The present invention provides devices and methods for detecting microorganisms associated with the infection of wounds. The rapid, reliable and accurate detection of microbial infections of a wound is is a vital part of both the treatment and prevention of infection.

A number of inventions have previously been disclosed which attempt to simplify and expedite the process of detection and identification of wound infections For instance Gorski et al. (WO2010/085755) disclose polymeric indicator films and pH indicating wraps for detecting the presence of metabolic byproduct in microorganisms that can infect wounds.

The present invention represents advances that have been made by the inventors in view of the devices disclosed in WO2013/083993. This document concerns small-scale devices for detecting, identifying and/or quantifying microorganisms in a sample wherein the devices comprise a pore containing means for reporting the presence of a microorganism. The reporting means comprises a solid or semi-solid substrate, a metabolic indicator and a media and/or nutrients that support or encourage microbial growth but which does not activate the indicator in the absence of a microorganism. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), MTT (3 -(4, 5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) are the preferred indicators disclosed in WO2013/083993.

The inventors realised that the technology disclosed in WO2013/083993 could be adapted and refined to provide an improved device which can be used to detect for the presence of microorganisms that cause wounds to become infected. In particular, the device may be used to detect the presence of specific types or species of microorganisms in the environment of a wound.

In a first aspect, the invention provides a device for detecting and/or identifying microorganisms in the environment of a wound, said device comprising a substrate that has disposed on or in it at least one reporting means, said reporting means comprising a carrier substance in which there is dispersed a metabolic indicator and optionally a selection factor;

wherein the selection factor permits growth of specific microorganisms within the reporting means such that the metabolic indicator is only activated by preselected microorganisms.

The devices provided by this invention (or methods described herein) may be used to identify, detect and/or quantify microorganisms present in, or around, a wound.

In a preferred embodiment of the invention the device may be used by placing one or more devices in contact with wound tissue and placing a wound dressing over the top. The device may be removed (and checked for activation) when the dressing is changed.

In another preferred embodiment of the invention the device is incorporated in a wound dressing. The wound dressing should be formed such that when the dressing is applied to a wound that the reporting means is brought into contact with, or brought into the proximity of, the wound and under conditions suitable to permit passage of any microorganisms present in the wound to the reporting means.

In an alternative embodiment the device is not placed in contact with the wound or incorporated in a wound dressing. When this is the case, a sample from the wound (e.g. a swab of the wound bed or a biopsy of soft tissue or bone) may be used to inoculate the device. This allows for circumstances where direct contact of the device with a wound is not possible or desirable. After a suitable incubation period the device can be examined to determine whether or not the metabolic indicator has been activated and thereby indicate whether or not any microorganisms are present in, or around, the wound. A particular advantage of the devices according to the present invention is that the reporting means may comprise selection factors which enable a clear, all or nothing activation of the metabolic indicator at a pre-determined threshold concentration of microorganism. A further advantage of devices according to the present invention is that selection factors may be chosen that make it possible to discriminate between the types of microorganism that may have colonised the wound.

This invention provides an elegant, inexpensive technology providing realtime, clear and simply understood information to an untrained operator of the device, a clinician examining a wound dressing comprising devices according to the invention or even a wearer of a dressing comprising the medical device. The selection factor within the reporting means allows selective microbial growth within the device to allow the safe, limited and controlled growth of challenge organisms of interest giving rise to the same clear all or nothing response, once a trigger dose of microorganisms that are resistant to the selection factor has been reached. The inventors have established that the triggering of metabolic indicators in devices according to the invention may be regulated by varying the amount of, or type of, selection factor that is included in the reporting means. The selection factor selectively retards the growth of certain microorganisms within the reporting means. Accordingly, the device may only be triggered in response to microorganisms that are resistant to the selection factor. By way of example only, an antibiotic may be selected as a selection factor that has broad spectrum activity against bacteria in general and Staphylococcus aureus in particular. Devices with reporting means containing such an antibiotic are useful for distinguishing between Staphylococcus aureus that are sensitive to the antibiotic and strains that are resistant (e.g. MRSA).

The device provided by the first aspect of this invention represents a significant improvement over the prior art as it is enables a user to rapidly and clearly identify whether or not there is infection of a wound and furthermore to discriminate between the types of microorganism present without a need for additional tests or operator intervention. The devices according to the first aspect of the invention may be adapted such that the "triggering" of the device will occur depending upon whether or not a type of microorganism that is of interest to a user is present in the sample. Throughout this specification reference is made to "microorganisms" and this term should be understood as encompassing all life forms not visible to the naked eye. As such, the term "microorganism" may include, for example, bacteria, fungi, viruses, protozoa and algae. It is preferred that the devices described herein may be used to identify detect and/or quantify one or more microorganisms selected from the group consisting of, bacteria, fungi, protozoa and algae. It is preferred that the device is used to detect bacteria and in particular pathogenic bacteria.

It is most preferred that the devices are used to establish whether or not a wound is infected with Staphylococcus aureus (and particularly multiresistant Staphylococcus aureus - MRSA), Pseudomonas aeruginosa, Staphylococcus epidermidis, Streptococcus mitis, Streptococcus sanguis, Enterococcus faecium, Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Enterococcus faecalis, Klebsiella pneumoniae, Candida albicans, or gram negative bacil li.

Reporting Means

Reporting means according to the invention comprise a carrier substance in which there is dispersed a metabolic indicator and optionally a selection factor. Carrier substance

The carrier substance may be any solid or semi-solid (e.g. a gel) substance that is a capable of retaining the metabolic indicator, selection factor (if present) and any other components of the reporting means; while allowing micro-organisms to enter the reporting means and activate the metabolic indicator.

It is preferred that the carrier substance is a semi-sold or gel.

In some embodiments the carrier substance is agar or agarose. The precise quantity being determined by the degree of solidity required. By way of example, the reporting means may comprise 0.1%-1.5% w/v agar mix, preferably 0.75%-1.4% w/v agar mix, more preferably 0.9%-l . l% w/v agar mix and most preferably about 1% w/v agar mix. Insofar as agarose is concerned, the reporting means may comprise 0.3%-1.7% w/v agarose mix, preferably 0.5%-1.3% w/v agarose mix, more preferably 0.75%-1.25% w/v agarose mix and most preferably about 1.0% w/v agarose mix. It is most preferred that the carrier substance is an alginate. Alginate is a linear copolymer with homopolymeric blocks of (l-4)-linked β-D-mannuronate and epimera-L-guluronate (G)residues:

A number of salts of alginic acid may be used to form carrier reporting means according to the present invention although it is preferred that sodium alginate (XaCfJ i-O:.) is used

Reporting means according to the present invention are preferably formed by combining relevant components with Sodium Alginate and Calicium Carbonate to form gel bead reporting means. The inventors have found that an important balance is required to ensure that the gel beads have a consistency which allows microrganisms to enter the reporting means and activate the indicator while at the same time retaining the components of the reporting means within the bead. Alginate reporting means according to the invention may comprise 1.0 - 5.0% (w/v) alginate. It is preferred that such reporting means comprise 2.0 - 4.0% (w/v) alginate and most preferred that the reporting means comprise about 3.0% (w/v) alginate (e.g. 3.0 ±0.5 % (w/v) alginate).

Alginate reporting means are preferably 20-50μ1 in volume, more preferably 30-40μ1 in volume and most preferably 32-37μ1 in volume.

The alginate reporting means may be substantially spherical and preferably have a diameter of between 0.5 and 5mm. It is preferred that the beads are between 2.0 and 4.0mm in diameter and most preferred the beads have a diameter of about 3mm.

A preferred alginate reporting means is described in more detail in Example 2. Metabolic Indicators

The reporting means may comprise a metabolic indicator comprising components that report the presence of a microorganism by way of a colour change reaction. Without wishing to be bound by any particular theory, it is well known that microorganisms can be distinguished on the basis of the various biochemical pathways they express and/or metabolites they produce. As such, the reporting means may comprise one or more indicators capable of reporting the presence of a microbial biochemical pathway and/or metabolite. For example, the reporting means may comprise one or more compounds which are metabolised by one or more microorganisms to yield a detectable (for example optically detectable) substance.

The reporting means may comprise one or more indicator(s) which report the presence of microorganisms. For example, the reporting means may comprise one or more metabolic indicators which report the presence of living organisms/cells. Examples of metabolic indicators that may be used include Resazurim (e.g. Alamar blue) and 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red).

