GB2340235A - Monitoring bacterial contamination of a wound involving assay of adenosine triphosphate - Google Patents

Description

2340235 METHOD OF MONITORING BACTERIAL CONTAMINATION OF A WOUND The

present invention relates to a method of monitoring the bacterial contamination of a wound by monitoring the ATP concentration of wound fluid or exudate. The present 5 invention also relates to devices and kits for use in such methods.

In mammals, injury triggers an organised complex cascade of cellular and biochemical events that result in a healed wound. Wound healing is a complex dynamic process that results in the restoration of anatomic continuity and function; an ideally healed wound is one that has returned to normal anatomic structure, function and appearance.

Infection of wounds by bacteria delays the healing process, since bacteria compete for nutrients and oxygen with macrophages and fibroblasts, whose activity are essential for the healing of the wound. Infection results when bacteria achieve dominance over the systemic and local factors of host resistance. Infection is therefore a manifestation of a disturbed host/bacteria equilibrium in favour of the invading bacteria. This elicits a systemic septic response, and also inhibits the multiple processes involved in wound healing. Lastly, infection can result in a prolonged inflammatory phase and thus slow healing, or may cause further necrosis of the wound. The granulation phase of the healing process will begin only after the infection has subsided.

The persistent presence of bacteria in injured tissue results in the prolonged elevation of proinflammatory cytokines such as interleukin-1 and tumour necrosis factor alpha (T'NF-(x). This in turn causes increases in the levels of matrix metal loproteinas es, a decreased level of tissue inhibitors to the metalloproteinases JIMP), and a decreased production of growth factors.

Chronically contaminated wounds all contain a tissue bacterial flora (see, for example, Miller and Gilchrist, "Understanding wound cleaning and infection", Professional Nurse 1998, published by MacMillan Magazines Ltd.). These bacteria may be indigenous to the patient or might be exogenous to the wound. Closure, or eventual healing of the wound is often based on a physician's ability to control the level of this bacterial flora.

2 Current methods used to identify bacterial infection rely mainly on judgement of the odour and appearance of a wound. With experience, it is possible to identify an infection in a wound by certain chemical signs such as redness or pain. Some clinicians take swabs that are then cultured in the laboratory to identify specific organisms, but this technique takes time.

There is thus a long felt need for a diagnostic aid that would assist in the monitoring of the bacterial contamination of a wound. Such a diagnostic would enable small-scale bacterial infection to be treated before wound chronicity sets in and would also allow the monitoring of an infected wound to assess the success of an anti-bacteri'al treatment.

It has been discovered that the adenosine tniphosphate (ATP) concentration of a wound correlates closely with the level of bacterial contamination. According to the present invention, there is therefore provided a method of monitoring the bacterial contamination of a wound comprising monitoring the ATP concentration of wound fluid. The measurement of the ATP concentration of the wound fluid allows the level of bacterial contamination to be accurately assessed. The step of monitoring is preferably carried out on wound fluid that has been removed from the body of the patient, but can also be performed on wound fluid in situ.

Any type of wound may be diagnosed for infection according to the method of the present invention. For example, the wound may be an acute wound such as an acute traumatic laceration, perhaps resulting from an intentional operative incision, or the wound may be a chronic wound. The method of the invention is envisaged as being most useful in the diagnosis of bacterial contamination of a chronic wound. Preferably, the chronic wound is selected from the group consisting of venous ulcers, pressure sores, decubitis ulcers, diabetic ulcers and chronic ulcers of unknown aetiology.

According to the method of the present invention, the diagnostic assay is designed so as to provide a correlation between a measurable signal and the size of the bacterial population that is associated with clinical infection. Under present clinical standards, bacterial infection of wounds is generally taken to be 105CfU/CM2. Accordingly, the I 3 system should be set up so that a detectable response is tripped at around 104C fu/CM2 bacteria so as to give a warning of bacterial contamination. The ATP concentration correlated with such a level of bacteria is far in excess of the normal ATP turnover in the wound; accordingly, background ATP concentrations do not distort the signal to a 5 significant extent.

As used herein, the term wound fluid is meant to refer to the exudate that is secreted or discharged by cells in the environment of the wound. This fluid contains cells, both living and dead, and a variety of inflammatory cy-tokines.

By ATP concentration is meant the free concentration of ATP in the wound fluid. The ATP concentration may be assessed in situ, or alternatively a sample of wound fluid may be taken as a clinical swab or as a fluid sample.

