HK1094320B - Antiseptic compositions, methods and systems - Google Patents

Description

Antimicrobial compositions, methods and systems

Field of the invention

The invention relates toAntimicrobial compositions, methods and systems for use in a variety of medical applications, as well as disinfection applications in general, including industrial and environmental disinfection applications. The compositions of the present invention have antimicrobial, antifungal, antiviral and anti-amoebic properties and are also useful as anticoagulants. Specific salts of ethylenediaminetetraacetic acid (EDTA) and compositions (C)₁₀H₁₂N₂Na₄O₈) Used at specific concentrations and pH levels. Exemplary applications include inhibiting, reducing or eliminating the presence of microbial and/or fungal microorganisms on a surface, in a solution, or in a complex form, such as in a biofilm. Exemplary methods include providing an antimicrobial coating on the surface of an object to reduce the incidence of infection, and inhibiting the proliferation of, reducing or eliminating the microbial population by rinsing, soaking and/or rinsing the object and/or surface with an antimicrobial solution.

Description of background of the invention and Prior Art

Infection is a significant problem in many areas where sanitary conditions are important, such as in healthcare. Problematic infections may result from bacterial, fungal, amoebic, protozoan and/or viral microorganisms. Once an infection is established, challenges are encountered in both preventing the infection and reducing or eliminating the infection. The infected environment may include the surface of an object, fluids and fluid conduits, and/or humans or animals.

Alcohol solutions and isopropyl alcohol wipes are commonly used to disinfect surfaces and have been shown to have antimicrobial activity. A 70% isopropanol solution was found to have the most effective antimicrobial inhibitory effect. Alcohol solutions of this concentration are quite expensive and evaporate rapidly, greatly impairing their efficacy and increasing their cost. Moreover, while isopropyl alcohol solutions are useful on surfaces, including human skin, and in various medical applications, alcohol solutions of such concentrations cannot be applied to humans for medical purposes, other than on the body surface.

In the healthcare field, various types of infections and causes are common and often result in longer hospitalizations, resulting in higher hospital costs. Even worse, over 90,000 patient deaths per year are attributed to nosocomial infections-i.e., infections acquired in hospitals or other healthcare settings. Infection monitoring for hospitals has become an integral part of hospital practice. Studies conducted over 20 years prior to the center for disease control and prevention (CDC) demonstrated the efficacy of these monitoring activities in reducing the incidence of nosocomial infections. Despite the concern over nosocomial infections, however, the infection rate is not significantly reduced and nosocomial infections remain a significant risk and a significant health problem.

A problematic source of infection in the medical and veterinary fields is found in catheters, and especially in indwelling catheters. Catheters have become a necessity in critical care patients, however, the interior of the catheter is often the primary source of infection. Catheters are used for the transport of fluids, blood products, drugs, and nutrients, as well as for hemodialysis, hemofiltration, peritoneal dialysis, blood sample extraction, monitoring of patient condition, and the like. Percutaneous catheters are often infected by skin penetration of the catheter. Seventy percent (70%) percent of nosocomial blood infections have been found to occur in patients using a central venous catheter. Daouicher et al, New Engl. J. Med.340: 1-8(1999).

In particular, in some methods, the catheter must be implanted and remain implanted in the patient for a relatively long period of time, e.g., more than 30 days. Intravenous (IV) treatment catheters and urinary catheters are typically implanted for a considerable period of time. Such catheters cannot be frequently removed and implanted due to trauma to the insertion area, and pain to the patient. Catheter-produced bacteria have been shown to be a major source of urinary tract infection. Patients receiving shallow skin insertion of a central catheter during pregnancy have also been found to be at significant risk of infectious complications. Ogura et al, am.J.Obstet.Gynecol.188 (5): 1223-5(2003). In addition, central venous catheter infection, which leads to catheter-related sepsis, has been cited as the most frequent complication during home venous access nutrition. Reimund et al, clinical Nutrition, 21 (1): 33-38(2002). Due to the risk of infection, catheter procedures may be limited to emergency situations when the procedure is absolutely necessary. This seriously jeopardizes the health of the patient.

Following most prescribed medical procedures involving catheters, the catheters are flushed with saline and then filled with a liquid, such as saline or heparin solution, to prevent blood from clotting within the catheter, inhibit backflow of the patient's blood into the catheter, and prevent gas from entering the catheter. The liquid used to flush the catheter is referred to as "lock-flush", while the liquid used to fill the catheter after flushing or during periods of inactivity is referred to as "lock" solution.

Traditionally, catheters have been blocked with saline or heparin solutions that provide anticoagulant activity. Heparin and saline are sometimes used in combination. Saline is commonly used to occlude short term peripheral intravenous catheters, but has no anticoagulant or antimicrobial activity. Heparin solutions are commonly used for catheters that occlude blood vessels. Heparin has anticoagulant activity, but it has no antimicrobial effect and does not prevent or alleviate infection. There is also a strong suggestion that blocking heparin solutions can contribute to heparin-induced thrombocytopenia, a serious bleeding complication that occurs in the type of patient receiving heparin injections.

Recommended catheter lock solutions include Taurolidine, citric acid and sodium citrate. A recent publication (Kidney International, sept.2002) describes the use of 70% alcohol solutions as a blocking solution for the subcutaneous injection catheter port. The use of alcohol as a blocking solution is problematic because it is not an anticoagulant and because there are risks associated with the entry of such a solution into the blood. The inventors have not perceived any evidence that the 70% alcohol solution has any biofilm killing activity.

A trend and recommendation emerging from the infectious disease Center (CID) is to systematically treat existing catheter infections with specific or broad-range antibiotics. The use of antibiotics in a blocking solution to prevent infection is not recommended. The use of antibiotics to treat existing catheter infections carries certain risks, including: (1) risk of developing antibiotic resistant strains; (2) antibiotics are not able to kill sessile, or deep biofilm, bacteria, which may require the use of toxic concentrations of antibiotics; and (3) the high cost of extended antibiotic therapy. Catheters coated with an antibacterial or antibiotic material are useful. However, these coated catheters may provide only limited protection for a relatively short period of time.

In general, free plankton may be sensitive to antibiotics. However, bacteria and fungi may become immune to antibiotics by accumulating on the surface and producing a sticky protective substance, often referred to as Extracellular Polymer (EPS), to form a biofilm. As the microorganisms proliferate, more than 50 genetic upregulations or downregulations may occur, leading to the formation of more antibiotic resistant microbial biofilms. Two thirds of the bacterial infections encountered by physicians are due to biofilms. Netting, Science News, 160: 28(2001).

Biofilm formation is a genetically controlled process in the life cycle of bacteria that produces many changes in the cellular physiology of microorganisms, often including an increase in antibiotic resistance (up to 100 to 1000 fold) compared to growth under planktonic (free-floating) conditions. As the microorganisms grow, the problems of overcrowding and nutrient shortage arise, causing shedding of the microorganisms to find new locations and resources. These newly shed microorganisms quickly revert to their initial free-planktonic phase and are again susceptible to antibiotics. However, free-plankton may enter the patient's blood causing blood infections that produce clinical signs, e.g., fever, and more serious infection-related symptoms. The liferafts of biofilms may slough off and attach to tissue surfaces, such as heart valves, causing biofilm proliferation and serious problems, such as endocarditis.

In industrial environments, biofilm formation is common and is commonly referred to as biofouling. For example, biofilm growth on mechanical structures, such as filtration devices, is a major cause of biological contamination in drinking water distribution systems. Biofilm formation in industrial environments can lead to material degradation, product contamination, mechanical blockage and heat transfer resistance of process systems. Biofilm formation and the resulting contamination are also a common problem in the food preparation and processing industry.

For more complex cases, conventional susceptibility tests only measure free-planktonic antibiotic susceptibility, not microorganisms in the biofilm state. As a result, the administration of doses of antibiotics to a patient, such as through a catheter, rarely has the intended effect on microorganisms at the biofilm stage that may be present in the catheter anyway. Biofilm microorganisms may continue to shed more plankton or may go dormant and proliferate later as a significant recurrent infection.

In order to eradicate biofilm microorganisms through the use of antibiotics, the laboratory must determine the concentration of antibiotic required to kill the microorganism at a particular genetic biofilm stage. Highly specialized equipment is required to provide the minimum concentration for biofilm elimination. Furthermore, current diagnostic protocols are very time consuming and often fail to obtain results for many days, e.g., 5 days. Such time periods clearly do not allow for rapid treatment of the infection. This delay, and the justified fear of infection, may lead to overuse of the broad spectrum antibiotic and continued unnecessary catheter removal and replacement procedures. The overuse of broad-spectrum antibiotics can lead to the development of antibiotic-resistant strains that cannot be effectively treated. Unnecessary catheter removal and replacement is painful, costly, and may result in damage and damage to the tissue at the site of catheter insertion.

Antibiotic resistance of biofilms, as well as complications of antibiotic use, such as the risk of developing antibiotic-resistant strains, have made antibiotic treatment an undesirable option. Thus, antibiotic use is limited to symptomatic infections and prophylactic antibiotics are not generally used for pre-contamination prevention. Because biofilms may act as a barrier to the selective phenotype of most antibiotics, catheters must often be removed in order to eliminate catheter-related infections. Removal and replacement of the catheter is time consuming for the patient, creates stress on the patient, and complicates medical treatment. Accordingly, there is a need for a convenient and effective method for killing microorganisms, and particularly those present inside a catheter, without requiring removal of the catheter from the body.

In addition to bacterial and fungal infections, amoebic infections can be very serious and painful and can be life threatening. For example, several species of acanthamoeba have been found to infect humans. Acanthamoeba are found worldwide in soil and dust, and in fresh water sources as well as brackish and sea water. They are often found in heating, ventilation and air conditioning elements, humidifiers, dialysis elements and contact lens paraphernalia. In addition to microbial and fungal infections, acanthamoeba infections may also be common in medical and dental devices, including toothbrushes, dentures and other dental appliances, and the like. Acanthamoeba infections often result from improper storage, handling and sterilization of contact lenses and other medical devices in contact with the human body, and acanthamoeba may enter the skin through incisions, wounds, nostrils, eyes, etc.

There are many different kinds of microorganisms that cause problematic infections, including various bacteria and fungi. However, current methods of eliminating infections typically employ solutions that are effective against a limited number of different microorganisms. Root et al (Antimicrobial Agents and Chemotherapy, 32: 1627-1633(1988)) describe the use of disodium EDTA in vitro against specific catheter-associated isolates of Staphylococcus epidermidis pathogens.

