Biofilm and methods of use

Abstract

An Artificial Test Soil may include: a base medium and Klebsiella pneumoniae.

Description

CLAIM OF PRIORITY AND CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 641,681, filed on May 2, 2024, entitled “BIOFILM AND METHODS OF USE,” which is incorporated by reference herein in its entirety. BACKGROUND

[0002] Proper reprocessing, which includes cleaning and / or disinfection / sterilization of medical devices is critical to ensure patient safety. SUMMARY OF THE INVENTION

[0003] In some aspects, the techniques described herein relate to a biofilm including: a base medium and Klebsiella pneumoniae.

[0004] In some aspects, the techniques described herein relate to a method of evaluating a cleaning method on a device including: applying an biofilm including base medium and Klebsiella pneumoniae to the device; allowing the biofilm to develop on the device; subjecting the device to the cleaning method to be evaluated; and determining the presence or absence of Klebsiella pneumoniae on the device. DETAILED DESCRIPTION OF THE INVENTION

[0005] Reference will now be made in detail to certain aspects of the disclosed subject matter. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0006] Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%)andthe sub-ranges (e.g., 0.1%to0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.

[0007] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. The statement “at least one of A and B” has the same meaning as “A, B, or A and B.” In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. A comma can be used as a delimiter or digit group separator to the left or right of a decimal mark; for example, “0.000,1” is equivalent to “0.0001.” All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0008] In the methods described herein, the acts can be carried out in any order without departing from the principles of the invention, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0009] The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range. The term “substantially” as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. The term “substantially free of’ as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that about 0 wt% to about 5 wt% of the composition is the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than or equal to about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%.

[0010] This disclosure relates to the technical field of biofilm generation and evaluation on medical devices, with a focus on creating clinically relevant and challenging biofilms for the assessment of cleaning methods. Previous approaches, such as those described in ISO 15883 Annex F for polytetrafluoroethylene channels, have provided foundational knowledge but come with limitations. These methods often require extensive manual intervention, are limited in their applicability across different device sizes, and may not produce biofilms that adequately simulate clinical challenges.

[0011] The prior methods faced challenges in terms of space requirements, hands-on time, maintenance, and the robustness of the resulting biofilm. Specifically, the need for daily maintenance, the risk of user error, and the inability to produce a sufficiently challenging biofilm for testing cleaning methods were significant shortcomings.

[0012] The present disclosure addresses these challenges by introducing a method and composition for generating and evaluating biofilms in or on medical devices. The invention utilizes a unique biofilm construction that can include a matrix material such as an artificial test soil and a test bacteria distributed about the matrix. The matrix material can include an artificial test soil and the bacteria can be Klebsiella pneumoniae. Other examples of bacteria can include Pseudomonas aeruginosa, Escherichia coll, Staphylococcus aureus. Enterococcus faecalis. In general, the biofilm can include any gram-positive or gram-negative bacteria that is desired.

[0013] The biofilm can be applied across all polytetrafluoroethylene channel sizes and lengths in a medical device or medical device mock-up. This method is designed to be cost-effective, userfriendly, and capable of producing a robust, confluent biofilm that is challenging enough to test the efficacy of cleaning methods. The biofilm can be incubated at temperatures ranging from about 20 °C to about 40 °C, with an optimal range of about 25 °C to about 35 °C, and is in contact with a fluoropolymer, specifically polytetrafluoroethylene. Additionally, the composition may include serum blood, such as sheep blood, to enhance the clinical relevance of the biofilm.

[0014] This disclosure is particularly significant for the medical device industry, offering a more efficient, reliable, and comprehensive platform to evaluate cleaning and sterilization methods, thereby enhancing device safety and infection control measures.

[0015] The present disclosure relates to a biofilm for simulated use testing and cleaning validation studies of medical devices. Examples of medical devices can include a flexible endoscope, a narrowed lumened accessory device, or a balloon catheter. The artificial test soil can include a base medium. The artificial test soil may optionally contain bilirubin and / or mucin or carbohydrate. The artificial test soil can further include a serum blood. The artificial test soil is contacted with the bacterial and subjected to growth conditions. The growing biofilm can also be placed in contact with a fluoropolymer. An example of a suitable fluoropolymer is a polytetrafluoroethylene. Polytetrafluoroethylene is useful for testing because polytetrafluoroethylene channels are commonly found in medical devices such as flexible endoscopes, and the interaction between the biofilm and the fluoropolymer surface is significant for evaluating cleaning efficacy.

