WO2016156773A1

WO2016156773A1 - Microbial sensing devices

Info

Publication number: WO2016156773A1
Application number: PCT/GB2016/000062
Authority: WO
Inventors: Curtis DOSBSON; Nishal GOVINDJI-BHATT
Original assignee: Microbiosensor Limited
Priority date: 2015-03-30
Filing date: 2016-03-29
Publication date: 2016-10-06
Also published as: WO2016156773A8; GB201505387D0

Abstract

The invention concerns devices for detecting and/or identifying microorganisms in the environment of a wound. The devices comprise a surface with at least one reporting means disposed on it which comprises a metabolic indicator and a selection factor. The selection factor selectively permits growth of microorganisms within the pore and as result the metabolic indicator may only activated by preselected microorganisms. In some embodiments the selection factor restricts device activation to predetermined threshold concentrations of microorganism. The devices are particularly useful for helping a clinician to understand the sensitivity of infecting micro-organisms in a wound to selection factors (which may be antibiotics) that are included in the reporting means. This understanding helps when deciding upon a therapeutic strategy for treating or preventing wound infections.

Description

MICROBIAL SENSING DEVICES

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 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 microbes in a variety of situations. For instance Acousta et al. (WO2005/095635) disclose a food microbiological sensing device, in which a housing comprising a pH sensitive material with opposing first and second surfaces are held near a possible source of microbial contamination, allowing gases released by the microorganisms present in the food sample to pass through the material and cause a colour change.

Sophisticated microarray technologies have also been disclosed which could provide diagnostic information regarding infectious agents, through molecular interactions of nucleic acids in clinical samples with arrays of molecular probes incorporated into test chips; such samples would be applied to these devices by a skilled operator, and detected by additional equipment or chemistry (US2002095073A1).

An improvement in the methods available for diagnosing antibiotic resistant infection has been disclosed by Frimodt-Moller, in which agar plates are divided into segments containing differing antibiotics or inhibitory agents; this can allow the identification of organisms in laboratories through recognition of particular growth patterns (WO200926920 Al).

However, none of the methods described above provide a small "micro" sensor which might be incorporated directly into a wound dressing, or attached discretely to a surface of a dressing, allowing on-going monitoring of levels or types of microbial contamination in the vicinity of a wound and without any need for operator intervention. Several technologies might be adaptable for use in this way, however these have significant limitations. For instance, Swanson and Rimmer l describe a responsive hydrogel approach, in which a fluorochrome is incorporated into a medical device hydrogel, which is responsive to a pH change caused by the presence of infectious agents (WO2008/059274). Booher (WO2006/133430) describes an apparatus for detecting microbial growth beneath a wound dressing, in which gases produced by microbial contamination pass into the device leading to a change in the colour of a pH sensitive dye. However all these approaches rely on a method which is not specific to microorganisms, and which requires an infection to produce a marked changed in the pH of patient tissues or gas production by microorganisms.

Two prior art approaches involve an approach that is more specific for microbial detection by utilising a layer which is degradable by microorganisms, as the means of detection. Ferguson et al. (US2004/0043422 Al) disclose a device in which a pH sensitive / signalling layer is protected by a microbially degradable layer in contact with a patient. In the event that the device becomes colonised, the degradable layer is broken down and the signalling layer undergoes a colour change to indicate the presence of microbes. Martin et al (US 2008/0057534 Al) also make use of a microbially degradable layer, in this case uncovering a previously "hidden" graphic symbol. Both these devices are capable of triggering partially, and thereby producing an unclear signal to the operator. They cannot be used to provide diagnostic information about the nature of the infection, or an indication of the numbers of infectious agents present. They are not capable of amplifying the signal produced by a challenge dose, and therefore cannot be adapted for use in situations where lower levels of infectious agents are to be detected. Furthermore the prior art devices cannot discriminate between the types of microorganisms that can activate such devices.

The present invention seeks to obviate one or more of the deficiencies associated with the prior art. 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. The inventors realised that the technology disclosed in WO2013/083993 could be adapted and refined to provide a device that can be used in particular 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 surface and wherein the surface has disposed on or in it at least one reporting means comprising:

a metabolic indicator; and

a selection factor;

wherein the selection factor permits growth of specific microorganisms within the pore 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 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 formed into a wound dressing. When this is the case a swab of the surface of a wound 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 period of incubation 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 comprises 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 epidermitus, Streptococcus mitis, Streptococcus sanguis, Enterococcus faecium, Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Enterococcus faecalis, Klebsiella pneumonia, Candida albicans, or gram negative bacil li . 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 may be used according to the invention. It is useful because the inventors have appreciated that it has minimal activity for limiting microbial growth, replication or viability. Accordingly MTS can be used in embodiments where the threshold concentration of microorganisms required for triggering a device is required to be low.

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is another preferred indicator for use according to the invention. It is particularly useful because the inventors have appreciated that it has activity for limiting microbial growth, replication or viability. Accordingly the concentration of MTT can be manipulated to additionally control the threshold of microorganisms required to trigger a device.

Embodiments of this invention in which MTT is exploited in the reporting means, may comprise at least about \0μ§/πύ, 20μg/ml, 30μg/ml, 40μg/ml, 50μg/ml, 75μg/ml, 100μg/ml, 125μg/ml, 150μg/ml, 2(^g/ml, 250μg ml, 300μg/ml, 350μg/ml, 40⁽^g/ml, 45⁽^g/ml, 500pg/ml, 55( g/ml, βθθμ^ηιΐ, 750μg/ml or about l,00(^g/ml MTT. Preferably the reporting means comprises 100-75(^g/ml and more preferably about 400-60(^g/ml of MTT. In a preferred embodiment about 50C^g/ml of MTT is used. About the same quantities of MTS may be employed when it is used instead of MTT.

According to one embodiment of the invention, the inventors have found that a combination of tetrazolium salts may be used to make reporting means according to the invention. For instance MTS may be combined with MTT. Accordingly reporting means of devices may comprise 100-750 g /ml MTS and 100- 750 g /ml MTT and more preferably may comprise 400-600 μg /ml MTS and 150- 500 μg /ml MTT.

