Materials and methods for assessing and mapping microbes and microbial biofilms on wounds

Abstract

The subject invention provides point-of-care assays for assessing the topographical distribution of microbial biofilm and/or specific microorganisms in wounds.

Claims

1. A method of mapping spatial distribution of microorganism(s) and/or biofilm(s) of said microorganism(s) in a wound comprising: applying a cationic membrane to the wound, wherein the membrane non-selectively adsorbs negatively-charged biological molecules when applied to the wound and wherein the membrane does not contain any ligand that is specific to a microbial marker; removing the cationic membrane from the wound; and applying a stain to the cationic membrane after removing the cationic membrane from the wound wherein the stain binds to negatively-charged biological molecules that have bound to the membrane, thereby mapping the spatial distribution of the microorganism(s) and/or biofilm(s).

2. The method, according to claim 1, further comprising applying a blocking agent to the cationic membrane after removing the cationic membrane from the wound and before applying the stain to the cationic membrane.

3. The method, according to claim 1, wherein the membrane is a nylon membrane.

4. The method, according to claim 1, further comprising washing the cationic membrane after applying stain to the membrane.

5. The method, according to claim 1, wherein the method is completed in 5 minutes or less.

6. The method of claim 4, comprising washing the membrane with a solution including a weak acid and an alcohol, after applying the stain.

7. The method of claim 6, wherein the weak acid is acetic acid and the alcohol is methanol or ethanol.

8. The method, according to claim 1, wherein the stain is ruthenium red.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawings(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

(2) FIGS. 1A-1C show a schematic of the topographical wound map assay of the subject invention. FIG. 1A shows a wound map affinity membrane blot that contains the bound ligand (e.g. antibody) being applied to the wound surface. In FIG. 1B the ligand binds the unique marker for the target microorganism or biofilm (e.g., polysaccharide of biofilm matrix). FIG. 1C shows the blot is then transferred into a first chamber that contains a solution of the same (or different) ligand that is labeled with a reporter (e.g. alkaline phosphatase enzyme). After a short incubation time (10 minutes) the wound map blot is transferred into the second chamber to wash out any unbound labeled-ligand and, after rinsing, the blot is transferred into the final chamber that contains the substrate for the enzyme of the labeled-ligand. After a short period of development (5 minutes) the wound map blot strip is rinsed and the areas of wound surface that contained the microorganism or biofilm are revealed as uniquely colored areas.

(3) FIG. 2A-H illustrates blots of the top (wound bed) or bottom of porcine skin explants onto HYBOND-N+ membrane and stained with 5 mg/ml Alcian Blue and washed with PBS. 2A) Blot of the bottom of an explant with 3 day mature PAO1 biofilm. 2B) Blot of the top of an explant with 3 day mature PAO1 biofilm. 2C) Second blot of the top of an explant with 3 day mature PAO1 biofilm after blotting on spot B. 2D) Blot of the top of an explant with 3 day mature PAO1 biofilm. 2E) Blot of the top of an explant with 1 day immature PAO1 biofilm. 2F) Blot of the bottom of an explant with 1 day immature PAO1 biofilm. 2G). Blot of the top of an unsterile explant (negative control). 2H) Blot of the bottom of an unsterile explant (negative control).

(4) FIG. 3A-F illustrates blots of the top (wound bed) or bottom of porcine explants onto HYBOND-N+ membrane and stained with 5 mg/ml Ruthenium Red and washed with PBS. 3A) Blot of the bottom of an explant with 3 day mature PAO1 biofilm. 3B) Blot of the top of an explant with 3 day mature PAO1 biofilm. 3C) Second blot of the top of an explant with 3 day mature PAO1 biofilm after blotting on spot B. 3D) Blot of the top of an explant with 3 day mature PAUL biofilm. 3E) Blot of the top of an explant with 1 day immature PAO1 biofilm. 3F). Blot of the top of an unsterile explant (negative control).

(5) FIG. 4 illustrates an embodiment of a rapid, point of care matrix metalloproteinase (MMP) detector in accordance with the subject invention.

(6) FIG. 5A-C illustrates dot blots of Alginic acid. A solution of 5 mg/ml Alginic acid was 2-fold serially diluted and 2 l of each dilution was dotted onto HYBOND-N+ membrane and stained with 5 mg/ml of Alcian Blue or Ruthedium Red. 5A) Stained dot blot washed with PBS with 5 ul/ml Tween-80. 5B) Stained dot blot washed with PBS with 0.1% SDS. 5C) Stained dot blot washed with 40% methanol with 10% acetic acid solution.

