US20130272922A1

US20130272922A1 - Antimicrobial composition and methods for using same

Info

Publication number: US20130272922A1
Application number: US13/847,876
Authority: US
Inventors: Matthew Franco Myntti
Original assignee: Next Science LLC
Current assignee: Next Science IP Holdings Pty Ltd
Priority date: 2008-10-06
Filing date: 2013-03-20
Publication date: 2013-10-17
Also published as: WO2010042427A2; EP2346324A2; US8940792B2; EP2346324A4; US20100086576A1; US20130079407A1; WO2010042427A3

Abstract

An aqueous composition adapted to kill bacteria in both planktonic and biofilm states is lethal toward a wide spectrum of gram positive and gram negative bacteria as well as other microbes. The composition, which is slightly to moderately acidic, includes a significant amount of one or more surfactants and large amounts of osmotically active solutes. The composition can be applied directly to a site of bacterial growth. Even when the bacteria is in biofilm form, the surfactant component(s) begin to kill the bacteria before the macro-molecular matrix is removed or dislodged from the site.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No. 13/679,994 filed 16 Nov. 2012, presently pending, which is a continuation of U.S. patent application Ser. No. 12/573,340 filed 5 Oct. 2009, presently pending, which claimed the benefit of U.S. provisional application no. 61/103,214 filed 6 Oct. 2008, the entire disclosures of which are incorporated herein by reference.

BACKGROUND INFORMATION

Bacteria is found virtually everywhere and is responsible for a significant amount of disease and infection. Ridding surfaces of bacteria is desirable to reduce human exposure. Bacteria in normal environments have developed self preservation mechanisms and are therefore extremely difficult to remove and/or eradicate.
Bacteria can be found in both planktonic and biofilm forms. In the biofilm form, they interact with surfaces and form surface colonies which adhere to a surface and continue to grow. The bacteria produce exopolysaccharide (EPS) and/or extracellularpoly-saccharide (ECPS) macromolecules that keep them attached to the surface and form a protective film that is effective against many forms of attack. Protection most likely can be attributed to the small diameter of the flow channels in the matrix, which restricts the size of molecules that can transport to the underlying bacteria, and consumption of biocides through interactions with portions of the EPS/ECPS macromolecular matrix.
Additionally, the bacteria in biofilm form are down-regulated (sessile) and not actively dividing. This makes them resistant to attack by a large group of antibiotics and antimicrobials, which attack the bacteria during the active parts of their lifecycle, e.g., cell division.
Due to the protection afforded by the macromolecular matrix and their down-regulated state, bacteria in a biofilm are very difficult to treat. The types of biocides and antimicrobials that are effective in treating bacteria in this form are strongly acidic, oxidizing, and toxic, often involving halogen atoms, oxygen atoms, or both. Common examples include concentrated bleach, strong mineral acids (e.g., HCl) and hydrogen peroxide. Commonly, large dosages of such chemicals are allowed to contact the biofilm for extended amounts of time (up to 24 hours in some circumstances), which makes them impractical for many applications.
Recent developments have involved formulations intended for use against compromised animal/human tissue which, accordingly, are intentionally gentle so as to prevent damage or irritation to compromised tissue. These formulations solvate the bio-film matrix so that still-living bacteria can be rinsed or otherwise removed from infected tissue. The concentrations of active ingredients in these formulations are too low to effectively kill the bacteria in the biofilm, and are thus ill suited for surface disinfection.
A solution that can disrupt the macromolecular matrix, or bypass and/or disable the defenses inherent in these matrices, allowing lethal doses of the antimicrobial ingredients in the solution to access and kill the bacteria in their biofilm and sessile states, remains desirable. Such a solution that is not particularly acidic or caustic (i.e., about 3≦pH≦9) and has little to no toxicity would be particularly advantageous.