The ATP concentration of the wound fluid may be monitored by any method known to those of skill in the art. Suitable methods include those utilising chemical or enzymelinked reactions, or by conjugating the reaction of ATP to immunological, fluorogenic, chemiluminescent, chromogenic or radioactive detection mechanisms.

Reactions in which ATP participates as a substrate are usually driven in the direction leading to hydrolysis of ATP. The chemical energy released from hydrolysis is then utillsed under physiological conditions in active transport, or may be converted to mechanical, light or electrical energy, or may be released as heat. Accordingly, any chemical reaction to which ATP concentration can be stoichiometrically coupled may be used in the method of the present invention.

It is envisaged that the most convenient method of ATP detection will involve the use of enzymes whose reaction with substrate is driven by ATP and which either themselves, or through coupled reactions give a detectable signal that is proportionate to ATP levels.

Preferably, ATP concentration is coupled to the generation of a detectable signal through the action of enzymes that dissipate the energy of ATP hydrolysis as light or electrochemical energy. Suitable enzymatic reactions include, but are not limited to the luciferase-luciferin reaction, the hydrolysis of ATP by alkaline phosphatase.

4 Alkaline phosphatase (ALP) is an enzyme that catalyses the hydrolysis of orthophosphoric monoester to an alcohol and orthophosphate. ATP is a substrate of this enzyme. There are many commercially available ALP assays that couple the activity of this enzyme to ATP concentration by electrochemical modulation. For example, the well-known electrochemical luminescence (ECL) detection method (Amersham) may be used to couple the ALP-catalysed hydrolysis of ATP to give a measurable signal that is proportionate to the levels of ATP in wound fluid. The "Alkphos direct" labelling and detection systems (Amersharn Life Sciences) are of particular usefulness in this regard.

The method of the present invention may also utilise the luciferaseluciferin reaction.

This assay is particularly applicable to the method of the present invention due to its simplicity and sensitivity. The luciferase enzyme catalyses reactions in which a singlet intermediate is formed in high quantum yield. This intermediate decays, simultaneously 2+ omitting visible light. In the presence of luciferin (D-LH2), ATP-Mg, and molecular oxygen, luciferase catalyses the production of light according to the following reactions determined by DeLuca et al. Method EnzMol., vol. LIM, pages 3-15 (1978); M921 + 7 D - LH2 + ATP ≤> E.LH2AMP + PPi E.LH2AMP + 02 => E.P + AMP + C02 + LIGHT The first reaction involves the activation of luciferin and hydrolysis of ATP resulting in the formation of an enzyme-bound luciferyl adenylate (E. LH2AMP). The second reaction requires molecular oxygen and leads to the formation of enzyme-bound excited product, which subsequently decomposes to give off light and enzyme-bound product. Quantitative liberation Of C02 from the luciferin during the reaction is also observed.

Coenzyme A may be used as a co-factor in this reaction; in the presence of coenzyme A, oxidation occurs from luciferyl-CoA with more favourable total kinetics, resulting in the generation of constant light intensity that is proportionate to the ATP concentration.

In order to measure the generation of light, a luminometer may be used. However, it will be apparent to the skilled artisan that this assay is easily adaptable to measurement of light generation in scintillation counters or using photographic film.

To allow measurement of the ATP concentration in a wound, a sample of wound fluid must be added to the ATP assay system. Measurement may either be made in situ, or fluid may be removed from the wound for subsequent ATP measurement. The decision as to which method is used will depend upon the type of wound in question.

For example, in the case of surface-exposed wounds, a clinical swab, dressing, "dipstick" or other biosensor device may be applied directly to the surface of the wound. This device should contain all of the required components of the ATP-linked reaction so that the reaction itself may proceed in situ. The device can then be removed fi7om the wound and the signal measured by the appropriate means. In many cases, a physician may not actually require an accurate assessment of the precise degree of bacterial infection, but may just wish to know whether there is a sufficient degree of infection to warrant prophylactic action, In these cases, visible assessment of the dressing may be sufficient to allow identification of the specific areas of infection. Unnecessary treatment of healthy granulating tissue can then be avoided.

A dressing that allows mapping of the infected areas of a wound will be preferable in certain instances. Diagnostic wound mapping sheets that could be adapted to the methods of the present invention are described in co-pending application GB 9705081.9 filed on 12th March 1997, the entire content of which is hereby incorporated by reference.

