Prevention and / or treatment of wound infection

Abstract

A composition comprising Cutibacterium acnes (also known as Propionibacterium acnes or Corynebacterium parvum), C. acnes culture supernatant and / or a bioactive extract or metabolite obtainable from C.

Description

This application relates to prevention and / or treatment of wound infection. In particular it relates to a composition for use in a method of medical treatment, in particular for treating or preventing wound infection. The NHS spends £5 billion annually in wound care and wound management. The spending is likely to increase year on year due to the pressures of the antibiotic resistance crisis and the routine failure of frontline wound therapeutics. The use of antibiotics to treat wound infections is limited by the emergence of multidrug or pandrug resistant wound pathogens. This is a particular issue for hospital associated infections which typically have a broad resistance profile. The prophylactic use of antibiotics to prevent wound infection is also perpetuating this problem. It is known to provide wound dressings augmented with substances (for example, antibiotics or other substances) to prevent or treat wound infection. However, many of the wound dressings that are in clinical use are ineffective against biofilm-forming pathogens, or they contain compounds such as silver which have antimicrobial properties but can also inhibit or slow wound healing. The present invention seeks to provide improved prevention and / or treatment of wound infection. According to another aspect of the present invention, there is provided a composition including Cutibacterium acnes and / or Cutibacterium acnes culture supernatant, and / or a bioactive extract of Cutibacterium acnes culture and / or supernatant for use in a method of treating or preventing a skin wound infection by disrupting the virulence of a pathogen, wherein the pathogen is Pseudomonas aeruginosa and the composition is for application to the wound. The C. acnes cells may be live or dead. The wound may be a burn. In some embodiments, the C. acnes strain may be Phylotype I. In other embodiments it may be Phylotype II. According to another aspect of the present invention, there is provided a formulation for application to a human skin wound including a composition as specified above in combination with a dermatologically compatible carrier. The carrier may be an ointment, a gel, or a cream. According to another aspect of the present invention, there is provided an augmented wound dressing including a composition or a formulation as specified above. Also disclosed is a method of making a formulation as specified above, including incorporating a therapeutically effective amount of a composition as specified above into a dermatologically compatible carrier. The amount of the composition to be incorporated into the dermatologically compatible carrier can be determined on the basis of the wound type and its size and depth, and the type of dressing to which it is eventually applied. Also disclosed is a method of making an augmented wound dressing including applying a composition as specified above or a formulation as specified above to a dressing for treating a skin wound, where applying can mean incorporating, impregnating, saturating, or any other suitable method of introducing the composition or formulation to a skin wound dressing. Also disclosed is a method of preventing or treating wound infection including applying a composition, a formulation and / or an augmented wound dressing as specified above to a skin wound of a patient in need thereof. The skilled person will appreciate that the dressing will be changed at appropriate intervals until the wound has healed and / or until infection is no longer a risk. Embodiments of the present invention are described below, by way of example only, with reference to the accompanying drawings, in which: Figures 1 to 5 are schematic illustrations of embodiments of wound dressings; Figure 6 illustrates infection progression in an invertebrate burn wound model; Figure 7 is a graph showing the effect of C. acnes CCUG38584 on P. aeruginosa challenged insect larvae in the model illustrated in Figure 6; Figure 8 is a graph showing the effect of C. acnes CCUG48370 on P. aeruginosa challenged insect larvae in the model illustrated in Figure 6; and Figure 9 is a graph showing the effect of C. acnes CCUG6369 on P. aeruginosa challenged insect larvae in the model illustrated in Figure 6. It is now well established that a healthy gut microbiome