It is preferred that the metabolic indicator is activated by an enzyme endogenous to the micro-organism being detected and more preferred that the indicator is activated by the action of a cellular reductase (e.g. an NAD(P)H reductase). For example, the reporting means may comprise a tetrazolium salt. Yellow tetrazolium salts are reduced to purple formazan in living cells and preferred indicators for use in the reporting means of the devices provided by this invention may include, for example, XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H- tetrazolium-5-carboxanilide), MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), MTT (3 -(4, 5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) or water soluble tetrazolium salts (WST) such as WST-1, WST-3, WST-4, WST-5, WST-7, WST-8, WST-9, WST-10 or WST-11. Alternatively, other tetrazolium salts may be used including indonitrotetrazolium chloride (INT), Nitrobluetetrazolium (NBT), Tetranitro blue tetrazolium (TNBT), Thiocarbamyl nitro blue tetrazolium (TCNBT), Tetrazolium red (TR), Tetrazolium Violet (TV) or Neotetrazolium chloride (NTC). MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-( 4- sulfophenyl)-2H-tetrazolium), MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) and mixtures thereof may be used according to the invention.

It is preferred that a water soluble tetrazolium salt (WST) selected from WST-1, WST-3, WST-4, WST-5, WST-7, WST-8, WST-9, WST-10 or WST-11 is used as the indicator.

It is most preferred that the indicator is WST-9 or a derivative thereof. WST-9 has the chemical formula: 2-(4-Nitrophenyl)-5-phenyl-3-[4-(4- sulfophenylazo)-2-sulfophenyl]-2H-tetrazolium, monosodium salt and the chemical structure:

WST-9 is preferably included in the reporting means at concentrations greater than 0. ImM and more preferably at concentrations greater than 0.75mM. For instance, WST-9 may be used in in the reporting means in the range of 0.75 -lO.OOmM, and more preferably in the range 1.0 - 6.0mM. Most preferred concentration of WST-9 in the reporting means are 1.5mM, 6.0mM and particularly about 3mM (e.g. 3.0 ± l .OmM).

The reporting means may comprise other colour indicators or dyes including, for example, one or more of the following: Crystal violet, Carbol fuchsine, Safronin, Nigrosin, Indian ink, Iodine, Ziehl-Neelsen, Haemotoxylin, Eosin Y/Eosin yellowish, Papanicolaou, Orange G, Light green SF yellowish, Bismarck brown Y, Nile blue/Nile blue A, Nile red/Nile blue oxazone, Mason's trichome, Romanowsky, Wright's, Jenner's, Leishman, Giemsa, Silver, Sudan III, Sudan IV, Oil red O, Sudan Black B, Conklin, Malachite green, Osmium tetroxide/Tetraoxide, Rhodamine, Acridine Orange, Carmine, Coomassie blue, DAPI, Eosin B, Ethidium bromide, Acid fuchsine, Hoechst, Methylene green, Methylene blue, Neutral red/Toluylene red, or HDTMA/CTAB.

Indicators for use according to the invention may comprise chromagenic substances, otherwise known as chromagens. The term "chromagen" may be used to describe any compound that can be metabolised or converted into a pigment or dye. The pigment or dye may result from a reaction between a metabolite produced by the microorganism and a chromagen contained within the medium. Suitable chromagens may include, for example, a-D-galactopyranoside, β-D-galactopyranoside, β-D- cellobioside, N-acetyl- -D-galactosaminide, a-D-glucopyranoside, β-D- glucopyranoside, N-acetyl- -D-glucosaminide, β-D-fucopyranoside, β-D- xylopyranoside and/or 5-bromo-6-chloro-3-indoxyl- -D-glucopyranoside (X-Gal). The chromagenic substances listed above may be exploited to facilitate the detection of microorganisms which express the enzyme β-glucosidase. It should be understood that this is not an exhaustive list of the chromagenic substances that may be used in the reporting means of the device provided by this invention and one of skill will be familiar with suitable alternatives.

It will be appreciated that the reporting means may comprise a chemiluminescent, bioluminescent or fluorescent indicator.

A significant advantage of many devices according to the invention is that they may be read 'by eye'. However, under some circumstances (e.g. when a chemiluminescent, bioluminescent or fluorescent indicator is used or when very faint colour changes may occur), it may be desirable to 'read' the reporting means by something other than a human eye. Therefore, in one embodiment, a scanner or detector may be used. In this case it may be desirable to incorporate a 'label' reader (e.g. a barcode) to simplify the process of indicating when a particular sensor had been examined. For instance, a nurse may scan a device according to the invention incorporated in a medical device on a hospital ward to see if it triggered. The scanner would record the barcode and result to indicate on the patient record what has been done.

Selection Factors

By "selection factor" we mean an agent that may be incorporated within the reporting means that will arrest replication, decrease growth or increase death of selected microorganisms but may permit replication, growth or survival of other microorganisms. It will be appreciated that a sufficient amount of the selection factor should be included in the reporting means that will prevent any activation of the device by microorganisms that are sensitive to it.

The selection factors can have a broad or narrow spectrum of activity and one skilled in the art will appreciate the activity of specific factors should be taken into account when designing devices according to the invention.

1. Selection Factors used to regulate a threshold for activation of devices according to the invention.

In one embodiment of the invention selection factors may be used to set a threshold concentration of microorganisms for activation of the reporting means. Accordingly, a concentration of a selection factor may be chosen that will only allow microorganisms to activate the device at a predetermined level of infection (e.g. cells/ml or a defined number of CFUs). Alternatively, the reporting means may contain a concentration of a factor that is at such a high level that, for all practical purposes, activation of the device will not be caused by microorganisms that are sensitive to that factor.

Factors that regulate the threshold for activation are also referred to herein as threshold factors. By "threshold factor" we mean an agent that may be incorporated within the reporting means that will either limit replication of microorganisms within the reporting means, arrest replication, retard growth or cause the death of microorganisms (i.e. a biocide). The choice of threshold factor and the amount used will depend upon the threshold concentration of microorganisms that is required for activation of a device according to the invention. This threshold will in turn be set by the desired use for the device as discussed in more detail below Devices according to the first aspect of the invention are adaptable such that the "triggering" of the device may be adjusted depending open the concentration of microorganism that may be expected in the wound environment in which the device is placed or the concentration of microorganism in a sample (e.g. wound exudate) which is of interest. It will be appreciated that an increase in the amount of threshold factor contained within the reporting means will result in the arrest of microbial replication, a decrease in microbial growth or an increase in microbial death. This will limit the exposure of the metabolic indicator in the reporting means to the microorganisms in the sample and will limit activation of the indicator. Accordingly, a greater titre of microorganisms will be required to overcome the effect of the threshold factor and allow activation of the indicator in the device. The inventors have appreciated that the inclusion of a threshold factor in the device according to the invention has the effect that a threshold concentration of microorganisms that will trigger the device by activating the metabolic indicator will increase proportionate to the concentration of threshold factor in the device. Devices with no or low concentrations of threshold factor are triggered at relatively low microbial titres whereas devices comprising higher concentrations of threshold factor are triggered at relatively higher microbial titres. It will be appreciated that a number of devices with a range of concentrations of threshold factor may be employed to allow quantification of the levels of microorganisms.

A wide range of agents may be used as threshold factors. It is preferred that the agent has broad spectrum activity for retarding the growth or killing microorganisms. The threshold factor may be active against fungi, algae and bacteria. It is preferred that the threshold factor has broad spectrum activity against bacteria. It will be appreciated that threshold factors may be used that have narrow spectrum activity (for instance an agent that only has antibiotic activity against a limited number of bacterium). However narrow spectrum agents are mostly useful as threshold factors when samples are tested that are only likely to contain microorganisms that are sensitive to such agents or are more useful for selecting between micro-organisms as discussed below. Samples containing unknown microorganisms or those containing a variety of microorganism may well contain microbes that will readily grow in the reporting means because they are insensitive to the narrow spectrum agent. Accordingly, the metabolic indicator could be triggered in such devices- irrespective of the inclusion of the narrow spectrum agent. It will therefore be appreciated that the choice of narrow spectrum agents (e.g. some antibiotics) as threshold factors will require careful consideration. This is because resistant strains of bacteria have developed against many of the antibiotics that are in clinical use. Accordingly, narrow spectrum agents are likely to be most useful for selecting between micro-organisms (as discussed below) rather than as broadly acting threshold factors.

