WO1994000015A1

WO1994000015A1 - Biocidal antimetabolite compositions with particular activity against microorganisms with a high propensity to reproduce (infectivity)

Info

Publication number: WO1994000015A1
Application number: PCT/US1993/006126
Authority: WO
Inventors: Richard J. Craft; William A. Curby; Indu Parikh; Brian W. Sheldon
Original assignee: Bioxy Inc
Current assignee: Bioxy Inc
Priority date: 1992-06-25
Filing date: 1993-06-25
Publication date: 1994-01-06
Anticipated expiration: 1994-12-25
Also published as: AU4544893A

Abstract

This invention relates to stable biocidal antimetabolite compositions comprising water, a source of chlorite ions, a source of chloride ions and a source of chlorate ions in combination with one or more components selected from the group comprising chelators, phosphates, soaps, detergents, and/or surfactants, including but not limited to, disodium EDTA, monobasic and dibasic sodium phosphates, trisodium phosphate, and sodium lauryl sulfate. The antimetabolite compositions produced thereby are very effective biocides and have selective biocidal action, based on concentration and exposure time, against living cells including Gram negative and Gram positive bacteria and other pathogens and show more effectiveness against those populations which have strong infectivities (propensities to reproduce) or which have been stressed prior to contact with the antimetabolite compounds.

Description

BIOCIDAL ANTIMETABOLITE COMPOSITIONS WITH PARTICULAR ACTIVITY
AGAINST MICROORGANISMS WITH A HIGH PROPENSITY TO REPRODUCE
(INFECTIVITY)
BACKGROUND OF THE INVENTION
This invention relates to biocidal antimetabolite compositions. The invention relates to a method for disinfection using antimetabolite compounds which are very effective biocides and have selective biocidal action, based on concentration and exposure time, against living cells and microorganisms including Gram negative and Gram positive bacteria and other pathogens, and which show particular effectiveness against those populations of pathogens having strong propensities to reproduce (infectivities). Such pathogens are contacted with compositions that form on mixing various chlorine containing compounds with water and various chelators, phosphates, soaps, detergents and/or surfactants.

The invention further relates to the treatment of hosts infected with such pathogens by administration of the composition to said hosts.

Previous art has taught, and it has long been known that various chlorine-containing compounds can be used as biocidal agents. Indeed, the literature contains many references to the use of chlorine gas, various chlorites and even chlorine dioxide as materials that can be used to kill microorganisms. Chlorine dioxide has been suggested as a strong oxidizing agent that is especially useful to kill various microorganisms. Many patents teach that chlorine dioxide is an effective microbicide and that it is also a powerful and effective oxidizing agent. Thus, there is a wide variety of patents and prior art references which describe the production of chlorine dioxide and/or the use of stabilized chlorine dioxide solutions.

The prior art describes many processes for the direct use of chlorine dioxide as a biocide. The prior art also describes many processes for the production of stabilized chlorine dioxide by the addition of a variety of inorganic compounds such as inorganic boron and/or various peroxides, including hydrogen peroxide.

In spite of the large number of prior art references to chlorine dioxide, it has many shortcomings because it is a potentially hazardous material which is generally difficult to produce and apply where needed. Chlorine dioxide is also corrosive, and its formation requires considerable amounts of acid which add to its corrosive properties. Furthermore, it has limited effective biocide action; does not have selective biocidal action, based on concentration and exposure time, against living cells including Gram negative and Gram positive bacteria and other pathogens; and has limited biocidal action against growing cell populations which have high infectivities.

In contrast, Gordon, U.S. Patent No. 4,880,638, teaches that compositions which do not produce measurable amounts of chlorine dioxide, but do regenerate premicrobial interactive intermediates, including but not limited to chlor-halogen and oxy-halogen reactants, also exhibited microbicidal properties.

Gordon teaches that a composition which forms upon mixing specific materials has an unexpectedly high biocidal effectiveness. This composition forms upon mixing water, a source of chlorite ions and a source of chlorate ions wherein the molar ratio of chlorite ions to chlorate ions is in the range from about 2:1 to about 1000:1. This composition also requires chloride ion wherein the ratio of chlorite ion to chloride ion is at least about 0.1:1 but may be as large as 100:1. This combination of chlorite ion, chlorate ion and chloride ion results in the biocidal activity heretofore reported and documented by Gordon.