EDTA is traditionally used as a metal chelator, but also for various purposes with other active compounds. EDTA is often used at low concentrations as an in vitro anticoagulant for blood sample collection and testing, as an antioxidant synergist, and may also be added to solutions, for example, as a chelating agent, stabilizer, or preservative for pharmaceutical preparations. EDTA may exist in various forms, some of which are in the form of sodium salts, such as disodium, trisodium, tetrasodium salts, others are metal chelates such as iron, copper, magnesium, and the like. Certain forms of EDTA, in combination with other substances as adjuvants, are used in compositions for treating infected catheters. When used in a clinical setting, or in a composition for human or animal use, the solution is typically adjusted to a physiological, or neutral, pH range.

PCT publication WO 02/05188 describes the combination of alcohols with additives such as the non-sodium salt form of EDTA. PCT publication WO 00/72906 a1 describes antimicrobial agents, e.g., antibiotics, in combination with a second group of agents, which may be lyophilized mixtures of the non-sodium salt forms of EDTA, as chelating agents for catheter flushing. In U.S. patent No. 5,688,516, compositions having an anticoagulant, a chelating agent such as EDTA, and an antimicrobial agent such as minocycline are described for coating medical devices and inhibiting catheter infection. In the specifically described examples, the disodium form of EDTA is adjusted to a physiological pH of 7.4 for use in the composition. PCT publication WO 99/09997 describes the use of antifungal agents in combination with chelating agents such as EDTA to treat fungal infections.

Other areas where infection is problematic include medical devices and materials used in connection with the eye, such as contact lenses, scleral buckles, suture materials, intraocular lenses, and the like. In particular, the development of methods for disinfecting artificial eyes, such as contact lenses, is being emphasized. Bacterial biofilms may be involved in eye infections and allow bacteria to be present on abiotic surfaces that come into contact with the eye or that are transplanted into the eye. Biofilms may also form on biological surfaces of the eye. Zegans et al, DNA Cell biol., 21: 415-20(2002). Several forms of keratitis may also originate from the protozoan amoeba, which may contaminate the lens disinfecting liquid. Ophthalmic formulations of tetrasodium and alkali metal salts of EDTA, buffered to pH 6-8, are described in U.S. Pat. No. 5,300,296 for disinfection of contact lenses. Ophthalmic compositions of EDTA and other substances, such as cyclodextrin and boric acid, are described in U.S. patent No. 5,998,488.

In the dental field, items placed in the oral cavity, such as dental tools, and dental and orthodontic devices such as holders, bridges, dentures, and the like, need to be maintained in sterile conditions, particularly during storage and prior to placement in the oral cavity. In addition, the infection can be transferred to the blood and become severe. The use of EDTA with other active ingredients in denture cleansing compositions is described in us patent 6,077,501.

The water supply is also prone to microbial and other types of infection. Water storage devices, as well as water supply and drainage conduits, are often infected. The interior surfaces of liquid holding barrels in medical and dental offices provide an environment suitable for microbial infection and growth, and microbial adhesion and highly protective biofilm formation are often problematic in liquid storage and supply devices.

Improved methods and compositions for preventing and destroying infections in a variety of environments are needed. Such an antimicrobial solution would have broad spectrum antimicrobial properties. In particular, the solution should be able to penetrate the protective film to eliminate microorganisms in the protective film. The methods and solutions should be safe enough to be used as a prophylactic measure, as well as to treat an existing infection.

Summary of The Invention

The present invention provides an antimicrobial solution comprising, consisting essentially of, or consisting of one or more pHs above physiological pH, and a defined concentration of EDTA salt. The present inventors have found that certain EDTA compositions have potent antibacterial activity and function as broad-spectrum antimicrobials, as well as fungicides against many pathogenic yeast strains. The EDTA compositions of the present invention are also very effective in killing pathogenic biofilm microorganisms, in reducing and eliminating existing biofilms, and in preventing the formation of protective films. The EDTA compositions and combinations of the present invention further exhibit anti-protozoal and anti-amoebae activity. According to published reports, it is further expected that the EDTA composition of the present invention exhibits antiviral activity.

The EDTA formulations of the present invention are safe for human administration, and are biocompatible and non-corrosive. They may also have anticoagulant properties and thus may be useful in the prevention and/or treatment of various catheter-related infections. The antimicrobial solutions of the present invention have many applications, including use as occlusive and occlusive irrigation solutions for various catheters, as preservatives or solutions for disinfecting various medical, dental and veterinary devices, instruments and other objects, surfaces, and the like. In addition, they can be used for sterilization in industrial, and food preparation and handling equipment.

The antimicrobial compositions of the present invention may be used prophylactically to prevent infection, or to reduce and/or eliminate existing or established infections. Methods of preventing or treating an object or surface having an undesirable microbial infection are provided, such methods comprising contacting the surface or object with a composition of the invention. The compositions of the invention, therefore, are useful for inhibiting the growth and proliferation of microbial populations and/or fungal pathogens, including inhibiting the formation and proliferation of biofilms; inhibiting the growth and proliferation of protozoan populations; inhibiting the growth and proliferation of amoeba populations; and prevention of amoebic infections, especially acanthamoeba infections.

The invention also provides methods of substantially eliminating microbial populations, including planktonic microbial populations and microbial populations in the form of biofilms, and methods of substantially eliminating acanthamoeba populations. Such methods comprise contacting an infected object or surface, or an object or surface suspected of being infected, with a composition of the present invention. Depending on the antimicrobial composition used in the different methods, different contact times may be required to inhibit formation and proliferation of different populations, and/or to substantially eliminate different populations. Suitable contact times for the different compositions are provided in the examples and can also be determined by routine experimentation.

In one embodiment, the antimicrobial composition of the present invention has at least 4, preferably at least 5 of the following characteristics: anticoagulant properties; inhibitory and/or bactericidal activity against a broad spectrum of planktonic forms of bacteria; inhibitory and/or bactericidal activity against a broad spectrum of fungal pathogens; inhibitory and/or bactericidal activity against broad spectrum sessile, or biofilm forms of bacteria; inhibitory activity against protozoal infection; inhibitory activity against acanthamoeba infection; safe and biocompatible, at least in moderate volumes, for the patient in contact; safe and biocompatible, at least in moderate volumes, with the patient's blood; safe and compatible for industrial objects and surfaces.

Soluble salts of EDTA are useful in the compositions of the invention. The sodium salts of EDTA are generally available and commonly used include the disodium, trisodium and tetrasodium salts, but other EDTA salts may be used, including ammonium, diammonium, potassium, dipotassium, copper disodium, magnesium disodium, iron sodium salts, and combinations thereof, so long as they have the desired antibacterial, fungicidal, antiprotozoal and/or antimicrobial properties and are sufficiently soluble in the desired solvent. Different compositions of EDTA salts may be used and are preferred for specific applications. Importantly, in most embodiments, the compositions and methods of the present invention do not use traditional antibiotic agents and therefore do not promote the production of antibiotic resistant microorganisms.

The antimicrobial compositions of the present invention comprise, consist of, or consist essentially of, one or more solutions of EDTA salt at a pH above physiological pH. Preferably, such compositions have a pH of at least 8.0. Preferably, the pH of the composition of the invention is in the range of 8.5 to 12.5, 9.5 to 11.5, or 10.5 to 11.5. Salts of EDTA are typically present in an amount of at least 0.01% w/v. In a preferred embodiment, the composition of the invention comprises at least one salt of EDTA in an amount of from 0.2% to 10% w/v, more preferably from 0.2% to 6.0% w/v, most preferably from 0.2% to 4.0% w/v.

In a particular embodiment, the composition of the invention comprises, or consists essentially of, a solvent and at least one salt of EDTA at a concentration of 0.01% to 10% w/v, wherein the solution has a pH of at least 9.0 and possesses bactericidal activity against a broad spectrum of bacteria. Preferably, the at least one salt of EDTA is trisodium or tetrasodium EDTA. The solvent is typically selected from the group consisting of water, brine, alcohol and combinations thereof. In one embodiment, the solvent is a combination of water and ethanol.

The antimicrobial compositions of the present invention are useful as occlusive and occlusive irrigation solutions for various types of internal access catheters, including vascular catheters for delivering fluids, blood products, drugs and nutrients, draining fluids or blood, dialysis, monitoring patient condition, and the like. The antimicrobial solutions of the present invention may also be used as occlusive and occlusive irrigation solutions for catheters, nasal tubes, throat tubes, and the like. The general solution parameters described below are suitable for these uses. In one embodiment, the antimicrobial solution comprises, consists of, or consists essentially of, one or more sodium salts of EDTA at a pH above physiological pH for maintaining patency of an internal intravascular access device. Methods for disinfecting catheters and other medical tubes, such as nasal tubes and the like, are also provided and include contacting the catheter or other medical tube with the disinfecting composition of the invention.

In another embodiment, the antimicrobial compositions of the present invention are provided which comprise, consist of, or consist essentially of, one or more sodium salts of EDTA, which has a pH greater than physiological pH, as a disinfecting solution for use in medical devices such as dentures and other dental, orthodontic and/or periodontal devices, for use in contact lenses and other optical devices, for use in medical and veterinary instruments, devices, and the like, and as a disinfecting solution for the disinfection of surfaces and objects. Also provided are methods of sterilizing such devices comprising contacting the devices with the antimicrobial compositions of the present invention. In general, the antimicrobial compositions of the present invention are useful as soaking solutions for dental, orthodontic and periodontal devices, including toothbrushes, and also as soaking solutions for contact lenses and other optical devices, as well as medical and veterinary instruments, devices, and the like. For these applications, the antimicrobial compositions of the present invention are typically formulated as a solution, or provided in dry form, to which a suitable solvent is added to form a solution.

In another embodiment, the antimicrobial compositions of the present invention are formulated for use in solutions, gels, emulsions and other articles designed for topical application, as preservatives, wipes, antimicrobial therapeutics and the like. The antimicrobial compositions of the present invention are also useful as antimicrobial agents in connection with bandages, dressings, wound healing agents and devices, and the like. In a related embodiment, coverings for wound healing, such as bandages and dressings, are provided wherein the covering is impregnated with one or more compositions of the present invention.

The antimicrobial compositions of the present invention may also be used in industrial settings such as water storage and distribution systems, water purification, humidification and dehumidification devices, and in food preparation, processing and packaging devices to inhibit, reduce or substantially eliminate free floating and sessile forms of microbial populations, as well as many fungi, amoeba and protozoan populations. Industrial equipment and surfaces may be contacted with, rinsed with, or soaked in the antimicrobial compositions of the present invention. Also provided are timed release antimicrobial composition formulations to provide treatment over time, particularly at sites that are often difficult to reach.

Drawings

FIGS. 1A-1D show that the resistance measured using the agar dilution method is essentially determined by dipotassium EDTA; diammonium EDTA; disodium EDTA; trisodium EDTA; and tetrasodium EDTA salt solutions for various gram-positive and gram-negative bacterial microorganisms. Bacterial microorganisms are isolated from catheter-related infections in human patients. Experimental techniques are described in example 1.