plays a key role in human health. However, relatively little work has been carried out on the role the human skin microbiome plays in maintaining health. Over the last couple of years, the capacity to use probiotics to promote wound healing has been demonstrated in vitro and in vivo. The majority of these studies have focused on gut bacteria, such as Lactobacilli and bifidobacteria, with clinical trials taking place for Lactobacillus plantarum (Peral et al. (2009), Lukic etal. (2017)). It was hypothesised that these would compete with pathogenic bacteria, modify the wound environment and promote tissue repair. However, these studies were too small to allow appropriate statistical analysis, and there has been no approval for use in a clinical setting. The skin microbiome is a major area of research focus with respect to chronic skin conditions such as psoriasis, eczema and acne. However, comparatively little is known about how the native skin microbiome responds to burn wound trauma, despite numerous studies exploring the impact of burn wound trauma on the gut microbiome (Lima etal., 2021). The main burn wound microbiome study performed in humans has suggested that there are no differences in microbial abundance or composition between burn sites and unaffected areas (Liu etal., 2018). Another study exploring the human burn wound microbiome suggested that the commensal microbial community was significantly altered as a result of burn wound trauma compared to controls. This study uncovered several bacterial taxa that correlated with improved clinical outcomes. However, a key limitation of this study is that it analysed a single snapshot of the microbial community at a specific time and as such gave no insights into the temporal community kinetics or the impact on the microbiome at undamaged sites (Plichta etal., 2017). A more recent study of the human burn wound microbiota reported a reduction in alpha diversity within the wounds and a reduction in the abundance of common skin commensals such as Cutibacterium acnes and Staphylococcus epidermidis. However this study was limited to only 10 patients {Lima et al., 2021). Other studies in animal models also support the hypothesis that the commensal bacteria that recolonise the burn wound can influence clinical outcomes (Sanjar et a!., 2020). C. acnes (previously known as Propionibacterium acnes) is an anaerobic, Gram-positive bacterium. It is largely commensal and can be found in the skin flora present on most healthy adult humans' skin. It is, however, also implicated in the inflammation of hair follicles, and under certain conditions, notably in adolescents, specific strains are known to be pathogenic, being associated with skin conditions such as acne vulgaris. Embodiments of the invention relate to a composition including C. acnes, C. acnes culture supernatant, and / or a bioactive extract or metabolites obtainable from C. acnes culture / supernatant for use in a method of medical treatment, in particular for treating or preventing wound infection. The wound may be a burn. Bioactive non-pathogenic strains could be used, potentially as an alternative to antibiotics, to prevent wound infection and promote wound healing. In embodiments, the bacterial cells themselves may not be necessary for the anti-virulence activity. Rather, the cell free supernatant that the C. acnes strain has grown in (or bioactive extracts obtainable therefrom) could be used to disrupt virulence. The present inventors have found that infection, in particular by Gramnegative bacteria such as Pseudomonas aeruginosa, can be prevented by C. acnes. An invertebrate in vivo burn wound model (Maslova et al. (2020)) was used to screen a range of different skin commensals to determine if any could prevent subsequent infection with Pseudomonas aeruginosa, a common Gram-negative opportunistic pathogen. This model has been shown to recapitulate the hallmarks of burn wound trauma and infection seen in higher models (such as mammalian models). It therefore addresses a major limitation to the development of wound probiotics, by providing an alternative to mammalian in vivo models to enable high-throughput screening of potential probiotics. From these screens, the inventors identified C. acnes as a promising candidate for a wound treatment probiotic. C. acnes, when applied directly to a wound, can improve survival and disable virulence by P. aeruginosa, one of the most prevalent burn wound pathogens. C. acnes, and / or components produced thereby, can help prevent infection developing