Test populations of living cells were selected to demonstrate the broad application of the cell-killing ability of compounds taught by Gordon. Bacterial populations were selected as demonstration models because bacterial cells reproduce and divide into new cells in time intervals on the order of minutes and hours, rather than days or weeks. It is also possible with current technology to know exactly how many bacterial cells are in a growth medium, and how many are dividing at any instant.

At an exact time, a specific concentration of a candidate cellkill chemical or chemicals can be put into contact with the bacterial cells. The contact period can be controlled precisely.

The demands for nutrition, growth and metabolism of these living cells are clearly different; however, the killing speed and concentration of compound needed for killing was shown to be independent of cell type.

The composition of Gordon, however, while providing a great improvement over the prior art chlorine dioxide formulations requires relatively large concentrations of reagents for efficacy. Furthermore, the biocide of Gordon does not distinguish between different microorganisms and their respective growth and metabolism requirements. The Gordon composition does not lend itself to control of its effect based on manipulation of factors such as concentration and exposure time.

The compositions of the invention represent an improvement over those of Gordon. Compositions are disclosed herein which contain little or no chlorine dioxide or stabilized chlorine dioxide and which exhibit biocidal synergism which is developed between the chlorite ion, chlorate ion and chloride ion when mixed in accordance with the procedures and in the ratios described herein. As will be more fully discussed hereinafter, the instant disclosure provides for mixing various components so as to provide a "redox buffered" equilibrium of intermediate antimetabolite species in combination with one or more agents from the group consisting of chelators, phosphates, soaps, detergents, and/or surfactants, including but not limited to disodium EDTA, monobasic and dibasic sodium phosphates, trisodium phosphate and sodium lauryl sulfate.

These combinations are extremely effective as biocidal agents. In other words, the instant invention is aimed at the formation of such intermediate antimetabolite species without the production of chlorine dioxide as taught by the prior art.

The addition of various chelators, phosphates, soaps1 detergents and/or surfactants assists in cellular wall penetration and trans location across the cellular membrane of the formed biocidal antimetabolite intermediates. This improves in a synergistic manner the efficacy of the compositions as biocides. The formation of biocidal intermediates may be triggered by a factor or factors inherent in the target organisms themselves. As a consequence of adding one or more surface-reactant and/or viscosity-altering compounds to act in concert with the aforementioned intermediates, improved disinfectant compositions are generated.

The improved disinfectant compositions produce antimetabolites which are very effective biocides and which have selective biocidal action, based on concentration and exposure time, against aggressively growing cells and microorganisms including Gram negative and
Gram positive bacteria and other pathogens having strong infectivities. Said compositions may enhance the formation and/or capture of the particularly effective antimetabolite intermediates. In any event, the inventive compositions have a biocidal synergism and unique, unexpected biocidal activity heretofore unreported and undocumented.

In view of the foregoing it is readily apparent that it is desirable to have improved biocidal compositions that are safe, easy to prepare and economical. It is also readily apparent that it is desirable to have improved methods of disinfecting various articles for which there are limited safe and effective disinfectants. Such improved methods would involve the use of biocidal compositions which have very high kill rates of microorganisms and which have selective biocidal action, based on concentration and exposure time, against virulent cells and microorganisms including Gram negative and Gram positive bacteria and other pathogens having a strong infectivity. It is also highly desirable to have stable biocidal compositions that can be stored over long periods of time without appreciable loss of their biocidal properties.

SUMMARY OF THE INVENTION
Accordingly, it is an object of this invention to provide an improved composition that is safe and economical to use; has outstanding cellular antimetabolite disinfection action; and is a very effective biocide with selective biocidal action, based on concentration and exposure time, against living cells and microorganisms including Gram negative and Gram positive bacteria and other pathogens having a high infectivity. It is another objective of this invention to provide an improved method of disinfecting articles by enhancing the transport and formation of biocidal compositions into microorganisms' cells.

This enhanced transport of the biocidal composition is achieved by the addition of one or more agents from the group consisting chelators, phosphates, soaps, detergents, and/or surfactants, including but not limited to disodium EDTA, monobasic and dibasic sodium phosphates, trisodium phosphate and sodium lauryl sulfate. It is a further objective of the invention to provide a biocidal composition of enhanced stability over that of previously known compositions. The inventive compositions can be stored indefinitely without appreciable loss of their potency.