FIG. 2 shows MIC and MBC concentrations of various fungal microorganisms for different formulations of EDTA-resistant using the agar dilution method. Experimental techniques are described in example 1. Fungal microorganisms are collected from human patient samples.

FIGS. 3A and 3B show anti-EDTA consisting essentially of disodium copper EDTA; EDTA magnesium disodium; MIC and MBC data for gram positive and gram negative bacterial microorganisms in EDTA salt solution consisting of sodium iron EDTA. The bacterial microorganisms are isolated from catheter-related infections in human patients. Experimental techniques are described in example 1.

FIGS. 4A-4C show anti-EDTA consisting essentially of disodium copper EDTA and tetrasodium EDTA; MIC and MBC data for various gram-positive and gram-negative bacterial microorganisms of copper disodium EDTA and dipotassium EDTA and a combined EDTA salt solution consisting of copper disodium EDTA and diammonium EDTA. The bacterial microorganisms are isolated from catheter-related infections in human patients. Experimental techniques are described in example 1.

FIGS. 5A-5C show anti-EDTA formation substantially from tetrasodium EDTA and diammonium EDTA; tetrasodium and dipotassium EDTA; and MIC and MBC data for various gram-positive and gram-negative bacterial microorganisms of a combined EDTA salt solution consisting of diammonium EDTA and dipotassium. The bacterial microorganisms are isolated from catheter-related infections in human patients. Experimental techniques are described in example 1.

FIG. 6 shows the Minimum Biofilm Elimination Concentration (MBEC) values, expressed in mg/ml tetrasodium EDTA (w/v), for various microorganisms determined using the method described in example 2.

FIG. 7 shows the results of a test conducted with a renal hemodialysis catheter infected with a 40mg/ml (w/v) concentration of an antimicrobial composition consisting essentially of tetrasodium EDTA.

FIG. 8 shows the results of an experiment in which an infected renal hemodialysis catheter, as well as an arterial and a venous catheter, were treated with a 20-100mg/ml (w/v) concentration of an antimicrobial composition consisting essentially of tetrasodium EDTA.

Detailed Description

EDTA is used in many compositions at low concentrations in combination with other active ingredients as a stabilizer or preservative. The antimicrobial compositions of the present invention comprise relatively high concentrations of EDTA, preferably at least 0.01% EDTA salt, by weight per volume of solution (w/v), and may comprise up to 15% (w/v) EDTA salt. For many applications, the antimicrobial compositions of the present invention preferably include at least 0.1% (w/v) and less than 10% (w/v) of an EDTA salt, more preferably 0.1% (w/v) to 8.0% (w/v) of an EDTA salt, and most preferably 0.1% to 6.0% of an EDTA salt. Exemplary compositions include 3.6-4.4% (w/v) aqueous solutions of EDTA salts, or 0.01-0.2% (w/v) solutions of mixtures of water and ethanol of EDTA salts, as described below.

The concentration of EDTA salt required for various applications depends on the EDTA salt, or combination of salts, used, the type of infection being treated, to some extent, the solvent used for the antimicrobial composition. For example, when using an aqueous solvent comprising ethanol, the concentration of EDTA salt required to provide the desired level of activity can be reduced relative to the concentration of EDTA salt used in compositions in which water is the solvent. Antimicrobial compositions comprising one or more salts of EDTA have demonstrated inhibitory and/or bactericidal efficacy at concentration ranges of 0.01% to 30% or higher as shown by the exemplary data provided below. The "effective" concentration of EDTA salt required for inhibitory, bactericidal, fungicidal, biofilm elimination, and other uses in the antimicrobial compositions of the present invention can be determined by routine experimentation as described in the examples provided below.

The British Pharmacopoeia (BP) specifies a pH of 4.0 to 5.5 for a 5% disodium EDTA solution. BP also specifies a pH range of 7.0 to 8.0 for trisodium EDTA solution. The pH of other aqueous solutions of EDTA are provided in example 10 below. At physiological pH, EDTA sodium salt is present as disodium EDTA in combination with trisodium, and trisodium EDTA salt is predominant. In the united states, the drug EDTA "disodium" is prepared for injection, typically titrated with sodium hydroxide to a pH of 6.5 to 7.5. At this pH, the EDTA solution actually comprises mainly trisodium EDTA, and the proportion of disodium salts is low. Other compositions including sodium salts of EDTA for medical or healthcare applications are typically adjusted to substantially physiological pH.

In certain embodiments, the antimicrobial compositions of the present invention comprise, consist essentially of, or consist of, a solution of sodium EDTA (or combination of sodium EDTA salts) at a pH above physiological, preferably at a pH above 8.0, at a pH above 8.5, at a pH above 9, at a pH above 9.5, or at a pH above 10. In other embodiments, the antimicrobial compositions of the present invention comprise, consist essentially of, or consist of a solution of sodium EDTA (or combination of sodium EDTA salts) at a pH of 8.5 to 12.5, at a pH of 9.5 to 11.5, or at a pH of 10.5 to 11.5. As used herein, the term "EDTA salt" may refer to a monovalent salt, such as the disodium, trisodium or tetrasodium salt, or another EDTA salt form, or it may refer to a combination of these salts. The composition of the EDTA salt depends on the EDTA salt used to formulate the composition, as well as the pH of the composition. For the antimicrobial compositions of the present invention (specified above) that include sodium EDTA in the desired pH range, sodium EDTA is present primarily as trisodium and tetrasodium salts.

In one embodiment, the antimicrobial composition of the present invention comprises or consists essentially of a combination of at least trisodium EDTA and tetrasodium salt. In another embodiment, the antimicrobial composition of the present invention comprises or consists essentially of a combination of at least trisodium EDTA and tetrasodium salt, wherein at least 10% of the EDTA in the composition is present as the tetrasodium salt. In other embodiments, the antimicrobial compositions of the present invention comprise or consist essentially of a combination of at least trisodium EDTA and tetrasodium salt, wherein at least 50% or at least 60% of the EDTA in the composition is present as the trisodium salt. In another embodiment, the antimicrobial composition of the present invention comprises or consists essentially of a combination of disodium EDTA, trisodium and tetrasodium EDTA, wherein less than 10% of the EDTA in the composition is present as the disodium salt.

Antimicrobial compositions comprising, consisting essentially of, or consisting of EDTA salts other than, or in addition to, EDTA sodium salts have different "effective" pH ranges. The "effective" pH range of the desired EDTA salt in the antimicrobial compositions of the present invention for inhibition, disinfection, sterilization, biofilm eradication, and other uses can be determined by routine experimentation.

In some embodiments, as noted above, the antimicrobial compositions of the present invention are comprised of EDTA salt and the antimicrobial solution is comprised of EDTA salt dissolved in a solvent, typically an aqueous solvent such as water or saline. In other embodiments, as noted above, the antimicrobial compositions of the present invention consist essentially of EDTA salt, typically dissolved in an aqueous solvent such as water or saline. The antimicrobial compositions of the present invention, which consist essentially of EDTA salt, or a combination of EDTA salts, are substantially free of other active agents having significant antimicrobial and/or antifungal activity. In this context, significant antimicrobial and/or antifungal activity means that an aqueous solution of the EDTA sodium salt composition has at least 50% antimicrobial and/or antifungal activity at a concentration of 4.0% at ph 10.5.

In some embodiments, the antimicrobial compositions of the present invention include EDTA salt at a specified concentration, at a specified pH range, and may include materials containing active ingredients in addition to EDTA salt as described above. Other antimicrobial or bactericidal biological components may be incorporated into the antimicrobial compositions of the present invention, but traditional antibiotic and pesticide applications are generally precluded as a result of the scare effects of antibiotic-producing and bactericidal-resistant microorganisms. In some embodiments, the antimicrobial compositions of the present invention comprising EDTA salt at the specified concentrations and pH ranges are substantially free of other active agents having significant antimicrobial and/or antifungal activity.

Other active and inactive ingredients may also be incorporated into the antimicrobial compositions of the present invention so long as they do not adversely affect the activity and/or stability of the EDTA salt. Proteolytic agents may also be incorporated into the antimicrobial composition for certain applications. Antibacterial compositions prepared for topical application, for example, may include various ointments, creams and skin care compositions such as aloe vera (aloe vera), and the like. The antimicrobial compositions of the present invention provided in solution formulations may also include other active and inactive ingredients, so long as they do not negatively interfere with the activity and/or stability of the EDTA salt.

The compositions of the present invention may be applied in solution or dry form. In solution, the EDTA salt is preferably dissolved in a solvent, which may include an aqueous solution, such as water or saline, or another biocompatible solution in which the EDTA salt is soluble. Other solvents, including alcohol solutions, may also be used. In one embodiment, the EDTA salt composition of the present invention may be formulated in a mixture of water and ethanol. Such solutions are highly efficient and can be prepared by preparing a concentrated stock solution of EDTA salt in water and then introducing ethanol at the desired concentration. Concentrations of EDTA salt of about 0.01% to 10%, w/v are suitable, and concentrations of ethanol of about 0.1% to about 10% v/v provide effective antimicrobial compositions. In some embodiments, EDTA salt concentrations of about 2mg/ml (0.2% w/v) and ethanol concentrations of about 1% (v/v) in water are very effective against a broad spectrum of strains. When sodium EDTA is used, the pH ranges for these antimicrobial compositions are as described above. Biocompatible non-aqueous solvents may also be used, so long as the EDTA salt is soluble and remains in solution during storage and use.

The EDTA solutions of the present invention are preferably provided in sterile and non-pyrogenic form and may be packaged in any convenient manner. In some embodiments, the antimicrobial EDTA compositions of the present invention may be provided with or as part of a medical device, such as in a pre-filled syringe or other medical device. The compositions may be prepared under aseptic conditions, or they may be sterilized after preparation and/or packaging, using any of a variety of suitable sterilization techniques. Vials, syringes or containers may be provided for single or multiple uses of the EDTA solution. The system of the invention comprises such a vial, syringe or container containing the EDTA solution of the invention.

The compositions of the present invention may also be provided in a substantially "dry" form, such as a substantially dry coating on the surface of a barrel or catheter, or medical or industrial instrument such as a catheter or tube, or container, and the like. This substantially dry form of the EDTA composition of the present invention may be provided as a powder or lyophilized form, which may be reconstituted by the addition of a solvent to form a solution. The EDTA composition is provided in a substantially dry form, either as a coating, or may be incorporated into a gel or other type of carrier, or encapsulated, or packaged, and provided on a surface as a coating or in a container. Such an EDTA composition of the present invention may be prepared in substantially dry form in the presence of a solution, such that the substantially dry composition forms an EDTA solution having the composition and characteristics described above. In certain embodiments, different encapsulation or storage techniques may be used in order to achieve effective time release of EDTA upon extended exposure to the solution. In this embodiment, a substantially dry EDTA solution may provide antimicrobial activity over an extended period of time and/or upon multiple exposures to the solution.

