Quantifying endotoxin burden in bacterial biofilms

Abstract

Disclosed herein are methods for quantifying total endotoxin load in a biofilm sample. Also provided are methods for identifying a Gram-negative biofilm-derived bacterial infection. The disclosed methods more accurately define the actual total endotoxin level and are able to detect the presence of endotoxin in a given biofilm volume with higher resolution than current extraction techniques.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 007,430, filed April 9, 2020, the entirety of which is hereby incorporated by reference. TECHNICAL FIELD

[0003] The present disclosure relates to methods of quantifying endotoxins in a biofilm. BACKGROUND

[0004] Currently, many medical devices, such as, for example, dural replacement patches, hernia meshes, and wound dressings, are manufactured from bacterial-derived cellulose, which is a form of biofilm. Certain bacteria used to produce cellulose include gram-negative bacteria. One issue unique to gram-negative bacteria is the presence of endotoxins. Endotoxins, also known as lipopolysaccharides, are part of the bacterial cell wall structure of gram-negative bacteria. They are known pyrogens and, when released from the cell wall, such as during cell division or cell death, are contributors to the inflammatory process in the body.

[0005] To obtain regulatory approval, gram-negative bacteria or products produced with gram-negative bacteria must be treated to render them non-pyrogenic.

[0006] The FDA-approved bacterial endotoxin test (BET) standard is the Limulus Amoebocyte Lysate (LAL) assay. This test for medical devices is outlined in ANSI / AAMI ST72:2011, USP <85>, and USP <161>. The assay involves contacting the sample with amoebocyte lysate from the horseshoe crab (Limulus Polyphemus). The assay has three variants, including a gel-clot technique, a turbidimetric technique, and a chromogenic technique. The gel-clot method is the simplest and most widely used LAL test and relies on the formation of a gel. The turbidimetric technique relies on the development of turbidity in the sample after endogenous substrate cleavage. The chromogenic technique is based on the development of color in the sample after cleavage of a synthetic peptide-chromogen complex. In the event of conflicting test results, the gel-clot test is considered the method of choice.

[0007] Currently, BET on medical devices relies on an indirect extraction method to quantify endotoxin units (EU). The sample is typically immersed in non-pyrogenic water and heated to near body temperature (e.g., 37°C) for at least one hour. The extract in the water is taken, and the amount of endotoxin is tested with a LAL assay. From this indirect measurement, the EU value of the medical device is determined. Depending on the intended use of the medical device, the FDA sets a threshold EU value that is acceptable.

[0008] From a clinical perspective, biofilms are not readily detected in patients with biofilm-derived bacterial infections. To identify antibiotic treatment courses, tissue samples from patients are typically cultured and then bacterial growth is identified. This process is time consuming and only measures the number of viable bacterial cells that are able to grow on selected agar under selected incubation conditions. Under certain conditions, no surviving gram-negative bacteria from biofilm samples are detected. In the case of infections derived from gram-negative biofilms, the presence of endotoxin can provide an alternative method to identify the presence of biofilm infections in patients. SUMMARY

[0009] The present disclosure relates to methods for quantifying endotoxin in biofilms. The methods disclosed herein more accurately define the actual total endotoxin level and are able to detect the presence of endotoxin in a given biofilm volume at much lower levels than with current extraction techniques. Thus, the methods described herein are an improvement over the current state of the art methods and can additionally be used to determine potential biofilm-derived infections in patients with gram-negative bacterial infections.

[0010] According to the present disclosure, a method for quantifying endotoxin concentration in a biofilm includes the following steps:

[0011] digesting the biofilm sample with an enzyme to form a digested biofilm sample;

[0012] separating the digested biofilm sample into a supernatant and a cell pellet;

[0013] combining the cell pellet with a cell lysis agent to release endotoxin and form an endotoxin suspension;

[0014] concentrating the endotoxin suspension into an endotoxin sample and a supernatant of the suspension; and,

[0015] performing a bacterial endotoxin test (BET) assay on the endotoxin sample to obtain a sample endotoxin value.

[0016] According to certain embodiments, the biofilm is composed of cellulose; preferably, the biofilm is substantially composed of bacterial-derived nanocellulose (BNC). According to the present disclosure, the biofilm is at least partially composed of a microorganism that produces endotoxin (e.g., a gram-negative bacteria). In preferred embodiments, the gram-negative bacteria is from the genus Gluconacetobacter.