SUMMARY OF THE INVENTION

Provided herein is an aqueous composition adapted to kill bacteria in both planktonic and biofilm states. In addition to being lethal toward a wide spectrum of gram positive and gram negative bacteria, the composition also exhibits lethality toward other microbes such as viruses, fungi, molds, yeasts, and bacterial spores.
Broadly, the aqueous composition includes a significant amount of one or more surfactants and large amounts of osmotically active solutes. The pH of the composition preferably is moderately low (about 4≦pH≦6), although higher concentrations of acidic components can be employed. At least some of the osmotically active solutes include the dissociation product(s) of one or more acids that are effective at interrupting or breaking ionic crosslinks in the macromolecular matrix of the biofilm, which facilitates passage of the solutes and surfactant through the matrix to the bacteria entrained therein and/or protected thereby.
The antimicrobial composition, which is adapted for use against bacteria in a biofilm that includes a macromolecular matrix, can consist essentially of water, dissociation product(s) of one or more organic acids, and at least 1% by weight of one or more surfactants (based on the total weight of the composition). The composition can have an osmolarity of at least 3 Osm and a pH of no more than 6.5.
In certain embodiments of the foregoing, the acid(s) can include or consist of one or more organic polyacids.
In at least some embodiments, a portion of the osmotically active solutes result from dissociation of one or more alkali metal salts of the one or more polyacids, the use of which can provide a buffered composition, i.e., a composition which resists significant changes in pH when, for example, some of the hydronium ions are consumed in the crosslink interruption just described.
In at least some embodiments, the composition includes no biocidal additives; in other words, the ingredients just described alone are sufficient to provide significant biocidal activity. Additionally or alternatively, the composition can contain no active ingredients other than acid(s) and surfactant(s).
Without wishing to be bound by theory, the combination of high tonicity (high osmolar concentration) and large amounts of surfactant is believed to induce bacterial membrane leakage, leading to cell lysis. While the ingredients used to prepare such compositions typically are ineffective as bactericides when used at concentrations commonly employed in commercial products, an appropriately formulated composition has been found to be very effective at breaking down or bypassing and disabling biofilm defenses, thereby allowing the composition to access and kill the bacteria, even when it is in a sessile state.
The aqueous composition is lethal toward planktonic and bacterial cells with high efficacy. Advantageously, in many embodiments, it is non-toxic or, at worst, has low toxicity.
Also provided are methods of making and using the foregoing composition. In at least one such method, application of a composition of the type described above to a biofilm can provide at least a 3 log reduction in the number of live bacteria after a residence time of 5 minutes.
To assist in understanding the following description of various embodiments, certain definitions are provided immediately below. These are intended to apply through-out unless the surrounding text explicitly indicates a contrary intention:

- “microbe” means any type of microorganism including, but not limited to, bacteria, viruses, fungi, viroids, prions, and the like;
- “antimicrobial agent” means a substance having the ability to cause greater than a 90% (1 log) reduction, preferably at least a 99% (2 log) reduction in the number of one or more of microbes including, but not limited to, bacteria selected from Staphylococcus aureus and Pseudomonas aeruginosa;
- “active antimicrobial agent” means an antimicrobial agent that is effective only or primarily during the active parts of the lifecycle, e.g., cell division, of a microbe;
- “biofilm” means a community of microbes, particularly bacteria and fungi, attached to a surface with the community members being contained in and/or protected by a self-generated macromolecular matrix;
- “residence time” means the amount of time that an antimicrobial agent is allowed to contact a bacterial biofilm;
- “entrenched biofilm” is a biofilm that has reached a steady state mass after a growth period of two or more days;
- “buffer” means a compound or mixture of compounds having an ability to maintain the pH of a solution to which it is added within relatively narrow limits;
- “buffer precursor” means a compound that, when added to a mixture containing an acid, results in a buffer;
- “polyacid” means a compound having at least two carboxyl groups and specifically includes dicarboxylic acids, tricarboxylic acids, etc.;
- “biocompatible” means presenting no significant, long-term deleterious effects on or in a mammalian species;
- “biodegradation” means transformation, via enzymatic, chemical or physical in vivo processes, of a chemical into smaller chemical species;
- “biosorption” means absorption of a material into the body of a mammalian species;
- “soil load” means a solution of one or more organic and/or inorganic substances added to the suspension of a test organism to simulate the presence of body secretions, excretions, and the like; and
- “inoculum” means a solution containing bacteria, growth solution (e.g., tryptic soy broth) and protein soil load.