[0016] As an example, the artificial test soil can include about 5 / o v / v to about 30 / o v / v base medium about 10 % v / v to about 20 % v / v, less than, equal to, or greater than about 5 % v / v, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 ,or about 30 % v / v. Serum can range from about 1% v / v to about 20% v / v, about 5% v / v to about 10% v / v, less than, equal to, or greater than about 1 % v / v, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 % v / v. If present, the bilirubin will be in the amount of about 1,000 nmoles / ml and the mucin or carbohydrate will be in the amount up to 10,000 pg / ml.

[0017] To grow a biofdm, the artificial test soil and bacteria can be incubated at a temperature in a range of from 20 °C to about 40 °C, about 25 °C to about 35 °C, less than, equal to, or greater than about 20 °C, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or about 40 °C.

[0018] The base medium can be any medium that mimics physiological human fluids. In some examples, the base medium is an organic soil, such as ATS2015. ATS2015 includes contaminants remaining on surgical instruments after clinical use, including protein (~33mg / ml, BCA assay), hemoglobin (~2.5mg / ml, TMB assay) and carbohydrates (~1 Img / ml, Phenolsulfuric acid assay), measured without the addition of sheep’s blood. These quantities are reflective of the “worst case” levels found to remain on instruments after patient use, but before reprocessing. The FDA and other international regulatory agencies specify that simulated-use testing should approximate as closely as possible the actual soiling the instrument is exposed to during clinical use. The bacteria that are ultimately used are added to the base medium separately.

[0019] Optionally, the serum can be added. That serum can be used from a variety of serum sources; an example of serum is calf serum. The blood can be from any source such as whole blood. A particularly suitable blood is sterile sheep blood. The Artificial Test Soil may optionally contain bilirubin, for example when preparing a soil that mimics the gastrointestinal site. When present, the bilirubin can be derived from oxgall bile such as bovine oxgall bile. The Artificial Test Soil may also optionally contain a carbohydrate source. When present, the carbohydrate can be derived from L-Glutamine, glucose or oxgall bile. The Artificial Test Soil may also contain a source of sodium which is added to the RPMI base to provide osmolarity that mimics physiological solutions. The sodium source may be derived from sodium chloride, sodium bicarbonate and / or sodium pyruvate.

[0020] The biofilms of the present disclosure are based on data representing the “worstcase” soil levels from patient-used medical devices and therefore represents a standardized soil formulation that would be applicable to a wide range of medical device simulated-use testing.

[0021] In a particular example, the present disclosure provides a biofilm that mimics the gastrointestinal site comprising: RPMI 1640, LPS, Calf Serum, Bovine Oxgall, Sterile Sheep Blood, Sodium Bicarbonate, Sodium-Pyvuvate, and L-Glutamine. In another example, the present disclosure provides a biofilm that mimics soil related to the lung including RPMI 1640, LPS, Calf Serum, Sterile Sheep Blood, Sodium Bicarbonate, Sodium-Pyvuvate, and L-Glutamine.

[0022] The need for an appropriate biofilm challenge has been recognized by governing agencies such as the FDA, but an acceptable standardized test soil, used as a matrix, has not been achievable due to lack of data for patient-used worst-case soil determinations. Because the components used in making the Artificial Test Soil formulations are well defined; RPMI is tissue culture medium that is chemically defined, and the other components including serum, blood, oxgall bile and purified endotoxin are all commercially available, standardization of the Artificial Test Soil formulations is feasible. Each batch of a particular Artificial Test Soil formulation would be analyzed to ensure it was within 5% of the expected values as outlined in Example 2. Other Artificial Test Soil formulations may be prepared as needed within the ranges of the components outlined and / or other physiological parameters that might be measured from medical device samples after patient-use.