When the reporting means is disposed on the surface as a powder it is preferred that each reporting means comprises 0.5 - 1,000 μg of powder, preferably 0.25 - 500 μg of powder, and more preferably 0.5 - 100 μg of powder. Such powders may be hydrated in volumes of approximately 1-200μΙ. of fluid (e.g. wound exudate) to give concentrations of indicator as discussed above. For instance preferred hydrated reporting means may comprise a final concentration of about 500μg/ml of MTT in a final volume of about l-200uL.

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-P-D-galactosaminide, a-D-glucopyranoside, β-D- glucopyranoside, N-acetyl-p-D-glucosaminide, β-D-fucopyranoside, β-D- xylopyranoside and/or 5-bromo-6-chloro-3-indoxyl-P-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 microsensor 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. 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 ApoEi_{41 -}i49 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 pneumonia.

(c) Triclosan is effective against staph, species and Klebsiella pneumonia 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- 1 ,3 -dioxane

• Dimethyldioctadecylammonium chloride

• Cetnmide

· 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 DBNPA (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 10⁷ 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 > 10⁶ - 10⁷ CFU / 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. 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 presense 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 antibiotics 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 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 750 g/mL (for instance about 500μg/ml). Media and/or nutrients that support or encourage microbial growth.

The inventors realised that in some embodiments of the invention that the device should further comprise a media and/or nutrients that support or encourage microbial growth. The media and/or nutrients may be used to encourage the microorganism to grow or multiple such that the device may be triggered. For instance such media may be helpful for detecting microorganisms when they are found in low numbers and, unless they are encouraged to grow, would not be present in a wound in sufficient numbers to trigger a device. Therefore according to a preferred embodiment of the invention there is provided a device for detecting and/or identifying microorganisms in the environment of a wound, said device comprising a surface and wherein the surface has disposed on or in it at least one reporting means comprising:

a metabolic indicator;

a media and/or nutrients that support or encourage microbial growth; and a selection factor;

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

microorganisms.

During development work the inventors found that a number of factors can potentially lead to the degradation of the reporting means or can lead to the false triggering (i.e. the generation of the reporting signal in the absence of a micro- organism) of the metabolic indicator. The inventors found that selection of an inappropriate media and/or nutrients that support or encourage microbial growth was a significant factor.

The inventors established that a media and/or nutrients should be chosen that: (i) maintains viable micro-organisms in the reporting means and in some embodiments supports or encourages microbial growth and/or division;

(ii) takes into account whether a narrow spectrum or broad spectrum of microorganisms needs to be maintained and detected;

(iii) will not result in false triggering of the indicator in the absence of microorganisms;

(iv) preferably is capable of permitting selective and sensitive sensing of microorganisms in volumes of reporting means that are less than 500μ1 and more preferably less than about 50μ1; and

(v) will not degrade or inactivate the threshold factor or selection factor contained within the reporting means.

The inventors applied a great deal of inventive endeavour to select media and/or nutrients with the correct balance of features (i)-(v) above. When tetrazolium salts are used as the metabolic indicator, the inventors found that these indicators are inappropriately converted to formazan by a number of commonly used media/broths without exposure to any microorganisms (i.e. (iii) above was at issue). They were able to assay this by measuring, over time, the Optical Density (at 570nm) of media solutions mixed with the tetrazolium salts. Tests were carried out 24°C, 30°C, 40°C 60°C or 80°C and for 1 hours, 2hours, 8 hours, overnight (10-20 hours) and 24 hours. Media/broths may be discounted that turned black (i.e. formazan was formed) when the broth containing the indicator was incubated under these conditions A media and/or nutrients used in the reporting means is preferably a media or broth that does not cause the conversion of a tetrazolium salt into formazan when the media or broth is incubated with a tetrazolium salt at 40°C over night. Alternatively the media and/or nutrients used in the reporting means is preferably a media or broth that does not cause the conversion of a tetrazolium salt into formazan when the media or broth is incubated with a tetrazolium salt at 24°C for 8 hours or 24 hours. It is more preferred that the media or broth does 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. Of the media/broths which resulted in no, or irrelevant triggering, the suitability of the broths had to be further considered in view of (i), (ii), (iv) and (v) above. For instance the inventors found that only a small number of broths were suitable for incorporating in a reporting means that comprised a tetrazolium salt (e.g. MTT or MTS) and which was required to sense a broad spectrum of microorganisms and in which an antibiotic was used as a selection factor. Examples of such broths include Mueller Hinton and Wilkins Chalgren broths.

Surface

The surface 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 surface is a membrane or film

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

Surface membranes/films and/or the barrier membrane 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 or within the wound dressing has been activated. Preferably such membranes comprise the outermost component of a wound dressing when applied to the skin. Transparent barrier layers are also referred to herein as visualizing sheets. These transparent layers may optionally be covered with a protective sheet. Preferably the protective sheet is opaque and may be removed when a user wishes to observe the reporting means.

Membranes, films and sheets which may be 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-1, 4 -phenylene) , poly (oxycarbonyloxy- 1,4 (1 , 4- phenyleneisopropylidene-1, 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, poly ally lbenzene, polyvinylbutyl ether, polyvinyl formate, polyvinyl phenyl ether, polynorbornadine , polycarbonate, hydrophobic polyesters and polyurethanes, and mixtures thereof. Reporting means can be applied to membrane/film surfaces in a number of ways. For instance the reporting means can be sprayed as a powder (e.g. using a micro compressed air sprayer). Alternatively 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). By way of example, figure 1 illustrates how reporting means (some applied as dots others arranged as concentric rings) may be sprayed in an array onto a surface that is incorporated into a foam pad dressing. It will also be appreciated that different reporting means may be sprayed onto the same surface. Thus, by way of further example, the "dot" reporting means shown in figure 1 may be applied to the surface by one spraying device and subsequently the "concentric ring" reporting means (e.g. comprising a different selection factor) may be sprayed using a second spraying device.