[0017] In certain embodiments, the digestion step can include a heating step. In certain embodiments, the enzyme used for digestion includes a cellulase. In additional embodiments, the cell lysis agent comprises a chelating agent and a salt.

[0018] In certain embodiments, the method can further comprise the step of diluting the endotoxin sample prior to the step of performing the BET assay. According to additional embodiments, the method can comprise the step of adding a beta-glucan inhibitor to the endotoxin sample. In additional embodiments, the method can further comprise performing a colony forming unit (CFU) assessment on the biofilm sample to obtain a CFU value, such that a relationship between the endotoxin value and the CFU value can be determined.

[0019] The present disclosure further discloses a method for clinically identifying a gram-negative biofilm-derived bacterial infection. The method comprises:

[0020] enzymatically digesting a mammalian tissue sample to form a digested tissue sample;

[0021] separating the digested tissue sample into a supernatant and a cell pellet;

[0022] combining the cell pellet with a cell lysis reagent to release an amount of endotoxin, wherein the amount of endotoxin has a lower limit of zero;

[0023] concentrating the amount of endotoxin, and

[0024] performing a bacterial endotoxin test (BET) assay on the concentration to obtain a tissue sample endotoxin value.

[0025] In certain embodiments, the biofilm is comprised of a pathogenic microorganism, for example, a gram-negative bacteria from the Enterobacteriaceae family. DETAILED DESCRIPTION

[0026] In this document, the terms "a" or "an" are used, unless otherwise indicated, to include one and also multiple, and the term "or" is used, unless otherwise indicated, to mean "and / or". In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise expressly defined, is for the purpose of description only and not of limitation. If a range is expressed, another embodiment includes from one particular value and / or to another particular value. Similarly, where "about" is utilized to indicate that a value is an approximation, it is understood that the precise value constitutes another embodiment. All ranges include the endpoints and are combinable. Additionally, reference to values stated in a range includes each and every value within that range. It is also to be understood that certain features of the application described herein in the context of separate embodiments can also be provided in combination in a single embodiment. Conversely, various features of the application, which are, for brevity, described in the context of separate embodiments, can also be provided separately or in any suitable subcombination.

[0027] The present disclosure relates to a method for quantifying total endotoxin load in a biofilm sample. The method can comprise:

[0028] digesting the biofilm sample with an enzyme to form a digested biofilm sample;

[0029] separating the digested biofilm sample into a supernatant and a cell pellet;

[0030] combining the cell pellet with a cell lysis solution to release endotoxin and form an endotoxin suspension;

[0031] concentrating the endotoxin suspension into an endotoxin sample and a supernatant;

[0032] performing a bacterial endotoxin test assay on the endotoxin sample to obtain a sample endotoxin value.

[0033] According to certain embodiments, the biofilm consists of cellulose. With respect to the manufacture of an implantable medical device, in certain embodiments, the biofilm consists essentially of bacterial-derived nanocellulose (BNC). According to the present disclosure, the biofilm consists at least in part of a microorganism that produces endotoxin (e.g., a gram-negative bacterium). In preferred embodiments, the gram-negative bacterium is Gluconacetobacter. Other gram-negative bacteria known for producing cellulose can include, for example, Azotobacter, Rhizobium, Agrobacterium, Pseudomonas, Salmonella, or Alcaligenes.

[0034] According to certain embodiments, the biofilm sample is a BNC film. According to certain embodiments, the cellulose content of the sample film can be about 1% to about 50%, for example, about 5%, 10%, 20%, 25%, or 40%.

[0035] It should be noted that the materials and equipment used to perform the processes described herein should preferably be depyrogenated prior to use to avoid contamination and potentially cause false positive results.

[0036] The present disclosure describes a step of digesting a biofilm sample with an enzyme to form a digested biofilm sample. The purpose of the digestion step is to break down the extracellular matrix of the cellulose that entraps the bacteria contained therein and thus make the bacterial cell wall more accessible for the subsequent cell lysis step. Although the digestion step is described in detail below in the context of enzymatic digestion, it should be understood that the digestion step can be accomplished chemically, for example, with a low concentration of acid. In certain embodiments, the enzyme used is a cellulase. The amount of enzyme used can be determined based on the amount of biofilm to be digested. A suitable ratio of enzyme to biofilm sample can be about 1 : 10 to about 1 :20. Preferably, the enzyme composition is prepared in a depyrogenated flask or beaker using high purity water (HPW). The enzyme composition can be vortexed as appropriate. Furthermore, in certain embodiments, the enzyme composition can be filtered prior to use, for example, with a 0.2 pm sterile filter.