Hereinthroughout, pH values are those which can be obtained from any of a variety of potentiometric techniques employing a properly calibrated electrode.
The relevant portion(s) of any specifically referenced patent and/or published patent application is/are incorporated herein by reference.

DETAILED DESCRIPTION

A composition according to the present invention can result, after no more than 10 minutes residence time, in at least 6 log (99.9999%) reductions in the number of bacteria in an entrenched biofilm. Embodiments of the composition which are non-toxic if ingested can result, after no more than 10 minutes residence time, in at least 4 log (99.99%) reductions in the number of bacteria in an entrenched biofilm. The foregoing compare to commercially available bactericides, where non-toxic versions show less than a 1 log (90%) reduction and toxic versions show 1 to 2 log (90-99%) reductions, with the latter exhibiting a much higher level of toxicity levels than any embodiment of the presently provided composition.
The composition can contain as few as three ingredients: water, the dissociation product(s) of at least one acid, and at least one surfactant, each of which generally is considered to be biocompatible. The dissociation product(s) of one or more alkali metal salts of organic acids can be included in some embodiments. Certain embodiments of the composition employ no active biocides.
Essentially any source of water can be used, although those that are relatively free of bacteria without advance treatment are preferred. The water need not be distilled, deionized, etc., although such treatments certainly are not excluded. To enhance solubility of one or more of the other components of the composition, the water can be heated.
The composition has a pH less than 7. Without wishing to be bound by theory, acidic protons (i.e., hydronium ions) are believed to be involved in breaking ionic cross-links in the macromolecular matrix of a biofilm.
Increases in the concentration of hydronium ions, i.e., reductions in pH, generally correspond with enhanced efficacy of the composition. This effect may not be linear, i.e., the enhancement in efficacy may be asymptotic past a certain hydronium ion concentration. As long as the pH of the composition is greater than ˜3, the composition generally will be biocompatible; specifically, external exposure will result in no long-term negative dermal effects and ingestion can result biodegradation and/or biosorption, particularly if diluted with water soon after ingestion. If the pH is greater than ˜4, accidental inhalation or exposure to an aerosolized version of the composition should not result in laryngospasms or other throat-related damage. However, even those embodiments of the composition having a pH below ˜4 are believed to be significantly less toxic than presently available commercial products.
The pH of the composition is less than 7.0, generally less than 6.6, less than 6.4, less than 6.2, less than 6.0, less than 5.8, less than 5.6, less than 5.4, less than 5.2, less than 5.0, less than 4.8, less than 4.6, less than 4.4, less than 4.2, less than 4.0, less than 3.8, less than 3.6, less than 3.6, less than 3.4, less than 3.2, or even less than 3.0; in terms of ranges, the pH of the composition can be from ˜2 to ˜6.7, from ˜2.5 to ˜6.5, from ˜2.7 to ˜6.3, from ˜3 to ˜6, from ˜3.3 to ˜5.7, or from ˜3.5 to ˜5.5.
Acidity is achieved by adding to water (or vice versa) one or more acids, specifically strong (mineral) acids such as HCl, H₂SO₄, H₃PO₄, HNO₃, H₃BO₃, and the like or, preferably, organic acids, particularly organic polyacids. Examples of organic acids include monoprotic acids such as formic acid, acetic acid and substituted variants (e.g., hydroxy-acetic acid, chloroacetic acid, dichloroacetic acid, phenylacetic acid, and the like), propanoic acid and substituted variants (e.g., lactic acid, pyruvic acid, and the like), any of a variety of benzoic acids (e.g., mandelic acid, chloromandelic acid, salicylic acid, and the like), glucuronic acid, and the like; diprotic acids such as oxalic acid and substituted variants (e.g., oxamic acid), butanedioic acid and substituted variants (e.g., malic acid, aspartic acid, tartaric acid, citramalic acid, and the like), pentanedioic