in wounds by disabling the mechanisms that pathogens use to establish infection. C. acnes has previously been shown to be able to influence the skin microbiome in hair follicles through the production of cutimycin, a thiopeptide antibiotic, which has activity against Gram-positive bacteria and is capable of killing Staphylococcus species (Claesen etal. (2020)). However, thiopeptide antibiotics are ineffective against Gram-negative bacteria. The bacterial cells could be directly applied to the wound. Prior probiotic treatment of wounds has taken the form of direct application of a probiotic to the wound bed, saturation of a standard wound dressing with the probiotic, integration of the probiotic into a gel that is applied to the wound, or the integration of the probiotic into a cream / lotion (Huseini etal. (2012), Jones et al. / 2012), Peral etal. / 2009), DiMarzio et al. (2008)). Any or all of these application methods may be used with the C. acnes probiotics and / or bioactive extracts disclosed herein as exemplified below. It is proposed that C. acnes can be used in a number of different applications to treat or to prevent wound infection. This can include, but is not limited to, the direct application of the bacteria to the wound or the application of spent growth medium (culture supernatant) from the bacterium or the application of bioactive metabolites produced by the bacteria to the wound. The bacteria, culture supernatant or metabolites may be impregnated into a vehicle (for example, an ointment, a cream, or a wound dressing). Embodiments of the composition may be applied to the burn or other wound in the form of an impregnated or otherwise augmented wound dressing (such as a sterile pad or compress), a lotion, ointment, gel, or a cream that is applied to the wound after injury. These applications will minimise the probability of infection developing as the mechanisms through which a pathogen can establish infection will be disabled. A composition according to embodiments may comprise C. acnes cells (live or dead), cellular contents, cell free supernatant or bioactive components derived from the cell free supernatant. Genetic elements associated with bioactivity can be amplified and subcloned into a heterologous host for upscaled production, or genetically manipulated either through evolved or targeted mutagenesis to increase the production of the bioactive elements or to enhance the bioactivity of the strain and / or cell free supernatant or to increase production of the bioactive components. C. acnes compositions for use in the methods described herein can be prepared by liquid or plate culture, for example. Preparation of liquid culture of C. acnes can include the steps of: 1) Inoculating a suitable liquid culture medium, for example, TSB, with C. acnes] and 2) Incubating at 37°C in anaerobic conditions, for example for 48-72 hours. Plate culture can include: 1) Inoculating blood agar (including, for example, TSA and horse blood) with C. acnes; and 2) Incubating in anaerobic conditions, for example for 48-72 hours. A composition including C. acnes supernatant for use in the methods described herein can be prepared for example by a method including the following steps: 1) Preparing a liquid culture as set out above; 2) Pelleting the bacterial cells using a centrifuge; and 3) Filtering the supernatant. The composition may be incorporated into a formulation by impregnating or otherwise combining with a vehicle such as an ointment, gel or cream. In practice, the vehicle is seeded with live or dead C. acnes cells prepared as described herein, or a component of the vehicle is C. acnes cell free supernatant or a bioactive component (e.g. metabolite) derived from the cell culture or cell free supernatant. The cells, supernatant or bioactive component may be included alone, or in any combination, with or without other anti-infective agents. It is envisaged that a wound dressing be impregnated with a seed culture of bioactive strains of C. acnes. This would allow prolonged and stable colonisation of the wound bed by C. acnes. This wound dressing can take the form of a hydrogel, gauze, films, foams, hydrocolloids or composites augmented with C. acnes. Equally these dressings could be saturated or augmented with C. acnes cell free supernatant or the bioactive components derived from the cell free supernatant. The augmented dressings can be used to prevent infection by application to the wound immediately post-trauma. Equally they can be used to treat infection either singularly or in