Alternatively capsules comprising powdered reporting means can be applied to the surface, It will be appreciated that the capsule should allow microorganisms, and if necessary wound exudate, to enter the capsule and trigger the device. Alternatively the capsule may be a dissolvable capsule which dissolves upon contact with wound exudate.

It will be appreciated that the reporting means does not have to be in powder form. Accordingly a compressed tablet, a viscous medium in which the reporting means is embedded, or a reporting means combined with a rehydrating gel medium (e.g. alginate) may be applied to the surface instead.

It will be appreciated that the volume of reporting means applied to a 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. Typically an individual reporting means will have a volume of 1 -200μί.

2. Embodiments wherein the surface comprises a pore

In other embodiments the substrate has a surface defining a pore for containing the reporting means. Reporting means contained within pores preferably also comprise a solid or semisolid substrate. Devices which do not include selection factors are disclosed in WO2013/083993. These devices may be adapted to form devices according to this embodiment of the invention and the devices disclosed in WO2013/083993 are incorporated by reference herein. The metabolic indicator contained within pores of devices according to this embodiment of the invention may be as described above or as described in WO2013/083993.

The selection factor within pores of devices according to this embodiment of the invention may be as described above.

The media and/or nutrients that support or encourage microbial growth described above or as described in WO2013/083993 may be included when the device is an embodiment of the invention which includes such media and or nutrients in the reporting means.

By "pore" we mean any recess within the surface of the device that may retain the reporting means. It will be appreciated that a pore may be a cylindrical recess within the surface with a circular aperture for contact with the sample. However pores as referred to herein may also define more complex recesses within the surface of the device. For instance the pore may comprise a complex pattern that is etched or engraved into the surface as discussed below.

Preferred devices according to this embodiment of the invention may have a reporting means which comprises: a solid or semi-solid substrate; a metabolic indicator for reporting the presence of living organisms or cells; media and/or nutrients that support or encourage microbial growth and a selection factor for allowing growth of selected microorganisms. The inventors have surprisingly found that such reporting means: (a) provide real-time, clear and simply understood information on microbial contamination to an untrained operator, untrained wearer of a medical device, or untrained user of an area being monitored for microbial contamination;

(b) can be incorporated in small volumes into miniature devices according to the invention (also referred to herein as "microsensors") while retaining suitable sensitivity and selectivity for reporting microbial contamination; and

(c) the type of selection factor in the reporting means can be altered to regulate what type of microorganism can activate the device and also to regulate the threshold concentration of microorganisms required to activate the device.

Preferred devices comprise a reporting means that comprises a solid or semi- solid substrate which is agar or agarose. The precise quantity being determined by the degree of substrate 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%- 1.1% 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.

The pores of devices according to this embodiment of the invention may be cylindrical and have a diameter of less than 10mm and preferably a diameter of less than 5mm. Preferred cylindrical pores have a diameter of between 0.5 and 4mm and may have a diameter of 4, 2, 1 or 0.5mm. It will be appreciated that it is preferred that, for ease of use, that the pore is not so small that it will be invisible to the naked eye. Preferred cylindrical pores are less than 10mm deep, more preferably less than 5mm deep and preferably about 2mm deep or less. Preferred cylindrical pores may be 4x2mm, 2x2mm or lxlmm (diameter x depth).

The inventors have been able to devise miniaturised devices (i.e "microsensors") that have applications that could not be envisaged for the microbial sensors that are known to the art that tend to be large and or require a skilled user. In this embodiment, miniaturization has been made possible by developing pores with a limited volume. Significant inventive endeavour has been employed to develop small devices which comprise suitably sensitive and selective reporting means. Devices according to the invention may typically have pores which are a few millimetres deep or even only a few micrometres deep. Accordingly a typical pore will be adapted to have a volume of less than 500μ1, less than 250μ1, less than ΙΟΟμΙ, less than 50μ1 or less than 5μ1. In one embodiment of the invention, a pore may be portioned such that it will have a volume of about 25 μΐ which may be adapted to contain about 20μ1 of a reporting means and about 5μ1 of a liquid containment layer (see below). In another preferred embodiment a pore may be portioned such that it will have a volume of about 6.2μ1 which may be adapted to contain about 5 μΐ of a reporting means and about 1.2μ1 of a liquid containment layer. In another preferred embodiment a pore may be portioned such that it will have a volume of about 1.6μ1 which may be adapted to contain about 1.4 μΐ of a reporting means and about 0.2μ1 of a liquid containment layer.

The device may also comprise a containment layer as discussed above. The containment layer may be located between where the wound/wound sample will be located when in use and the reporting means in the pore. The layer should be adapted such as to allow microorganisms to enter the reporting means but substantially retain the components of the reporting means within the pore. The layer may be effective for maintaining the viability of the reporting means such that the device will have a shelf- life of several weeks, several months or more preferably a shelf-life of one, two or more years.

The containment layer is preferably contained within the pore and lies over the top of the reporting means. In some embodiments the pore may be contained within a depression, recess or dimple in the sampling surface of the device. In such embodiments the containment layer may be positioned in the depression, recess or dimple above the pore.

In one embodiment the containment layer may prevent any material contained within the reporting means becoming desiccated and/or prevent diffusion of low molecular weight constituents out of the reporting means. The containment layer may comprise a viscous liquid approximately 1 - 1,000 μηι thick or deep between where the sample will be located (when in use) and the reporting means. In preferred embodiments, where a cylindrical pore is l-2mm deep the barrier/containment layer will typically be 100-400μπι thick/deep. The containment layer may be alginate based, pectin based, hyaluronic acid, glycerol or cellulose based. In a preferred embodiment the containment layer is carboxymethyl cellulose (CMC). By way of example the inventors have found that 5μ1 of 5% carboxymethyl cellulose in PBS represents an effective containment layer for a 4x2mm cylindrical pore with a volume of about 25μ1. The remaining 20μ1 of such a pore may comprise the reporting means (e.g. 20μ1 of a Wilkins Chalgren Agarose containing 500μg ml MTS and 512 μg mL methicillin (as a selection factor).