Compositions comprising EDTA have a very positive safety profile in medical use and administration to humans. Up to 3000mg disodium EDTA is infused daily, 3 hours, at doses up to 3000mg, for the treatment of hypercalcemia in humans, and is well tolerated. EDTA salts may also be present in many solutions for medical and human health applications, along with other components, and have been determined to be safe for use in vitro and in vivo. EDTA salts are available at reasonable prices and are stable in solution over time.

The formulation and production of the antimicrobial compositions of the present invention is generally straightforward. In one embodiment, the antimicrobial compositions desired for the present invention may be formulated by dissolving at least one EDTA salt in an aqueous solvent, such as purified water, to achieve the desired concentration and adjusting the pH of the EDTA salt solution to the desired pH. In other embodiments, the antimicrobial compositions desired for the present invention may be formulated by dissolving at least one salt of EDTA in a solvent, wherein the EDTA salt or combination of salts is soluble to provide a concentrated, dissolved EDTA salt solution. Additional solvents or components may then be added. Alternatively, the dissolved EDTA salt composition may be formulated in forms other than solutions, such as topical preparations. The antimicrobial solution may then be sterilized using conventional methods, such as autoclaving, ultraviolet irradiation, filtration, ultrafiltration, and/or other methods. The preferred osmolarity range of the EDTA solution is 240-500mOsM/Kg, more preferably 300-420 mOsm/Kg. The solution is preferably prepared using USP materials.

Antimicrobial compositions consisting essentially of, or consisting of, tri-or tetrasodium salts, or mixtures thereof, are preferred for many applications and can be prepared using sodium EDTA salts other than tri-and tetrasodium salts, such as disodium EDTA, are readily available. The disodium EDTA solution has a pH in solution that is below the desired pH range for the composition of the invention. However, after adjusting the pH to the desired range using a pH adjusting substance such as sodium hydroxide, sodium acetate or other well known pH adjusting agents, the EDTA solution prepared using the disodium salt is converted to the preferred combined di, tri and/or tetrasodium EDTA composition of the present invention. Accordingly, various forms and combinations of EDTA salts may be used to prepare the EDTA compositions of the present invention, provided that the pH of the composition is adjusted to the desired pH range prior to use. In one embodiment, an antimicrobial composition consisting essentially of a mixture of tri-and tetrasodium EDTA may be provided by dissolving disodium EDTA in an aqueous solution on a 3% -5% weight/volume basis and adding an amount of sodium hydroxide sufficient to provide the desired pH of 8.5 to 12.0.

The antimicrobial compositions of the present invention comprising, consisting essentially of, or consisting of at least one salt of EDTA as described above may also be used in a number of other applications. EDTA solutions can be used as an antimicrobial solution for soaking, rinsing, or contacting medical, dental, and veterinary surfaces and objects. The EDTA solutions of the present invention, for example, may be used to store and/or disinfect contact lenses and other optical devices; for storing and/or sterilizing dental devices such as dentures, bridges, holders, toothbrushes, etc.; and for storing and/or sterilizing medical, dental and veterinary devices and instruments. In these applications, the device or surface may be contacted with, or soaked in, an EDTA solution of the invention for a time sufficient to substantially eliminate microbial and/or fungicidal infections. The EDTA compositions of the present invention are additionally useful for disinfecting water and other liquid supply lines. The liquid supply line may be sterilized by intermittently flushing the line with the EDTA composition of the invention. Also, the EDTA compositions of the present invention can be used to eliminate biofilms in water supply and storage facilities, and microorganisms (including some viruses and protozoa) and eubacteria populations.

A number of experimental tests and methods have been performed to determine their characteristics and their efficacy as antimicrobial compositions using the EDTA-containing compositions of the present invention. Several experimental methods are described in detail below. These methods and experimental results are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

Example 1

The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) data for microorganisms resistant to various EDTA formulations were determined using the agar dilution method

The Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of different gram-positive and gram-negative bacterial and yeast microorganisms were determined for several different EDTA formulations using the agar dilution method described below. Different microorganisms were also tested for MIC and MBC for EDTA salt combinations.

Gram-positive and gram-negative bacterial microorganisms were isolated from human patients with catheter-associated infections to ensure that bacterial staining was actively pathogenic and is a common type of human catheter-associated bacterial infection. Yeast microorganisms were collected from patients with severe septicemic infection. Microorganisms were catalogued and deposited in the Peter Kite laboratory at the university of Leeds.

Various EDTA salt solutions and combinations of EDTA salt solutions were prepared by dissolving the corresponding reagent grade EDTA salt in distilled water to form the desired EDTA salt concentration (w/v). Concentrated EDTA salt stock solutions were prepared for each EDTA salt or combination of EDTA salts and used to determine the MIC and MBC of various microorganisms. The tetra and trisodium EDTA solution is prepared using tetra and trisodium EDTA salt instead of disodium EDTA, and adjusting the pH of the solution to achieve the desired pH range. The EDTA salt solution was sterilized prior to use and stored at 4 ℃.

Protocol for agar dilution

Preparation of agar

● 2 liters of nutrient agar was placed in a steam bath and held for approximately 1 hour (until melted).

● the agar was cooled to 50 ℃.

● 20 sterilized (125ml) glass bottles were collected and 100ml of nutrient agar was dispensed into each glass bottle. Using a 200mg/mL stock solution, add 0.5, 1.0, 1.5, 2.0, 4, 6, 8, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 and 100mg/mL tetrasodium EDTA (or other EDTA salt or combination of EDTA salts tested) to the vial.

● 20mL of agar was poured into sterilized petri dishes and allowed to group. Pour into another 3 plates. Plates were labeled with the concentration of EDTA they contained. This operation was performed for each concentration.

● the plates can then be stored in a 4 ℃ refrigerator until use.

Inoculation plate

● cultures of 23 gram positive and 19 gram negative microorganisms were cultured overnight in nutrient broth.

● Each culture was diluted to 106cfu/mL using Phosphate Buffered Saline (PBS).

● Each plate was inoculated with 21 microorganisms using an automated plate inoculator.

Plates were incubated overnight at ● 37 ℃.

● the next day the growth was either + or-scored.

● cultures were transferred from the original plates to fresh Cled agar plates using sterile filter paper to determine MBC.

Replica plates were incubated overnight at 3537 ℃.

● the next day the growth was either + or-scored. MIC and MBC are referred to as the lowest concentrations without growth.

The results are shown in FIGS. 1A-5C. FIGS. 1A-1D show that many gram-positive and gram-negative microorganisms are resistant to the action of dipotassium EDTA; diammonium EDTA; disodium EDTA; MIC and MBC data (expressed as mg/ml EDTA solution, w/v) for an EDTA salt solution consisting of trisodium EDTA and tetrasodium EDTA.

FIG. 2 shows yeast resistance to a composition consisting essentially of tetrasodium EDTA; dipotassium EDTA; MIC and MBC data for EDTA salt solutions consisting of diammonium EDTA (expressed as mg/ml EDTA solution, w/v).

FIGS. 3A and 3B show that gram-positive and gram-negative microorganisms are resistant to the action of essentially disodium copper EDTA; MIC and MBC data for EDTA salt solutions consisting of magnesium diammonium EDTA and iron sodium EDTA (expressed as mg/ml EDTA solution, w/v).

FIGS. 4A-4C show that gram-positive and gram-negative microorganisms are resistant to the interaction of essentially disodium and tetrasodium copper EDTA; EDTA copper disodium and EDTA dipotassium; MIC and MBC data for a combination EDTA salt solution of copper disodium EDTA and diammonium EDTA (expressed as mg/ml EDTA solution, w/v).

FIGS. 5A-5C show that gram-positive and gram-negative microorganisms are resistant to microorganisms consisting essentially of tetrasodium EDTA and diammonium; tetrasodium and dipotassium EDTA; MIC and MBC data for a combined EDTA salt solution of diammonium EDTA and dipotassium (expressed as mg/ml EDTA solution, w/v).

Several EDTA salts in combination with EDTA salts are effective at inhibiting and/or eliminating a broad spectrum of bacterial strains at reasonable concentrations. Previous medical tests and applications in humans and animals have established an excellent biocompatibility profile for sodium EDTA, although the biocompatibility of other EDTA salts has not been established. EDTA tetra and trisodium salts appear to be the most effective against a broad spectrum of pathogenic bacteria. Furthermore, their biocompatibility for human and veterinary applications has been, or can be readily determined, and they can be cost effective and are stable. In addition, tetrasodium EDTA has activity as an anticoagulant and is highly soluble in aqueous solvents. Based on these factors and experiments described above, EDTA tetra and trisodium salts were selected as the most promising candidates for the antimicrobial compositions of the invention.

Example 2

Determination of Minimum Biofilm Elimination Concentration (MBEC) data for tetrasodium EDTA-resistant microorganisms using a modified Calgary device method

Biofilm formation is an important factor in bacterial contamination. An effective antimicrobial composition preferably has the ability to reduce biofilm proliferation, or prevent or inhibit biofilm formation. We therefore tested 4 candidate tetrasodium EDTA antimicrobial solutions to determine if it could prevent or inhibit biofilm formation. The Minimum Biofilm Elimination Concentration (MBEC) of various microorganisms resistant to tetrasodium EDTA was determined using the modified Calgary device method. Calgary method is described in Olsen et al, Canadian Journal of dimensional Research, 66: 86-92(2002) and us patent 6,599,714. The method and results are described below.

The tetrasodium EDTA salt solution was prepared by dissolving reagent grade tetrasodium EDTA salt in distilled water to form the desired concentration (w/v) of the EDTA salt. A concentrated EDTA tetrasodium salt mother liquor was prepared for the determination of MBEC of various microorganisms in either sessile, or biofilm form. The tetrasodium EDTA solution was sterilized and stored at 4 ℃ prior to use.

Method

Forming a biological film:

● Muller Hinton overnight broth of 100ml of the desired microorganism was used.

● transfer 200. mu.L into all wells of a 96 well microtiter plate. Placed on a cap with 96 needles. Incubate at 37 ℃ for 24 hours on an orbital shaker at 200 rpm.

Susceptibility test:

● use the biofilm formed above.

● the lid (with the pins) is placed in a new 96 well microtiter plate containing 250. mu.L of the test reagent at the desired concentration. Incubate at 37 ℃ for 1 to 2 hours. (not on a shaker).

● at 1, 3, 6, and 24 hour intervals, 4 pins per concentration were removed from the lid by inserting a screwdriver and pulling the pins into the holes.