Preferred threshold factors for use according to the invention are biocides and other antimicrobial agents with broad spectrum activity selected from Polyhexanide (polyhexamethylene biguanide, PHMB), Chlorhexidine, Quaternary ammonium compounds (including enzylkonium chloride, polyquaternium compounds, didecyldimethylammonium chloride), antimicrobial peptides (including peptides based on tandem repeats of ApoE _{141 -149} and derivatives thereof ), Triclosan, cetrimonium salts (such as Cetrimide (Cetrimonium bromide), cetrimonium chloride and cetrimonium stearate) and tellurite.

It will be appreciated that a skilled person will select a threshold factor at least in part in view of the nature of the sample to which the device will be exposed (e.g. in view of the type and quantity of microorganisms expected in a wound). Choice of a threshold factor may therefore depend upon the known selectivity of the agent used. The selectivity of threshold factors according to the invention include:

(a) PHMB has broad spectrum activity and is effective against both Gram- negative and Gram-positive bacteria, including Staph, species and Pseudomonas (as well as non-bacterial microorganisms)

(b) Chlorhexidine, although broad-spectrum, may be less useful for samples that may comprise some Gram-negative bacteria such as Klebsiella pneumoniae.

(c) Triclosan is effective against staph, species and Klebsiella pneumoniae but less effective against Pseudomonas aeruginosa.

(d) Anti-infective peptides, such as cathelicidins, defensins, protegrins, magainins, dermaseptin, melittin, cecropin and peptides derived from apolipoprotein E and apolipoprotein B HSPG-binding domains have similar efficacy to PHMB but with lower cytotoxicity for the user.

(e) A number of quaternary ammonium compounds may be used.

(f) Tellurite is a tellurium compound that is useful because it is highly toxic to most microorganisms although some Staphylococci are resistant.

Examples of quaternary ammonium compounds ((e) above) include:

• Alkyltrimethylammonium salts: cetyl trimethylammonium bromide (CTAB) a.k.a. hexadecyl trimethyl ammonium bromide, cetyl trimethylammonium

chloride (CTAC)

• Cetylpyridinium chloride (CPC)

• Benzalkonium chloride (BAC) - this has been mentioned

• Benzethonium chloride (BZT)

• 5-Bromo-5-nitro-L3-dioxane

· Dimethyldioctadecylammonium chloride

• Cetrimide

• Dioctadecyldimethylammonium bromide (DODAB)

Cetrimide (Cetrimonium bromide) is a preferred quaternary ammonium compound for use as a threshold factor. It is an antiseptic used in agars and is selective for Pseudomonas. It is also used in certain cosmetics and suited to uses where devices come into human contact. Cetrimide is closely related to cetrimonium chloride and cetrimonium stearate which may also be used as threshold factors. Other preferred threshold factors are Isothiazolin based biocides,

Glutaraldehyde based biocides and DB PA (2,2-dibromo-3-nitrilopropionamide).

PHMB is a most preferred threshold factor for use according to the invention. When antibiotics are used as threshold factors, it will be appreciated that the factor will primarily be effective against bacteria. Furthermore, the antibiotic should be selected in view of any resistance that is known for a given antibiotic. Such information can be checked at http://antibiotics.toku-e.com/. The inventors found that there is a relationship between the concentration of microorganism and concentration of a threshold factor in the reporting means. This means that it is possible to arrange for a device to trigger at a preordained concentration of microorganism. It will be appreciated that this means devices according to the invention may be used to quantify the amount of microorganism. This is of use for monitoring the levels of microorganisms in a wound environment and providing an indication when levels of microbe have reached a higher level than that normally expected to be found. Therefore, devices according to the invention may be used for signalling an elevation in levels of microbes above pre-determined concentrations. For example, a wound dressing might normally be adjacent to relatively high levels of normal skin fauna; however an increase of such fauna to levels of around 10 ⁷ CFU / ml or higher would be clinically important, and it is therefore preferred that the threshold for triggering devices according to the invention are set at such levels when they are to be used in conjunction with wound dressings. For instance, devices for use in monitoring abnormally high infection in wound care products applied to chronic ulcers may be required to be activated at concentrations of microorganisms of > 10 ⁶ - 10 ⁷ CFU7 ml.

The exact concentration of threshold factor can be easily established by employing standard experimental techniques that titre threshold factor concentration against differing concentrations of target microorganisms. Examples 3 and 4 outline preferred approaches.

2. Selection Factors used to discriminate between microorganisms

In preferred embodiments selection factors are incorporated in devices according to the invention that are suitable for allowing discrimination between different types of microorganism.

In some embodiments the selection factor may be an agent with broad spectrum activity for retarding the growth of microorganisms or for killing microorganisms. The agent may be active against more than one type of microorganism. For instance, it may be active against fungi, viruses and bacteria. Under the circumstances where devices are formed with reporting means having a concentration of such factors that set a very high threshold for activation, it will be appreciated that such devices will be of interest to a user because the microorganisms that activate the device will be rare or virulent organisms that are resistant to the chosen agent. In a preferred embodiment a selection factor is chosen that has broad spectrum activity against bacteria, but which is selective for bacteria over other types of microorganism.

In other preferred embodiments the selection factor may be an agent with narrow spectrum activity (for instance an agent that only has antibiotic activity against a limited number of species of bacteria). Such narrow spectrum selection factors are useful as selection factors when a device is designed where the user expects a sample to contain a specific microorganism but is uninterested in that microorganism and wishes to specifically prevent it from activating a device. Under these circumstances the device will be activated by other microorganisms in the sample that are not influenced by the narrow spectrum agent.

It will be appreciated that a skilled person will chose a selection factor at least in part in view of the nature of the environment or sample to which the device will be exposed (e.g. in view of the type and quantity of microorganisms expected in a wound).

Preferred narrow spectrum selection factors include certain antibiotics (discussed below) although other agents may be used. These include metabolic inhibitors specific to particular microorganisms and specific enzyme inhibitors. Cetrimonium salts (such as Cetrimide (Cetrimonium bromide), cetrimonium chloride and cetrimonium stearate) are useful as narrow spectrum selection factors. Cetrimide is an antiseptic used in agars and is selective for inhibiting the growth of Pseudomonas. It is also used in certain cosmetics and may therefore be suited to uses where devices come into human contact. Cetrimide is closely related to cetrimonium chloride and cetrimonium stearate which may also be used as selection factors.

Antibiotics are preferred selection factors for incorporation into reporting means of devices according to the invention. When antibiotics are used, it will be appreciated that the antibiotic will primarily be effective against bacteria. Antibiotics are useful because they may be used to discriminate between bacteria on the basis of any resistance that is known for a given antibiotic. Such information is well known to the art and can be accessed at http://antibiotics.toku-e.com/.

Devices comprising reporting means that include an antibiotic should contain a sufficient amount of the antibiotic that will prevent the growth or kill non-resistant bacteria. This means non-resistant bacteria will not be responsible for device activation whereas device activation will occur in the presence of bacteria that are resistant to, or insensitive to, the chosen antibiotic.

A skilled person will appreciate that antibiotics are grouped into classes of agent with associated selectivity. As well as penicillin and methicillin (which can be used as selection factors on the basis of their well-known resistances), other preferred antibiotics for use according to the invention include:

Fusidin (Fusidic acid): a bacteriostatic antibiotic which is effective primarily against Gram-positive bacteria. Accordingly, it will prevent activation of devices by Gram-positive bacteria but will permit activation by Gram-negative bacteria

Cephalosporins: First generation cephalosporins are primarily effective against Gram-positive bacteria. Following generations have increased activity against Gram- negative bacteria but may have reduced effectiveness against Gram-positive bacteria.

Carbapenems: These are a class of broad spectrum antibiotics that are highly resistant to most β-lactamases and can therefore be used to prevent activation of devices by many types of bacterium that express β-lactamases and which are typically resistant to penicillins etc.

Sodium Nalidixate: A narrow spectrum agent that may be used to prevent Pseudomonas activating devices according to the invention.

