CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Non-provisional Application no. 62/406,524 filed on October 11, 2016, the entire contents of which are hereby incorporated by reference.

BACKGROUND

[0002] This disclosure relates to a composition for biofilm disruption, methods of manufacture thereof, and to articles comprising the same.

[0003] Biofilms are communities of microorganisms (also referred to herein as microbes) protected by a self-synthesized layer of complex polysaccharides, proteins, lipids and extracellular DNA, collectively called the extracellular polymeric substance (EPS). Biofilms form when a microbe (or a group of microbes in an aggregate or cluster) irreversibly attaches itself to a surface and commences cell division and recruitment of other

microorganisms by providing more diverse adhesion sites to a substrate. Being in a biofilm provides these microorganisms with a host of advantages, including, but not limited to: physical protection from the host immune system and antimicrobials/antibiotics, retention of water and tolerance to desiccation, nutrient sorption and storage, high extracellular enzymatic activity, adhesion to the infection site and cell aggregation, leading to a coordination of their virulence factor expression via quorum sensing.

[0004] Particularly troubling to the medical field is that it has been estimated that as much as 80% of all human bacterial infections are biofilm-associated, including more than 90% of all chronic wound infections. Additionally, the biofilm mode of microbial life is responsible for up to a 1000-fold increase in antibiotic tolerance due to the physical impedance and enzymatic inactivation of the drugs, coupled with lowered metabolic rates in many biofilm-associated cells. Thus, biofilm infections are highly recalcitrant and are associated with chronic, non-healing infections.

[0005] Traditionally, infections have been treated by directly targeting the causative pathogens. However, biofilms change the game by providing microorganisms with greatly increased protection from antimicrobials, causing the effective concentrations to be elevated to dangerous levels. Therefore, some researchers have switched their focus to anti-biofilm agents that deny the pathogens the protection of the biofilm, thereby increasing the effectiveness of traditional, antimicrobial therapies. In order to effect this, it is desirable to determine the types, quantities and species of microorganisms that inhabit the biofilm.

[0006] One such avenue of research has been the testing of compounds and strategies that lead to a dispersal event: dispersal agents. Nearly all mature biofilms undergo dispersal, which can be divided into two main subtypes: passive and active, both of which result in the release of planktonic, free-floating cells into the environment.

[0007] Passive dispersal simply refers to a physical sloughing event brought on by external forces such as fluid and solid shear, and mechanical interventions (e.g., tooth brushing). For example, a biofilm streamer may be torn off of the main mass of the biofilm by the flow of interstitial fluid or due to physical abrasion by a surgeon. This results in aggregates of microorganisms that cannot be accurately identified or quantified. Typically inaccurate disaggregation results in under-representing the true microbial load.

[0008] Active dispersal, on the other hand, refers to dispersal events triggered by the biofilm microbes themselves in response to environmental changes such as nutrient starvation, toxic byproducts, bacteriophages, phagocyte challenge, antimicrobial stress, and unfavorable oxygen levels. Thus, active dispersal is a vital stage in the life-cycle of a biofilm that contributes to bacterial survival and disease progression. Active dispersal in

uncontrolled situations does not facilitate quantification or understanding of the biofilm composition.

[0009] Microbial quantification has relied on culture methods to grow microscopic organisms into larger colonies or plaques. This method relies also on suspending organisms in liquid and serially diluting the organisms until only about 1 to 100 organisms remain in solution. Once these relatively few organisms remain in solution, they may be spread onto semi-solid growth media and allowed to form colonies or plaques. This process was relatively straight-forward until research demonstrated how intensely dense and compact biofilm-embedded microorganisms are.

[0010] Quantification of biofilm-embedded microorganisms is difficult because generating a suspension of single cells is extremely difficult when microorganisms use protein appendages or other extracellular components to aggregate together. Mechanical shaking, mixing, and sonication attempts to produce a homogenous single-cell mixture are inadequate. Microorganism clusters withstand vigorous shaking and maintain clusters of even 100 to 1000 cells. These clusters of varying sizes are then diluted numerous times and plated on culture medium to enumerate the cells at a given dilution. This does not result in complete disaggregation of the cluster. Often one colony (counted as 1 microorganism) comprising 100 to 1000 cells is dropped onto the culture medium. If the cluster is fully disaggregated the 100 to 1000 cells would have been broken apart homogeneously and diluted and plated such that 1 cell accounted for the production of 1 new colony.