[0023] The biofilm of the present disclosure can be used to evaluate the efficiency of a reprocessing method on a device, such as a medical device. Accordingly, the present disclosure provides a method of evaluating a reprocessing method on a device comprising: (a) growing a biofilm on a device, the biofilm includes a bacteria and a matrix (the matrix may include an artificial test soil comprising base medium, serum, blood and / or endotoxin) to the device; (b) subjecting the device to the reprocessing method to be evaluated; and (c) determining the presence or absence of at least one contaminant on the device.

[0024] The term “contaminant” means any material that should not be present if the reprocessing method worked efficiently. The presence of a contaminant indicates that the process is not efficient or the operator has not properly performed the process. Examples of contaminants that may be detected include blood or hemoglobin, protein, glucose, endotoxin, bilirubin, sodium and chloride ions and leukocyte esterase. Preferably, the presence of more than one contaminant is evaluated.

[0025] The phrase “reprocessing method” means any process that is purported to clean and / or disinfect / sterilize a device and includes: (a) cleaning the device to remove soil, and / or (b) disinfection / sterilization which kills microorganisms. The process can include apparatuses such as washers / disinfectors or autoclaves (which may be automated cleaning systems), sterilizers and / or compositions such as detergents, disinfectants and sterilants. A person skilled in the art would appreciate that reprocessing of previously used medical devices includes cleaning of the device, which is followed by disinfection / sterilization.

[0026] In one example, the biofdm can be used to evaluate the efficiency of a reprocessing method on the killing and / or removal of microorganism(s). In some examples, a reprocessing method can include hand cleaning the medical device.

[0027] Accordingly, the present disclosure provides a method of determining whether a reprocessing method can kill and / or remove a microorganism on a device comprising: (a) inoculating a biofilm on a device, the biofilm include a bacteria and a matrix (the matrix may include an artificial test soil comprising base medium, serum, blood and / or endotoxin); (b) applying the inoculated biofilm to the device; (c) subjecting the device to the reprocessing method to be evaluated; and (d) determining the presence or absence of the microorganism on the device. The presence of a microorganism indicates that the process is not efficient or the operator has not properly performed the method.

[0028] The above described methods have a variety of applications some of which are outlined below. In one example, the biofilms of the invention can be used as standardized test soils by manufacturers when testing equipment such as cleaner / disinfectors and sterilizers, as well as for medical devices where reprocessing protocols need to be developed. Examples of medical devices include flexible endoscopes, narrow lumened accessory devices used in gastrointestinal or respiratory tract such as cannulatomes, balloon catheters or other accessory devices used for endoscopic procedures. In addition, the biofilms can be used by manufacturers needing to perform simulated-use evaluations for comparative testing of detergents, disinfectants or sterilants for medical device reprocessing.

[0029] For microbial killing-efficiency testing, the test microorganisms would be present in the biofilm at a desired concentration (most commonly 106 viable organisms / device) and inoculated onto the medical device (or appropriate test carrier) for evaluation. The microorganisms can be allowed to grow as a biolfilm in the device. The biofilms can grow at a rate in a range of from about 0.10 cm / s to about 0.50 cm / s, about 0.15 cm / s to about 0.22 cm / s, less than, equal to, or greater than aboutO.10 cm / s, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or about 0.50 cm / s.

[0030] The proposed applications would include; inoculation of the Artificial Test Soil (with or without microorganisms) onto a test medical device followed by up to 24 hours of drying to provide a “worst-case” challenge. Following the guidelines outlined in AAMI TIR#12 (1994), the Artificial Test Soil would act as the inorganic / organic challenge for this protocol. After air drying on the test carrier, the soil may also be fixed using 2.5% glutaraldehyde to provide a greater challenge for washing / detergent testing. This mimics what might occur in actual patient-use when devices are not adequately cleaned and are high-level disinfected using glutaraldehyde (2.0 to 2.5%) solutions. Furthermore, the Artificial Test Soil can be made up to contain up to 400 ppm hard water to meet FDA requirements for hard water testing. Because the composition of the soil is multi-facetted, it allows manufacturers to assess a wide variety of factors including; protein, hemoglobin (blood), bilirubin, carbohydrate, endotoxin, sodium and chloride ions. This allows manufacturers to assess relative efficacy of a reprocessing method on various soil components.