Nalidixic acid: A synthetic quinolone antibiotic that may be used to prevent activation of devices by Aeromonas hydrophila, Clostridium and Haemophilus but permits activation with Bifidobacteria, Lactobacillus, Pseudomonas and Staphylococcus

Quinolone antibiotics: Target DNA gyrase in Gram-negative bacteria and topoisomerase IV in Gram-positive. Bacteriostatic and then bactericidal at higher concentrations. Widely used in treatment of hospital acquired infections associated with urinary catheters.

Polymixin B sulphate: Antibiotic used primarily for Gram-negative infections, it is bactericidal against nearly all Gram-negative bacilli, with the exception of the Proteus group.

Table 1 outlines other factors that may be considered when selecting a factor for using according to the invention.

Table 1:

In a preferred embodiment, devices may be exposed to chronic ulcers and particularly diabetic foot ulcers or exudates therefrom. Such ulcers are typically treated with cephalosporin antibiotics, specifically, cephalexin; beta lactam antibiotics such as amoxicillin; clavulanic acid, fluoroquinolone antibacterial agents such as moxifloxacin; and clindamycin. Devices used to sample diabetic ulcers preferably include one or more of these antibiotics as selection factors.

In another preferred embodiment, devices may be exposed to burn wounds. Broad spectrum penicillins such as piperacillin and carbapenems such as meropenem may be used to treat Gram-negative and Gram-positive infections of burn wounds. Invasive Candida infections of such wounds are treated with clotrimazole or ciclopiroxolamine creams. Infection with filamentous fungi can be treated with amphotericin B. Devices comprising such antimicrobial agents are preferably used with samples associated with burn wounds.

Device according to the invention may also be used to detect microbial infection of venous wounds, pressure ulcers, cavity wounds, bleeding wounds, surgical wounds. MRSA is a common problem with wounds. Therefore MRSA selective devices are preferred devices according to the invention.

The exact concentration of selection factor required to prevent activation of devices by the selected microorganism(s) can be easily established by employing standard experimental techniques that titre the selection factor concentration against differing concentrations of target microorganisms (e.g. see Examples 2 or 4). Antibiotics used in reporting means should be included at concentrations of antibiotic that are sufficient to retard bacterial growth to such an extent that the device will not trigger. The exact concentration required will depend on the antibiotic used but will typically be up to 2.5mg/mL and may be 750 μg/mL (for instance about 500μg/ml).

It will be appreciated that preferred antibiotics for inclusion in a reporting means are those which are used in treating wound infections. A clinician can observe whether or not such antibiotics prevent triggering of the metabolic indicator and can then select an appropriate therapeutic regimen. Preferred antibiotics for inclusion in the reporting means are vancomycin, tobramycin flucloxacillin, Cephalexin, Ciprofloxacin, Clindamycin and Co-amoxyclav. In a preferred embodiment a device is formed which contains 5 reporting means, A first reporting means comprises no selection factor (a growth control) and the other four reporting means may comprise a sufficient amount of Cephalexin, Ciprofloxacin, Clindamycin and Co-amoxyclav (respectively) for killing sensitive bacterium. By way of example, the inventors have found 2.05 mg/mL Cephalexin, 0.52 mg/mL Ciprofloxacin, 2.05 mg/mL Clindamycin and 0.1 mg/mL Co-amoxyclav to be sufficient amounts.

Other components of reporting means

The inventors have found that activation of metabolic indicators is optimised

(with respect to threshold for activation and intensity of colour) when an electron mediator is included in the reporting means that will promote the activity of redox enzyme systems. It is most preferred that the electron mediator promotes the activity of cellular reductase such as NAD(P)H reductase.

Examples of electron mediators are well known to the art. For instance, the electron mediators listed by Fultz and Durst (Analytica Chimica Acta 140 (1992) 1- 18). Preferred electron mediators are Phenazine methosulphate (PMS) and derivatives thereof (e.g. Phenazine ethosulphate. A most preferred electron mediator for inclusion in the reporting means is l-Methoxy-5-methylphenazinium methyl sulfate (mPMS). mPMS is preferably included in the reporting means at concentrations greater than O. lmM. For instance, mPMS may be used in in the reporting means in the range of 0.1 - 0.8mM, and more preferably in the range 0.2 - 0.6mM. Most preferred concentration of mPMS in the reporting means are 0.3mM, 0.4mM, 0.5mM and 0.6mM. and particularly about 0.3mM (e.g. 0.3 ± O. lmM). Preferred reporting means according to in the invention comprises an alginate as the carrier substance and a tetrazolium salt as the metabolic indicator. It is more preferred that such reporting means additionally comprise an electron mediator.

In one preferred embodiment the reporting means comprises an alginate and WST-9. It is more preferred that such reporting means additionally comprise an electron mediator. In another preferred embodiment the reporting means comprises an alginate,

WST-9 and mPMS.

In another preferred embodiment the reporting means comprises 2-4% (w/v) alginate, 1.5-6.0mM WST-9 and 0.2-0.6mM mPMS.

In a most preferred embodiment the reporting means comprises 3% (w/v) alginate 3.0mM WST-9 and 0.3mM mPMS.

It will be appreciated that each of these embodiments may optionally contain a selection factor. Reporting means without a selection factor represent positive control reporting means in which micro-organisms will grow unhindered and which will be expected to activate in the presence of micro-organisms. Antibiotics are preferred selection factors which may be included in reporting means. Examples of preferred antibiotics which may be included include Tobramycin, Vancomycin, Cephalexin, Ciprofloxacin, Clindamycin: and Co-amoxyclav.

It will be appreciated that it may be desirable to include a bacterial growth media and/or nutrients in the reporting means. When this is the case it is preferred that a media or nutrients is used that does not cause the conversion of a tetrazolium salt into formazan when incubated with a tetrazolium salt at 40°C overnight. Alternatively, the media and/or nutrients used in the reporting means is preferably do not cause the conversion of a tetrazolium salt into formazan when the incubated with a tetrazolium salt at 24°C for 8 hours or 24 hours. It is more preferred that the media and/or nutrients do not cause the conversion of a tetrazolium salt into formazan when the media or broth is incubated with a tetrazolium salt at 24°C or 40°C for 1 or 2 hours. The inventors found that many media and/or nutrients caused false triggering of metabolic indicators. Of the media and/or nutrients which resulted in no, or irrelevant triggering, their suitability had to be further assessed. For instance the inventors found that only a small number of broths were suitable for incorporating in a reporting means that comprised a WST-9 which was required to sense a broad spectrum of microorganisms and in which an antibiotic was also used. Examples of such many media and/or nutrients include Mueller Hinton and Wilkins Chalgren media. It is most preferred that Wilkins Chalgren media is used in the reporting means.

Reporting means according to the invention are particularly useful because, in the absence of a selection factor, they are sensitive to a broad spectrum of microorganisms (and particularly a broad spectrum of bacterium); and the threshold titre of microorganisms for activation of such reporting means is comparable to the titre found in the environment of an infected wound. Establishing a suitable reporting means required significant inventive endeavour because the inventors found that many dye stuffs that were candidate metabolic indicators for use according to the inventor would not trigger or false triggered when exposed to microorganisms.

Substrate

The substrate may be a membrane or film upon which at least one reporting means is affixed or alternatively may be thick enough to contain a pore which may be used to retain the reporting means in the device.

1. Embodiments wherein the reporting means is retained within the substrate

In one embodiment the substrate is thick enough to retain the reporting means within the substrate.

Such devices may comprise a substrate fabricated from (or comprising) a substrate comprising or consisting of any suitable polymeric, plastic, metal, resin and/or composite material(s). One of skill will be familiar with these types of material but exemplary materials for use as substrates may include, for example, Poly(2- hydroxyethyl methacrylate) (pHEMA), Polyethylene glycol (PEG), poly(lactic-co- glycolic acid) (PLGA), Poly(methyl methacrylate) (PMMA), Polydimethylsiloxane (PDMS), Poly-vinyl chloride (PVC) and/or Polyurethane (PU), polyethylene terephthalate (PET), methacrylamide derivatives such as Q9, sulphopropyl acrylates, cyclic olefins such as Topas, and/or polysulphone (PS). One of skill will readily appreciate that hydrogels comprising pHEMA may additionally include copolymers such as for example, methacrylic acid (MA), N-vinyl-2-pyrrolidone (NVC) or carboxymethyl cellulose (CMC).