[0037] According to the present disclosure, the digestion step can also include a heating step. For example, during the digestion step, the biofilm sample can be placed in a container with the enzyme composition used for digestion and heated in, for example, a water bath. A suitable temperature range for digesting the biofilm can be 20 °C to 60 °C. A preferred temperature range for digesting a cellulose biofilm sample is 45 °C to 55 °C. According to certain embodiments, the digestion step can be from about 10 hours to about 48 hours. A suitable range for digesting a cellulose biofilm can be from about 10 hours to about 18 hours, for example, 15 hours to 16 hours. It will be appreciated that the digestion can occur with heating throughout the step, or alternatively, the period of heating can be shorter than the length of the digestion step.

[0038] After digestion of the biofilm is complete, the method includes a step of separating the digested biofilm sample into a supernatant and a cell pellet. In certain embodiments, the separation step includes centrifuging the digested sample. Depending on the total volume of the digested biofilm sample, the sample can be homogenized (e.g., vortexed) and can be portioned into smaller containers for centrifugation. Further, the sample can be centrifuged one or more times to fully concentrate the bacterial cells and form a cell pellet. Where the sample has been portioned for centrifugation, the method must include a step of recombining the sample. According to further embodiments, the method can also include removing (e.g., aspirating) the supernatant prior to the step of combining the cell pellet with a lysing agent.

[0039] After the step of separating the cell pellet is complete, the method includes a step of combining the cell pellet with a cell lysis solution to release endotoxin and form an endotoxin suspension. In certain embodiments, the cell lysis solution has a buffered solution with a pH of 5.5 to 9, preferably a pH of 6 to 8. According to certain embodiments, the cell lysis solution includes a chelating agent. Suitable chelating agents can include, for example, EDTA and EGTA. According to certain additional embodiments, the cell lysis solution can include a salt, such as Tris-HCl, Triton X, Tween, or sodium dodecyl sulfate (SDS).

[0040] According to certain embodiments, the cell pellet can be broken up and the cells uniformly redistributed in the cell lysis solution by, for example, vortexing or other agitation process. In certain embodiments, the homogenized cells in the solution can be heated as the cell lysis process occurs. A suitable temperature range for the heating step can be from about 20 °C to about 37 °C, for example, 20 °C to 25 °C. Additionally, the homogenized cells in the cell lysis solution can be continuously agitated as the cell lysis process occurs. For example, the sample can be placed in a heated water bath shaker anywhere from about 37 °C for a gentle agitation for about 10 minutes to about 45 minutes.

[0041] Once the step of lysing the cells of the sample is complete, the endotoxins present in the cell wall will be released, thereby forming an endotoxin suspension. Accordingly, the method further comprises the step of concentrating the endotoxin suspension into an endotoxin sample and a suspension supernatant. According to one embodiment, this can be performed by using centrifugation as previously described. Additionally, once the endotoxin suspension is concentrated into an endotoxin sample and a supernatant, the supernatant can be removed (e.g., by aspiration) prior to testing the endotoxin sample.

[0042] Once the endotoxin sample is obtained, the method comprises the step of performing a bacterial endotoxin test (BET) assay on the endotoxin sample to obtain an endotoxin value for the sample. According to certain embodiments, the BET assay is a Limulus amebocyte lysate (LAL) assay. The LAL assay is a U.S. Food and Drug Administration (FDA) approved standard for endotoxin testing. There are three variants of acceptable LAL assays: gel-clot, turbidimetric, and chromogenic.

[0043] According to certain embodiments, it is preferred that a beta-glucan inhibitor be added prior to performing the BET test. In certain more preferred embodiments, the beta-glucan inhibitor is added immediately prior to the BET test. In the LAL testing method, beta-glucans can interfere with the measurement of endotoxins by giving false positives. Beta-glucans are present in cellulose and, although the digestion step can eliminate their presence in the subsequent testing of the endotoxin sample, there is a possibility that a non-negligible amount still remains in the sample to be tested. Accordingly, the addition of a beta-glucan inhibitor to the sample reduces the likelihood of false positives in the BET assay. According to certain embodiments, the beta-glucan inhibitor is an endotoxin-specific buffer commercially available (ES buffer).