(7) FIG. 6A-D illustrates a wound map of Pseudomonas aeruginosa biofilm on porcine skin explants. 6A) Blot of the top of a pig skin explant with one day immature Pseudomonas aeruginosa PAO1 biofilm. Negative controls include 6B) blot of the top of a sterilized porcine skin explant without a biofilm; 6C) blot of top of unsterilized porcine skin explant; and 6D) blot of bottom of unsterilized skin explant.

(8) FIG. 7 illustrates colorimetric detection of Pseudomonas aeruginosa PAO1 biofilm extracellular polysaccharides (EPS) matrix.

(9) FIGS. 8A-8B illustrate microscopic cryosections of dye staining Pseudomonas aeruginosa biofilms on pig skin.

(10) FIG. 9 illustrates microscopic cryosections of dye staining Pseudomonas aeruginosa biofilms on pig skin.

(11) FIGS. 10A-B illustrate immunodetection of Pseudomonas aeruginosa biofilm on porcine explant biofilm with fluorescent antibodies to polyalginic acid, where the cryosections were incubated with a 1:100 dilution of 1 mg/mL anti-alginate antibody in blocking buffer for two hours at 4 C. They were then rinsed with PBS w/0.1% Tween 20 and incubated for one hour with 1:1000 dilution fluorescent anti-human antibody, then rinsed again and visualized under a fluorescent microscope. 10A is a dark field fluorescent microscopic image of cryosection of pig skin explant with mature Pseudomonas aeruginosa biofilm immunostained with antibody to polyalginic acid antibody. 10B is bright field microscopic image of the same cryosection of 10A.

(12) FIG. 11 illustrates bright field microscopic images of paraffin sections of dye staining Psuedomonas aeruginosa biofilms on pig skin. Ruthenium Red 0.1%-0.5% gluteraldehyde in 100 mM cacodylate buffer was prepared by adding 0.03 g ruthenium red and 0.16 mL gluteraldehyde to cacodylate buffer and bringing to 30 mL with buffer. Cacodylate buffer (100 mM) was prepared by adding 4.2782 g cacodylic acid to 200 mL dlH.sub.2O. Slides were deparaffinized. Ruthenium red solution was added for one hour. Slide was rinsed with cacodylate buffer for two minutes.

(13) FIG. 12A-B illustrates wound map of Pseudomonas aeruginosa biofilm on porcine skin wounds. 12A illustrates wound map twenty minutes after inoculating planktonic bacteria. 12B illustrates wound map twenty-four hour after inoculating plantktonic bacteria.

DETAILED DISCLOSURE

(14) Bacterial colonization, particularly the presence of microbial biofilm, is one of the primary factors that can cause delayed wound healing. Also, the increased resistance of biofilm to antimicrobial treatments, relative to planktonic organisms, has been well documented. Unfortunately, the need for reassessing the efficacy of current antimicrobial treatments and to develop new treatment strategies specific for managing microbial biofilm in wounds, particularly chronic wounds, has only recently become appreciated.

(15) The subject invention provides point-of-care methods for assessing the topographical distribution of microbial biofilm and/or specific microorganisms. Advantageously, the assays of the subject invention can be used to identify the location of biofilm and/or microbes on a wound, as well as to provide information about the chemical and/or biological characteristics of the biofilm and microbes.

(16) Microbial biofilm distribution on wounds is a dynamic condition. Knowing the topographical location within the wound of microbial biofilm and/or microorganisms enables the health care provider to make informed decisions on the appropriate treatment strategies to be applied to the wound in a specific localized manner. The point-of-care topographical biofilm wound map of the subject invention provides health care workers (e.g. physicians, nurses, and others) immediate information on the microbial condition of the wound, thereby assisting and justifying the choice of treatment methods employed to promote wound healing.

(17) Furthermore, the technology of the subject invention is amenable to archiving (e.g. digital photography) of the topographical data in the patient's care record, thereby facilitating long term comparative assessment.

(18) In a preferred embodiment, the topographical assay involves taking an impression of the wound and processing the impression in order to produce a two dimensional map of the location of microbial biofilm and/or specific microorganisms (class, genera, species etc.) on the wound. Thus, the use of this device can aid in, for example, chronic wound treatment.

(19) The molecule(s) targeted for detection and/or measurement can be polysaccharides or glycoproteins that contribute to the formation of biofilms. The primary targets, used to indicate the general presence of microbial biofilm, are preferably components found in the extracellular matrix of microbial biofilm (e.g. polyanionic bacterial exopolysaccharides such as poly--(1-6)-N-acetyl-D-glucosamine, alginic acid, etc.).