The substrate may comprise a hydrophobic polymeric material. One of skill will be familiar with these types of material but exemplary materials for use may include silicone elastomers, poly(vinyl chloride), polystyrene and styrene-containing block copolymers, polyurethanes, epoxy resins, hydrophobic acrylic and methacrylic polymers with Tg>r.T. (e.g. poly(methyl methacrylate)), polycarbonates, semicrystalline or elastomeric polyolefins (e.g. ethylene-propylene rubbers, polyolefin elastomers), ethylene - vinyl acetate copolymers, polyesters such as poly(ethylene terephtalate), or polymers of lactic and/or glycolic acid, and related polyesters (e.g. poly(caprolactone), poly(2-dioxanone)).

In other embodiments, a polymeric material for use as the substrate may comprise a densely cross-linked hydrogel. Suitable hydrogels may include those based on poly(2-hydroxy ethyl methacrylate) (pHEMA).

Further exemplary materials for use in forming the substrate component of the devices provided by this invention may include other monomers in combination with HEMA, such as methacrylic acid (MA), N-vinyl-2-pyrrolidone (NVP), acrylamide (AM), N-substituted acrylamides. Other exemplary materials may include synthetic monomers other than HEMA, such as poly(ethylene glycol) (PEG) acrylated or methacrylated derivatives, or be based on polysaccharide backbones, such as derivatives of cellulose or starch.

In a preferred embodiment the substrate is an elastomer and preferably a silicone elastomer. Silicones are particularly useful for use as a substrate because it is advantageously inert, clear (enabling a user to view changes in the reporting means), durable, flexible, water proof, biocompatible and may be easily wiped clean. An example of a silicone that may be used according to the invention is Sylgard® 184. A skilled person will appreciate that a number of functionally equivalent silicones are available. Devices according to this embodiment may be made to whatever size, and contain as may reporting means as may be required.

According to one embodiment the device may comprise a substantially disk shaped substrate which may be 0.1 - 2.0 cm thick and is more preferably 0.1 to 0.5cm thick. Such disks may have a diameter of 0.5 to 3 cms. The disk may contain multiple reporting means and preferably comprises at least one growth control reporting means (i.e. one that contains no selection factor) and at least one reporting means containing a selection factor. The reporting means are preferably alginate bead reporting means and are preferably 30-40μ1 in volume and most preferably about 32-37μ1 in volume. Such alginate reporting means may be substantially spherical and preferably have a diameter of about 3mm.

A preferred device according to this embodiment of the invention is illustrated in Figure 1(a) and shown in figures 3 and 4.

2. Embodiments wherein the substrate is a membrane or film

The substrate may comprise a thin layer, membrane or film upon which at least one reporting means, and preferably an array of reporting means are arranged. In some embodiments such substrates are impermeable to the compounds contained within the reporting means to prevent leaching of the reporting means and microorganisms across the thin layer, membrane or film. Alternatively, the substrate to which the reporting means is affixed is not impermeable itself. In this case the substrate may be closely associated with a barrier membrane/sheet that prevents leaching of the reporting means and microorganisms (see below).

Thin layer/membrane/film substrates may be transparent, hydrophobic, polymeric membranes that acts as a barrier to the outside environment, but at the same time are sufficiently transparent to allow an observer to monitor whether or not the reporting means within the device has been activated. Preferably such substrates may comprise the outermost component of a wound dressing when applied to the skin.

Thin layer, membranes of film substrates may comprise the materials described above for other substrates. It is preferred that thin layer, membrane or film substrates used in accordance with the present invention include but are not limited to poly (vinylidene fluoride) , poly (vinylidene chloride), phenoxy resins, butadiene/styrene copolymers, butadiene/methylstyrene copolymers, poly (meth) acrylates, butadiene/acrylonitrile copolymers, ethylene/propylene copolymers, polybutadiene, polyisoprene, poly (oxy-2, 6-dimethyl-l, 4 -phenylene) , poly (oxycarbonyloxy- 1,4 (1 , 4-phenyleneisopropylidene-l, 4-phenylene) , acrylonitrile styrene copolymers, acrylonitrile/methyl acrylate/butadiene copolymers, acrylonitrile/styrene/butadiene copolymers, poly-1- vinylaphthalene, polyvinylphenyl ketone, poly-p- xylylenedodecanedioate, poly-tetramethylene octenediamide, polytetramethylene terephthalene, poly-trimethylene-3 , 3'- dibenzoate, poly-terephthallic anhydride, poly-4-methyl- diamine, polyvinylene carbonate, polyvinylene laurate, polyisoprpenyl acetate, polyallylbenzene, polyvinylbutyl ether, polyvinyl formate, polyvinyl phenyl ether, polynorbornadine , polycarbonate, hydrophobic polyesters and polyurethanes (PU), and mixtures thereof.

In a preferred embodiment the substrate is a sheet of polyurethane (PU) on to which the reporting means may be affixed. Such PU sheets may be transparent or opaque. Opaque PU sheets should have a transparent sheet fixed over the top of the reporting means to allow the reporting means to be viewed by a user.

In a preferred embodiment thin layer/membrane/film substrates are perforated sheets wherein the perforations are arranged to retain the reporting means. In a most preferred embodiment the substrate is a perforated sheet of PU. Example 4 describes how a device comprising a perforated PU sheet as a substrate may be made and figure 1(b) illustrates such a device.

Reporting means can be applied to thin layer, membrane/film substrates in a number of ways. For instance, printing devices may be used to apply the reporting means. It will be appreciated that spraying/printing in this manner can be finely controlled to allow for reporting means to be applied to the surface in a wide variety of shapes and also as arrays of reporting means (which may define detailed patterns). It will be appreciated that a variety of reporting means (e.g. ones with or without selection factors and/or ones with a variety of selection factor may be sprayed onto the same surface).

It will be appreciated that the volume of reporting means applied to a thin layer/ membrane/film surface will depend upon the size required when a triggered device is to be visualized and also on the shape/pattern that the reporting means will define. Preferred reporting means for use according to this embodiment are the alginate beads discussed above.

Other components of the device

Devices according to the invention may additionally include a physical barrier that acts to retain the reporting means, and the constituents thereof, within, or on, the substrate. Such a physical barrier may be located between where the wound will be located when in use and the reporting means. The physical barrier should be adapted such as to allow microorganisms to enter the reporting means but substantially retain the components of the reporting means within the substrate. Preferred barriers are meshes with a mesh pore size that is large enough to allow bacteria into the reporting means but small enough to prevent dislodgement of the reporting means and ingress of larger cells (e.g. mammalian cells) or particles. Preferred meshes may have a mesh pore size of 1-1,000μm, preferably a mesh pore size of 50-500μm, more preferably a mesh pore size of 75-200μm and most preferably a mesh pore size of approximately 100μm. Typically a mesh will be less than 500μm thick. Meshes may be manufactured from PMMA, PET or polypropylene and functionally equivalent polymers. A most preferred mesh has a mesh size of approximately 100μm and may be fabricated from 150μιη thick PMMA. Devices according to the invention may also comprise one or more protective sheets. Such sheets may be made from the materials used to make the substrate. In a preferred embodiment the sheets are transparent to allow viewing of the reporting means and/or are impermeable. Figure 1(b) (as discussed in more detail below) illustrates a device with a protective sheet of silicone which overlays a perforated substrate and the reporting means retained within the substrate.

The device may also comprise a peel away sheet which is stuck over the reporting means (and any physical barrier over laying the reporting means). The peel away sheet acts to keep the device sterile and hydrated and should be peeled away when the device is used to allow any microorganisms in the fluid being sampled to interact with the reporting means. A most preferred device comprises a perforated sheet substrate that retains reporting means within the perforations. On a viewing side the reporting means and substrate are overlayed by a sheet of transparent silicone and on the side to be exposed to a wound or wound fluid the substrate and reporting means are overlayed by a peel away sheet and optionally a physical barrier to retain the reporting means in place.

A most preferred device comprising a perforated sheet substrate, a protective sheet, a physical barrier and a peel away sheet is illustrated in figure 1(b) and discussed in more detail below. Arrays of Reporting Means

Reporting means may be arranged in arrays. Such arrays may comprise a plurality of devices arranged in a grid formation or other pattern. Alternatively such arrays may comprise a grid formation or other pattern of a plurality of reporting means arranged on or within the substrate of a device.