acid and substituted variants (e.g., glutamic acid, 2-ketoglutaric acid, and the like), hexanedioic acid and substituted variants (e.g., mucic acid), butenedioic acid (both cis and trans isomers), iminodiacetic acid, phthalic acid, and the like; triprotic acids such as citric acid, 2-methylpropane-1,2,3-tricarboxylic acid, benzenetricarboxylic acid, nitrilotriacetic acid, and the like; tetraprotic acids such as prehnitic acid, pyromellitic acid, and the like; and even higher degree acids (e.g., penta-, hexa-, heptaprotic, etc.). Where a tri-, tetra-, or higher acid is used, one or more of the carboxyl protons can be replaced by cationic atoms or groups (e.g., alkali metal ions), which can be the same or different.
In certain embodiments, preference can be given to those organic acids which are, or can be made to be, highly soluble in water; acids that include groups that enhance solubility in water (e.g., hydroxyl groups), examples of which include tartaric acid, citric acid, and citramalic acid, can be preferred in some circumstances. In these and/or other embodiments, preference can be given to those organic acids which are biocompatible; many of the organic acids listed above are used in preparing or treating food products, personal care products, and the like. Alternatively or additionally, preference can be given to those organic acids which can act to chelate the metallic cations ionic involved in crosslinking the macromolecular matrix of the biofilm.
The surfactant component can be added to water before, after or at the same time as the acid(s).
Essentially any material having surface active properties in water can be employed, although those that bear some type of ionic charge are expected to have enhanced antimicrobial efficacy because such charges, when brought into contact with a bacteria, are believed to lead to more effective cell membrane disruption and, ultimately, to cell leakage and lysis. This type of antimicrobial process can kill even sessile bacteria because it does not involve or entail disruption of a cellular process.
Potentially useful anionic surfactants include, but are not limited to, sodium chenodeoxycholate, N-lauroylsarcosine sodium salt, lithium dodecyl sulfate, 1-octane-sulfonic acid sodium salt, sodium cholate hydrate, sodium deoxycholate, sodium dodecyl sulfate, sodium glycodeoxycholate, sodium lauryl sulfate, and the alkyl phosphates set forth in U.S. Pat. No. 6,610,314. Potentially useful cationic surfactants include, but are not limited to, hexadecylpyridinium chloride monohydrate and hexadecyltrimethylammonium bromide, with the latter being a preferred material. Potentially useful nonionic surfactants include, but are not limited to, polyoxyethyleneglycol dodecyl ether, N-decanoyl-N-methyl-glucamine, digitonin, n-dodecyl B-D-maltoside, octyl B-D-glucopyranoside, octylphenol ethoxylate, polyoxyethylene (8) isooctyl phenyl ether, polyoxyethylene sorbitan mono-laurate, and polyoxyethylene (20) sorbitan monooleate. Useful zwitterionic surfactants include but are not limited to 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate, 3-(decyldimethylammonio) propanesulfonate inner salt, and N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate. For other potentially useful materials, the interested reader is directed to any of a variety of other sources including, for example, U.S. Pat. Nos. 4,107,328 and 6,953,772 as well as U.S. Pat. Publ. No. 2007/0264310.
The composition contains a sufficient amount (expressed in terms of weight/moles or concentration) of surfactant to interrupt or rupture bacterial cell walls. This amount can vary widely based on a variety of factors including, for example, the age of the biofilm (particularly whether it is entrenched, a factor which relates to the type of proteins and mass of the macromolecular matrix), size of the biofilm, amount of surface soiling, the species of bacteria, whether more than one type of bacteria is present, the solubility of the surfactant(s), and the like. The amount of surfactant generally constitutes greater than ˜0.2%, typically at least ˜0.5%, more typically at least ˜0.7%, often at least ˜0.9%, and preferably at least 1% of the composition (with all being weight percentages based on the total weight of the composition), with the upper limit being defined by the solubility limits of the particular surfactant(s) chosen. Some surfactants can permit extremely high loading levels, e.g., at least 5%, at least 10%, at least 12%, at least 15%, at least 17%, at least 20%, or even on the order of ˜25% or more (with all being weight percentages based on the total weight of the composition). Any of the foregoing minimum amounts can be combined with any of the foregoing maximum amounts to provide an exemplary range of potential amounts of surfactant.