(20) The use of reporter-ligands to specific microbial markers (solely or in addition to reporter-ligands to general microbial biofilm extracellular matrix targets) allows the presence of specific microbial classes, genera, and/or species to be located on the biofilm wound map.

(21) The detection ligand molecule(s) can be monoclonal or polyclonal antibodies, DNA aptamers, protein aptamers, phage display, or any other macromolecular recognition that currently exists or will exist. The reporter molecule(s) will be fluorescent, chemiluminescent, chromogenic, or any other detectable signal.

(22) In one embodiment, the subject invention can be used to detect Pseudomonas aeruginosa biofilm on a skin wound using a cationic membrane that binds anions such as polyalginic acid that make up the majority of the biofilm exopolymeric matrix. After blotting the membrane onto the wound with the biofilm, the membrane can be stained with a cationic red dye molecule that binds to the biofilm matrix. Planktonic P. aeruginosa bacteria (single cells) on wounds that are blotted with the cationic membrane and stained do not retain any red dye. Thus, the method is specific for the biofilm exopolymeric matrix.

(23) Assays can be developed for naked eye or quantitative assessment using well-established, relatively inexpensive technical and non-technical personnel.

(24) The appropriate methods of visual assessment and data recording of the biofilm wound map can be correlated with the reporter molecules and assay membranes used. The physical embodiment of the topographical wound map microbial biofilm reporter assay device correlates with the optimal means of assessing the target molecules.

(25) In a second embodiment, the subject invention provides a very, simple, easy and quick wound map procedure. In this embodiment the membrane does not contain antibodies to the biofilm components. Instead, the membrane binds, for example, proteins, polysaccharides, and DNA in a non-selective manner. The polyanionic polysaccharide matrix of the biofilm is then detected by staining with polycationic dye molecules. The dye may be, for example, alcian blue or ruthenium red.

(26) In yet another embodiment, biomolecules are bound to a non-specific membrane but then specific ligands are used to identify target molecules that have bound to the membrane. The specific ligand, may be, for example, antibodies, aptamers, or other macromolecular recognition entities.

(27) In a third embodiment, for wounds in which a topographical map assay device would be impractical or unfeasible, a point-of-care bacteria and bacterial biofilm wound assay using a single sampling platform, such as a swab, is provided. Such a device is amenable for assessment of targets other that outer dermal wounds (e.g. eye, ear, etc). FIG. 4 illustrates an example of a rapid, point-of-care sampling platform for indicating matrix metalloproteinase (MMP) detection. A movable wound swab 15 is provided for application to a wound to retrieve a sample. The swab 15 includes a hollow swab shaft 10 connected to buffer in an upper reservoir. A snap valve 5 releases the buffer from the upper reservoir into the hollow swab shaft 10. The buffer preferably contains a substrate. When the buffer enters the hollow swab shaft 10, it preferably elutes the wound fluid sample from the swab 15. When the reaction is complete, preferably after a specified period of time, such as, for example, 5, 10, or 15 minutes, the swab 15 penetrates beyond a foil seal separating the swab 15 from an inhibitor 20 to stop the reaction. The inhibitor 20 is preferably a protease inhibitor.

(28) In a further embodiment, the subject invention provides a kit for wound mapping. In one embodiment, the kit comprises a membrane as described herein and instructions for use of the membrane to map wounds.

(29) By using the assays of the subject invention, the caregiver is able to assess the biological activity present in the actual wound bed. Further, the caregiver is more readily able to see the direct impact of various treatments on the wound.

(30) Advantageously, a picture of the wound environment serves as justification for applying more advanced wound management technologies on a case-by-case basis (i.e. advance personalized medicine).

(31) The assays of the subject invention are amenable to a number of readily available technologies for assessment and archiving of the topographical data. For example, chromogenic-luminescence-, or fluorescence-based detection methods may be used in conjunction with digital photography for sensitive, intuitive observation and storage of patient care records. Finally, in addition to describing the topographical distribution of biofilm and/or microbes, the system can be adapted to assess multiple analytes (i.e. protease, etc.), thus providing a more complete assessment of the wound bed.

(32) Upon conducting the simple procedures of the subject invention, the healthcare professional has very important information not only to treat the condition in an as-needed manner, but also to design and justify subsequent and related treatments, as required by the majority of insurance corporations.

(33) The assays of the subject invention can also be used prior to the application of therapy to ensure that the recipient site is conducive to the therapy (e.g. any treatment applied to the site will not be adversely affected by the presence of biofilm or microbes).

