⁴ CFU/cm 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 =5x10 ³ CFU/cm 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) Density /ratio S. aureus growth inhibition (mean % (SD))

C. accolens at »10 ³ CFU/cm 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)

Ratio is expressed as the quantity of C. accolens : S. pasteuri : S. epidermidis

As shown in Table IX, the results obtained with C. propmquum 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 <5x10 ³ CFU/cm ² , and for ratios 1 : 0.1 : 0.1 and 1 : 0.2 : 0.2 at a density of 10 ³ CFU/cm 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 ³ CFU/cm ² ). Increasing either the ratio (e.g. to 1 : 0.02 : 0.02) or the density (e.g. to 5x10 ³ ) increased anti-S. aureus activity, inhibiting S. aureus growth by approximately 89% and 93%, respectively. Table IX. C. propinquum/S. pasteuri/S. epidermidis combination at various ratios and densities

Example 8: C. accolens forms a biofilm in the presence of S. pasteuri

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

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. 50μΙ of the suspension (approx. 2x10 ⁶ CFU) of C. accolens AF2345 was distributed in 12 wells (row A); 50μΙ of a 1 :100 dilution (approx. 2x10 ⁴ CFU) was distributed in 12 wells (row B); 50 μΙ of the suspension of S. epidermidis AF2302 (approx. 2x10 ⁶ CFU) was distributed in columns 1 , 2, 3, 7, 8, and 9; 50 μΙ of the suspension of S. pasteuri AF2653 (approx. 2x10 ⁶ CFU) was distributed in columns 4, 5, 6, 7, 8, and 9. Columns 10, 1 1 , and 12 contain C. accolens AF2345 suspended in 100μΙ of culture medium.

The plates were centrifuged, the suspension saline was removed and 100μΙ of TSB were added to each well. After 24 hours of incubation, 50μΙ 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. 200μΙ of saline was added to each well and the plate immersed in an ultrasound bath to disperse and resuspend the biofilm. For each well, 5μΙ (1/40 ^th ) was transferred to 95μΙ of saline (1 /800 ^th ). 5μΙ of this suspension was then transferred to a fresh plate containing 95μΙ of saline (1/16000 ^th ). 10 μ I 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 (128μg/ml).

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

Results

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).

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

The same experiment presented in Example 8 was performed by replacing C. accolens with C. propinquum. Materials and methods

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

Results

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

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

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 μΙ_ of the undiluted C. accolens suspension and 50 μΙ_ of a 10 ^"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 μΙ_ 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 μΙ_ 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 200μΙ_ of a 30% acetic acid solution prepared in distilled water. 100 μΙ_ 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 595nm.

Results

As shown in Figure 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. pasteurilS. epidermidis.

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 : Reconstituted lyophilized bacteria prevent S. aureus colonization

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

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 ³ CFU/cm ² , 10 ² CFU/cm ² or 10 ¹ CFU/cm ² by swabbing 300 μΙ_ of the suspension on the surface of the agar plate. 300 μΙ of sterile water was inoculated as negative control. One 50mm 0.2 μηη 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 μΙ 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 μΙ_ 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

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 X: Anti-S. aureus activity of fresh or reconstituted C. accolens/S. pasteurilS. epidermidis combinations at various densities.

'Student's t-test (p<0.05 for significance).

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 12: 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.

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 Figure 5A). 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

Vials of lyophilized C. accolens strain AF2345, S. pasteuri strain AF2652 and S. epidermidis strain AF2302 were prepared as described in Example 1 1. Nine evenly spaced 4.5mm diameter holes were punched in hydrocolloid sterile patches, each hole constituting a 30μΙ 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 3x10 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 ⁸ CFU/mL. C. accolens, S. pasteuri and S. epidermidis alone or in various combinations were added to the skin wells at a dose of 2x10 ⁶ CFU per species per well and S. aureus at a dose of 5x10 ³ CFU per well. The hydrocolloid patches and the surrounding skin was then covered by sterile polyurethane adhesive surgical incision film (Figure 5A). 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 1X Itaq Universal Probes supermix (Biorad), 500 nM for each primer, 250 nM probe and DNA template in a final volume of 20 μΙ. 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 [(Threshold - CT)/ 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

Figure 5B shows the [(Threshold - CT)/ 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.

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

Conclusion

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. 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), greatly reduces colonization by S. aureus in vivo.

These Examples provide the first evidence that a composition comprising 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 of both MSSA and MRSA S. aureus colonization. REFERENCES

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