● pins of 3 concentrations were placed in 5ml PBS wash and inverted once.

● Place 3 pins in 3mL PBS and sonicate for 15 minutes. Filter out 2 μ L onto 3X CLED plates and spread using a sterile plastic spreader. Incubate overnight at 37 ℃. The number of clones was read the next day.

● the remaining active pins (at each concentration) were placed in 600. mu.L of 4% formaldehyde saline for SEM.

FIG. 6 shows the MBEC values, expressed in mg/ml tetrasodium EDTA (w/v), of various microorganisms determined using this method. The results demonstrate that 40mg/ml tetrasodium EDTA (4% w/v) is an effective biofilm elimination concentration for all tested microbiota.

Exemplary data for various microorganisms generated by MBEC experiments are provided below. Tetrasodium EDTA was used in all experiments, which was performed in triplicate.

Table 1: microorganisms: coli, 250e

EDTA concentration mg/mL	Number of clones/mL after 1 hour	Number of clones/mL after 3 hours	Number of clones/mL after 6 hours	Number of clones/mL after 24 hours
					0	40152	53285	64234	6133
	48175	62044	56934	4960
						43796	61314	76642	5120
5	0	520	80	0
						0	540	80	0
	0	620	133	730
					10	0	0	0	0
	0	0	0	0
						0	0	0	0
15	0	0	0	0
						0	0	0	0
	0	0	0	0
					20	0	0	0	0
	0	0	0	0
						0	0	0	0

Coli, MBEC 10mg/mL tetrasodium EDTA.

Table 2: microorganisms: j26 Pseudomonas aeruginosa

EDTA concentration mg/mL	Number of clones/mL after 1 hour	Number of clones/mL after 3 hours	Number of clones/mL after 6 hours	Number of clones/mL after 24 hours
					0	86861	4400	92701	66667
	89781	3060	79562	35036

EDTA concentration mg/mL	Number of clones/mL after 1 hour	Number of clones/mL after 3 hours	Number of clones/mL after 6 hours	Number of clones/mL after 24 hours
						94891	3080	83212	41606
5	0	0	0	0
						0	0	0	0
	0	0	0	0
					10	0	0	0	0
	0	0	0	0
						0	0	0	0
15	0	0	0	0
						0	0	0	0
	0	0	0	0
					20	0	0	0	0
	0	0	0	0
						0	0	0	0

For J26 pseudomonas aeruginosa, MBEC ═ 5mg/mL tetrasodium EDTA.

Table 3: microorganisms: 292 Enterobacter cloacae

EDTA concentration mg/mL	Number of clones/mL after 1 hour	Number of clones/mL after 3 hours	Number of clones/mL after 6 hours	Number of clones/mL after 24 hours
					0	1.00E+06	103704	94444	91241
	1.00E+06	118519	131481	116667
						1.00E+06	107407	100000	131481
5	69343	35036	36496	0
						67153	15974	32197	0

EDTA concentration mg/mL	Number of clones/mL after 1 hour	Number of clones/mL after 3 hours	Number of clones/mL after 6 hours	Number of clones/mL after 24 hours
						67153	19697	39416	0
10	38686	12035	80	0
						42336	17803	219	0
	40909	18561	0	0
					15	8000	8133	379	0
	8533	8133	219	0
						7467	8267	133	0
20	13786	2840	0	0
						12473	2820	0	0
	14661	2600	0	0

For 292 enterobacter cloacae, MBEC ═ 5mg/mL tetrasodium EDTA.

Table 4: microorganisms: enterococcus sp.

EDTA concentration mg/mL	Number of clones/mL after 1 hour	Number of clones/mL after 3 hours	Number of clones/mL after 6 hours	Number of clones/mL after 24 hours
					0	5600	3520	4000	6133
	8133	3980	3440	4720
						6800	3920	3760	4640
5	1380	780	80	0
						1160	580	100	0
	1140	500	120	0
					10	40	0	0	0
	100	0	0	0

EDTA concentration mg/mL	Number of clones/mL after 1 hour	Number of clones/mL after 3 hours	Number of clones/mL after 6 hours	Number of clones/mL after 24 hours
						20	0	0	0
15	40	0	20	0
						0	0	0	0
	80	0	20	0
					20	1480	730	160	0
	1560	379	160	0
						2000	320	140	0

For h.enterococcus sp, MBEC ═ 5mg/mL tetrasodium EDTA.

Table 5: microorganisms: j22 Enterobacter cloacae

EDTA concentration mg/mL	Number of clones/mL after 1 hour	Number of clones/mL after 3 hours	Number of clones/mL after 6 hours	Number of clones/mL after 24 hours
					0	124074	107407	105556	101852
	116667	91241	105556	120370
						112963	98540	92701	100000
5	6400	267	2040	0
						5200	133	2160	0
	8933	379	1820	0
					10	3540	1920	267	80
	3040	2900	160	1532
						3760	2340	219	800
15	2620	1560	740	0
						2100	1740	720	0
	2720	1580	920	0

EDTA concentration mg/mL	Number of clones/mL after 1 hour	Number of clones/mL after 3 hours	Number of clones/mL after 6 hours	Number of clones/mL after 24 hours
					20	2040	80	960	0
	2360	1460	840	0
						1620	133	560	0

For J22 enterobacter cloacae, MBEC 15mg/mL tetrasodium EDTA.

Table 6: microorganisms: r81 Proteus vulgaris

EDTA concentration mg/mL	Number of clones/mL after 1 hour	Number of clones/mL after 3 hours	Number of clones/mL after 6 hours	Number of clones/mL after 24 hours
					0	62044	81752	112963	59259
	55474	73723	103704	68519
						54015	78832	107407	59124
5	3160	160	3460	0
						4000	400	3120	0
	4000	160	3140	0
					10	1520	730	400	0
	1920	533	1460	0
						1900	438	160	0
15	2960	379	1100	0
						2580	80	780	0
	2560	400	1220	0
					20	4560	400	1520	0
	4480	320	1280	0
						2820	240	720	0

For R81 proteus vulgaris, MBEC ═ 5mg/mL tetrasodium EDTA.

Example 3

External catheter sealing treatment method for patient positive catheter

Catheter lock treatment methods using a candidate 40mg/ml (4% w/v) tetrasodium EDTA solution were developed and used to test hemodialysis catheters for various samples positive for microbial infection. Catheters determined to have microbial infection were subjected to catheter occlusion treatment using tetrasodium EDTA and colony counts were performed at different time points. In the first experiment, all catheters were treated with 4% w/v tetrasodium EDTA, while in the other experiment, catheters were treated with varying concentrations of tetrasodium EDTA solution. Tetrasodium EDTA solution was prepared and stored as described in examples 1 and 2 above. The methods and results are described below.

The method comprises the following steps:

● renal hemodialysis catheters suspected of having infection were screened for removal by flushing 1mL of sterile phosphate buffered saline along each lumen. Quantitative culture was performed using 1 and 10 μ L aliquots that were spread onto blood agar plates and incubated.

● catheters were first stored at 4 ℃ until after screening, and the external lumen was sterilized by wiping with alcohol.

● prior to the blocking treatment test, the screened positive catheters were blocked with nutrient broth using a 5mL syringe and incubated overnight at 37 ℃ to ensure biofilm viability and to ensure total replication of all luminal surfaces with infected microorganisms.

● after overnight incubation, each catheter lumen was rinsed with 5mL of sterile saline and 21 cm pieces were excised distally, each in 1mL of 1M sterile calcium chloride (for neutralization reagents), one for Scanning Electron Microscopy (SEM) and the other for culture in sterile universal containers.

● for the incubation procedure, all were placed in a sonication tank for 15 minutes at room temperature and then vortexed for 20 seconds.

● quantitative cultures were performed using 1. mu.l and 10. mu.l aliquots placed on blood agar plates and spread by sterile plastic L-bars, incubated overnight at 37 ℃ and colony counts performed the next day.

● catheters were rinsed and blocked with a suitable concentration of tetrasodium EDTA blocking solution and incubated at 37 deg.C for 18 hours.

● at 3, 6 and 18 hour incubations, 21 cm pieces of the distal end of the catheter were cut and neutralized in 1mL of 1M sterile calcium chloride solution.

● at each time interval, a quantitative counting procedure was followed, as described previously, and 1 fragment was used for SEM.

17 infected renal hemodialysis catheters were treated with an antibacterial composition consisting of tetrasodium EDTA at a concentration of 40mg/ml (4% w/v). The results are shown in fig. 7. Another 10 infected renal catheters, as well as one arterial and one venous catheter, were treated with an antibacterial composition consisting of tetrasodium EDTA, at a concentration of 20-100mg/ml (2-10% w/v). The results are shown in fig. 8.

The results demonstrate that after 24 hours of treatment, 40mg/ml (4% w/v) tetrasodium EDTA is effective at killing or significantly reducing the majority of the microbial population. Tetrasodium EDTA at such concentrations is safe for use in humans and other animals and is considered effective and therefore is a desirable concentration for use in the antimicrobial compositions and methods of the present invention.

Example 4

Effect of tetrasodium EDTA on Acanthamoeba and Effect of Tetrasodium EDTA-treated Klebsiella on Acanthamoeba

Several species of acanthamoeba can infect humans. Acanthamoeba infections are often the result of improper storage of contact lenses and other medical devices that come into contact with the human body. Acanthamoeba feed on bacterial flora and are resistant to many treatments. The effect of tetrasodium EDTA prepared as described above on the acanthamoeba flora was tested as follows. Tetrasodium EDTA composition was also prepared using Pages saline and normal saline as solvents. The effect of tetrasodium EDTA-treated klebsiella against acanthamoeba was also experimentally tested using the following method.

Effect of tetrasodium EDTA on Acanthamoeba

The method comprises the following steps:

● before testing, fresh blood agar plates were incubated with Klebsiella edgewarsii for 18 hours at 37 ℃.

● use tetrasodium EDTA stock (100mg/mL) to reach concentrations of 22 and 44mg/mL in Page's saline.

● A9 mL concentration of each liquid was placed in a sterile glass tube. 9mL of sterile Page's saline was placed in another sterile glass tube as a control.

● A suspension of grams Klebsiella edgewarsii was prepared in 6mL of sterile Page's saline. Adjusted to McFarland standard 5.

● add 1mL of this suspension to each serial dilution and control. Each concentration is now 20 and 40mg/mL depending on the dilution factor of the Klebsiella suspension. The control still contained no tetrasodium EDTA. All concentrations were repeated in physiological saline.

● were vortex mixed. Each tube now contains a klebsiella suspension of McFarland 0.5.

● the surface of the entire acanthamoeba plate was scraped and suspended in 1.5mL of Page's saline. Vortex.

● Add 200. mu.L of Acanthamoeba suspension to each serial dilution and control.