[0031] In another example, the biofilm can also be used as a positive control in a test kit for cleaning validation that can be used in hospitals and other institutions to: 1) ensure adequacy of cleaning of medical devices as part of a quality assurance program, and 2) to ensure staff training competency. Accordingly, the present invention provides a cleaning validation test kit comprising the artificial test soil of the invention and an indicator to determine if the contamination has been adequately removed from the medical device. The indicator can be any indicator that can detect the presence of a contaminant. In some examples, more than one indicator is used, each one detecting the presence of a different contaminant. In some examples, a single indicator strip is used that detects the presence of more than one contaminant. Examples of the latter include a chemi-strip that can detect blood, protein and leukocyte esterase.

[0032] Using the test kit of the invention, samples are taken of the cleaned medical device, and tested using Chemi-strips for blood, glucose, protein and leukocyte esterase. The result from the cleaned device is compared to the positive control which consists of the Artificial Test Soil. Any medical device samples that flag any of the tests as positive should be recleaned before proceeding to the disinfection and / or sterilization step of device reprocessing. The clinical applicability of this test kit has been validated using in-use samples.

[0033] An advantage of the cleaning validation kit of the invention is that more than one parameter is evaluated, making it more sensitive than only testing for one soil parameter. In addition, the enzymatic detergents frequently used for reprocessing of medical devices will cause the leukocyte esterase and protein tests to flag as positive. Thus, the kit can not only ensure that patient material has been adequately cleaned, it can also provide some assurance that rinsing has been adequate to remove any residual enzymatic detergent. Currently there are no test methods that allow such determinations to be made. The test kit of the invention provides a wider range of test parameters (blood, protein and leukocyte esterase) compared to the few published methods that detect protein only. In addition, the test kit can detect if there is residual enzymatic detergent (of formulations containing enzymes in concentrations adequate to trigger the protein and / or leukocyte esterase flags) remaining which is something other test kits cannot do. Furthermore, the benchmarks to validate the usefulness of the Chemistrip test method for protein and hemoglobin has been provided for the kit whereas similar validations are not provided for other published methods. The advantages of the biofilm and the test kit method are outlined below: 1) The biofdm is reproducible and mimics “worst-case” soil levels expected from patientuse of medical devices. 2) The biofdm can be used for training of staff involved in medical device reprocessing and using the biofilm as the device soil reduces the risk of trainee exposure to infectious material during training. 3) The biofdm can be used for simulated-use testing with or without test organisms (e.g., same soil for cleaning validation testing and microbial load reduction testing). 4) Allows quantitative relative assessment of protein, blood, carbohydrate, bilirubin, sodium / chloride ions and endotoxin by standard chemistry assays. Examples

[0034] Various aspects of the present invention can be better understood by reference to the following Examples which are offered by way of illustration. The present invention is not limited to the Examples given herein.

[0035] The following protocols will be used to assess the artificial test soil’s ability to function. Example 1: For Instrument Channels (using full-strength Artificial Test Soil): 1. From the stock plate, subculture Klebsiella pneumoniae ATCC 700603 on nutrient agar. Create 1-2 lawn plates with a sterile cotton swab. 2. Incubate the plates at 37°C for 24 hours. 3. Using these fresh subculture plates, resuspend a loopful of Klebsiella pneumoniae in 10 mL sterile water. Vortex to mix thoroughly. 4. OD540 of this resuspension will be approximately 1.0 5. Store lawn plates in a refrigerator. 6. Rehydrate a vial of Artificial Test Soil with 7-8 mL of resuspended culture. Shake and vortex vigorously to mix well. 7. Transfer resuspended Artificial Test Soil-culture mixture to a new sterile cup. 8. Rinse out the Artificial Test Soil vial with the remaining culture and add to the cup. a. If several vials of Artificial Test Soil are used, repeat steps 6-8 and pool the Artificial Test Soil-culture mixtures. 9. Using aseptic technique, attach silicone segment to polytetrafluoroethylene channel. 10. Add the appropriate volume of Artificial Test Soil-culture mixture to the channels and close the loop by attaching the silicone segment to the other end of the polytetrafluoroethylene channel. a. For larger channels, a serological pipette can be used to inject culture into the channel. 11. Any remaining Artificial Test Soil-culture can be discarded. 12. Insert the polytetrafluoroethylene biofilm loop into a peristaltic pump with the correct silicone # settings and flow rate programmed. 13. Circulate the biofilm for 4 days. 14. After 4 days, stop the pump, remove the channels, and purge the channels with ~25 mb sterile water, purge with air. Example 2: For Air / Water Channels (using diluted Artificial Test Soil): 1. From a stock plate, subculture Klebsiella pneumoniae ATCC 700603 on nutrient agar. Create 1-2 lawn plates with a sterile cotton swab. 2. Incubate the plates at 37°C for 24 hours. 3. Using these fresh subculture plates, resuspend a loopful of Klebsiella pneumoniae in 9 mL sterile water, pH 7.0. Vortex to mix thoroughly. 4. OD540 of this resuspension should be around 1.0 5. Store lawn plates in a refrigerator. 6. Rehydrate a vial of Artificial Test Soil with 8 mL of sterile water, pH 7.0. Shake and vortex vigorously to mix well. 7. Add 1 mL of the hydrated Artificial Test Soil to 9 mL Klebsiella culture tube (this dilutes the Artificial Test Soil 1:10). Mix well. 8. Carefully, and using aseptic technique, attach silicone segment to polytetrafluoroethylene channel. 9. Add the appropriate volume of Artificial Test Soil-culture mixture to the channels and close the loop by attaching the silicone segment to the other end of the polytetrafluoroethylene channel. 10. Insert the polytetrafluoroethylene biofilm loop into a peristaltic pump with the correct silicone # settings and flow rate programmed. 11. Circulate the biofilm for 6 days. There is no culture refresh.