Immobilisation of reaction components onto a dipstick, wound mapping sheet or other solid or gel substrate offers the opportunity of performing a more quantitative measurement. For example, in the case of a reaction linked to the generation of a colour, or of light, the device may be transferred to a spectrometer, a luminometer or a scintillation counter. Suitable methods of analysis will be apparent to those of skill in the art.

6 Immobilisation of the reaction components to a small biosensor device will also have the advantage that less of the components (such as enzyme and substrate) are needed. The device will thus be less expensive to manufacture than a dressing that needs to have a large surface area in order to allow the mapping of a large wound area.

Methods for the incorporation of the components of the assay reaction onto a clinical dressing, "dipstick", sheet or other biosensor are routine in the art. See for example Fdgerstam and Karlsson (1994) Immunochemistty, 949-970.

The ATP concentration of a wound may alternatively be measured in an aqueous assay system. Wound fluid may be extracted directly from the environment of the wound or can be washed off the wound using a saline buffer. The resulting solution can then be assayed for ATP concentration in a test tube or in a microassay plate. Clearly, this will allow a more accurate assessment of the exact levels of bacteria in a wound, Such a method will be preferable for use in cases in which the wound is too small or too inaccessible to allow access of a diagnostic device such as a dipstick. This method has the additional advantage that the wound exudate sample may be diluted. Under most diagnosis methods, dilution will still allow accurate assessment of ATP amount, yet will enable an operative to assess the concentration of a wound from which it is not possible to extract more than a few microlitres of exudate. Such wounds will include small or internal ulcers and pressure sores.

It will be clear that an aqueous assay system is more applicable to use in a laboratory environment, whereas a wound dressing containing the necessary reaction components will be more suitable for use in a hospital or domestic environment.

Specific embodiments of the present invention will now be described in more detail, by way of example, with reference to the accompanying drawings, in which:- BRIEF DESCRIPTION OF THE FIGURES

Figure I shows a luciferase calibration curve at high concentrations of ATP.

Figure 2 shows a luciferase calibration curve at low concentrations of ATP.

7 Figure 3 shows the stability of luciferase at different temperatures.

Figure 4 shows the elucidation of ATP concentration for different amounts of E, coli using a luciferase assay, EXAMPLES Materials and stock solutions pH 7.8 Luciferase storage buffer ml stock containing:- 0.298 g of 25 mM4-(2-hydroxyethyl)-l- piperazine ethanesulfonic acid (HEPES) 0.051 g of 5niM MgC12 0.077 g of 10 mM dithiothreitol (DTT) 12.5 jil of Triton X-100 (RTM) ml of Glycerol pH 7.8 Luciferase reaction buffer ml stock containing:- 0.298g of 25 mM HEPES 0.05 1 g of 5 mM MgC12 0.077g of 10 mM DTT Luciferase 0 Iml of 32 iM Luciferase stock:- 2 mg of solid Luciferase (Sigma L9506) I ml of pH 7.8 Luciferase storage buffer - Iml of 32 nM Luciferase:- 10 gl of 32 gM Luciferase 990 jil of pH 7.8 Luciferase storage buffer Luciferin 0 1 ml of 18 mM Luciferin stock:- 5 mg of solid Luciferin (Sigma L6152) I ml of pH 7.8 Spectroscopy buffer - I ml of 0.9 mM Luciferin containing:- 50 RI of 18 mM Luciferin stock I ml of pH 7.8 Spectroscopy buffer pH 7 ATP 1 ml of 10mMATP containing:- 0.055 g of solid ATP ml of spectroscopy buffer Serial dilutions were carried out to product ATP concentrations ranging from 10tM - I OOgM to produce a calibration curve with the different ATP concentrations.

8 E.coli DH5a E.coli was supplied by the Biochemistry department, from The University of Birmingham Absorbance I 5xlO 8 cells of bacteria/ml gl of stock; 990 pl sterile water: Absorbance 0.267 = l.3xlO8 cells of E. colilml E. coli ranged between: 0-20 gl (0 - 2.6xlO8 cells of E.colilml) Example 1:-Establishing the optimal conditions for the luciferase assay A calibration curve was developed for the luciferase assay under optimal conditions. This curve gave information regarding the minimum ATP concentration necessary for detection.