Devices according to this embodiment of invention may additionally include a physical barrier that acts to retain the reporting means within the pore. Such a physical barrier may be located between where the sample will be located when in use and the reporting means. Alternatively the barrier may be between where the sample will be located when in use and the containment layer (if present) or the barrier may be between the containment layer and the reporting means. The physical barrier may be contained within the pore and lie over the top of the reporting means. In preferred embodiments the pore may be contained within a depression, recess or dimple in the sampling surface of the device. In this case it is preferred that the reporting means and containment layer are contained within the pore and the physical barrier is positioned over the pore and fixed within the depression, recess or dimple.

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 pore. 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 1-1,000μπι, preferably a mesh pore size of 50-500μηι, more preferably a mesh pore size of 75-200μπι and most preferably a mesh pore size of approximately ΙΟΟμπι. Typically a mesh will be less than 500μπι thick. Meshes may be manufactured from PMMA, PET or polypropylene and functionally equivalent polymers. A most preferred mesh has a mesh size of approximately ΙΟΟμπι and may be fabricated from 150μπι thick PMMA.

Devices according to this embodiment of the invention may be independent devices which essentially comprise a housing for the pore; the pore with reporting means contained therein; and any containment layer or physical barrier. The housing is preferably formed to enable or encourage microorganisms to enter the pore. The housing may be adapted as discussed above in connection with the first aspect of the invention. Examples of such devices are shown in figure 3.

Preferred devices may comprise independent units that may comprise a moulded plastic house containing a pore. The pores in such devices may be 2x2mm and in a preferred embodiment have lxl mm pores.

Most preferred reporting means for use in pore containing devices comprise a solution of 1.0% agarose in Wilkins Chalgren broth containing 50(^g/ml MTT and an antibiotic selection factor (e.g. about 512 μg/mL methicillin). When the pore is lxlmm the volume of the pore in such devices is about 1.6μ1 and may contain about 1.4 μΐ of a reporting means and about 0.2μ1 of a liquid containment layer (e.g. 5% carboxymethyl cellulose). A mesh may also be fitted above the containment layer to retain the reporting means in the pore. Arrays of Reporting Means

Reporting means according to the first aspect of the invention may be arranged in arrays. Such arrays may comprise a plurality of pore containing devices arranged in a grid formation or other pattern. Alternatively such arrays may comprise a grid formation or other pattern of reporting means arranged on devices with membrane or film surfaces.

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 a most preferred embodiment such arrays are incorporated within wound dressing. Such wound dressings may incorporate an array of reporting means on a membrane or film surface. Alternatively a number of pore containing devices may be woven or otherwise fixed into a wound dressing.

Arrays incorporated in a wound dressing preferably include reporting means containing 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 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 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. Accordingly 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 (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 dressing. Such uses of the devices represent a most preferred embodiment of the invention and are discussed below.

In preferred embodiments that employ a tetrazolium dye as the indicator, activation of the devices within the dressing will result in the production of dark dots (or other pattern) in the dressing (for devices containing selection factors to which the microorganisms in the sample are resistant) or 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 dressing 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.

An array according to the invention that has been incorporated into a wound dressing is illustrated in figure 1 and discussed in more detail below.

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 surface with an array of reporting means. In another embodiment the wound dressing comprises an array of pore containing devices. Wound dressings comprising a device with a film or membrane surface with an array or reporting means

A device with a film or membrane surface 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 reportmg 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 1-1,000μηι, preferably a mesh pore size of 50-500μπι, more preferably a mesh pore size of 75-200μπι and most preferably a mesh pore size of approximately ΙΟΟμιη. Typically a mesh will be less than 500μπι thick. Meshes may be manufactured from PMMA, PET or polypropylene and functionally equivalent polymers. A most preferred mesh has a mesh size of approximately ΙΟΟμπι and may be fabricated from 150μπι 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, and particularly when the viewing sheet and/or membrane surface is/are permeable, 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.

Preferred embodiments of devices, or components of a wound dressing, which comprises combinations of barriers/meshes, visualising sheets and protective sheets are illustrated in figure 2 and described below. Wound dressings comprising an array of pore bearing devices

Individual pore bearing devices may be embedded or woven into the fabric of the dressing and/or affixed by adhesive or may be affixed to a membrane or film surface as discussed above.

The pore bearing devices may be combined with barriers/meshes, visualising sheets and protective sheets are discussed above.

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.

Alternatively the devices may 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. 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 f 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: is an illustrative photograph of a wound dressing incorporating a device with an array of reporting means affixed to a membrane surface in which some of the reporting means have been activated.

Figure 2: illustrates sections of devices according to a number of embodiments of the invention that may be incorporated in wound dressings.

Figure 3: illustrates a section through an array of pore bearing devices according the invention that may be incorporated in wound dressings

Figure 4A-C: views of an embodiment of devices according to the invention which incorporate pores.

Figure 5: represents results illustrating how a selection factor which is known to be ineffective against MRSA but effective against sensitive Staphylococcus aureus can be used in the development of devices according to the invention that may be selectively activated (discussed in Example 1).

Figure 6: is an illustrative example of results obtained by culturing varying numbers of CFUs of Pseudomonas aeruginosa with a range of concentrations of PHMB as discussed in Example 2

Figure 7: represents photographs of devices comprising methicillin as a selection factor which were exposed to Staphylococcus aureus or MRSA as discussed in Example 3;

Figure 8: is a photograph illustrating some of the data showing the effect of PHMB on the activation of devices exposed to Staphylococcus aureus as discussed in Example 4; and

Figure 9: represents photographs of devices challenged with agents used in wound care or with Pseudomonas aeruginosa as discussed in Example 5. Figure 1 is a photograph of a wound dressing 1 with a top most layer formed of a device according to the invention which comprises a membrane surface 2 onto which are applied arrays 3 of reporting means (numbered 1 -5 in the photograph) that are arranged as a grid of 4x4 arrays on the dressing. Each array comprises five reporting means that are printed or sprayed onto the surface as concentric rings 4. Each concentric ring is in turn arranged such that it encircles five further reporting means that appear as dots 5. Each of the reporting means 4 and 5 is of sufficient size that it can be visualised by the naked eye when the indicator contained therein has been triggered (the darker coloured rings or dots) or not (the lighter coloured rings or dots).