Such arrays preferably comprise more than one type of reporting means which contain a range of selection factors that will provide detailed information on the numbers and types of microorganisms found in a wound environment. It will be appreciated that when there is more than one type of reporting means that at least one of the types of reporting means may comprise no selection factor. Such reporting means represent a control whereby the indicator should be triggered in the presence of any microorganism. In one embodiment such arrays are incorporated within a wound dressing. Such wound dressings may incorporate an array of reporting means on a membrane or film substrate. Alternatively, a number of devices may be woven or otherwise fixed into a wound dressing. Alternatively, a device bearing an array of reporting means (e.g. the devices illustrated in Figure 1(a)) may be placed in contact with the wound tissue and covered over by a conventional dressing.

Arrays preferably include a plurality of reporting means such that the array includes a range of antibiotic selection factors that will discriminate between microorganisms that may be present in the wound environment.

Such arrays may be used to identify which microorganisms, or types of microorganisms, are contained within the sample and can assist when deciding how the wound should be clinically managed. Such arrays are particularly helpful when deciding what antibiotics should be used to treat an infected wound. Arrays may be set up with a range of reporting means containing candidate antibiotics for treatment of the wound. Any activated reporting means in the array will have activated because the sample contains microorganisms that are resistant to the specific selection factor/antibiotic in the reporting means of such devices. However the presence of non- activated devices in the array will suggest that the selection factor/antibiotic in the reporting means of such devices was able to prevent the growth of microorganisms in the sample and those antibiotics may therefore be useful for treating the infected wound. A clinician may use an array of devices according to the invention to identify the most suitable antibiotic(s) for treating an infection. The array has the great advantage that it can provide such information rapidly and in the immediate vicinity of a subject needing treatment. Devices according to the invention obviate the need to wait for a laboratory to identify the species causing an infection (i.e. following the taking of a swab and sending it off to be cultured). The inventors realised that devices according to the invention may be used in this way for more broadly identifying suitable antimicrobial agents for treating a range of microbial infections and not just limited in the context of management a wound.

In preferred embodiments that employ a tetrazolium dye (e.g. WST-9) as the metabolic indicator, activation of the devices will result in the production of purple/dark dots (or other pattern) for devices containing selection factors to which the microorganisms in the sample are resistant; or light/yellow/orange/colourless dots for devices containing selection factors to which the microorganisms in the sample are sensitive. This will indicate to a clinician or a user of the device (or dressing containing the device) whether or not there is microbial contamination of at least the dressing and possibly also the wound itself; and, if so, what sort of microorganism is causing the infection and which type of antibiotic may be best suited for treating the infection. Wound Dressings

According to a second aspect of the invention there is provided a wound dressing comprising at least one device according to the first aspect of the invention.

In one embodiment the wound dressing comprises a device with a film or membrane substrate with an array of reporting means. In another embodiment the wound dressing comprises an array of devices comprising silicone substrates.

Wound dressings comprising a device with a film or membrane substrate with an array or reporting means

A device with a thin layer, film or membrane substrate having an array of reporting means may be incorporated into a wound dressing by a number of ways.

Devices according to this embodiment may include or be placed in contact with a physical barrier which, in the wound dressing, will preferably lie more proximal to the wound than the membrane or film bearing the reporting means. The physical barrier should be adapted such as to allow microorganisms to enter the reporting means but substantially retain the components of the reporting means on the surface of the device. Preferred barriers are meshes with a mesh pore size that is large enough to allow bacteria into the reporting means but small enough to prevent dislodgement of the reporting means and ingress of larger cells (e.g. mammalian cells) or particles. Preferred meshes may have a mesh pore size of 1-1,000μm, preferably a mesh pore size of 50-500μm, more preferably a mesh pore size of 75-200μm and most preferably a mesh pore size of approximately 100μm. Typically a mesh will be less than 500μm thick. Meshes may be manufactured from PMMA, PET or polypropylene and functionally equivalent polymers. A most preferred mesh has a mesh size of approximately 100μιη and may be fabricated from 150μιη thick PMMA. The meshes are preferably semi-permeable. Alternatively, the physical barrier may be a semipermeable membrane which is adapted such as to allow microorganisms to enter the reporting means but substantially retain the components of the reporting means on the surface of the device. Such membranes may comprise a sheet component of a wound dressing.

Devices according to this embodiment may further comprise or be placed in contact with a viewing sheet which, in the wound dressing, will preferably lie distal to the wound. The viewing sheet should be transparent in order that triggered reporting means may be viewed through it. The viewing sheet may also act as a barrier to prevent reporting means and/or exudate leaching out of the device/dressing. In some embodiments the outer layer of the device or dressing may comprise a protective sheet. The protective sheet may be removed to allow visualization of the reporting means below (whether directly or through a viewing sheet). In some embodiments, and particularly when there is no other non-permeable layers between the wound and the environment, the protective layer may be removed and will assist in the hydration of the reporting means arranged on the surface of the device.

Devices according to the invention are preferably incorporated in foam wound dressings and may be readily incorporated into foam dressings such as Allevyn/Aquacel Foam or Tegaderm foam.

The devices may also be incorporated into hydrocolloid dressings (e.g. Tegaderm Hydrocolloid/Suprasorb/Replicare); hydrogel dressings (e.g. Flexigel/Intrasite Gel/Derma-Gel/Nu-Gel); alginate dressings (e.g. Algisite/Tegaderm Aliginate); hydrofiber dressings (e.g. Aquacel Hydrofiber); transparent film dressings (e.g. from Tegaderm); or composite dressings (e.g. Alldress/Opsite)

Wound dressings according to the invention may be used with any animal of veterinary interest but are preferably for use on a human being. In a preferred embodiment, wound dressings according to the invention comprise an array of reporting means with a range of antibiotic selection factors that will discriminate between microorganisms that may be present in the wound environment. Such arrays are most preferably used for deciding which antibiotics may be used to treat an infected wound.

In preferred embodiments arrays of reporting means are arranged in a wound dressing wherein the array comprises at least two types of reporting means. These reporting means are characterised by the fact they contain different candidate antibiotics for treatment of the wound to which the dressing is applied. Any activated devices in the array will have activated because the sample contains microorganisms that are resistant to the specific selection factor/antibiotic in the reporting means of such devices. However the presence of non-activated reporting means in the array will suggest that the selection factor/antibiotic in the reporting means was able to prevent the growth of microorganisms in the wound and those antibiotics may therefore be useful for treating the infected wound.

A clinician may use the information provided by the pattern of activation in the array of reporting means within the dressing to rapidly obtain clinically relevant information and thereby quickly decide on a regimen for treating any infection of the wound to which the dressing was applied. Devices according to the invention obviate the need to wait for a laboratory to identify the microbial species causing an infection (i.e. following the taking of a swab and sending it off to be cultured and organisms identified) and therefore mean that an infection can be treated much more quickly and efficiently.

Use of Devices according to the invention

In a clinical setting the device may be put in place following cleaning and debriding of a wound. It will be appreciated that devices may be used that are either incorporated in a wound dressing or distinct devices may be used that are placed in contact with the wounded tissue and a suitable conventional dressing is placed over the top. Devices should be placed in the wound environment such that any microorganisms may enter the reporting means. This may be achieved by ensuring the devices are in contact with the tissue and/or ensuring that wound exudate may travel (e.g. soaked up by surround dressing) to the reporting means.

In a preferred embodiment the reporting means are alginate beads and the device is a discrete unit. Such devices are placed such that reporting means is placed in direct contact with the wound surface (or, if a mesh is used as a physical barrier, the mesh is in direct contact with the wound surface with the reporting means lying immediately below it). As soon as the device is placed in position, the wound should be dressed as per normal clinical practise.

A clinician will decide how long a dressing should be in place and the device should be read when the dressing is changed in the normal course of wound management. It is preferred that devices according to the invention are left exposed to wound tissue/exudate for at least 4 hours, preferably at least 8hours and more preferably at least 12 hours. For instance, devices may stay in contact with the wound tissue/bone and exudate for 24 hours or longer if the wound is next re-dressed after more than 24 hours. The device should be observed for clinically relevant levels of microbial growth (indicated by the growth control reporting means changing to a dark formazan colour) and antibacterial sensitivities (by observing changes in reporting means containing selection factors) when the dressing is changed.