In certain embodiments, the surfactant(s) can be the only antimicrobial agents in the composition, specifically, the composition can be free of active antimicrobial agents.
The lethality of the surfactant component(s) is increased and/or enhanced when the composition has at least moderate effective solute concentrations (tonicity). (In biological applications, a 0.9% (by wt.) saline solution, which is ˜0.3 Osm, typically is considered to be have moderate tonicity, while a 3% (by wt.) saline solution, which is ˜0.9 Osm, generally is considered to be hypertonic.) Without wishing to be bound by theory, compositions having higher tonicities may exert higher osmotic pressure to the bacterial cell wall, which increases its susceptibility to interruption by surfactant.
The osmolarity of the composition generally increases in proportion with the amount of acid(s) employed, with the osmolarity maximum for a given composition primarily being a function of the solubility limits of the specific acid, i.e., the point at which the acid(s) begin to no longer be soluble. An obvious corollary to increased levels of acid(s) in the composition is higher concentrations of hydronium ions, i.e., low pH values. As noted previously, some end-use applications can call for a composition with only a moderately low pH. To increase the osmolarity of a composition without depressing its pH past a desired target, one or more types of other water soluble compounds can be included. Such compounds, upon dissociation, increase the effective amount of solutes in the composition without greatly impacting the molar concentration of hydronium ions while, simultaneously, providing a buffer system in the composition.
One approach to achieve increased tonicity of the composition is by adding large amounts of ionic compounds (salts); see, e.g., U.S. Pat. No. 7,090,882. Where one or more organic acids are used in the composition, a preferred approach to increasing tonicity involves inclusion of salt(s) of one or more the acid(s) or the salt(s) of one or more other organic acids. For example, where the composition includes x moles of an acid, a many fold excess (e.g., 3x-10x, preferably at least 5x or even at least 8x) of one or more salts of that acid also can be included. The identity of the countercation portion of the salt is not believed to be particularly critical, with common examples including ammonium ions and alkali metals. Where a polyacid is used, all or fewer than all of the carboxyl H atoms can be replaced with cationic atoms or groups, which can be the same or different. For example, mono-, di- and trisodium citrate all constitute potentially useful buffer precursors. However, because trisodium citrate has three available basic sites, it has a theoretical buffering capacity up to 50% greater than that of disodium citrate (which has two such sites) and up to 200% greater than that of sodium citrate (which has only one such site).
Regardless of how achieved, the tonicity of the composition is at least moderately high, with an osmolarity of at least about 1 Osm being preferred for most applications. Depending on particular end-use application, the composition can have any of the following concentrations: at least ˜1.5 Osm, at least ˜1.75 Osm, at least ˜2.0 Osm, at least ˜2.25 Osm, at least ˜2.5 Osm, at least ˜2.75 Osm, at least ˜3.0 Osm, at least ˜3.25 Osm, at least ˜3.5 Osm, at least ˜3.75 Osm, at least ˜4.0 Osm, and even at least ˜4.25 Osm. Certain embodiments of the composition can exhibit solute concentrations of 1 to 5 Osm, 1.2 to 4.5 Osm, 1.4 to 4.4 Osm, 1.6 to 4.3 Osm, 1.8 to 4.2 Osm, 1.9 to 4.1 Osm, and 2 to 4 Osm; other potentially useful ranges include 3-5 Osm, 2.5-4.5 Osm, 3-4.5 Osm, 3.5-5 Osm, 3.25-4.5 Osm, and the like.