[0044] According to further embodiments, the method can additionally comprise performing a colony forming unit count on the biofilm sample in order to obtain a CFU value for determining the amount of viable bacterial cells in the sample. Being able to correlate the CFU value of the sample with the endotoxin value of the sample is beneficial for the BET analysis. According to certain embodiments, the method further comprises determining the molecular weight of the endotoxin such that the measured EU value provides a known viable cell count of bacteria in the sample.

[0045] The present disclosure further describes methods for identifying a Gram-negative biofilm-derived infection in a mammal. The ability to rapidly identify the type of biofilm-derived infection provides a significant clinical improvement in diagnosing the infection and providing appropriate treatment, over existing methods that rely on culturing to identify the specific type of pathogenic bacteria causing the infection. With respect to the pathogenic bacteria, the biofilm can at least partially consist of bacteria from the Enterobacteriaceae family, which can include, for example, Salmonella, Escherichia coli, Klebsiella, Shigella, Enterobacter, and Citrobacter. Other Gram-negative microorganisms are known and within the scope of the present disclosure.

[0046] According to the present disclosure, a method for clinically identifying a gram-negative biofilm-derived bacterial infection is described, the method comprising the steps of:

[0047] enzymatically digesting a biofilm sample to form a digested biofilm sample;

[0048] separating the digested biofilm sample into a supernatant and a cell pellet;

[0049] combining the cell pellet with a cell lysis reagent to release an amount of endotoxin, wherein the amount of endotoxin has a lower limit of zero;

[0050] concentrating the amount of endotoxin, and

[0051] performing a bacterial endotoxin test (BET) assay on the concentration to obtain a sample endotoxin value.

[0052] According to certain embodiments, the biofilm sample is a mammalian tissue sample. In other words, a biopsy of tissue (e.g., blood, bone, muscle) can be taken from a patient suspected of having a biofilm-derived bacterial infection. In certain other embodiments, the source of the suspected biofilm-derived infection can be from an infected implanted medical device within the patient, such as, for example, a joint prosthesis, a bone fixation device, a soft tissue device (e.g., a hernia mesh), or other implanted medical device well known to those skilled in the art. In such cases, the biofilm sample can be taken from the surface of the suspected infected device. In certain embodiments, the surface biofilm sample is extracted from the implanted medical device while the device is still implanted in the patient. In certain other embodiments, the biofilm sample is collected after the device is removed from the patient.

[0053] In embodiments in which the sample endotoxin value is greater than zero, it can be determined that the biofilm at least partially comprises a pathogenic gram-negative bacterium. According to additional embodiments, determining the endotoxin value can comprise determining an EU / mL value. As previously explained, a CFU to EU correlation can exist, such that determining an EU / mL value can provide therapeutic medical personnel with information about the virulence of any determined gram-negative bacterial colony. Accordingly, an additional step can comprise administering one or more therapeutic agents effective to reduce the presence of the pathogenic gram-negative bacterium. Thus, the method provides a clinical benefit of preventing the unnecessary administration of broad-spectrum antibiotics or other types of therapeutic agents that can be administered without knowing that a gram-negative pathogen is present in the biofilm, e.g., antibiotics that would otherwise be ineffective in treating the gram-negative infection.

[0054] According to certain embodiments, the molecular weight of the endotoxin of the pathogenic gram-negative bacterium can be specific to individual bacterial species, such that the method can further comprise identifying the pathogenic bacterium from the sample endotoxin value.

[0055] SUMMARY

[0056] SYNTHECEL dura repair (Depuy Synthes) is an implantable medical device in the form of a nanocellulose biofilm made from a gram-negative bacterium, Gluconacetobacter xylinus.

[0057] PTS (portable test system) (Charles River) and calibrated test cassettes were used to evaluate each method. The assay must meet acceptance criteria such as a coefficient of variation of less than 25%, a spike recovery of 50-200%, and an acceptable start time provided by the compliance certificate of the test cassette.

[0058] In addition, in certain test runs, a traditional cell viability method was used to estimate the total amount of viable cells at each process point.

[0059] Example 1

[0060] Preparation of cellulase digestion enzymes

[0061] Dilute 10 mL of cellulase (cellulase from Trichodema reesei, Sigma Aldrich) in 90 mL of high purity water (HPW). Vortex the mixture to ensure homogeneity. Filter the dilution into a depyrogenated flask (V = 1000 ml) using a 0.2 uM sterile filter. Cover the flask with depyrogenated foil or paraffin film until needed for use.