combination with standard antibiotic therapy. Figures 1 to 5 illustrate some exemplary types of augmented wound dressing that can be used as a prophylactic (on an uninfected wound) or for treatment of an infected wound. Augmented wound dressings can be made by applying a composition or formulation including C. acnes, C. acnes culture supernatant, and / or a bioactive extract or metabolite obtainable from C. acnes culture and / or supernatant to a skin wound dressing, which may be of known type. Figure 1 illustrates a simple single layer cell dressing (100). In this example, a wound dressing of known type (130) (for example, a sterile pad or compress, a hydrogel, gauze, a film, foam, hydrocolloid or composite) is impregnated or saturated with alive or dead C. acnes cells (110) to seed a wound bed. Figure 2 illustrates a bi-layer cell dressing (100). The dressing comprises or consists of two layers: an upper layer (130) composed of a wound dressing of known type, and a lower layer (140), which has a less dense dressing. The lower layer is impregnated or saturated with alive or dead C. acnes cells (110). This dressing provides two advantages: increased protection from potential sources of infection in the upper layer and increased dispersal of the C. acnes into the wound microenvironment via the less dense lower layer. Figure 3 also illustrates a wound dressing (100) that includes C. acnes cells (110). The C. acnes cells are sealed in a nutrient permeable compartment (150) within a wound dressing of known type (130). A wound sealing membrane (160), such as a Tegaderm(RTM) dressing is provided at the side of the dressing (100) to be applied to the wound. A bioactive component or metabolite produced by the C. acnes cells is able to diffuse through the wound sealing membrane into the wound bed and treat or prevent infection of the wound. Figure 4 illustrates a bioactive wound dressing (100) that, in preferred embodiments, does not contain C. acnes cells. A dressing matrix (170) of known type (such as for example gauze or silicone dressings) is formulated or saturated with a bioactive component derived from C. acnes cell free supernatant. Figure 5 illustrates a wound dressing (100) in which a wound dressing matrix (170) of known type has been impregnated or saturated with C. acnes cell free supernatant. For example, the matrix (170) could be a hydrogel in which the aqueous component is the C. acnes cell free supernatant. The described probiotic finds use in a clinical capacity and also has the potential to be used as an off-the-shelf skin probiotic to treat minor cuts or burns. It takes advantage of the therapeutic potential of a commensal bacterium that is commonly found on the skin of healthy individuals. The probiotic provides an alternative to the use of ineffective wound dressings or the topical application of antibiotics, both of which are ineffective against resistant pathogens, which cause many hospital-associated wound infections. Further, it would reduce the use of antibiotics on wound patients, potentially helping to stem the impact of antibiotic resistance. A probiotic wound treatment based on C. acnes as described herein has cost advantages, being relatively cheap to produce compared to a novel antibiotic. Example 1 - Preparation of C. acnes culture Examples of how to prepare C. acnes for use in the methods described herein are now described. Liquid culture 1. 15ml of Tryptic Soy Broth (TSB) or suitable equivalent is aliquoted into 50ml Falcon tubes. 2. -80°C cultures are inoculated into said Falcon tubes with TSB broth. 3. The Falcon tubes are placed into a hermetic chamber vertically with an Anaerogen sachet. 4. The hermetic chamber is incubated at 37°C for 48-72 hours. Plate culture 1. Blood agar is prepared by melting Tryptic Soy Agar (TSA) and adding defibrinated horse blood in the proportion 1:19 blood:TSA. A suitable equivalent may also be used. 2. -80°C cultures are inoculated onto the blood agar plates. 3. The plates are placed into a hermetic chamber with Anaerogen sachet. 4. The hermetic chamber is incubated at 37°C for 48-72 hours. Example 2 - Preparation of C. acnes cell free supernatant 1. 48hr Falcon tubes with grown cultures are taken out of the hermetic chamber. 2. They are spun down at 5,000 rpm for 10 minutes at room temperature in a centrifuge. 