While the characteristics of the acid(s), surfactant(s) and optional (non-acid) water soluble compounds have been described in isolation, combinations of individual values or ranges for one component can be provided in conjunction with values or ranges for one or both of the other components. For example, a biocompatible composition (i.e., minimal toxicity) can entail a pH maintained above ˜4, an effective solute concentration of at least ˜0.10 Osm, and large amounts of one or more surfactant(s). Increasing the pH to ˜6 or higher can increase biocompatibility of the composition but, simultaneously, decrease its efficacy in killing microbes; conversely, decreasing the pH below ˜4 and/or increasing the osmolarity of the composition can increase its antimicrobial capacity while, simultaneously, reducing its biocompatibility. Generally, no particular benefit is seen from reducing the amount of surfactant employed, although too high of an amount can present toxicity concerns in some circumstances.
The composition can be prepared in a number of ways. Description of an exemplary method follows.
Acid (e.g., anhydrous citric acid), optional buffer precursor (e.g., a citric acid salt such as sodium citrate dihydrate), and sufficient water to constitute ˜80% of the calculated desired volume. This solution can be stirred and/or heated to promote solution of the acid and optional buffer precursor. The desired amount of surfactant(s) then can be added before additional water is added to bring the composition close to the calculated volume. Once stirring, if used, is complete, sufficient water is added so as to bring the composition to the calculated value. Advantageously, no special conditions or containers are needed to store the composition for an extended time, although refrigeration can be used if desired.
A variety of additives and adjuvants can be included to make a composition more amenable for use in a particular end-use application with negatively affecting its efficacy in a substantial manner. Examples include, but are not limited to, emollients, fungicides, fragrances, pigments, dyes, defoamers, foaming agents, flavors, abrasives, bleaching agents, preservatives (e.g., antioxidants) and the like.
The composition does not require inclusion of an active antimicrobial agent for efficacy, but such materials can be included in certain embodiments. For example, one or more of bleach, any of a variety of phenols, aldehydes, quaternary ammonium compounds, etc., can be added.
The composition conveniently can be provided as a solution, although other forms might be desirable for certain end-use applications. Accordingly, the composition can provided as a soluble powder (for subsequent dilution, an option which can reduce transportation costs), a slurry, or a thicker form such as a gel or paste (which might be particularly useful for providing increased residence times). For the latter, the composition can include additional ingredients such as a coalescent (e.g., polyvinylpyrrolidone).
Embodiments of the composition can provide very large reductions in the number of bacteria, even with extremely short residence times. For example, a composition having high concentrations of surfactant (e.g., 1.5-2.5% by wt.) and total solutes (e.g., 3-4 Osm) can provide a 2, 3 or 4 log (99.99%) reduction in the number of bacteria in an entrenched biofilm with a 3, 4, 5, 7, 8, 9, or 10 minute residence time and a 3, 4, 5, or 6 log (99.9999%) reduction in the number of planktonic bacteria with a mere 30-second residence time.
Quantitative Carrier Testing (ASTM E2197) is designed to determine the contact time necessary to eradicate from a surface (e.g., countertops, sinks, bathroom fixtures, and the like) bacteria in a soil-loaded inoculum. In this test, bacteria combined with a soil loading and a 10 μL inoculum is placed on a stainless steel carrier disk. After the inoculate is allowed to dry completely, 50 μL of antimicrobial treatment composition is applied and allowed to stay in place for the desired treatment time, after which dilution with a saline dilution is performed. The following are results achieved from Quantitative Carrier Testing using 5% soil load and a 3 minute residence time (with TDTMAB representing tetradecyltrimethylammonium bromide):