[0011] Accurate quantification of biofilm-contained microorganisms is therefore desirable for efficient research and development in biotechnology sectors and valid diagnoses in the clinical sector. Where clinical diagnosis is concerned, poor quantification or inaccurate speciation could result in deciding not to prescribe or to prescribe wrong antibiotics because an infection is either not identified or is instead misidentified. Clinically, if the correct microorganisms are not identified due to poor disaggregation, the appropriate antibiotics may not be used. For research and development of biotechnologies, misrepresenting microbial loads in animal and bench studies can result in failure to recover microorganisms for development or device-based claims. Standardized biofilm quantification test methods that rely on mechanical methods of disruption to determine product efficacy will produce inaccurate data for generation of anti-biofilm or antimicrobial claims.

[0012] To develop appropriate anti-biofilm therapeutics and methods of combating the development of biofilms, accurate methods are desirable to evaluate the biofilm to determine the types of microbes that form the biofilm. It is also desirable to perform the dispersal (also referred to herein as disaggregation) in a controlled environment to facilitate an understanding of the types of microbes and the quantities of microbes that form the biofilm.

SUMMARY

[0013] Disclosed herein is a composition for disaggregating biofilms comprising a plurality of enzymes; a calcium salt that prevents activity inhibition in the enzymes; a surfactant; and a pH adjusting additive.

[0014] Disclosed herein is a method comprising mixing a plurality of enzymes; a calcium salt; a surfactant; a pH adjusting additive and water to form a disaggregation composition.

BRIEF DESCRIPTION OF THE FIGURES

[0015] Figure 1 is a graph that depicts the efficacy of the previous method of quantification using only mechanical disaggregation versus the inventive composition. DETAILED DESCRIPTION

Definitions

[0016] A biofilm is a dense colonization of organisms encased in an extracellular matrix commonly tolerant of antimicrobial compounds and antiseptic agents.

[0017] A microorganism is a small single-cell organism invisible to the naked eye.

[0018] A composition is a mixture of components contained in a single solution.

[0019] Disaggregation as defined herein includes the breaking up of complexes of multiple small units into individual units capable of being suspended in a homogeneous solution.

[0020] Disclosed herein is a composition (also referred to herein as a composition) that is designed to break apart (disaggregate or disperse) microorganisms contained in a biofilm. Disclosed herein too is a method of manufacturing and using the composition. The biofilm may contain either a single species or multiple species that differ from one another. This disaggregation of microorganisms occurs while maintaining microbial viability and individual microbial cell characteristics of the biofilm. In an embodiment, the composition comprises enzymes that allow for accurate quantification of microorganisms present in biofilms after disaggregation.

[0021] Biofilm quantification (while preserving the viability of colony forming units of microorganisms) is generally challenging due to the clumping characteristics of organisms embedded in or on biofilms. This quantification is useful for accurate clinical diagnosis using patient biopsy samples, animal samples for either veterinary clinical practice or preclinical research animals, and in vitro biotechnology research and development.

[0022] Tissue-embedded biofilms (i.e., biofilms that are embedded in tissues in the bodies of living beings or in tissues outside the bodies of living beings contained in a laboratory setting) may be quantified with a composition of tissue-degrading enzymes that permits disaggregation of microbes that comprise cells of non-microbial origin. Standard test methods include disaggregating biofilms using mechanical methods. However, the use of a composition that comprises enzymes would provide more accurate information than from existing mechanical methods. This composition may be used on biofilm from in vitro experiments or in in vivo or ex vivo tissue-embedded biofilms.

[0023] In an embodiment, the composition for disaggregation of a biofilm comprises a plurality of enzymes, a calcium salt for maintaining the activity of at least one of the enzymes in the plurality of enzymes, a surfactant, and a pH adjusting additive. The enzyme may target one or more of proteins, polysaccharides, nucleic acids, starches, or collagens. Combinations of enzymes may also be used. All of the foregoing ingredients of the composition are dispersed in primarily in water to form a single phase solution. Other solvents and additives may be added to the composition as desired.