● the tubes were placed in an incubator at 30 ℃ for 24 hours.

● after incubation, the tubes were centrifuged at 3000rpm for 10 minutes.

● the supernatant was decanted and the pellet resuspended.

● duplicate 10. mu.L of each dilution and control were placed on non-nutrient agar plates with Klebsiella lawn. A groove was cut along the center of each plate to prevent movement and 10 μ Ι _ of dilution to be tested was placed on each side.

● Each inoculation site was marked with a black marker.

The plates were incubated at ● 30 ℃ for 72 hours.

● the growth of Acanthamoeba was examined by direct plate visualization starting from each inoculation site using a magnifying eyepiece light microscope of X10.

Table 7: growth after 24 hours incubation with tetrasodium EDTA

Concentration of EDTA mg/mL (solution)	Growth of Acanthamoeba
		0(Pages saline)	+++
0(Pages saline)	+++
		20(Pages brine)	++
20(Pages brine)	++
		40(Pages brine)	-

Concentration of EDTA mg/mL (solution)	Growth of Acanthamoeba
		40Pages salt water)	-
0 (physiological saline)	+++
		0 (physiological saline)	+++
20 (physiological saline)	++
		20 (physiological saline)	++
40 (physiological saline)	-
		40 (physiological saline)	++

Table 8: growth after 24 hours incubation with tetrasodium EDTA (replicates)

Concentration of EDTA mg/mL	Growth of Acanthamoeba
		0(Pages saline)	+++
0(Pages saline)	+++
		20(Pages brine)	++
20(Pages brine)	+ + (existence of nutrient)
		40(Pages brine)	-
40Pages salt water)	-
		0 (physiological saline)	+++
0 (physiological saline)	+++
		20 (physiological saline)	--
20 (physiological saline)	--
		40 (physiological saline)	+++
40 (physiological saline)	+ + (existence of nutrient)

Table 9: growth after 48 hours incubation with tetrasodium EDTA

Concentration of EDTA mg/mL	Growth of Acanthamoeba
		0(Pages saline)	+ + + + (for Ying)Existence of health preserving
0(Pages saline)	+ + + + + (existence of nutrient)
		20(Pages brine)	-
20(Pages brine)	-
		40(Pages brine)	-
40(Pages brine)	-
		0 (physiological saline)	+++
0 (physiological saline)	+++
		20 (physiological saline)	-
20 (physiological saline)	-
		40 (physiological saline)	-
40 (physiological saline)	-

The results demonstrate that 20-40mg/ml (2-4% w/v) tetrasodium EDTA is effective in reducing or substantially eliminating Acanthamoeba populations after exposure to Pages and physiological saline for 48 hours. Tetrasodium EDTA composition prepared using water as the solvent was also effective (data not shown).

These results indicate that the antimicrobial compositions of the present invention are suitable for use as soaking solutions for a variety of medical devices and devices, including contact lenses, and dental, orthodontic and/or periodontic devices. The antimicrobial compositions of the present invention are effective in substantially eliminating acanthamoeba populations in other applications, including in fresh water and seawater storage and distribution systems, in heating, ventilation, and air conditioning units, humidifiers, dialysis units, and the like.

Acanthamoeba feed on bacterial populations. We therefore tested whether there was any effect on acanthamoeba feeding the treated bacterial populations with the antibacterial EDTA composition of the present invention.

Effect of tetrasodium EDTA-treated Klebsiella on Acanthamoeba

The method comprises the following steps:

● before testing, fresh blood agar plates were incubated with Klebsiella edgewards ii for 18 hours at 37 ℃.

● Tetrasodium EDTA stock (100mg/mL) was used to achieve concentrations of 22 and 44mg/mL in Page's saline.

● 9mL of each concentration was placed in a sterile glass test tube. 9mL of sterile Page's saline was placed in another sterile glass tube as a control.

● A suspension of Klebsiella edgewarsii was prepared with 6mL sterile Page's saline. Adjusted to McFarland standard 5.

● add 1mL of this suspension to each serial dilution and control. Each concentration is now 20 and 40mg/mL depending on the dilution factor of the Klebsiella suspension. The control still contained no tetrasodium EDTA. All concentrations were repeated in physiological saline.

● were vortex mixed. Each tube now contains a klebsiella suspension of McFarland 0.5.

Tubes were incubated overnight at ● 37 ℃.

● the next day, the tubes were centrifuged at 300rpm for 10 minutes. Pouring out the supernatant; 10mL of fresh saline or Page's saline was added, resuspended and recentrifuged. The supernatant was decanted and resuspended in 1mL saline or Page's saline.

● the surface of the entire acanthamoeba plate was scraped and suspended in 1.5mL of 5mL Page's saline. Vortex.

● mu.L of the acanthamoeba suspension was added to 3 tubes containing 9mL of saline, and 3 tubes containing 3mL of Page's saline. The EDTA concentration for incubation with klebsiella was labeled for each tube.

● Add 1mL of resuspended Klebsiella to the appropriate tube containing Acanthamoeba.

● the tubes were placed in an incubator at 30 ℃ for 24 hours.

● set up another set of tubes to incubate Klebsiella with EDTA overnight at 37 ℃ as before.

●, each tube containing acanthamoeba was centrifuged at 3000rpm for 10 minutes.

● the supernatant was decanted and the pellet resuspended.

● duplicate 10. mu.l of each dilution and control were placed on non-nutrient agar plates (incubated without EDTA) with Klebsiella lawn. A groove was cut along the center of each plate to prevent movement and 10 μ Ι _ of dilution to be tested was placed on each side.

● Each inoculation site was marked with a black marker.

The plates were incubated at ● 30 ℃.

● the growth of Acanthamoeba was examined by direct plate visualization starting from each inoculation site using a magnifying eyepiece light microscope of X10.

● the remaining acanthamoeba suspension was placed in a new set of tubes containing fresh saline or fresh Page's saline.

● and resuspend Klebsiella which had been incubated overnight with EDTA as before and added to each appropriate tube containing Acanthamoeba.

The tubes were incubated at ● 30 ℃ overnight.

● after incubation, each tube was centrifuged at 3000rpm for 10 minutes.

● the supernatant was decanted and the pellet resuspended.

● duplicate 10. mu.l of each dilution and control were placed on non-nutrient agar plates (incubated without EDTA) with Klebsiella lawn. A groove was cut along the center of each plate to prevent movement and 10 μ Ι _ of dilution to be tested was placed on each side.

● Each inoculation site was marked with a black marker.

The plates were incubated at ● 30 ℃.

● Using a magnifying eyepiece light microscope of X10, the growth of Acanthamoeba was examined starting from each inoculation site by direct plate visualization.

Table 10: growth of Acanthamoeba after 24 hours incubation with Klebsiella

(Pre-incubation with EDTA)

Concentration of EDTA mg/mL	Growth of Acanthamoeba
		0(Pages saline)	+++
0(Pages saline)	--
		20(Pages brine)	++
20(Pages brine)	++
		40(Pages brine)	++
40(Pages brine)	-
		0 (physiological saline)	+++
0 (physiological saline)	+++
		20 (physiological saline)	++
20 (physiological saline)	++
		40 (physiological saline)	-
40 (physiological saline)	-

Table 11: growth of Acanthamoeba after 48 hours incubation with Klebsiella

(Pre-incubation with EDTA)

Concentration of EDTA mg/mL	Growth of acanthamoeba
		0(Pages saline)	+++
0(Pages saline)	+++
		20(Pages brine)	+
20(Pages brine)	-
		40(Pages brine)	-

Concentration of EDTA mg/mL	Growth of acanthamoeba
		40(Pages brine)	-
0 (physiological saline)	+++
		0 (raw)Saline water)	+++
20 (physiological saline)	-
		20 (physiological saline)	-
40 (physiological saline)	-
		40 (physiological saline)	-

These results demonstrate that the growth of acanthamoebae can be inhibited and that the acanthamoebae population can be substantially eliminated by treating the population of bacteria on which acanthamoebae feeds with the antibacterial EDTA composition of the invention. Antibacterial EDTA compositions having tetrasodium EDTA concentrations of 20-40mg/mL (2-4% w/v) are effective. This demonstrates that the antimicrobial compositions of the present invention can be used in applications such as soaking solutions for various medical instruments and equipment, including contact lenses and dental/orthodontic/periodontic devices, as well as in other applications such as fresh water and seawater storage and dispensing systems, in heating, ventilation and air conditioner devices, humidifiers, dialysis devices, and the like.

Example 5

These experiments were conducted to determine whether tetrasodium EDTA composition prevented adhesion and adherence to the silicon tubes of the microorganisms. If the attachment and adherence to microorganisms in a silicon cuvette can be prevented, the formation of biofilm can be reduced. The experimental protocols used and the results obtained are provided below.

The method comprises the following steps:

● A1 cm section of a silicon test tube was filled with molten wax to seal each lumen and hardened at 4 ℃.

● the 4 sections were placed in 30mL sterile Phosphate Buffered Saline (PBS) as a control. 8 sections were placed in 30mL of 4% tetrasodium EDTA.

● 30 min, 4 sections from PBS and 4 sections from 4% tetrasodium EDTA were placed in a heat-sealed clean-up container and allowed to dry.

● the remaining 4 sections were transferred to 30mL sterile PBS for rinsing and then allowed to air dry as described previously.

● Once dried, all 12 sections were placed in 105cfu/mL mixed microorganisms (overnight cultures of Klebsiella and CNS grown in nutrient broth at 37 ℃) and incubated at 37 ℃.

● 30 min later, 2 sections of each type were removed and rinsed in 2X 30mL sterile PBS. Air drying as described previously. Separate washing and drying conduits for each type were used to prevent contamination.

● Each section was placed in 1mL PBS in a centrifuge tube and sonicated in an ultrasonic water bath for 15 minutes.

● Filter out 50uL of each tube to place in log dilution on an automated plate inoculator in duplicate.

● A1/10 diluted aliquot of each tube was filtered off.

Plates were incubated overnight at ● 37 ℃. The clone numbers were read on an automated plate reader ProtocoL. Repeat after 6 hours.

The results for the control and EDTA treated catheter sections are shown below.

TABLE 12

Type of catheter section	Incubation time	Number of catheter sections	Number of net clones (cfu/mL)	1/10 clone number (cfu/mL)
					Control	30 minutes	1	240220	00
Control	30 minutes	2	280140	10
					Control	6 hours	1	14801120	00
Control	6 hours	2	52005467	78005800
					Air-dried EDTA	30 minutes	1	240400	1333800
Air-dried EDTA	30 minutes	2	267720	00
					Air-dried EDTA	6 hours	1	12801120	168008800
Air-dried EDTA	6 hours	2	22402340	2133316000
					Rinsing EDTA	30 minutes	1	267379	00
Rinsing EDTA	30 minutes	2	10400	00
					Rinsing EDTA	6 hours	1	19801740	960012800
Rinsing EDTA	6 hours	2	36003660	190008600

The results for the pure EDTA solution were found to be more reproducible, so these were analyzed further. Since the sections were placed in 1mL of solution, the counts per mL were equal to the counts per section.