- 0.021 M caprylyl sulfobetaine, 3.2 Osm, 6.5 pH—5.2 log reduction of pseudomonas
- 0.028 M caprylyl sulfobetaine, 3.6 Osm, 5.5 pH—3.1 log reduction of staph and 5.9 log reduction of pseudomonas
- 0.041 M TDTMAB, 3.5 Osm, 6.5 pH—5.1 log reduction of staph and 7.5 log reduction of pseudomonas
- 0.027 M TDTMAB, 3.5 Osm, 6.5 pH—4.9 log reduction of staph and 5.2 log reduction of pseudomonas
- 0.041 M TDTMAB, 1.8 Osm, 6.5 pH—3.2 log reduction of staph and 5.2 log reduction of pseudomonas
- 0.027 M TDTMAB, 1.8 Osm, 6.5 pH—3.2 log reduction of staph and 4.7 log reduction of pseudomonas
- 0.014 M TDTMAB, 1.8 Osm, 6.5 pH—2.9 log reduction of staph and 4.5 log reduction of pseudomonas
  Where the third composition was used again at a one-minute residence time, the results were a 5.1 log reduction of staph (i.e., no change, indicating that much of the killing of staph bacteria may occur in the first minute), and a 6.8 log reduction of pseudomonas.

An alternative test designed to show efficacy against an entrenched biofilm involves treatment of biofilm-forming bacterial strains grown over several days (typically in an incubator at 37° C. and aerobic conditions) in a drip-flow reactor, designed to model growth in a low shear environment. Bacteria are inoculated (e.g., on glass slides, optionally coated with hydroxyapatite) pre-coated with a sterile medium (e.g., trypticase soy broth). A coated slide then is inoculated with a culture of the biofilm-forming microbe of interest. The reactor is placed in a horizontal position (typically ˜2 hours) to promote bacterial attachment to the substrate before being inclined (e.g., to a 10° angle), with sterile medium dripping on the slides (typically at a rate of ˜2.78×10⁻³mL/sec). After a desired number of days of growth, flow of the sterile medium is halted, and the reactor is raised to horizontal. Antimicrobial composition is applied, while a control slide is treated with saline solution. After an amount of time (typically 5-10 minutes), the slides are rinsed with saline solution. Each slide is removed and placed in a sterile container, scraped, vortexed, and sonicated multiple times to harvest bacteria which are then incubated on plates for counting. Efficacy is calculated by subtracting the bacterial count on a treated slide from the bacterial count on a control (non-treated) slide. The following are results achieved from this test using a 5 minute residence time:

- 0.028 M caprylyl sulfobetaine, 3.5 Osm, 6.5 pH—2.8 log reduction of staph and 6.0 log reduction of pseudomonas
- 0.023 M octyl sulfobetaine, 3.5 Osm, 6.5 pH—1.4 log reduction of staph and 6.2 log reduction of pseudomonas
- 0.016 M TDTMAB, 0.8 Osm, 6.5 pH—3.0 log reduction of staph and 2.2 log reduction of pseudomonas
- 0.041 M TDTMAB, 3.5 Osm, 6.5 pH—8.9 log reduction of staph and 6.7 log reduction of pseudomonas