In use the dressing 1 is applied to a wound (e.g. a diabetic foot ulcer) and left in place for sufficient time for wound exudate to soak through absorbant layers (not shown in figure 1) of the dressing to the reporting means.

In the dressing illustrated in figure 1 the "dot" reporting means 5 contain a selection factor whereas the concentric ring reporting means 4 contain no selection factor and act as growth controls.

Concentric ring reporting means 4 which do not trigger (e.g. each of the rings in the array 6 in the bottom right hand corner of the dressing) indicate that no microorganisms were found in the area of the wound below that array. In contrast four out of five of the concentric ring reporting means are triggered in the arrays 7 in the top left hand quadrant of the dressing. This indicates that microorganisms were present in the area of the wound below that portion of the dressing 1.

Each dot reporting means 5 within the arrays contains a different selection factor. In the arrays 7 in the top left hand quadrant of the dressing it can be seen that three concentric ring reporting means 8 have been triggered, but the dot reporting means contained within them have not been triggered. This indicates that these parts of the arrays were exposed to microorganisms and that growth of those microorganisms was inhibited by the selection factor in the dots (which did not activate). Therefore those selection factors may be suitable therapeutic agents for treating the infection in the wound. In contrast the top part of these arrays 7 show both the concentric ring and dot reporting means are activated 9. This indicates that the microorganisms in the wound are resistant to the selection factor contained within the activated dot and informs the patient, clinician or other user of the dressing that this specific selection factor would not be a suitable candidate for treating the wound.

Figure 2 provides illustrative cross-sections of devices that are suitable for incorporation into the wound dressing illustrated in Figure 1. In this figure solid lines represent impermeable sheets/layers whereas dashed lines represent semi-permeable layers/sheets.

Figure 2a is a section passing through reporting means defining concentric rings 4 and dot reporting means 5 of an array 3. The reporting means are sprayed or printed as a dry powder onto a membrane surface 2. A mesh 10 is placed on the wound side of the device with semi-permeable parts of the mesh aligning over reporting means. The reporting means is overlaid with a non-permeable, transparent viewing sheet 11 and a non-permeable, opaque protective sheet 12. At the time of viewing, the protective sheet 12 can be peeled to visualize the reporting means 4, 5 Figure 2b represents an embodiment of the invention similar to the one illustrated in Figure 2a, but with a protective sheet 12 with semipermeable parts aligned over the reporting means 4, 5 and also a viewing sheet 11 with semipermeable parts aligned over the reporting means 4, 5 which can be peeled to visualize the devices. This embodiment would be appropriate where the device is on the outer surface of a dressed wound. It may be advantageous to allow exudate to be drawn through reporting means 4 and 5 and evaporated.

Figure 2c represents another embodiment of the invention similar to the one illustrated in Figure 2b, but with semi-permeable parts of the protective sheet 12 and viewing sheet 11 adjacent to the reporting means 4, 5; and with non-permeable parts aligned over reporting means 4, 5. In this embodiment the surface 2 is entirely semipermeable. Devices according to this embodiment of the invention allow greater volumes of exudate to be drawn around the reporting means but not through them. Figure 2d represents an embodiment of the invention wherein six devices 13 are embedded in wound dressing material 14 that is impermeable to bacteria. Each device 13 comprises a surface 2 which bares a reporting means 15. The wound facing surface of the dressing comprises a semi -permeable mesh 10 and the outer surface of the dressing comprises a non-permeable visualizing sheet 11 and a protective sheet 12. Exudate and bacteria are drawn up through cavities 16 in the wound dressing material 14 to interact with the devices 13. It will be appreciated that the devices 13 shown in this embodiment may be replaced with pore bearing devices according to the invention.

Figure 2e is a variant of the embodiment illustrated in Figure le wherein the surface 2 is sprayed/printed with a dehydrated material 17 in addition to a dry powder reporting means 4, 5 which is affixed above the material. In this embodiment the dehydrated material 17 is rehydrated upon contact with wound exudate and may comprise a media and/or nutrients that support or encourage microbial growth and/or may comprise a solid or semi-solid substrate as contemplated herein.

It will be appreciated that the reporting means illustrated in figures 2a - 2e need not be a dry powder. For instance the reporting means may be affixed to the surface as a tablet (i.e. a compressed powder). Furthermore the reporting means may be formed as a dissolvable capsule or embedded in a viscous material.

Figure 3 illustrates an array 3 of six devices 18 with pores that retain the reporting means. The pores are within the surface 2. In this example some of the reporting means are shown as triggered devices (dark shading) whereas others are untriggered (light shading). In this embodiment the top of the device is presented to the wound. The device comprises a mesh 10 with semipermeable parts aligned over the reporting means to allow wound exudate to enter the pores. Visualizing sheets and a protective sheets may be used but are not shown in this illustration.

In some embodiments of the invention (for instance the devices illustrated in Figures 2 and 3) the reporting means may be a combination of a metabolic indicator (e.g. MTT), a selection factor (e.g. an antibiotic), a media and/or nutrients that support or encourage microbial growth (e.g. Wilkins Chalgren broth mix) and a solid or semi- solid substrate (e.g. agarose). Such reporting means may be in powder form or may be hydrated.