Determination of ATP concentration with maximum absorbance Maximum absorbance for ATP detected by UN. spectrophotometer is approximately 0.6 U. 20 ATP 10 mM ATP gives an absorbance of 154, 40 i.M ATP, by Beer Lambert Law, would give an absorbance of 0.616. UN. spectrophotometer provided a maximum absorbance of a 40 [LM ATP in 25 Iml of pH 7.8 spectroscopy buffer, in a spectrum scanned between 220 and 300 rim. 40 jiM ATP provided A260.5 of 0.476 units [data not shown].

Luciferin 18 mM luciferin gives an absorbance of 347.7.

36 pM ATP, by Beer Lambert Law, would give an absorbance of 0.649.

UN. spectrophotometer provided a maximum absorbance of 36gM lucifenin, in I ml of pH 7.8 spectroscopy buffer, spectrum scanned between 250 and 350 run.

0 36 gM luciferin provided A329.6 of 0.573 units [data not shown].

9 Luciferase Calibration curve Having achieved the ideal ATP and luciferin concentrations for the assay, the reaction was initiated. A typical assay mixture contained I ml pH 7.8 luciferase reaction buffer, 0.32nM luciferase (10gl of 32nM) made in pH 7.8 luciferase storage buffer, and 9pM luciferin (10d of 0.9mM). Appropriate amounts of ATP ranging between 10-2 nM _ 104 nIM were added to initiate the reaction and record the maximum chemi luminescence.

Figure I shows a calibration curve with high ATP concentrations, and confirms a maximum activity at 50 gM ATP. Figure 2 shows lower ATP concentrations, however, this curve also confirms a detection limit of InM. The rate of ATP detected is clearly proportional to quantity of ATP.

Stability of luciferase The stability of the luciferase assay was investigated at different temperatures over a one week period in order to investigate the ideal temperature and length of storage for the assay.

Three aliquots of luciferase at a concentration of 32nM (330 gl) each stored at different temperatures [-20, 4' and 20'C] were developed to determine the activity of luciferase over a one week period. A maximum chemi luminescence was recorded when 25PI of 25iM ALP (630 nM) was taken at each temperature range and added into I ml pH 7.8 luciferase reaction buffer with 9 nM luciferin and 50 gM ATP. The measurements were repeated after storage for a further five days to allow ample time for any modification in luciferase activity.

A concentration of 32nM luciferase was found to be most stable at -20'C confirming a 37% luciferase degradation, over a one week period. Whereas, a 100% luciferase degradation at 20'C, and a 68% luciferase degradation was observed over a one week period [Figure 3].

Example 2: Testing luciferase assays on biological samples ATP concentrations were investigated in saliva and in an E. coli culture, using the formulated assay.

Detection of ATP in saliva and tooth plaque extractant A luciferase assay kit was developed in the addition of 0.32nm luciferase and 9iM luciferin in Iml pH 7.8 luciferase reaction buffer. Maximum chemiluminiscence was measured by the addition of 10 gl of saliva or tooth plaque dissolved in saliva fTom healthy individuals.

The same procedure was investigated by the addition of 10 jil of extractant (I% Tween 80) in saliva or tooth plaque (left for approximately 5 minutes), into the luciferase assay.

Approximately 80% increase in ATP detection in saliva was confirmed in the presence of extractant, compared to the absence of extractant. A control was investigated in the substitution of saliva or tooth plaque with sterile water, confirming undetectable ATP [Table I J.

Table 1. Levels of ATP in saliva & tooth plaque.

Sample (10 gl) ATP (nM Saliva 0 Tooth plaque 2.5 Saliva + 10 il extractant 2.5 Tooth plaque + I Opl extractant 5.0 Saliva + 20gl extractant 2.6 Saliva + 30g] extractant 2.6 Detection of ATP in different concentrations of extractant A luciferase assay kit was produced from the addition of 0.32 nM luciferase and 9 AM luciferin in I ml pH 7.8 luciferase reaction buffer. ATP detection was initiated in the addition of 10 pI of saliva and 10 gl of extractant (left for approximately 5 minutes), into the luciferase assay. The amount of extractant was varied between 10- 30 Al.

The addition of 10AI extractant to saliva was found to be suitable to give a maximum ATP detection. An increase in extractant has no relevant significance to the detection [Table I].

Detection of ATP in Escherichia coli (DH5(x) A560 was measured for 1/100 dilution of 10 A] Escherichia coli to calculate the concentration of the stock solution. A luciferase assay kit was produced in the addition of 0.36 AM luciferase and 9 nM luciferin into I ml pH 7.8 luciferase reaction buffer.