Alternatively, devices may be used in at the point of care (e.g. in a clinic or for home use) or in a laboratory setting without being placed directly into a wound. When this is the case a sample of wound material (e.g. tissue or exudate) may be taken directly from the wound or extracted from a used dressing. The sample may then be put in contact with reporting means in a device and the device should then be incubated to allow any microorganisms to infiltrate the reporting means and possibly cause a colour change in the metabolic indictor. It is preferred that the device is incubated at 37°C for at least 4 hours and preferably between 6 and 24 hours before observing the results. In one embodiment the device may be incubated overnight (typically 8-12 hours) before being observed. Further Aspects of the Invention

In a third aspect, the invention provides a method of analysing a wound or sample taken from a wound for the presence of microorganisms, said method comprising the steps of:

(a) contacting a device provided by the first aspect of the invention with a wound or sample to be analysed; and

(b) examining the reporting means to determine whether or not microorganisms are present in the wound or sample. The device should be exposed to a wound or incubated with the sample for a period of time and under conditions suitable to facilitate, encourage or cause any microorganisms present in the sample or wound to pass into the reporting means. Microorganisms that have successfully passed into the reporting means may be maintained or multiple in the Media and/or nutrients that support or encourage microbial growth. At this point, the device may be left in contact with the sample or wound to allow the reporting means to complete any reactions necessary to report the presence of microorganisms to the user. Alternatively, after a period of incubation with the sample, the device and sample/wound could be separated and the device incubated for a further period of time before the reporting means is examined.

According to a fourth aspect of the invention there is provide a method of selecting an antimicrobial agent that is suitable for treating a subject with a microbial infection of a wound or a subject who is suspected of having a microbial infection of a wound, the method comprising:

(a) exposing at least one device according to the first aspect of invention to a wound or a sample obtained from a wound of the subject wherein the reporting means of the device comprises a selection factor that is a candidate antimicrobial agent;

(b) incubating the at least one device for sufficient time and under suitable conditions to allow device activation; and

(c) selecting an antimicrobial agent that is suitable for treating the subject on the basis that the device comprising the selected agent remains unactivated after incubation step (b). The inventors realised that devices according to the invention may be used for identifying suitable antimicrobial agents for treating an individual. The methods have the great advantage that it will identify the correct agent for the specific infection in the subject of interest. In preferred embodiments this aspect of the invention represents a form of personalised medicine.

It will be appreciated that the fourth aspect of the invention may be carried out with wound dressings according to the second aspect of the invention as discussed above. Alternatively arrays of reporting means could be exposed to a sample taken from a wound (e.g. a swab of wound exudate) and the methods carried out in vitro on an array.

The subject may be any animal of veterinary interest but is preferably a human being.

The method may preferably be employed to select a medicament that is suitable for treating a subject which may have a fungal, viral, protozoan or bacterial infection. However it is preferred that the device or devices comprise candidate antibiotics and the method is for selecting a medicament that is suitable for treating a subject with a bacterial infection or who is suspected of having a bacterial infection,

In preferred embodiments the invention is substantially as described in the description and figures. DETAILED DESCRIPTION

The present invention will now be described in detail with reference to the following figures which show:

Figure 1 : illustrates to two embodiment of the device according to he invention. Figure 2: is photograph of a microtitre plate showing that a metabolic indicator

(WST-9) was activated in response to a variety of bacteria that can potentially infect a wound. Figure 3 is a photograph of devices according to the present invention, (a) shows a device comprising 5 reporting means which are unactivated; and (b) shows the same device following activation of the reporting means (containing the metabolic indicator WST-9) following exposure to infected wound fluid.

Figure 4: is a photograph of devices according to the invention wherein the reporting means in each device were exposed to simulated wound fluid (SWF) inoculated with a number of microorgansims (1 - 10); and wherein antibiotic resistance profiles for the organisms were established for (a) Cephalexin, (b) Ciprofloxacin, (c) Clindamycin and (d) CoAmoxyclav.

Figure 1(a) illustrates a device 1 according to the present invention which comprises a substantially disk shaped silicone substrate 2 which is about 0.5cm thick and has a diameter of about 3.0cms. The substrate comprises five reporting means 3, 4. A growth control reporting means (i.e. one that contains no selection factor) 3 is located centrally with four reporting means containing a selection factor 4 arranged around it. The reporting means 3,4 are alginate bead reporting means with a volume of 32-37μ1 and a diameter of about 3mm.

Figure 1(b) illustrates a cross section of part of a device 10 according to the present invention showing two reporting means 3, 4 retained within perforations of a substrate which is a perforated sheet of polyurethane 5. The cross-section shows a growth control reporting means (i.e. one that contains no selection factor) 3 and a reporting means containing a selection factor 4. The reporting means 3,4 are alginate bead reporting means with a volume of 32-37μl and a diameter of about 3mm.

A protective sheet of transparent silicone 6 overlays the reporting means 3, 4 and substrate 5 on a viewing side of the device of figure 1(b). The device also comprises a mesh 7 which may have a pore size of 75-200μm and which acts as physical barrier to retain the reporting means 3, 4 in place. The device further comprises a peel away sheet 8 which is stuck over the mesh 7. The peel away sheet 8 acts to keep the device sterile and hydrated and should be peeled away when the device is used to allow any microorganisms in the fluid being sampled to interact with the reporting means. The mesh 7 and peel away sheet 8 are shown in an exploded view although it will be appreciated that the mesh 7 will be suitably adhered to the substrate 5; and the peel away sheet 8 will be suitably adhered to the substrate 5 and/or mesh 7.

Figure 1(c) illustrates a plan view of a sheet of polyurethane 9 which is cut to contain an array of circular substrates 5 for use in devices according to the invention. The sheet 9 is first punch or dye cut to form holes 30, 40 which in devices according to the invention 10 will receive reporting means 3 and reporting means 4 respectively. The sheet is then kiss cut to form discrete substrates 5 each containing 5 holes 30, 40.

Individual substrates may be separated from the sheet 9 and a device according to the invention may be manually assembled. Alternatively, an intact sheet 9 containing multiple substrates 5 may be loaded with reporting means 3,4 and this may be done either manually or robotically. It will be appreciated that such sheets may overlay meshes 7 and peel away sheet 8 and a protective sheet of transparent silicone 6 can be overlayed once the reporting means have been loaded into the substrates. The individual devices 10 may then be punched out of the assembled sheets (through the kiss cuts for the substrates).

EXAMPLES

EXAMPLE 1: Identifying a Metabolic Indicator

The inventors tested a number of dyes to establish the most suitable dye for use in the detection of microorganisms in the wound environment.

Many tested compounds were found to be unresponsive to micro-organisms or false triggered in the presence of cells that may be found in a subject (e.g. leukocytes).

The inventors found that tetrazolium salts were most useful for use according to the invention. For instance, MTT and MTS were found to be effective. The most preferred metabolic indicator for use according to the invention were found to be Water Soluble Tetrazolium (WST) salts and WST-9 was found to be particularly useful. Figure 2 illustrates how WST-9 is reduced from a colourless/straw colour to a dark/purple formazan in the presence of eight different species of bacteria that can be found in the wound environment. It will be seen from Figure 2 that the optimal concentration of WST-9 was greater than 0.37mM and preferably 1.5mM or greater. The preferred concentration range of WST-9 in the reporting means is 1.5mM - 6mM and is preferably about 3mM.

Further optimization experiments were conducted in which it was found that WST-9 performed best when used with an electron mediator such as methoxy-PMS (mPMS). The optimal combination was found to be 3mM WST-9 + 0.3mM mPMS (data not shown).

EXAMPLE 2: Production of Alginate Bead Reporting Means for use according to the invention

The inventors developed methods for making alginate bead reporting means utilising Calcium Chloride and Sodium Alginate to form a 3% (w/v) alginate bead which comprises other components of the reporting means

An illustrative protocol for forming a most preferred reporting means was

2.1 Making up Sodium Alginate Solution

2.1.1 Add 100ml of sterile deionised water to a sterile vessel.

2.1.2 Add 3 g of Sodium Alginate, 180mg WST-9 and 10.8mg mPMS to the vessel

2.1.3 Mix and then to leave until all air bubbles have gone. This will be a stock solution (final concentrations 3mM WST-9 and 0.3mM mPMS).