Figures 4A and 4B show a single pore bearing device according to one embodiment of the invention 20 that may be woven into a wound dressing or affixed to a membrane of film for incorporation in a wound dressing. In this embodiment, the microsensor unit 20 comprises a pore 22 defined within the sampling surface 24 of a substrate 26. The pore 22 is 500μηι wide and extends 250um into the substrate 26. Pore 22 connects to a chamber 28 containing the reporting means 30. The chamber 28 is cylindrical and about 1mm in diameter and extends about 2mm into the substrate 24 (in other words the chamber is about 2mm deep). Between the pore 22 and the chamber 28 containing the reporting means 30, is provided a barrier or containment layer 32. This layer 32 prevents desiccation and egress of components of the reporting means 30 from the chamber 28 and is about 50μπι deep.

Figure 4C shows an array of pore bearing device units 20 in a microsensor array 40. In this embodiment, the array 40 comprises 8x8 pores comprising reporting means. In this embodiment, the distance between the central point (i.e. the centre of each pore 22 defined by the surface 24 of the substrate 26) of each microsensor unit 20 from the central point of a neighbouring microsensor unit 20, is about 2.5mm

EXAMPLES

The inventors realised that there were no commercially available products that were small and simple which may be used for detecting microbial contamination of the wound environment. In particular there were no devices available which could be used to distinguish between types of microorganism in the wound environment and/or in which the threshold for device activation could be sensitively controlled.

Initial proof of principle experiments (Examples 1 and 2) were conducted to establish whether or not reporting means could bs generated that could distinguish/select between microorganisms and also for which the threshold for triggering of such reporting means could be controlled. The inventors then proceeded to adapt the pore bearing devices described in WO2013/083993 to produce novel devices with reporting means comprising selection factors and other preferred components. These devices were then tested to establish whether or not they may be used to distinguish between microrganisms in the wound environment (Examples 3 and 4). Further experiments were conducted to test whether or not the reporting means were compatible with wound dressings (Example 5).

EXAMPLE 1: Testing a Selection Factor according to the invention

Initial proof of principle experiments were conducted to establish whether or not reporting means could be generated that could distinguish/select between microorganisms. Experiments were conducted using methicillin as a putative selection factor. The agent was chosen because MRSA, a common cause of mortality and morbidity especially in the nosocomial setting, is resistant to beta-lactam antibiotics, including methicillin, as well as the cephalosporins.

1.1 Materials and Methods

1.1.1 Production of a range of reporting means according to the invention

A stock solution of 2% agar in 2x Wilkins Chalgren broth was made and autoclaved.

After cooling (to 40°C), a sterile filtered stock solution of MTS was added to the agarose broth stock to a final concentration of 500μg ml. Methicillin was also added to aliquots of the resulting solution such that reporting means according to the invention were made with a range of final antibiotic concentrations of 0μg/mL, 50 g/mL, 75 g/mL and 100μg/mL.

The final reporting means comprised 1% agar in lx Wilkins Chalgren broth with 500μg/mL MTS and methicillin at a range of concentrations: 0μg mL, 50μg mL, 75 g/mL and 100μg/mL.

ΙΟΟμΙ of the reporting means was then added to the wells of a microtitre plate, in triplicate using a Gilson pipette and allowed to solidify for an hour at 4°C. 1.1.2 Test Organism

A culture of Staphylococcus aureus ATCC 6538 and a clinical isolate of MRS A was grown overnight in tryptone soya broth at 37°C, 200 rpm. A stock suspension of 10⁹ CFU/mL in PBS was prepared from the overnight culture.

A stock was made to 10⁹ CFU/mL for both cell types and ΙΟμΙ, (10⁷cells) was added in triplicate to wells of the microtitre plate prepared in 2.1.1.

The plates were then incubated at 30°C overnight after which the results were observed.

1.2 Results

Figure 5 is a photograph of an microtitre plate illustrating the triggering of reporting means according to the invention when incubated with Staphylococcus aureus or MRS A in the presense of methicillin. The top row of wells contained no microorganism (control), the middle row was inoculated with 10⁷ CFU Staphylococcus aureus and the bottom row 10⁷ CFU MRS A. The micro-organisms were tested with a range of concentrations of methicillin (from left to right in triplicate: 0 μg/rnL 50μg/mL,

or 100μg/mL of the antibiotic). It can be seen that the antibiotic acted as a selection factor according to the invention. Staphylococcus aureus ATCC 6538, as expected, was sensitive to the antibiotic; failed to thrive in the reporting means; and none of the reporting means activated (middle row) in devices comprising 50 g/mL, 75μg/mL or 100 g/mL of the antibiotic (the first three columns were controls comprising no antibiotic). In contrast MRSA was resistant to the antibiotic; thrived in the reporting means; and lead to activation of the device at all concentrations of the antibiotic tested (bottom row).

Similar results were obtained for other microorganisms and other selection factors according to the invention. 1.3 Discussion

These experiments demonstrate that reporting means can be produced that can be selectively activated by microorganisms by incorporating a selection factor according to the invention. The results inspired the inventors to develop devices according to the invention.

EXAMPLE 2: Testing a Threshold Factor according to the invention

The inventors also hypothesised that it may be possible to alter the threshold concentration of microorganism at which the indicator is triggered by including a factor that will retard microbial growth.

Experiments were conducted with PHMB as a putative threshold factor. The agent was chosen because it is known to have broad spectrum antimicrobial activity.

2.1 Materials and Methods

2.1.1 Production of a range of reporting means according to the invention

A stock solution of 2% agarose in 2x Wilkins Chalgren broth was made and autoclaved.

After cooling (to 40°C), a sterile filtered stock solution of MTS was added to the agarose/broth stock to a final concentration of 500μg/mL to. PHMB was also added to aliquots of the resulting solution such that reporting means according to the invention were made with a range of final PHMB concentrations.

The final reporting means comprised 1% agarose in lx Wilkins Chalgren broth with 500μ&ΊηΙ. MTS and PHMB at a range of concentrations: ^g/mL, 2μg/mL, 4μg/mL, 8 g/mL, 16 g/mL and 32μg/mL.