Maximum chemiluminescence was measured in the presence of 30 pl of extractant to varying amounts of 1.3 x 10 9 Ecoli, ranging between 0 gl, which was inserted in the luciferase assay. The corrected chemi luminescence was read off the calibration curve to determine the amount of ATP in different amounts of E.coli under investigation.

The controls involved were:

Absence of Ecoli & presence of extractant, Absence of extractant & absence of Ecoh.

DH5cc Ecoli (10 pil) has an absorbance of 0.267 at a wavelength of 560nm detected by a U.V. spectrometer.

Absorbance I 5x 108 cells of bacteria/ml Absorbance 0.267 1.3x 108 cells of E.colilml 12 ATP detection was found to be proportional to the quantity of E. coli added to the luciferase assay. 45nM ATP was detected in extractant in the absence of E. coli [Figure 4].

Detection of ATP in DH5oLE.coli in physiological conditions The above procedure was repeated under physiological conditions in the presence of I 0gl saliva into the E.coli and extractant.

ATP detection of E.coli in physiological conditions, such as in saliva, confirmed similar results to E.coli in culture as shown in Figure 4.

The designed luciferase assay was found to detect levels of ATP that correlated with the levels of bacteria in wound exudate. It is evident that there is some degree of product inhibition, and that the enzyme can turn over slowly in the present of ATP and excess luciferin (DeLuca, 1976).

When injecting ATP into the luciferase assay, there is a rapid flash and a very rapid decay to a very low level of luminescence. This rapid flash and decay to the low level, is due to at least two major factors, a) the activation of dehydroluciferin, which is present in the luciferase assay, and acts as a very potent inhibitor of the luciferase or, b) the presence of inorganic pyrophosphate, which destroys the pyrophosphate in the system and thus tends to allow the inhibiting reaction of dehydroluciferin to become much more favoured.

The enzyme in principle can be used to measure total ATP in a sample or it can be coupled to ATP-producing or consuming reactions. One can confirm that higher ATP results in faster decay and therefore greater inhibition. The sensitivity quoted by DeLuca et al, (1978) is such that as little as 10-14 Mol of ATP can be accurately measured. In this study, the minimum ATP detection was found to be IriM.

13 Luciferase is ideally stable at -200C, however, it must not be stored for longer than a couple of days, due to the rapid degradation of luciferase confirmed. The contents, cost, storage temperature and length of storage is summarised in Table 2.

Table 2 Luciferase assay kit Contents/assay Cost/100 (-20'C for 2 days) Assays (f) Iml pH7.8 Luciferase 1.29 reaction buffer 10pl 0.9M Luciferin in pH7.8 3.83 spectroscopy buffer lOgI 32nM Luciferase in pH7.8 1.45 Luciferase storage buffer 1 Opl Extractant 0.20 Total 6.77 It will be appreciated that modification of detail may be made without departing from the scope of the invention as defined in the accompanying claims.

14

Claims (9)

1. I A method of monitoring the bacterial contamination of a wound comprising monitoring the adenosine triphosphate (ATP) concentration of wound fluid removed from the wound.
2. 2 The method of claim I wherein the wound is a chronic wound.
3. 3 The method of claim 2, wherein the chronic wound is a chronic ulcer such as a dermal ulcer, venous ulcer, pressure sore or decubitis ulcer.
4. 4 The method according to any preceding claim wherein the ATP concentration is measured using an enzyme-coupled reaction.
5. 5 The method of claim 4 wherein the enzyme-coupled reaction is the luciferin luciferase reaction.
6. 6 The method of claim 4 wherein the enzyme-coupled reaction involves the use of alkaline phosphatase.
7. 7 The method according to either of claims 5 or 6, wherein the enzymecoupled reaction is monitored using colorimetry, luminescence, chemiluminescence or autoradiography.
8. 8 A wound dressing or biosensor for use in monitoring the bacterial contamination of a wound comprising an enzyme that hydrolyses ATP and a substrate of the enzyme.
9. 9 A wound dressing or biosensor according to claim 8, further comprising a reporter molecule.

   A diagnostic kit for use in monitoring the bacterial contamination of a wound comprising a wound dressing or biosensor according to either of claims 8 or 9.

   I 11 Use of a wound dressing according to either of claims 8 or 9 for use in the manufacture of a medicament for the diagnosis of bacterial contamination of a wound.