(34) In an embodiment that is specifically exemplified herein, the subject invention provides assays that can be used to determine and/or monitor the status of a wound. The assays are quick and easy-to-use. In specific embodiments, the assays can be carried out by, for example, a nurse utilizing either no instrumentation or only minimal instrumentation. In one embodiment, information about the status of a wound can be readily, easily and reliably generated in 30 minutes or less. In a preferred embodiment, the results are obtained in 15 minutes or less. Most preferably, the results are generated in 10 minutes or less.

(35) In a specific embodiment, the assays of the subject invention are utilized to assess the status of chronic wounds. As used herein, reference to chronic wounds refers to wounds that after 2 weeks are not healing properly.

(36) Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting.

Example 1Assay Chamber

(37) In one embodiment the subject invention provides an assay chamber for processing a topographical wound map microbial/microbial biofilm detector assay membrane (FIG. 1). An impression is taken of the wound using a membrane for obtaining a specimen of microbial biofilm from wounds (FIG. 1A). The impression can be used to produce copy blots using an appropriate membrane/device, or it can be processed directly. The wash chamber consists of separate chambers containing reaction and wash buffers to process the assay membrane.

Example 2Assay Cassette Method

(38) In one embodiment, the subject invention provides an assay cassette method for processing a topographical wound map membrane. An impression can be taken of the wound using a membrane for obtaining a specimen of microbial biofilm from wounds (FIG. 1A). The impression can be used to produce copy blots using appropriate membrane(s)/device or will be processed directly.

(39) The assay cassette may contain a fluid reservoir at the base containing a compressible material to hold the reaction buffer. The assay cassette may contain an upper dry wicking layer employed to pull fluid through the assay membrane to facilitate the assay reaction and to wash the assay membrane.

Example 3Assay Method

(40) FIG. 1A is a schematic of one embodiment of the subject invention. FIG. 1A shows a wound map affinity membrane blot that contains the bound ligand (e.g. antibody) to the unique marker of the microorganism (i.e. bacteria, fungi) or biofilm. This membrane is applied to the wound surface. The ligand (e.g. antibody) that is bonded onto the blot binds with the unique marker for the target microorganism or biofilm (e.g., polysaccharide of biofilm matrix). The blot is then transferred into the first chamber of a developing block that contains a solution of the same ligand that is labeled with a reporter (e.g. alkaline phosphatase enzyme). After a short incubation time (10 minutes) the wound map blot is transferred into the second chamber to wash out any unbound labeled-ligand and after a minute of rinsing the blot is transferred into the final third chamber that contains the substrate for the enzyme of the labeled-ligand.

(41) After a short period of development (5 minutes) the wound map blot strip is rinsed under running tap water and the areas of wound surface that contained the microorganism or biofilm are revealed as uniquely colored areas.

Example 4Alternative Assay Format

(42) In one embodiment, the subject invention provides an assay as follows: (1) A high capacity binding membrane is used to non-specifically adsorb biological molecules (including, for example, polysaccharides, DNA, proteins and lipids) on a wound. In a preferred embodiment, the membrane is a HIGHBOND nylon sheet. (2) The membrane is then submerged in a blocking agent. The blocking agent may be, for example, serum albumin or casein. The blocking agent coats any remaining binding sites on the membrane. (3) The membrane is then briefly submerged in (or sprayed with) a concentrated solution comprising a cationic dye. The membrane may be contacted with the dye(s) for, for example, 1 to 5 minutes, and preferably for about 2-3 minutes. In specific embodiment the dyes may be alcian blue and/or ruthenium red. (4) The membrane is then rinsed in a solution of salt and dilute acid, with a small amount of methanol or ethanol. In a specific embodiment, the salt solution can be around 0.9% sodium chloride, the acid may be acetic acid (or other acid of similar strength) and the alcohol can be around 1-2%. (5) The final step is to dry the membrane and observe the dye-stained area that corresponds to the area of the wound bed surface that contains a biofilm.

Example 5Assessment of Biofilm Detection: Polyanionic Exopolysaccharides

(43) Polyanionic exopolysaccharides found in biofilm exopolymeric matrix were assessed. Preferably, a membrane having a high positive charge (such as positively charged nylon or activated papers) is used as the target capture membrane. In contrast to nitrocellulose membranes and uncharged membranes that have negative charges or no charges, respectively, high positively charged membranes are able to tightly bind to the highly negatively charged polysaccharides and bacterial DNA that make up a majority of exopolymeric material of biofilm. See Table 1 below. In one embodiment, Amersham HYBOND-N+ (GE Healthcare), a cationic nylon membrane, was chosen as the target capture membrane.