[0062] Preparation of cell lysis solution

[0063] Dilute 0.2 mL of 0.5 M EDTA HC1 buffer (pH 8.0) and 1 mL of 1 M Tris HC1 buffer (pH 8.0) in 100 mL of HPW in a depyrogenated beaker (200 ml). Vortex the mixture to ensure homogeneity. Cover the beaker with depyrogenated foil or paraffin film until needed for use.

[0064] Preparation of biofilm samples

[0065] Obtain the harvested cellulose pellicle (Gluconacetobacter xylinus) sample and record the weight harvested. Place the pellicle on a depyrogenated steel plate and cut it into small pieces of approximately the same size and weight using depyrogenated scissors.

[0066] Then transfer the sample pellicle pieces to the cellulase flask.

[0067] The flask is covered and placed in a water bath shaker set at approximately 50°C to initiate cellulose digestion. The digestion time is approximately 12 hours.

[0068] A visual observation is made to ensure that all of the film material is digested and no sample pieces are observed. If necessary, the digestion time can be extended until no visible sample film pieces are present.

[0069] The digested sample material is then transferred to a sterile centrifuge container (approximately 500 mL) designated for use with the centrifuge. The sample material is preferably evenly distributed in the centrifuge container, or a blank water container is used to compensate for the sample container. The digest is centrifuged at 4000 RPM for 15 minutes.

[0070] After centrifugation is complete, the supernatant is aspirated from each centrifuge container without disturbing the cell pellet.

[0071] Next, 25 mL of HPW is added to each cell pellet and the cells are resuspended evenly. The cell mixture from each of the centrifuge containers in the centrifuge container is combined into one container. If necessary, approximately 5 mL of the 25 mL of HPW supplied is retained to remove any cell material that adheres to the sides of the centrifuge container.

[0072] The centrifugation process is optionally repeated an additional 2-3 times.

[0073] After the final centrifugation is complete, the supernatant is aspirated from each centrifuge container without disturbing the cell pellet.

[0074] Next, 50 mL of cell lysis solution is added to the cell pellet and the cells are resuspended evenly. The cell suspension is placed in a water bath shaker at a temperature of 36-38°C for approximately 15 minutes with a gentle agitation setting.

[0075] After the cell suspension is removed from the water bath shaker, it is again centrifuged at 4000 PRM for approximately 15 minutes. Once complete, the supernatant is aspirated from each centrifuge container ensuring that the cell pellet is not disturbed.

[0076] Next, 50 mL of HPW is added to each centrifuge container to resuspend the cells evenly.

[0077] Five separate samples of the cell suspension are taken and diluted in LAL Reagent Water (LRW) at a 1 : 100 dilution factor and vortexed for approximately 1 minute.

[0078] The samples are then tested with a Charles River Laboratories Portable Test System (PTS) (sensitivity: 1 EU / mL - 0.01 EU / mL, 5 EU / mL - 0.05 EU / mL) as explained below.

[0079] Endotoxin measurement

[0080] A valid test result is determined when the spiked recovery is between 50-200% and the coefficient of variation of the reaction time of the sample and positive product control (PPC) replicates is less than 25%.

[0081] After obtaining the sample value, the total sample value in EU is calculated using the following formula:

[0082] Calculated sample value = sample value * first dilution value * second dilution value * third dilution value

[0083] Table 1a: Endotoxin results

[0084] Sample Spiked recovery (%) Sample value (EU / mL) Calculated sample value (EU x 10 6 )]]> 1 82％ 275 1.572 2 133％ 1232 1.261 3 131％ 525 1.055 4 63％ 193 3.850 5 109％ 520 16.406

[0085] Table 1b: EU and CFU results

[0086]

[0087] The calculated sample value is compared to the estimated cell count in the cellulose film prior to processing. These values are comparable in magnitude.

[0088] Example 2 - Extraction Method

[0089] The sample cellulose film was grown to have a surface area of approximately 9 inches by 12 inches. The film was cut into approximately equal pieces and placed in LAL Reagent Water (LRW) for one hour at 37°C. The extract was removed from the LRW and tested using the LAL assay. The recorded EU values are shown below.

[0090] Table 2

[0091] Sample Sample value (EU / mL) Calculated value (EU / device) 1 <5.00* 200 2 0.45 18 3 1.57 62.8 4 1.7 68 5 5.55 222 6 2.69 107.6 7 0.96 38.4 8 0.588 23.52 9 0.4 16 10 0.45 18 11 0.45 18 12 0.88 35.2 13 0.044 1.76 14 0.08 3.2

[0092] *- The limit of detection for this kit is 5.0 EU / mL. The sample did not have a registered value, so this is the reported sample value.