[0024] In an embodiment, the protease in the composition comprises Proteinase K. Proteinase K cleaves peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids and is useful for general digestion of protein in biological samples. It is desirable for the Proteinase K to be purified to remove RNase (ribonuclease) and Dnase (deoxyribonuclease) activities. The Proteinase K is present in the composition to disrupt and cleave proteins present in the biofilm thus releasing the microorganisms to be homogeneously resuspended.

[0025] The Proteinase K is present in an amount of 0.01 to 15 milligrams per milliliter, preferably 0.05 to 10 milligrams per milliliter, 0.1 to 4 milligrams per milliliter and more preferably 0.3 to 1.0 milligrams per milliliter based on the amount of water in the composition.

[0026] A variety of polysaccharides hydrolases may optionally be used in the composition. An exemplary polysaccharide hydrolase is Dispersin B that is used to target polysaccharides present in the biofilm. Dispersin B (also known as DspB or DispersinB) is a 40 kDa glycoside hydrolase produced by the periodontal pathogen, Aggregatibacter actinomycetemcomitans. The bacteria secrete Dispersin B to release adherent cells from a mature biofilm colony by disrupting biofilm formation. The enzyme catalyzes the hydrolysis of linear polymers of N-acetyl-D-glucosamines found in the biofilm matrices. Poly-acetyl glucosamines are integral to the structural integrity of the biofilms of various Gram-positive bacteria and Gram-negative bacteria and are referred to as PIA (PNAG,PS/A) in

Staphylococcus species and PGA in Escherichia coli. By degrading the biofilm matrix, Dispersin B allows for the release of microorganisms that can adhere to adjacent new surfaces and extend the biofilm or start new colonies.

[0027] The Dispersin B is used in an amount of 0.01 to 150 micrograms per milliliter, preferably 0.05 to 125 micrograms per milliliter, and preferably 0.1 to 100 micrograms per milliliter of water in the composition.

[0028] The enzymes in the composition also include a deoxyribonuclease (Dnase). Dnase is any enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA. Deoxyribonucleases are one type of nucleases, a generic term for enzymes capable of hydro lyzing phosphodiester bonds that link nucleotides. [0029] In an exemplary embodiment, the enzyme is Deoxyribonuclease I (Dnase I). Dnase I is an endonuclease coded by the human gene Dnase 1. Dnase I is a nuclease that cleaves DNA preferentially at phosphodiester linkages adjacent to a pyrimidine nucleotide, yielding 5 '-phosphate-terminated polynucleotides with a free hydroxyl group on position 3', on average producing tetranucleotides. In an exemplary embodiment, the Dnase I is used to target nucleic acids present in the composition. In an exemplary embodiment, the Dnase I is used to target deoxyribonucleic acid (DNA).

[0030] The Dnase I is present in an amount of 0.1 to 20 kilo Units of activity

(U)/ milliliter. Units of activity are typically used to describe enzyme catalytic activity, where a unit (U) refers to the amount of enzyme that catalyzes the conversion of 1 micromole (μιηοΐε) of substrate per minute. Thus, 1 enzyme unit (U) = 1 micro mol per minute

(μιηοΐ/min), where μιηοΐ refers to the amount of substrate converted.

[0031] In another embodiment, the Dnase I is present in the composition in an amount of 1 to 50 micrograms per milliliter, preferably 2 to 40 micrograms per milliliter and more preferably 5 to 30 micrograms per milliliter of water in the composition.

[0032] Enzymes such as collagenase and amylase may also optionally be used in the composition if desired. Collagenase is used to target collagen present in the biofilm while amylase is used to target starch present in the biofilm. Collagen is present in an amount of 10 to 300 U/milliliter of water in the composition.

[0033] In an embodiment, the composition comprises a calcium salt to facilitate the protection of Dnase I from Proteinase K. In short, the calcium salt facilitates a reduction in the inhibition of specific activities of certain enzymes. In the presence of preservatives such as (Ethylenediaminetetraacetic acid) EDTA, Dnase I is rapidly inactivated by proteinase K. The presence of calcium salts prevents the inactivation of the Dnase I by proteinase K.

[0034] Examples of calcium salts are calcium chloride, calcium sulfate, calcium nitrate, calcium bicarbonate, calcium citrate, calcium phosphate, or a combination thereof.

[0035] Calcium salts may be used in amounts of 2 to 40 millilmolar (mM), preferably 3 to 30 millimolar, preferably 5 to 20 millimolar, and more preferably 7 to 13 millimolar. The calcium salt concentration is based on the amount of water in the composition.