(44) TABLE-US-00001 TABLE 1 Properties of Materials used for Immobilization of Nucleic Acids Supported Activated Nitrocellulose nitrocellulose Uncharged nylon Positively charged nylon papers Application ssDNA, RNA, ssDNA, RNA, protein ssDNA dsDNA, ssDNA, dsDNA, RNA, ssDNA, RNA protein RNA, protein protein Binding capacity (g 80-100 80-100 400-600 400-600 2-40 nucleic acid/cm.sup.2 Tensile strength Poor Good Good Good Good Mode of nucleic acid Noncovalent Noncovalent Covalent Covalent Covalent attachment.sup.a Lower size limit for 500 nt 500 nt 50 nt or bp 50 nt or bp 5 nt efficient nucleic acid retention Suitability for reprobing Poor (fragile) Poor (loss of signal) Good Good Good Commercial examples Schleicher & Schleicher & Schuell AMERSHAM Schleicher & Schuell Schleicher & Schuell BA83, BA-S; AMERSHAM HYBOND-N; Nytran; AMERSHAM Schuell APT BA85; HYBOND-C extra Stratagene HYBOND-N.sup.+; Bio- papers AMERSHAM Duralon-UV; DU Rad ZetaProbe; PALL HYBOND-C; Pont NEN Biodyne B; Du Pont NEN PALL Biodyne A GeneScreen GeneScreen Plus .sup.aAfter suitable immobilization procedure.

(45) Two cationic chromogenic dyes were chosen as detectors: Alcian Blue 8GX (FIG. 2) and Ruthenium Red (Sigma-Aldrich) (FIG. 3). Early immature 1 and mature 3 day P. aeruginosa PAO1 biofilm were cultured using a porcine skin explant biofilm model.

(46) The in vitro biofilm porcine skin explant model includes obtaining fresh pigskin, processing the skin by mechanical depilation, removal of excess fat below the epidermis, and mechanically creating 8 mm explants with 2 mm borehole partial thickness wound beds. The explants were washed with 10% bleach solution, sterilized 45 minutes with chlorine gas, and washed with sterile PBS.

(47) Explant wound beds were inoculated with 10 ul of Log phase bacterial culture of clinically relevant bacterial species (e.g., Pseudomonas aeruginosa, Staphylococcus aureus, etc.). The explants were then placed on soft 0.5% soft agar media containing appropriate antibiotics (to which the bacteria in planktonic form are not resistant) to prevent penetration of bacterial biofilm through the bottom of the explant. The bacteria were cultured for 3 to 5 days, with daily transfer to fresh media, to produce mature bacteria biofilm. The explants were treated overnight in liquid media containing 100 MIC of appropriate antibiotic to kill remaining planktonic bacteria, gently washed with sterile PBS, and used as desired (e.g., to assess antimicrobial efficacy of various treatments on immature and mature bacterial biofilm; as a pseudo biofilm infected chronic wound). In certain experiments, explants may then be sonicated in PBS with 5 ul/ml Tween-80 in order to obtain bacterial suspensions for spread plate analysis to determine relative CFU/ml. The explant or the sonicant bacterial suspension may also be assessed using microscopy.

(48) Unsterilized explants and explants in which PAO1 was grown were blotted, from both sides of the explant, onto the membrane. The blots were stained with 5 mg/ml of Alcian Blue (FIG. 2) or Ruthenium Red (FIG. 3) for 1 minute, and washed three times with phosphate buffered saline (PBS) for 30 minutes each. The results showed that, compared to the unsterilized skin control, both dyes can detect PAO1 biofilm using this membrane (FIGS. 2 and 3).

(49) Due to the high background, alternative wash solutions were tested: PBS with 5 ul/ml Tween-80; PBS with 0.1% SDS; 40% methanol with 10% acetic acid. Alginate is the primary polyanionic exopolysaccharide secreted by PAO1 and is the major component of its biofilm matrix. A solution of 5 mg/ml Alginic acid (Sigma-Aldrich) was 2-fold serially diluted and 2 l of each dilution was dotted onto cationic nylon membrane, stained with 5 mg/ml of Alcian Blue or Ruthenium Red for 1 minute, and washed three times 30 minutes each with one of the wash solutions. The result show that PBS alone (FIGS. 2 and 3) or with 0.1% SDS (FIG. 5A) had high background, PBS with 5 ul/ml Tween-80 had reduced Ruthenium red but not Alcian blue background (FIG. 5B), and 40% methanol with 10% acetic acid solution removed most of the background for both dyes (FIG. 5C). Ruthenium red precipitated during staining of the explant with biofilm blots causing uneven and discolored staining, particularly for the 3 day biofilm blots (FIG. 3), which was resolved before staining the unsterile skin blots (FIG. 3G) as well as the dot blots (FIG. 5).

(50) All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

(51) It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.