3. The supernatant is poured from these centrifuged cultures, into a syringe with a 0.2pm filter attached. The supernatant is then passed through the filter into a new sterile 50ml Falcon tube. 4. The supernatant is then kept at room temperature for the rest of the day. 5. The supernatant is best used freshly made on the same day. Example 3 - In vivo anti-virulence potential of C. acnes Three different strains of C. acnes (C. acnes CCUG38584 (ATCC 6922; NCTC 556), C. acnes CCUG6369 (ATCC 11828), C. acnes CCUG48370) were tested using the invertebrate in vivo burn wound model developed by Maslova et al. (2020). This enabled determination of the strain specificity of the anti-virulence effect and found that all three strains were capable of significantly reducing pathogenicity in the in vivo wound infection model. Galleria mellonella were obtained from LiveFood UK Ltd. (Somerset, United Kingdom). Only 6th instar larvae were used for experiments, which is the life stage at which they do not require feeding. Prior to use, larvae were stored at +4°C. Before the experiment, larvae were sorted into Petri dishes lined with filter paper (Whatman, Fisher, United Kingdom) at a maximum of 10 larvae per dish, and they were stored at +4°C until use. 70% ethanol was used to sterilise the larval body surface. Petri dishes were left open in a sterile environment to allow for the ethanol to evaporate after sterilisation. The burn was induced by using a heated steel element embedded in insulating material to consistently achieve approx. 2mm squared burn area. The burn instrument was heated in the middle flame of a Bunsen burner until it was red / white-hot and applied to the middle segment of G. mellonella's back for 4s. The bacterial infection was induced by applying lOpI of overnight bacterial culture, or by applying bacterial colonies. For the cocolonisation assay, the C. acnes colonies were inoculated first directly onto the wound and lOpI of a liquid overnight culture of a pathogenic bacteria (P. aeruginosa PA14) was applied straight away on top of it. Figure 6 illustrates infection progression in the G. mellonella burn wound model, demonstrating that it mimics the bacteraemia and sepsis seen in mouse models, with local tissue damage, biofilm formation at the wound site and subsequent dissemination from the wound to deeper tissue and larval haemolymph (blood). Over time, the G. mellonella larvae (10) become damaged due to infection progression (30) from the burn site (20). Infection progression was seen through melanisation of the larvae and subsequent loss of movement. Larvae were considered dead if they did not respond to manual stimulation with a pipette tip. Live and dead larvae counts were then used to create Kaplan Meier Survival curves (probability of survival). Figure 7 demonstrates the probiotic potential of C. acnes CCUG_38584. Topical treatment of larvae with C. acnes prior to subsequent challenge with P. aeruginosa PA14 (UCBPP_PA14) significantly improves survival compared to untreated larvae. Time measured in Hours. Differences in survival between PA14 only vs. CCUG_38584 significant at Log Rank p< 0.01. C. acnes CCUG_38584 alone did not have a significant impact on survival. JB represents the "just burn" control. Figure 8 demonstrates the probiotic potential of C. acnes CCUG_48370. Topical treatment of larvae with C. acnes prior to subsequent challenge with P. aeruginosa PA14 (UCBPP_PA14) significantly improves survival compared to untreated larvae. Time measured in Hours. Differences in survival between PA14 only vs. CCUG_48370 significant at Log Rank p< 0.01. C. acnes CCUG_48370 alone did not have a significant impact on survival. JB represents the "just burn" control. Figure 9 demonstrates the probiotic potential of C. acnes CCUG_6369. Topical treatment of larvae with C. acnes prior to subsequent challenge with P. aeruginosa PA14 significantly improves survival compared to untreated larvae. Time measured in Hours. Differences in survival between PA14 only vs. CCUG_6369 significant at Log Rank p< 0.05. C. acnes CCUG_6369 alone did not have a significant impact on survival. JB represents the "just burn" control. This work shows that the application of C. acnes to a burn wound can have a significant impact on improving the survival of G. mellonella larvae that are subsequently challenged with the burn wound pathogen P. aeruginosa (Figures 7, 8, and 9). Example 4 - C. acnes augmented wound dressing C. acnes cells or cell free supernatant can be used in a wound dressing, and in an embodiment is particularly envisaged for a wound dressing for burns. In an embodiment, such a C. acnes augmented wound dressing can be prepared as follows: 15ml of Tryptic Soy Broth (TSB) or suitable equivalent is aliquoted into 