ΙΟΟμΙ of the reporting means was then added to the wells of a microtitre plate, in triplicate using a Gilson pipette and allowed to solidify for an hour at 4°C. 2.1.2 Test Organism

A culture of Pseudomortas aeruginosa, ATCC 9027 was grown overnight in tryptone soya broth at 37°C, 200 rpm. A stock suspension of 10⁹ CFU/mL in PBS was prepared from the overnight culture.

10 fold serial dilutions were made (10⁹ - 10³ CFU/mL) of the stock suspension and ΙΟΟμί, of each dilution (10⁸ - 10² cells) was added in triplicate to wells of the microtitre plate prepared in 2.1.1. The plates were then incubated at 30°C overnight after which the results were observed.

2.2 Results

Figure 6 is an illustrative example of the results obtained by culturing varying numbers of CFUs (PBS control to 10⁸ CFUs) of Pseudomonas aeruginosa with a range of concentrations of PHMB (from left to right in triplicate: 0 μg mL, 8 g/mL, Ιόμ^ιηΙ. or 32μg/mL). It can be seen that the threshold for triggering the reporting means (i.e. the number of cells used to challenge the reporting means) increased proportionally to the concentration of PHMB. The relationship was linear and made the inventors realise that agents such as PHMB could be used as threshold factors according to the invention.

Similar results were obtained for other microorganisms and other threshold factors according to the invention.

2.3 Discussion

These experiments demonstrate that reporting means can be produced that can have the threshold for activation controlled by incorporating a threshold factor according to the invention. The results inspired the inventors to develop devices according to the invention. EXAMPLE 3: Testing the selectivit of devices according to the invention

The inventors proceeded to make and test devices according to the first aspect of the invention in which selection factors were incorporated in the reporting means. 3.1 Materials & methods

3.1.1 Device housing

Prototype device housings (defining the surface and pore of a device according to the invention) were manufactured by injection moulding using crystal polystyrene. The mould defined the housing, surface of the device and a 4x2mm cylindrical pore (diameter x depth). The overall dimensions of the device were 10mmxl0mmx3mm. The device was then sterilised.

3.1.2 Reporting Means

Wilkins Chalgren agar (1% agar) was made up and then autoclaved. After cooling (to 40°C), a sterile filtered stock solution of MTT and a stock solution of methicillin was added to final concentrations of 500μg/ml and 512 μg/ml respectively to form the reporting means.

3.1.3 Filling the device housing

20μ1 of the reporting means was then added to the pore of each device housing using a Gilson pipette and allowed to solidify for an hour at 4°C. 5μg/ml carboxycellulose (in PBS) was then placed on top of the reporting means as a containment layer in the top of the pore.

3.1.4 Test Organisms

A culture of Staphylococcus aureus ATCC 6538 and a clinical isolate of MRSA was grown overnight in tryptone soya broth at 37°C, 200 rpm. A stock suspension of 10⁹ CFU/mL in PBS was prepared from the overnight culture.

A stock was made to 10⁹ CFU/mL for both cell types and ΙΟμΙ, (10⁷cells) was added to devices. The devices were then incubated at 30°C overnight after which the results were observed. 3.2 Results

Figure 7 show two photographs of devices according to the invention that had been cultured with Staphylococcus aureus (top photograph) and MRS A (bottom photograph) and with reporting means containing 5^g ml methicillin (bottom row of each photograph) or no antibiotic (top row of each photograph). It can be seen that the antibiotic acted as a selection factor. Staphylococcus aureus ATCC 6538, as expected, was sensitive to the antibiotic. It failed to thrive in the reporting means comprising methicillin such that none of the devices activated (bottom row of the top photograph). In contrast MRSA was resistant to the antibiotic; thrived in the reporting means; and this lead to activation of the device in the presence of the antibiotic (bottom row of bottom photograph).

Similar results were obtained for other microorganisms and other selection factors according to the invention.

3.3 Discussion

These experiments demonstrate that devices according to the invention may be manufactured with a suitable selection factor (in this case methicillin) to distinguish between antibiotic sensitive Staphylococcus aureus and MRSA. It will be appreciated that such devices are useful for identifying what kind of microorganism may be infecting a wound.

EXAMPLE 4: Testing the threshold control of devices according to the invention

The inventors proceeded to make and test devices according to the invention in which threshold factors were incorporated in the reporting means. 4.1 Materials & methods

4.1.1 Device housing

Device housings (defining the surface and pore of a device according to the invention) were manufactured as described above in Example 3.1.1. 4.1.2 Filling the device housing

(a) Reporting means were made as described in Example 3.1.2 except PHMB was included to a final concentration of 0 g/mL (controls), 4μg mL, 6μg/mL, 8μg/mL, 32μ^ιη1-, and 128μg/mL instead of methicillin. (b) 20 μΐ of the reporting means was then added to the pore of each device housing using a Gilson pipette and allowed to solidify for an hour at 4°C. (c) 5μg/mL carboxycellulose (in PBS) was then placed on top of the reporting means as a containment layer in the top of the pore. 4.1.3 Test Organisms

Cultures of Pseudomonas aeruginosa (ATCC 9027) and Staphylococcus aureus (ATCC 6538) were grown overnight in tryptone soya broth at 37°C, 200 rpm. A stock suspension of 10⁹ CFU/mL in PBS was prepared from the overnight culture. 10 fold serial dilutions were made (10⁸ - 10⁴ CFU/ml) of the stock suspension and ΙΟμΙ of each dilution (10⁶ - 10² cells) was added in triplicate to devices prepared in 3.1.2.

The devices were then incubated at 30°C overnight after which the results were observed.