[0036] The protocols of Examples 1 and 2 are expected to show that the artificial test soils described herein are suitable for determining whether a medical device can be properly reprocessed.

[0037] The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the aspects of the present invention. Thus, it should be understood that although the present invention has been specifically disclosed by specific aspects and optional features, modification and variation of the concepts herein disclosed may be resorted to by those of ordinary skill in the art, and that such modifications and variations are considered to be within the scope of aspects of the present invention. Exemplary Aspects.

[0038] The following exemplary aspects are provided, the numbering of which is not to be construed as designating levels of importance:

[0039] Aspect 1 provides a biofilm: a base matrix and a bacteria selected from Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, or a mixture thereof.

[0040] Aspect 2 provides the biofilm of Aspect 1, wherein the matrix comprises an artificial test soil.

[0041] Aspect 3 provides the biofilm of Aspect 2, wherein the matrix comprises an artificial test soil, blood, or organic medium.

[0042] Aspect 4 provides the biofilm of any of Aspects 1-3, wherein the biofilm is incubated at a temperature in a range of from about 20 °C to about 40 °C.

[0043] Aspect 5 provides the biofilm of any of Aspects 1-4, wherein the biofilm is incubated at a temperature in a range of from about 25 °C to about 35 °C.

[0044] Aspect 6 provides the biofilm of any of Aspects 1-5, wherein the biofilm is placed in contact with a fluoropolymer.

[0045] Aspect 7 provides the biofilm of Aspect 6, wherein the fluoropolymer comprises poly tetrafluoroethyl ene.

[0046] Aspect 8 provides the biofilm of any of Aspects 1-7, wherein the matrix comprises an artificial test soil that comprises a serum blood.

[0047] Aspect 9 provides the biofilm of Aspect 8, wherein the serum blood is sheep blood.

[0048] Aspect 10 provides the biofilm of any of Aspects 1-9, wherien the bacteria is Klebsiella pneumoniae.

[0049] Aspect 11 provides a method of evaluating a cleaning method on a device comprising: applying a biofilm comprising base matrix and a bacteria selected from Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, or a mixture thereof to the device; allowing a biofilm to develop on the device; subjecting the device to the cleaning method to be evaluated; and determining the presence or absence of Klebsiella pneumoniae on the device.

[0050] Aspect 12 provides the method of Aspect 11, wherein the matrix comprises an artificial test soil.

[0051] Aspect 13 provides the method of Aspect 12, wherein the matrix comprises an artificial test soil, blood, or organic medium.

[0052] Aspect 14 provides the method of any of Aspects 11-13, wherein the biofilm is incubated at a temperature in a range of from about 20 °C to about 40 °C.