2.2 Making up Calcium Chloride Solution

2.2.1 Add 100ml of sterile deionised water to a sterile vessel.

2.2.2 Add 2.94g of Calcium Chloride, 180mg WST-9 and 10.8mg PMS to the vessel. 2.2.2 Mix and then to leave until all air bubbles have gone. This will be a stock solution (final concentrations 3mM WST-9 and 0.3mM mPMS).

2.3 Making a stock solutions containing Selection Factors

2.3.1 Dispense 10ml of stock solutions of sodium Alginate (2.1) and Calcium

Chloride (2.2) into separate vessels. 2.3.2 Add the required amount of antibiotic powder to each solution (Table 1). Equal amounts are to be added to both the Sodium Alginate and Calcium Chloride solutions.

2.3.3 Gently mix until the antibiotics are fully dissolved. Table 1. Amount of antibiotic to be added to 10ml Sodium Alginate and 10ml Calcium Chloride solutions.

2.4 Forming Alginate Beads Reporting means

2.4.1 To form a growth control reporting means: slowly pipette 32-37uL drops of the alginate solution (2.1) into the Calcium Chloride solution (2.2).

2.4.2 To form reporting means containing selection factors: slowly pipette 32-37uL drops of the alginate solution into the Calcium Chloride solution which were made according to 2.3.

2.4.3 Each drop will form a bead in the Calcium Chloride solution and the beads should be left to solidify for at least one hour before use.

EXAMPLE 3: Production of a Device According to the invention.

Devices were formed by inserting alginate bead reporting means (made according to Example 2) into a silicone substrate.

The inventors selected a silicone (Sylgard® 184 Silicone Elastomer) as a substrate for retaining the reporting means although it will be appreciated that alternative silicones and other elastomer substrates may also be used in the devices of the invention.

Sylgard® 184 Silicone Elastomer is supplied as two-part liquid component kits comprised of Part A/Part B to be mixed in a 10: 1 ratio by weight or volume. When liquid components are thoroughly mixed, the mixture cures to a flexible elastomer. The silicone was found to be useful for supporting a bead or beads (e.g. as made according to Example 2) and also for preventing water loss and diffusion of the water soluble components of the device.

3.1 Production of a silicone substrate

A silicone substrate was made by the following procedure:

1. Mix Part A/Part B in a 10: 1 ratio by weight or volume.

2. Add the curing agent as per manufacturer's instructions and mix well.

3. Centrifuge at 5000rpm for 3 minutes to remove any trapped air during the mixing process.

4. Carefully pour into a mould from low height, to prevent the addition of bubbles. The mould used in this example was disk shaped, approx. 0.5cm thick and had a diameter of approx. 3.0cm. The mould was also shaped such that 5 pores would be formed in the substrate that were arranged as illustrated in Figure 1(b).

5. Heat at 50°C for 2 hours to allow the silicone to cure.

6. Remove from mould.

3.2 Placing reporting means into the silicone substrate

3.2.1 The silicone substrate was placed into a separate sterile petri dish and labelled as appropriate.

3.2.2 Beads from 2.4.2 were poured out of the Calcium Chloride solution out of the universal tube into a sterile petri dish.

3.2.3 Using flamed forceps, beads were placed into the pores in the silicone substrate.

Figure 1(a) is a schematic and Figures 3 and 4 show photographs of devices prepared according to the methods of Examples 2 and 3.

EXAMPLE 4: Production of a thin sheet device according to the invention.

A polyurethane substrate (DermaBak ^® Advanced Dermal Materials Fl 10 foam membrane, 0.4mm thickness) was perforated with a grid of holes that were arranged to receive the alginate bead reporting means made according to Example 2 and cut to size.

The substrate was then placed on a flat surface for receiving the reporting means. Optionally the perforated substrate may be placed on top of (and adhered to) a polyethylene mesh (Delstar Technologies, H518) with pore size of 75-200μιη (which in use acts to retain the reporting means in the substrate). In turn the substrate (or mesh if in place) may be placed on top of (and adhered to) a peel away sheet (which acts to keep the device sterile and the reporting means hydrated). Once the substrate and any mesh or peel away strip are assembled, a growth control alginate bead (with no selection factor) was positioned in a central hole in the perforated substrate. Four alginate beads where then positioned in holes around the growth control alginate bead in a clockwise direction. These beads contained the antibiotics specified in Table 1 as selection factors.

A transparent, silicone of 60gsm thickness non-perforated protective layer is then placed over the substrate containing the reporting means and held in place by a solvent based adhesive on each layer. This transparent layer represents the viewing surface of the device.

The assembled devices are then inoculated from the exposed surface (i.e. the side of the device that does not have the silicone protective sheet.) EXAMPLE 5: Testing a Device According to the invention.

Experiments were conducted in which devices according to the invention (produced according to the methods in Example 3) were exposed to Simulated Wound Fluid containing bacteria extracted from wound dressings.

5.1 Methods

5.1.1 Materials

5.1.1.1 Devices were prepared according to the methods of Example 3 with 5 reporting means arranged such that 4 reporting means containing clindamycin, cephalexin, co-amoxyclav and ciprofloxacin were arranged around a central reporting means without a selection factor.

5.1.1.2 Simulated Wound Fluid (SWF) comprises of 0.45% NaCl, 0.05% peptone from gelatine digest, 50% foetal bovine serum and 49.5% water.

5.1.2 Protocol for Bacterial Extraction and Analysis from Wound Dressings

Worn wound dressings were obtained from a population of patients with chronic foot ulcers.

5.1.2.1. Cutting a sample from the dressing

Using flamed forceps wound dressings were placed into separate sterile petri dishes. 3x1 cm ² samples of the wound dressing were measured from separate areas of the dressing and cut out using flamed scissors.

5.1.2.2 Bacterial extraction from the wound dressing

Each 1cm ² dressing was transferred into a bijou containing 5ml simulated wound fluid (5.1.1.2) and vortexed for 1 minute. Flamed forceps were then used to squeeze, drain, and remove the dressing sample from the bijou.

Six serial dilutions (neat to 10 ^"6 ) in PBS where then made of some of the supernatant. The Miles and Misra technique was performed to validate the level of bacteria extracted from the wound dressing. Plates were incubated at 37°C overnight and colony counts validated the level of bacteria used to inoculate the alginate beads.

5.1.2.3 Inoculation of device

Devices were inoculated by pipetting 50μ1 of neat suspension from 5.1.2.2 directly onto the alginate beads within the devices. The devices were then incubated at 37°C, and time lapse camera used to take photographs over 24 hours.

5.2 Results

Figure 4 shows devices with alginate bead reporter means containing antibiotic selection factors (1 - coamoxyclav, 2 - clindamycin, 3 - ciprofloxacin, 4 - cephalexin) and a growth control bead in the centre (with no antibiotic) in the centre of the device which had be inoculated with simulated wound fluid (SWF) and incubated for 24 hours. The threshold for activation of reporting means was set at 10 ⁵ CFU/mL.

The device in Panel A was innoculated with uninfected SWF and it can be seen that none of the reporting means changed colour (i.e. they were unactivated).

Panels B-D were each exposed to 4 different wound dressing extract which had been prepared according to method 5.1.2. In each case the clinician had suspected that the wounds may be infected.

Panel B demonstrates the SWF contained a level of bacteria which was greater than 10 ⁵ CFU/mL, Furthermore the bacterium were resistant to coamoxyclav and clindamycin, but sensitive to ciprofloxacin and cephalexin. Panel C demonstrates the SWF contained a level of bacteria which was greater than 10 ⁵ CFU/mL, Furthermore the bacterium were resistant to clindamycin, but sensitive to coamoxyclav, ciprofloxacin and cephalexin

Panel D demonstrates the SWF contained a level of bacteria which was greater than 10 ⁵ CFU/mL, Furthermore the bacterium were resistant to coamoxyclav and cephalexin, but sensitive to clindamycin and ciprofloxacin.

It will be appreciated that from the photographs in Figure 4 that devices according to the invention will be of great value to clinicians managing wounds and particularly chronic wound such as leg and foot ulcers which are prone to infection. The devices provide a simple test for establishing if there is any infection (activation of the central reporting means) and also to inform the clinician which antibiotic may be best used for treating that wound.