4.2 Results

Table 2 provides details of the threshold concentrations of bacterium that caused device activation with varying concentrations of PHMB as a threshold factor. Table 2:

Threshold at which devices triggered (CFU/mL)

P. aeruginosa S. aureus

50(Vg/mL MTS + (^g/mL PHMB 10² 10²

500μg/mL MTS + 4μg/mL PHMB 10² 10²

5( g/mL MTS + 6μg/mL PHMB 10³ 10³

500μg/mL MTS + 8μg/mL PHMB 10³ 10³

50(^g/mL MTS + 32μg/mL PHMB 10³ 10⁷

500 g/mL MTS + 128 μg/mL PHMB 10⁷

Figure 8 is a photograph illustrating some of the data on which the Staphylococcus aureus data in Table 2 is based. From left to right the photograph shows triggering when no threshold factor was present (the first three columns of devices); triggering in the presence of 6μg/mL PHMB (the second three columns of devices); and triggering in the presence of 32μg/mL PHMB ((the second three columns of devices). The rows, in descending order, represent devices inoculated with 10⁷ CFU/mL, 10⁶ CFU/mL, 10⁵ CFU/mL, 10⁴ CFU/mL and 10³ CFU/mL Staphylococcus aureus respectively. It can be seen that 32μg/mL PHMB blocked device activation at all concentrations of bacterium; all devices triggered in the absence of PHMB; and 6μg mL PHMB blocked activation at 10³ CFU/mL, but device triggering did occur at CFUs above this threshold.

4.3 Discussion

These experiments demonstrate that devices according to the invention may be manufactured with varying concentrations of a selection factor acting as a threshold factor (in this case PHMB). Accordingly devices may be manufactured with preselected thresholds for activation by varying the concentration of the factor in the reporting means. It will be appreciated that such devices are useful for quantifying the amount of microorganism in the environment of a wound. EXAMPLE 5: Testing the compatibility of devices and reporting means with wound care products

5.1 Materials & methods

Devices were prepared as described in Examples 3 and 4 and challenged by exposure to an unused silver dressing, an unused iodine dressing, an iodine ointment and saline. Devices were exposed to 10^s CFU/ml Pseudomonas aeruginosa (ATCC 9027) (grown as described in Example 4.1.3) as a positive control (i.e. sufficient bacterium to trigger the device). 5.2 Results

Unused silver dressings, unused iodine dressings, iodine ointment and saline failed to trigger the devices. This validated the usefulness of devices according to the invention because there was no false triggering of the devices by the dressings, ointment etc that would be used by a clinician in the wound environment.

Figure 9(A) is an illustrative photograph of devices comprising 500μg/mL MTS (indicator) exposed, left to right, to an unused silver dressing, an unused iodine dressing, an iodine ointment, saline and 10⁵ CFU/ml Pseudomonas aeruginosa. Only the device exposed to Pseudomonas aeruginosa triggered (the darkened reporting means) whereas uninfected wound care products did not activate the devices.

The inventors also sterilised the devices (which will be required if the devices are to be incorporated in sterile wound care products) using ethylene oxide (preconditioning: 18.5 hours, 37.5°C - 44.8°C; sterilisation exposure: 2.5 hours, 50.5"C - 50.8°C; and aeration: 8 hours, 38.9°C - 42°C) to examine whether or not the sterilisation process would lead to false triggering of devices according to the invention. Figure 9B is an illustrative photograph of devices comprising 500μg/mL MTS (indicator) exposed to saline (top row - a negative control) or 10⁵ CFU/ml Pseudomonas aeruginosa (bottom row - a positive control) after sterilization of eth devices. Only the devices exposed to Pseudomonas aeruginosa triggered (the darkened reporting means) whereas, as expected, devices exposed to saline did not trigger. This illustrates that devices according to the invention are not compromised by the sort of sterilization procedures that are required when manufacturing a wound dressing 5.3 Discussion

These experiments demonstrate that devices according to the invention are compatible with wound care products and sterilization procedures that are required as a step in the manufacture of such products.

Claims

1. A device for detecting and/or identifying microorganisms in the environment of a wound, said device comprising a surface and wherein the surface has disposed on or in it at least one reporting means comprising:

a metabolic indicator; and

a selection factor;

microorganisms.

2. The device according to claim I wherein the metabolic indicator is a tetrazolium salt.

3. The device according to claim 2 wherein the tetrazolium salt is one of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylt3trazolium bromide), MTS (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium), a water soluble tetrazolium salt (WST) or combinations thereof.

4. The device according to any preceding claim wherein the selection factor selectively inhibits the growth of bacteria or selectively kills bacteria.

5. The device according to any preceding claim wherein the selection factor is an antibiotic.

6. The device according to claim 5 wherein the antibiotic is one of methicillin. co-amoxiclav, cephalexin, ciprofloxacin, clindamycin or tobramycin

7. The device according to any one of claims 1-3 wherein the selection factor selectively inhibits fungal growth.

8. The device according to claims 7 wherein the selection factor is amphotericin.

9. The device according to any preceding claim wherein the reporting means further comprises a media and/or nutrients that support or encourage microbial growth.

10. The device according claim 9 wherein the media and/or nutrients that support or encourage microbial growth is Mueller Hinton Broth or Wilkins Chalgren Broth.

11. The device according to any preceding claim wherein a plurality of reporting means are arranged on the surface and the surface comprises a membrane or film.

12. The device according to claim 11 wherein the plurality of reporting means are arranged on the surface in an array or a pattern.

13. The device according to claim 11 or 12 wherein the plurality of reporting means includes reporting means with differing selection factors and wherein said selection factors allow differentiation between or quantification of microorganisms exposed to the device.

14. The device according to any one of claims 1 - 10 wherein the surface defines a pore and said pore contains the reporting means.

15. The device according to claim 14 wherein the reporting means additionally comprises a solid or semi-solid substrate.

16. The device according to claim 15 wherein the solid or semi-solid substrate is agar or agarose.

17. The device according to any one of claims 14 - 16 wherein the pore has a volume of less than 250μ1.

18. An array of devices as defined by any one of claims 14 - 17.

19. The array according to claim 18 wherein the array of devices includes devices with reporting means with differing selection factors and wherein said selection factors allow differentiation between or quantification of microorganisms exposed to the device.

20. A wound dressing comprising a device or array of devices according to any preceding claim.

21. 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 any one of claims 1 -17 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;