[0093] Example 3: Extraction vs. Digestion / Lysis

[0094] The following test compares two samples taken from films grown under the same conditions. One sample was prepared and tested according to the extraction method of the prior art. The sample was immersed in LAL Reagent Water (LWW) for one hour at 37°C. The extract was sampled and diluted 1:100. The other sample was prepared according to the method described in Example 1.

[0095] Table 3

[0096] Sample EU value Extraction method (prior art) <0.5000* Example 1 sample 33.744

[0097] The limit of detection for this cartridge is 0.005 EU / mL at a 1:100 dilution, and the lowest result that the system can detect is 0.500 Eu / mL. Since this assay does not detect trace endotoxin, the lowest endotoxin value that it can report is 0.500 Eu / mL.

[0098] The assay is able to detect significantly more lipopolysaccharide in a sample by adding a cell lysis technique when the biofilm is subjected to complete enzymatic digestion. For example, in this particular comparative example, the Example 1 method achieved a sensitivity that was 65 times greater than the prior art extraction method. According to certain embodiments, the method of the present application can increase sensitivity by at least 25%, 30%, 35%, 40%, 50%, or up to about 100% as compared to the extraction method.

Claims

1. A non-diagnostic method for quantifying endotoxin concentration in a biofilm sample, comprising: digesting the biofilm sample with a cellulase enzyme to break down the extracellular matrix of cellulose and form a digested biofilm sample; separating the digested biofilm sample into a supernatant and a cell pellet; combining the cell pellet with a cell lysing agent to release endotoxin and form an endotoxin suspension; concentrating the endotoxin suspension into an endotoxin sample and a supernatant of the suspension; and, performing a bacterial endotoxin test (BET) assay on the endotoxin sample to obtain a sample endotoxin value.

2. The method of claim 1, wherein the digesting step further comprises a heating step.

3. The method of claim 2, wherein the heating step is performed at a temperature of 48°C to 52°C.

4. The method of any one of claims 1-3, wherein the digesting step is performed over a period of 12 hours to 24 hours.

5. The method of any one of claims 1-3, wherein the separating step comprises centrifuging the digested biofilm sample.

6. The method of any one of claims 1-3, wherein the cell lysing agent comprises a chelating agent and a salt.

7. The method of claim 6, wherein the chelating agent comprises EDTA-HCl.

8. The method of claim 6, wherein the salt comprises Tris-HCl.

9. The method of any one of claims 1-3, wherein the step of combining the cell pellet with the cell lysing agent comprises suspending cells from the cell pellet in the cell lysing agent.

10. The method of claim 9, wherein the step of suspending cells from the cell pellet in the cell lysing agent comprises heating to a temperature of 22°C to 26°C.

11. The method of claim 9, further comprising stirring for a period of 10 minutes to 30 minutes.

12. The method of any one of claims 1-3, wherein the step of concentrating the endotoxin suspension comprises centrifuging the endotoxin suspension.

13. The method of any one of claims 1-3, further comprising the step of diluting the endotoxin sample prior to performing the BET assay step.

14. The method of any one of claims 1-3, wherein the BET assay is a Limulus amebocyte lysate (LAL) assay.

15. The method of claim 14, wherein the LAL assay is a gel-clot assay.

16. The method of claim 14, wherein the LAL assay is a turbidimetric assay.

17. The method of claim 14, wherein the LAL assay is a chromogenic assay.

18. The method of any one of claims 1-3, further comprising the step of adding a beta-glucan inhibitor to the endotoxin sample.

19. The method of any one of claims 1-3, further comprising performing a colony forming unit (CFU) assessment on the biofilm sample to obtain a CFU value, such that a relationship between the endotoxin value and the CFU value can be determined.

20. A non-diagnostic method of identifying a Gram-negative biofilm-derived bacterial infection comprising: enzymatically digesting a biofilm sample with a cellulase enzyme to break down the extracellular matrix of cellulose and form a digested biofilm sample; separating the digested biofilm sample into a supernatant and a cell pellet; combining the cell pellet with a cell lysing agent to release an amount of endotoxin, wherein the amount of endotoxin has a lower limit of zero; concentrating the amount of endotoxin, and conducting a bacterial endotoxin test (BET) assay on the concentration to obtain a sample endotoxin value.

21. The method of claim 20, wherein the cell sample is removed from the surface of an implanted medical device.

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