50ml Falcon tubes. -80°C stock cultures of the relevant C. acnes strain are inoculated into said Falcon tubes with TSB broth. The Falcon tubes are placed into a hermetic chamber vertically with an Anaerogen sachet or an equivalent anaerobic incubation chamber. The cultures are incubated at 37°C for 48-72 hours. Following this, cultures are spun down in a centrifuge for 15 minutes at <8,000 rotations per minute. The supernatant is then removed and can be used directly to saturate a standard woven gauze (cotton or suitable alternative). For a live culture preparation, the remaining cell pellet is resuspended in 1 ml of phosphate buffered saline solution to achieve a completely homogenous solution. This solution of live cells can then be applied directly to a standard woven cotton gauze dressing to achieve complete saturation typically (1 ml / cm2) (Peral et al., 2009). For optimal efficacy this dressing would then be applied immediately to the affected region of skin. This application would facilitate seeding of the wound bed with C. acnes. Alternatively, the dressing could be stored in sterile conditions that would preserve bacterial viability (4°C) for a defined period of time before being applied to the wound. It is suggested that a fresh dressing be applied daily and fresh bacterial cultures used for each dressing. The application of C. acnes as a probiotic to wounds (and / or more specifically to burn wounds) to prevent infection, in particular by common Gram-negative opportunistic pathogens has not previously been described. The principle supporting this application is that C. acnes can be used as probiotics and applied to wounds to prevent infection and to promote healing. It has been demonstrated that the most common burn wound pathogen P. aeruginosa's virulence is significantly disrupted when C. acnes is applied to a burn wound prior to pathogen challenge. It is envisaged that the active component of C. acnes bioactivity and / or the genes responsible for this, could be used in therapeutics rather than whole bacteria. Upscaled production of the bioactive component could be achieved by cloning the genes responsible for the activity into another commensal bacterium to augment their activity or by cloning them into a heterologous expression host using known methods. All optional and preferred features and modifications of the described embodiments and dependent claims are usable in all aspects of the invention taught herein. Furthermore, the individual features of the 09 12 24 dependent claims, as well as all optional and preferred features and modifications of the described embodiments are combinable and interchangeable with one another. It will be appreciated by those skilled in the art that changes could be 5 made to the embodiments and examples described above without departing from the broad inventive concept. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the claims. 10 References Claesen etal. (2020) Sci. Transl. Med. 12, eaay5445 DiMarzio etal. (2008) Int. J Immunopathol. Pharmacol. 21, 137-43. 15 Huseini etal. (2012) Burns 38, 719-23. Jones etal. (2012) Int. Wound J. 9, 330-43. Lima etal. (2021) Sci. Rep. 11, 10667 Liu etal. (2018) Wound Repair Regen. 26, 182-91 Lukic etal. (2017) Wound Repair Regen. 25, 912-22 20 Maslova etal. (2020) Front. Microbiol. 11, 998 Peral etal. (2009) Int. Wound J. 6, 73-81 Plichta et al. (2017) Shock 48, 441-8 Sanjar et al. (2020) J. Burn Care Res. 41, 347-58

Claims

1. A composition including C. acnes and / or C. acnes culture supernatant and / or a bioactive extract of C. acnes culture and / or supernatant for use in a method of treating or preventing a skin wound infection by disrupting the virulence of a pathogen, wherein the pathogen is Pseudomonas aeruginosa and the composition is for application to the wound.

2. A composition for use as claimed in claim 1, wherein the wound is a burn.

3. A composition for use as claimed in claim 1 or 2, wherein the C. acnes is C. acnes CCUG38584 (ATCC 6922;NCTC 556), C. acnes CCUG6369 (ATCC 11828), or C. acnes CCUG48370.

4. A formulation for application to a human skin wound including a composition for use as claimed in any preceding claim, in combination with a dermatologically compatible carrier.

5. A formulation as claimed in claim 4, wherein the carrier is an ointment, a gel, or a cream.

6. An augmented wound dressing including a composition for use as claimed in any of claims 1, 2 or 3 or a formulation for use as claimed in claim 4 or 5.

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