Watch 13

Type of catheter section	Average clone count after 30 min (cfu/section)	Average clone count after 6 hours (cfu/section)
			Control	880	3317
Air-dried EDTA	407	1745
			Rinsing EDTA	421	2745

TABLE 14

Type of catheter section	% reduction in mean cfu/section from control after 30 min%	% reduction in mean cfu/section from control after 6 hours%
			Air drying EDTA	53.8％	47.4％
Rinsing the EDTA	52.2％	17.3％

Repeat with klebsiella + CNS for 24 hours:

watch 15

Type of catheter section	Average clone count after 30 min (cfu/section)	Average clone count after 6 hours (cfu/section)	Average clone count after 24 hours (cfu/section)
				Control	377	9205	105806
Air drying EDTA	273	3720	70370
				Rinsing the EDTA	474	9499	77051

TABLE 16

Type of catheter section	% reduction in mean cfu/section from control after 30 min%	% reduction in mean cfu/section from control after 6 hours%	% reduction in mean cfu/section from control after 24 hours%
				Air drying EDTA	27.4％	59.6％	33.5％
Rinsing the EDTA	+25.7％	+31.9％	27.2％

+ represents the increase in mean cfu/section from control

Results for pseudomonas aeruginosa:

TABLE 17

Type of catheter section	Average clone count after 30 min (cfu/section)	Average clone count after 6 hours (cfu/section)	Average clone count after 24 hours (cfu/section)
				Control	6400	341994	1290000
Air drying EDTA	4108	30000	474494
				Rinsing the EDTA	5200	153758	1150000

Watch 18

Type of catheter section	% reduction in mean cfu/section from control after 30 min%	% reduction in mean cfu/section from control after 6 hours%	% reduction in mean cfu/section from control after 24 hours%
				Air drying EDTA	35.8	91.2	63.2
Rinsing the EDTA	18.8	55.0	10.9

These results demonstrate at least a short-term reduction of the bacterial population on air-dried and rinsed catheter sections.

Example 6

MBC values changed when tetrasodium EDTA was combined with ethanol

Solutions of EDTA alone, alcoholic alone, and EDTA/alcoholic solution were tested with water and ethanol formulated with a wide range of tetrasodium EDTA concentrations (0, 0.1, 0.5, 1, 2, 3, 4, and 8mg/ml, w/v) (to achieve final ethanol concentrations in water of 0, 0.1, 0.5, 1, 5, 10, 20, and 40%). A concentrated stock solution of tetrasodium EDTA is prepared in distilled water and ethanol is added to the concentrated aqueous stock solution to provide a suitable ethanol concentration.

The method comprises the following steps:

● the microorganisms were cultured in nutrient broth overnight at 37 ℃.

● stock solutions of alcohol and tetrasodium EDTA were used to fill a grid pattern of 96 well plates (1 per culture) using 0, 0.1, 0.5, 1, 2, 3, 4 and 8mg/ml tetrasodium w/v EDTA in 0, 0.1, 0.5, 1, 5, 10, 20 and 40% v/v alcohol in aqueous isopropanol solvent.

● Each well contained 150. mu.L of each dilution and 50. mu.L of 1X 10⁸cfu/mL of microorganisms.

● Each well contained 300. mu.L of nutrient broth by placing 96 pin lids on the plate (and into each well) and then transferring the lids to a 96 well plate over a period of 5 minutes, 6 hours, and 24 hours. Incubate overnight at 37 ℃. During the incubation period, each inoculum plate was incubated at 37 ℃.

The turbidity of each well was recorded after ● 24 hours.

The results for several microorganisms are shown below.

Watch 19

Microorganisms	MBC of tetrasodium EDTA (mg/mL)	MBC of alcohol (%)	MBC of tetrasodium EDTA (mg/mL) + alcohol (%)
				E.coli	3	10	0.5 and 0.5
Proteobacteria	3	10	2 and 1
				CNS(I)	8	10	2 and 1
Klebsiella sp	8	10	1 and 1
				Staphylococcus aureus	0.1	0.1	0.1 and 0.1
Pseudomonas sp	2	10	2 and 1
				CNS(II)	8	10	0.5 and 0.5

Tetrasodium EDTA solution dissolved in water kills the tested microorganisms more effectively than the ethanol (alone) solution. The alcohol solution combined with tetrasodium EDTA killed the tested microorganisms at the lowest concentration. A2 mg/ml (0.2w/v) tetrasodium EDTA solution in 1% alcohol provided excellent results and was bactericidal for all microorganisms tested. Such antimicrobial solutions are effective with tetrasodium EDTA and ethanol at concentrations lower than either the aqueous tetrasodium EDTA alone or the ethanolic solution of tetrasodium EDTA alone. Moreover, it can save cost, is safe and convenient for preparation and use. The antimicrobial compositions of the present invention for topical application thus comprise EDTA salt in a mixed aqueous solvent and ethanol.

Example 7

Solubility of tetrasodium EDTA in ethanol and Effect on pH

The solubility of tetrasodium EDTA in ethanol was tested, and the pH of various tetrasodium solutions in alcoholic solvents was measured.

The method comprises the following steps:

● duplicate portions of tetrasodium EDTA were weighed out through 1.5mL microcentrifuge tubes ranging in size from 10-100 mg. Add 1mL of 74% ethanol to each tube and vortex for 30 s.

● sterile distilled water 0.5ml was added to a double weight of tetrasodium EDTA and vortex mixed followed by 0.5ml of 74% ethanol.

● the pH of each tetrasodium EDTA tube was tested, where dissolution was observed.

The experimental results demonstrate that tetrasodium EDTA is completely insoluble in 74% ethanol solution. Furthermore, the results further demonstrate that tetrasodium EDTA remains in solution after the addition of ethanol when dissolved in distilled water at concentrations in the range of 10-100mg/ml, w/v. One preferred technique therefore involves first dissolving the EDTA salt in an aqueous solution and then adding ethanol or another solvent in which the EDTA salt is less soluble or insoluble. The EDTA salt solution prepared in this manner is expected to be stable over time. The measured pH values for the various solutions were as follows:

74% ethanol, pH 7.8 alone

pH of water 7.1

+10mg tetrasodium EDTA pH 9.0

+20mg tetrasodium EDTA pH 10.8

+40mg tetrasodium EDTA pH 11

+80mg tetrasodium EDTA pH 11.15

+100mg tetrasodium EDTA H11.25

Example 8

Effect of autoclaving at 121 ℃ on Tetrasodium EDTA solution

We tested the effect of autoclaving on tetrasodium EDTA solution to determine whether autoclaving can be used to sterilize tetrasodium EDTA solution prior to use.

The methods used and the results are described below.

The method comprises the following steps:

● 50 deg.C duplicate portions of 0, 20, 80 and 100mg/mL tetrasodium EDTA were prepared in sterile water and sterile, melt nutrient agar.

● one group was left at room temperature (no heating) and one group was autoclaved (heating).

● the next day, all agar bottles were placed in a steamer to melt for 40 minutes.

● measurement of diffusion band: using a cork punch, 2 holes were punched in 16 fresh blood agar plates.

● CNS suspensions of 0.5McFarland were prepared and plated through the plate using a sterile swab to produce a lawn.

● mu.l of each tetrasodium solution were pipetted into duplicate punched out holes and incubated overnight at 37 ℃.

● the diffusion bands were measured the next day and the results recorded.

The results of the measurements with the band sizes are shown below. Zones of control versus concentration are plotted to determine the actual EDTA concentration in the test sample, which is also shown below. These results demonstrate that autoclaving of tetrasodium EDTA compositions, either in sterile water or agar, does not substantially affect the antimicrobial activity of the tetrasodium EDTA composition.

Table 20: zone size in mm

Concentration of EDTA mg/mL	EDTA in sterile water (control)	EDTA in autoclaved Water	EDTA in agar	EDTA in autoclaved agar
					0	00	00	00	00
20	13.213.2	11.611.6	13.513.5	12.712.7
					80	16.116.1	15.215.2	17.217.2	15.315.3
100	17.117.1	17.017.0	17.117.1	16.416.4

Table 21: actual concentration of EDTA

Initial EDTA concentration mg/mL	EDTA in sterile Water (control)	EDTA in autoclaved Water	EDTA in agar	EDTA in autoclaved agar
					0	0	0	0	0
20	20	16	26	19
					80	80	60	101	62
100	100	98	100	83

Example 9

Effect of autoclaving at 121 ℃ on different EDTA formulations

We tested the effect of autoclaving on various formulations of EDTA solutions to determine whether autoclaving can be used to sterilize various EDTA solutions prior to use. The methods used and the results are described below.

The method comprises the following steps:

compounding agar

● A50 mL portion of nutrient agar solution was placed in a 7X 100mL sterile glass bottle.

● addition of EDTA-free powder to the first bottle (labeled 0)

Add 2000mg of EDTA powder to a second bottle (labeled 40mg/mLauto)

● Add 4000mg EDTA powder to the third bottle (labeled 80mg/mLauto)

● Add 5000mg of EDTA powder to the fourth bottle (labeled 100mg/mLauto)

● No EDTA was added to the fifth, sixth and seven bottles, (but they were non-autoclaved, labeled 40, 80 and 100mg/mL), and were left at room temperature.

● these procedures were performed for each EDTA formulation to be tested, and all bottles were autoclaved, auto-labeled, and autoclaved at 121 ℃ for 20 minutes.

● the next day all bottles were placed in a steam bath to melt the agar for pouring.

● once melted, it was allowed to cool to 50 ℃ before adding the appropriate amount of EDTA to the bottles marked as non-autoclaved. All bottles were ready for assay. .

Measuring a diffusion zone:

● Using a cork punch, 2 holes were punched in 7 fresh blood agar plates.

● CNS suspensions of 0.5McFarland were prepared and plated through the plate using a sterile swab to produce a lawn.

● mu.l were removed from each bottle into 2 separate "punched out wells" and incubated overnight at 37 ℃.

● this was done for each EDTA preparation.

● the diffusion bands were measured the next day and the results recorded. Two holes were used and 2 measurements were made for each zone.

Copper EDTA and iron EDTA solutions did not produce any zones. Therefore, the effect of heating on these solutions cannot be measured using this method. The zone sizes of the measured diammonium EDTA, dipotassium EDTA and magnesium EDTA solutions are provided below. Zone sizes of control as a function of concentration are plotted to determine the actual EDTA concentration in the test (heated) sample, and the results are provided below.