[0053] Aspect 15 provides the method of any of Aspects 11-14, wherein the biofilm is incubated at a temperature in a range of from about 25 °C to about 35 °C.

[0054] Aspect 16 provides the method of any of Aspects 11-15, wherein the matrix comprises an artificial test soil that comprises a serum blood.

[0055] Aspect 17 provides the method of Aspect 16, wherein the serum blood is sheep blood.

[0056] Aspect 18 provides the method of any of Aspects 11-17, wherein the biofilm is placed in contact with a fluoropolymer.

[0057] Aspect 19 provides the method of Aspect 18, wherein the fluoropolymer comprises polytetrafluoroethylene.

[0058] Aspect 20 provides the method of any of Aspects 11-19, wherein the device is a medical device.

[0059] Aspect 21 provides the method of any of Aspects 11-20, wherein the device comprises a flexible endoscope, a narrowed lumened accessory device, or a balloon catheter.

[0060] Aspect 22 provides the method of any of Aspects 11-21, wherein the device is an endoscope.

[0061] Aspect 23 provides the method of any of Aspects 11-22, wherein the cleaning method comprises a hand cleaning method.

[0062] Aspect 24 provides the method of any of Aspects 11-23, wherein the cleaning method comprises an automated cleaning method.

[0063] Aspect 25 provides the method of any of Aspects 11-24, wherein the cleaning method comprises the use of an autoclave.

[0064] Aspect 26 provides the method of any of Aspects 11-25, wherien the bacteria is Klebsiella pneumoniae.

Claims

1. A biofilm comprising a base matrix and a bacteria selected from Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, or a mixture thereof.

2. The biofilm of claim 1, wherein the matrix comprises an artificial test soil.

3. The biofilm of claim 2, wherein the matrix comprises an artificial test soil, blood, ororganic medium.

4. The biofilm of claim 1, wherein the biofilm is incubated at a temperature in a range of from about 20 °C to about 40 °C.

5. The biofilm of claim 1, wherein the biofilm is incubated at a temperature in a range of from about 25 °C to about 35 °C.

6. The biofilm of claim 1, wherein the biofilm is placed in contact with a fluoropolymer.

7. The biofilm of claim 6, wherein the fluoropolymer comprises polytetrafluoroethylene.

8. The biofilm of claim 1, wherein the matrix comprises an artificial test soil that comprisesa serum blood.

9. The biofilm of claim 8, wherein the serum blood is sheep blood.

10. The biofilm of claim 1, wherien the bacteria is Klebsiella pneumoniae.

11. A method of evaluating a cleaning method on a device comprising:applying a biofdm comprising a base matrix and a bacteria selected from Pseudomonas aeruginosa, Escherichia coll, Staphylococcus aureus. Enterococcus faecalis, Klebsiella pneumoniae, or a mixture thereof to the device;allowing a biofdm to develop on the device;subjecting the device to the cleaning method to be evaluated; anddetermining the presence or absence of the bacteria on the device.

12. The method of claim 11, wherein the matrix comprises an artificial test soil.

13. The method of claim 12, wherein the matrix comprises an artificial test soil, blood, ororganic medium.

14. The method of claim 11, wherein the biofdm is incubated at a temperature in a range of from about 20 °C to about 40 °C.

15. The method of claim 11, wherein the biofilm is incubated at a temperature in a range of from about 25 °C to about 35 °C.

16. The method of claim 11, wherein the matrix comprises an artificial test soil that comprises a serum blood.

17. The method of claim 16, wherein the serum blood is sheep blood.

18. The method of claim 11, wherein the biofilm is placed in contact with a fluoropolymer.

19. The method of claim 18, wherein the fluoropolymer comprises polytetrafluoroethylene.

20. The method of claim 11, wherein the device is a medical device.

21. The method of claim 11, wherein the device comprises a flexible endoscope, a narrowed lumened accessory device, or a balloon catheter.

22. The method of claim 11, wherein the device is an endoscope.

23. The method of claim 11, wherein the cleaning method comprises a hand cleaningmethod.

24. The method of claim 11, wherein the cleaning method comprises an automated cleaning method.

25. The method of claim 11, wherein the cleaning method comprises the use of an autoclave.

26. The method of claim 11, wherien the bacteria is Klebsiella pneumoniae.