[0036] Acids, bases and salts may also be used in the composition to maintain the pH of the composition to between 6.5 and 8. Examples of acids are phosphoric acid, sulfuric acid, nitric acid, acetic acid, hydrochloric acid or a combination thereof. Examples of bases are sodium hydroxide, potassium hydroxide, ammonium hydroxide, or a combination thereof. [0037] Salts may be added to maintain the pH of the composition. These are termed primary salts. In addition to the primary salts, secondary salts may also be added to neutralize the iodine and the chlorine. Examples of primary salts are sodium chlorides, sodium phosphates, sodium carbonates, sodium dihydrogen carbonates, sodium sulfate, sodium nitrate, or a combination thereof. In an exemplary embodiment, the salt is monosodium dihydrogen orthophosphate (NaH2P04), disodium hydrogen phosphate

(Na2HP04), or a combination thereof. The salt is added to the composition in an amount of 0.01 M to 0.2 M, preferably 0.05 M to 0.15 M. The salt concentration relates to water present in the composition.

[0038] Examples of secondary salts are sodium thiosulfate. The sodium thiosulfate is used to neutralize iodine and chlorine present in the composition as these two cytotoxic agents might be present in in vitro or in/ex vivo samples. The secondary salt is also operative to inactivate cytotoxic agents in the biofilm samples. The secondary salt (e.g., sodium thiosulfate) may be used in an amount of 1 to 5 grams per liter. The primary and secondary salt concentrations are with respect to the water content of the composition.

[0039] Surfactants may also be used in the composition. The surfactants include nonionic, cationic, anionic and zwitterionic surfactants that can be electron donating or electron accepting and can include cyclic, linear, or branched molecules. Many long chain alcohols exhibit some surfactant properties. Prominent among these are the fatty alcohols, cetyl alcohol, stearyl alcohol, and cetostearyl alcohol (consisting predominantly of cetyl and stearyl alcohols), and oleyl alcohol. Examples include polyethylene glycol alkyl ethers, octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether,

polypropylene glycol alkyl ethers, glucoside alkyl ethers, decyl glucoside, lauryl glucoside, octyl glucoside, polyethylene glycol octylphenyl ethers, Triton X-100, Tween-80, sodium thiosulfate, polyethylene glycol alkylphenyl ethers, Nonoxynol-9, glycerol alkyl esters, glyceryl laurate, polyoxyethylene glycol sorbitan alkyl esters, polysorbate, sorbitan alkyl esters, sorbitan trioleate, cocamide MEA, cocamide DEA, dodecyldimethylamine oxide, block copolymers of polyethylene glycol and polypropylene glycol, poloxamers,

polyethoxylated tallow amine (POEA), or a combination thereof.

[0040] Anionic surfactants contain anionic functional groups at their head, such as sulfate, sulfonate, phosphate, and carboxylates. Cationic surfactants include pH-dependent primary, secondary, or tertiary amines. Zwitterionic (amphoteric) surfactants have both cationic and anionic centers attached to the same molecule. The cationic part is based on primary, secondary, or tertiary amines or quaternary ammonium cations. The anionic part can be more variable and include sulfonates, as in the sultaines CHAPS (3-[(3- Cholamidopropyl)dimethylammonio]- 1 -propanesulfonate) and cocamidopropyl

hydroxysultaine. Betaines such as cocamidopropyl betaine have a carboxylate with the ammonium. The most common biological zwitterionic surfactants have a phosphate anion with an amine or ammonium, such as the phospholipids phosphatidylserine,

phosphatidylethanolamine, phosphatidylcholine, and sphingomyelins.

[0041] Preferred surfactants include succinimides, poly-isobutylene succinimide (e.g., Chevron product OLOA11000), octylamine, trioctylamine, Tween-80, S-49, sorbitan trioleate, sodium thio sulfate, non-ionic surfactants that include hydrophilic polyethylene oxide chains on a hydrocarbon oleophilic group.

[0042] The surfactants facilitate the formation of a composition in a single phase. The surfactant is added to the composition in an amount of 1 to 20 grams per liter, preferably 2 to 10 grams per liter of water.