Table 21: zone size (mm)

Concentration of EDTA mg/mL	Diammonium EDTA unheated	Diammonium EDTA heating	EDTA dipotassium salt is not heated	Heating of dipotassium EDTA	Magnesium EDTA unheated	EDTA magnesium heating
							0	0000	0000	0000	0000	0000	0000
40	18.318.318.318.3	17.917.917.917.9	16.216.216.216.2	15.515.515.515.5	6.86.86.86.8	10.610.610.610.6
							80	19.719.719.719.7	19.719.719.719.7	18.918.918.918.9	18.318.318.318.3	10.010.010.010.0	10.810.810.810.8
100	20.020.020.020.0	20.620.620.620.6	18.218.218.218.2	20.020.020.020.0	8.38.38.38.3	11.811.811.811.8

Table 22: actual values of autoclaved EDTA

Concentration of EDTA mg/mL	Diammonium EDTA heating	Heating of dipotassium EDTA	EDTA magnesium heating
				0	0	0	0
40	39	38	＞140
				80	80	71	＞140
100	150	＞140	＞140

The results demonstrate that autoclaving does not impair the efficacy of most EDTA salt compositions. Autoclaving of the antimicrobial compositions of the invention can therefore be carried out after preparation to provide sterile antimicrobial compositions.

Example 10

pH of EDTA salt, calcium chloride and sodium citrate

The pH of various EDTA salt, calcium chloride and sodium citrate solutions were measured at the specified concentrations using distilled water as a solvent. The results are shown below.

10% EDTA free acid pH 4.7

10% diammonium EDTA pH 4.38

10% calcium sodium EDTA pH 6.68

10% dipotassium EDTA pH 4.5

10% copper EDTA pH 6.15

10% tetrasodium EDTA pH 11.6

2% tetrasodium EDTA pH 11

Calcium chloride neutralized TS EDTA pH 7.3

Calcium chloride, 1 molar pH 3.8

50%, 25% sodium citrate pH 8.5

Example 11

Determining anticoagulant properties of EDTA solutions

The anticoagulant properties of EDTA solutions were confirmed using the following methods.

The method comprises the following steps:

● 100 μ l aliquots of a large concentration range (0.5-100mg/mL) tetrasodium or disodium EDTA solution, adjusted to pH 11.0-11.6, were placed in capped plastic tubes.

● fresh blood from healthy volunteers was added to an aliquot of each EDTA solution in 900. mu.l and gently mixed by inverting the blood tubes at regular intervals.

The results show that the clotting time of control tubes containing blood without EDTA solution was 10-23 minutes. The clotting times of the tubes containing the disodium EDTA solution were all over 5 days. The clotting time of tetrasodium EDTA tubes at concentrations greater than 1mg/mL exceeded 5 days. Tetrasodium EDTA tubes at a concentration of 0.5mg/mL coagulated within 28 minutes. Tetrasodium EDTA, at concentrations above 1mg/ml (1% w/v), may therefore be effective as an anticoagulant.

Example 12

Osmolarity of tetrasodium salt suspension

Osmolarity and erythrocyte lysis of various concentrations of tetrasodium EDTA solutions in water and saline were determined using standard experimental techniques. Lysis of red blood cells was determined by adding 50 μ l of EDTA blood to 2ml of each solution at each concentration for 2 hours. The plasma osmolality is 275-295 m/osmol.

TABLE 23

	Osmolarity (m/osmol)	Erythrocyte lysis
			2% tetrasodium EDTA in distilled water	140	++
4% tetrasodium EDTA in distilled water	277	+
			2% tetrasodium EDTA in physiological saline	219	+/-

	Osmolarity (m/osmol)	Erythrocyte lysis
			4% tetrasodium EDTA in physiological saline	588	-

Example 13

Efficacy of 3 EDTA salts on dissolution of Artificial Urinary Crystal (AUC)

One problem with urinary catheters is that urine crystals tend to accumulate on the catheter surface. The deposition of urinary crystals may promote colonization of microorganisms and/or biofilm formation, as well as reduce the flow rate of the catheter. Therefore, it is desirable to use a disinfecting composition with urinary catheters that reduces urine crystal formation. The efficacy of 3 solutions of EDTA salt for dissolving artificial urinary crystals was tested using the method described below.

Materials:

● 25ml artificial urine in a plastic universal container was incubated with urease for 7 days at 45 ℃.

● 100mg/ml diammonium EDTA, dipotassium and tetrasodium solutions.

The method comprises the following steps:

● the artificial urine crystals were centrifuged at 4000rpm for 2 minutes.

● the supernatant was poured slowly and the crystals were washed in water followed by centrifugation.

● the crystals were resuspended in water and 200. mu.l aliquots were loaded into 4 universal containers.

● at room temperature, 4ml of 100mg/ml each EDTA salt solution was added, as well as water as a control, to each universal container.

After ● 1, 2 and 3 hours, the dissolution of the crystals compared to the control was visually observed.

The results are shown below. All EDTA salt solutions reduced urine crystal deposition compared to aqueous solutions. Therefore, EDTA salt solutions are suitable for use in urinary catheters.

Watch 24

Solutions of	Deposition of crystals
		Water + AUC	+++++
Tetrasodium EDTA + AUC	++
		EDTA diammonium + AUC	+
EDTA dipotassium + AUC	+/-

All references, including patent documents and non-patent publications, cited herein are hereby incorporated by reference in their entirety.

While in the foregoing specification this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein can be varied considerably without departing from the basic principles of the invention.

Claims (36)

1. An antimicrobial composition comprising a solution of at least one salt of ethylenediaminetetraacetic acid, wherein the at least one salt of ethylenediaminetetraacetic acid comprises tetrasodium ethylenediaminetetraacetate in a concentration of at least 0.01% mg/ml and less than 15% mg/ml, wherein the antimicrobial composition has bactericidal activity against a broad spectrum of microorganisms, wherein the antimicrobial composition has a pH of at least 9.5, and wherein the antimicrobial composition is packaged in a sterile and non-pyrogenic form.

2. The composition of claim 1, wherein the pH of the composition is from 9.5 to 11.5.

3. The composition of claim 2, wherein the pH of the composition is from 10.5 to 11.5.

4. The composition of claim 1, wherein the at least one edetate is present at a concentration of 0.2% mg/ml to 10.0% mg/ml.

5. The composition of claim 4, wherein the at least one edetate is present at a concentration of 0.2% mg/ml to 6.0% mg/ml.

6. The composition of claim 5, wherein the at least one edetate is present at a concentration of 0.2% mg/ml to 4.0% mg/ml.

7. The composition of claim 1, wherein the solution is selected from the group consisting of: water; brine; an alcohol; and combinations thereof.

8. The composition of claim 7, wherein the solution is a combination of water and ethanol.

9. The composition of claim 8, wherein the solution comprises ethanol in an amount of 0.1% to 10% v/v.

10. The composition of claim 1, wherein the composition has bactericidal activity against bacteria in planktonic and sessile forms.

11. The composition of claim 1, wherein said composition further has at least one property selected from the group consisting of:

(a) anticoagulant activity;

(b) fungicidal activity against fungal pathogens;

(c) inhibitory activity against protozoal infection; and

(d) inhibitory activity against amoebic infection.

12. An antimicrobial solution in dry form which, upon reconstitution with a solution, forms the composition of claim 1.

13. A formulation of the composition of claim 1 suitable for topical application to surfaces and objects, wherein the formulation has a pH of at least 9.5.

14. An occlusive irrigation composition comprising a solution of at least one salt of ethylenediaminetetraacetic acid, wherein the at least one salt of ethylenediaminetetraacetic acid comprises tetrasodium ethylenediaminetetraacetate in a concentration of at least 0.01% mg/ml and less than 15% mg/ml, wherein the pH of the occlusive irrigation composition is at least 9.5, wherein the occlusive irrigation composition is packaged in a sterile and non-pyrogenic form, and wherein the biocompatibility of the occlusive irrigation composition is useful for internal access catheters, urinary catheters, nasal tubes, and pharyngeal tubes.

15. The composition of claim 12, wherein the solution is selected from the group consisting of: water; brine; an alcohol; and combinations thereof.

16. An antimicrobial composition comprising a solution of trisodium and tetrasodium salts of ethylenediaminetetraacetic acid at a concentration of at least 0.01% mg/ml and less than 15% mg/ml, wherein the antimicrobial composition has bactericidal activity against a broad spectrum of microorganisms, wherein the antimicrobial composition has a pH of at least 9.5, and wherein the antimicrobial composition is packaged in a sterile and non-pyrogenic form.

17. The composition of claim 16, wherein the pH of the composition is from 9.5 to 11.5.

18. The composition of claim 16, wherein the pH of the composition is from 10.5 to 11.5.

19. The composition of claim 16, wherein the at least one edetate is present at a concentration of 0.2% mg/ml to 10.0% mg/ml.

20. The composition of claim 19, wherein the at least one edetate is present at a concentration of 0.2% mg/ml to 6.0% mg/ml.

21. The composition of claim 20, wherein the at least one edetate is present at a concentration of 0.2% mg/ml to 4.0% mg/ml.

22. The composition of claim 16, wherein the solution is selected from the group consisting of: water; brine; an alcohol; and combinations thereof.

23. The composition of claim 22, wherein the solution is a combination of water and ethanol.

24. The composition of claim 22, wherein the solution comprises ethanol in an amount of 0.1% to 10% v/v.

25. The composition of claim 16, wherein the composition has bactericidal activity against bacteria in planktonic and sessile forms.

26. A method for inhibiting the growth and proliferation of at least one undesirable microbial population on a surface or within an object comprising contacting the surface or object with the composition of any one of claims 1-25.

27. The method of claim 26, wherein the unwanted microorganism is selected from the group consisting of: a microbial population.

28. The method of claim 26, wherein the undesirable microorganism is a fungal pathogen.

29. The method of claim 26, wherein the undesirable microorganism is a protozoan population.

30. The method of claim 26, wherein the undesirable microorganism is amoeba.

31. The method of claim 30, wherein the unwanted microorganism is acanthamoeba.

32. The method of claim 26, wherein the surface or object is selected from the group consisting of: a conduit; an intravascular device; an implanted medical device; medical and veterinary instruments; a contact lens; an optical implant; dental, orthodontic and periodontal devices; a water reservoir, dispensing and treatment device; an industrial device.

33. The method of claim 26, wherein the surface or object is a medical tube or catheter.

34. The method of claim 26, wherein the surface or object is food preparation and processing equipment.

35. A method for inhibiting biofilm growth and proliferation, comprising contacting a biofilm with a composition of any of claims 1-25.

36. A cover for use in wound healing, wherein the cover is soaked with the composition of any one of claims 1-25.