The foregoing data includes much of interest including, for example, a potential indication that pseudomonas, a gram negative bacteria, is more affected by the osmolarity of the composition than by the type or concentration of surfactant employed, while the opposite might be true for staph.
The foregoing levels of bactericidal activity are greater than for most strong chemical treatments in current use, even though the composition is far less toxic (i.e., more biocompatible) than those treatments. Generally, longer residence times can result in greater reductions in the number of bacteria, although the effect may be asymptotic.
The composition can be employed in a variety of ways. For example, when used to treat a biofilm on a surface (e.g., cutting board, counter, desk, etc.), the composition can be applied directly to the biofilm, optionally followed by physical rubbing or buffing, or the composition can be applied to the rubbing/buffing medium, e.g., cloth. Where a biofilm in an inaccessible area is to be treated, soaking or immersion of the biofilm in an excess of the composition can be performed for a time sufficient to essentially solvate the biofilm, which then can be flushed from the affected area. Regardless of contact method, the surfactant component(s) are believed to kill significant numbers of bacteria without a need for the bacteria to be removed from the biofilm or vice versa.
Due to the abundance of microbial contamination, the composition may find utility in a large number of potential uses including, but not limited to, household applications including non-compromised skin (hand, hair, and body washing), kitchen cleaning (countertop and surface cleaning, cleaning of food preparation utensils, dish washing, produce washing, etc.), bathroom cleaning (countertop and surface cleaning, fixture cleaning, toilet bowl cleaning and shower mildew eradication), and laundry area cleaning (including laundry detergent and diaper sterilization); commercial applications include livestock care (facility and equipment sterilization and dairy teat dip), produce sterilization (an alternative to irradiation, which can be particularly useful against e-coli, listeria, salmonella, Botulism, etc.), commercial kitchen (countertop and surface cleaning, food preparation utensil cleaning, storage equipment and facilities cleaning, dish washing and produce washing), mass food and beverage processing (processing and storage equipment cleaning, tank sterilization, cleaning of liquid transport lines, etc.), cleaning of water lines (e.g., for drinking water, dental offices, plumbing, and the like), and food and beverage transport (cleaning of tanker units for semi transport, cleaning of tanker cars for railroad transport, and cleaning of pipelines); and non-traditional uses such as denture cleaning, acne treatment, spermicides, laboratory equipment cleaning, laboratory surface cleaning, oil pipeline cleaning, and test article processing for biofilm attachment.
While various embodiments of the present invention have been provided, they are presented by way of example and not limitation. The following claims and their equivalents define the breadth and scope of the inventive methods and compositions, and the same are not to be limited by or to any of the foregoing exemplary embodiments.

Claims

That which is claimed is:

1. A method for treating a surface intended for contact with food, said method comprising applying to said surface a composition that comprises

1) water,

2) dissociation product of one or more biocompatible organic acids,

3) dissociation product of a buffer precursor, and

4) cationic surfactant,

said composition having a pH of from about 3.5 to about 5.5 inclusive and an effective solute concentration of from 1.8 to 4.2 Osm.

2. The method of claim 1 further comprising removing said composition.

3. The method of claim 1 wherein said applying step is accomplished by delivering said composition in or on a rubbing or buffing medium.

4. The method of claim 1 wherein said surface, prior to said applying step, comprises live bacteria, said bacteria optionally being present in a biofilm.

5. The method of claim 4 wherein said composition is capable of reducing the amount of live bacteria by at least 99.9% when said composition is permitted a 5-minute residence time on an entrenched biofilm tested in accordance with a drip-flow reactor test.

6. The method of claim 1 wherein said composition has a pH of less than 5.0.

7. The method of claim 1 wherein said composition has a pH of less than 4.6.

8. The method of claim 1 wherein said composition has a pH of less than 4.2.

9. The method of claim 1 wherein said composition has a solute concentration of from 2 to 4 Osm.

10. The method of claim 9 wherein said composition has an effective solute concentration of at least about 2.25 Osm.

11. The method of claim 1 wherein said one or more biocompatible organic acids comprises a polyacid.

12. The method of claim 11 wherein said buffer precursor is a salt of said polyacid.

13. The method of claim 1 wherein said buffer precursor comprises a salt of an organic acid.

14. A method for treating a surface intended for contact with food, said method comprising applying to said surface a composition that consists essentially of

1) water,

2) dissociation product of one or more biocompatible organic acids comprising citric acid,

3) dissociation product of a sodium salt of citric acid, and

4) cationic surfactant,

15. The method of claim 14 further comprising removing said composition.

16. The method of claim 14 wherein said applying step is accomplished by delivering said composition in or on a rubbing or buffing medium.

17. The method of claim 14 wherein said surface, prior to said applying step, comprises live bacteria, said bacteria optionally being present in a biofilm.

18. The method of claim 17 wherein said composition is capable of reducing the amount of live bacteria by at least 99.9% when said composition is permitted a 5-minute residence time on an entrenched biofilm tested in accordance with a drip-flow reactor test.

19. The method of claim 14 wherein said composition has a pH of less than 5.0.

20. The method of claim 14 wherein said composition has a pH of less than 4.6.