[0043] All of the foregoing ingredients are dispersed in water and form a single phase solution. All of the concentrations disclosed above relate to water. Other solvents may optionally be added to the water if desired so long as they are biocompatible and do not result in poisoning living beings. Examples of other solvents that may be used include ethanol, ethylene glycol, levulinic acid, or the like, or a combination thereof.

[0044] Table 1 lists the potential components, known molecular targets, and concentration ranges for an exemplary disaggregation composition. The appropriate components may be combined into an aqueous-based composition at appropriate

concentrations for efficacy against either known or unknown biofilm-embedded

microorganisms. The aqueous solution may contain appropriate buffers for enzyme interaction or material suspension. Another construction of the composition could be in a lyophilized form where the enzymes are more stable in a powder that is reconstituted in a buffer or appropriate liquid.

[0045] Table 1 shows the composition of one biofilm-disaggregation composition. Table 1

[0046] Table 2 reflects another composition that may be used for the disaggregation of biofilms.

Table 2

[0047] In one embodiment, in one method of manufacturing the composition (for disaggregating biofilms), the plurality of enzymes, the calcium salt for maintaining the activity of at least one of the enzymes in the plurality of enzymes, primary and secondary salts, the surfactant, and the pH adjusting additive are mixed together in a reactor. The ingredients listed above may be mixed for a period of 1 to 20 minutes at room temperature and stored at 4°C for use when desired. Certain ingredients used in the composition would be produced through biological production from bacteria or yeast and purified using typical protein production methods. Following production of individual components, the composition would be completed by mixing the individual components.

[0048] It is desirable for the composition after mixing to have a pH of 6.5 to 8.0, preferably 6.8 to 7.5.

[0049] Table 3 lists one method of using the composition for disaggregating a biofilm. The method for using the disaggregation composition will be to expose the biofilm- embedded microorganisms to the solution while applying mechanical disruption. For example, skin or soft tissue samples with suspected biofilm-embedded microorganisms will be dropped into the proposed disaggregation composition and the sample is blended, sonicated, and vortexed to homogenize the tissue and suspected microorganisms. The general description of methodology steps is summarized in Table 3. Table 3 provides a summary of the method using a disaggregation composition for quantification of biofilm- embedded microorganisms from tissue biopsy

Table 3

mixture. As seen in the Table 3, in Step 2, the mixture may be mixed in a mixer such as a sonicator or a vortex homogenizer. The mixture is incubated at a temperature of 37°C for 12 to 24 hours. The mixing in the mixer may be repeated several times. The mixture is then diluted in several steps up to a weight ratio of 1 : 10 in a phosphate-buffered saline solution. For example, the composition of a phosphate-buffered saline solution comprises sodium chloride, potassium chloride and sodium phosphates.

[0051] The dilution step may be conducted up to 8 times. In other words, the dilution step may be repeated up to 8 times till the desired dilution is achieved. The diluted sample is then spread on a substrate (a plate) for up to 3 times to achieve accurate quantification. Quantification is generally accomplished by enumeration of colonies after incubation at a selected temperature ranging between 22°C and 37°C for a chosen duration. The duration may be from 12 to 24 hours. This duration is sometimes referred to as an incubation period. [0052] The composition disclosed herein may be used advantageously over other processes for disaggregating biofilms. Previously used mechanical disaggregation is insufficient for complete disaggregation of biofilm-embedded microorganisms. Enzymatic methods for disaggregation or targeting a single molecule or organism-based matrix component will result in incomplete separation of microorganisms from other microorganism or other tissues. When chemicals are used to disaggregate the biofilm, microorganisms are killed and this precludes accurate quantification of the microorganisms in the biofilm.

[0053] The invention is described by the following non-limiting examples.

Example

[0054] This example was conducted with the formulation listed in Table 1 above to demonstrate the efficacy of disaggregating a biofilm that comprises microorganisms.

[0055] Figure 1 describes the efficacy of the previous method of quantification using only mechanical disaggregation. Control and anti-biofilm test surfaces were evaluated by bacterial loads in the experiment. The same bacterial load was applied to each test type, but only the samples with Proteinase K added to the quantification process resulted in accurate sampling. The difference between the control and control + Proteinase K is approximately 1.5 logs of bacteria meaning that 1.5 logs of bacteria are under-reported without the use of an enzymatic additive to disrupt the biofilm. Moreover, the anti-biofilm test surface was not able to demonstrate avoidance of biofilm without proteinase K.