It has now been found that a composition which forms on mixing water, a source of chlorite ions, a source of chloride ions, a source of chlorate ions and various chelators, phosphates, soaps, detergents and/or surfactants is a highly effective biocidal composition. Because of the unexpected, enhanced potency of the inventive compositions, amounts of the various components which are nontoxic to the host and inexpensive can be used to produce a formulation of maximum efficacy. The composition can also contain additional components to increase its biocidal properties selectively in certain environments; these additives are not inherently biocidal.

The compositions of this invention provide redox buffered stoichiometric solutions which contain little or no chlorine dioxide or so-called "stabilized" chlorine dioxide. Highly sensitive analytical measurements have been used which would detect as low as 1 ppm (parts per million) of chlorine dioxide or chlorine dioxide containing complexes in the solutions of this invention and no chlorine dioxide was detected. Instead, this invention describes a synergistic combination of safe chlorine compounds and membrane-altering agents which, when mixed in a specific range of ratios, results in unexpected, synergistic and selective antimicrobial activity.

As a consequence of adding one or more surface-reactant and/or viscosity altering compounds to act in concert with disinfectants which contain oxy-halogen, non-chlorine-dioxidegenerating intermediates, antimetabolites are produced which are very effective biocides and which also have increased selective biocidal action, based on concentration and exposure time, against living cells and microorganisms, including Gram negative and Gram positive bacteria and other pathogens having high infectivities. The inventive compositions should be effective, for example, against viruses. It would also be expected that the inventive compositions would be effective against cancer cells, which are known to be actively and rapidly dividing. The activity of the compositions is achieved without the necessity to produce chlorine dioxide per se.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the effect of the composition of Example 1 (identified as Salmide) on the growth of three bacterial strains.

Figure 2 shows the effect of Salmide on the growth of additional bacterial strains.

Figure 3 shows the effect of time of exposure to Salmide on the growth of S. tvphimurium.

Figure 4 shows the effect of concentration of Salmide on the growth of S. typhimurium.

Figure 5 compares the effect on the growth of Gram negative bacteria to the effect on Gram positive bacteria of a 30-minute exposure to Salmide.

Figure 6 compares the effect on the growth of Gram negative bacteria to the effect on Gram positive bacteria of a 60-minute exposure to Salmide.

Figure 7 shows the effect of the combination of sodium lauryl sulfate with Salmide on the growth of S. typhimurium.

Figure 8 shows the effect of the combination of ethylenediamine tetraacetic acid (EDTA) with Salmide on the growth of S. tvphimurium.

Figure 9 shows the effect of the combination of trisodium phosphate with Salmide on the growth of S. typhimurium.

Figure 10 shows the effect on chick body weight of Salmide and/or inoculation with S. typhimurium.

Figure 11 shows the effect on feed conversion ratios of chicks of administration of Salmide and/or inoculation with S.

tvphimurium.

Figure 12 shows the effect of different concentrations of
Salmide on S. tychimurium inoculated into chicks.

Figure 13 shows the effect of different concentrations of
Salmide on nitrogen excretion in chicks inoculated with S.

typhimurium.

Figure 14 shows the effect of different concentrations of
Salmide on rate of nitrogen retention in chicks inoculated with
S. tvphimurium.

Figure 15 shows the effect on rate of nitrogen retention in chicks inoculated with S. tvphimurium but not administered
Salmide.

Figure 16 shows the effect on excretion of chromium in chicks inoculated with S. typhimurium but not administered
Salmide.

Figure 17 shows the effect of different concentrations of
Salmide on S. tYDhimurium inoculated into chickens and subjected to moderate stress.

Figure 18 shows the effect of different concentrations of
Salmide on S. tvphimurium inoculated into chickens and subjected to heavy stress.

DESCRIPTION OF PREFERRED EMBODIMENTS
The instant invention provides for a composition that has improved, selective biocidal properties. Said improvement arises as a consequence of adding one or more surface-reactant and/or viscosity-altering compounds to act in concert with oxyhalogen, non-chlorine-dioxide-generating disinfectants to produce antimetabolites which are very effective biocides and have selective biocidal action, based on concentration and exposure time, against living cells and microorganisms including
Gram negative and Gram positive bacteria and other pathogens which have a high infectivity.

The composition forms upon mixing water with a source of chlorite ions, a source of chloride ions and a source of chlorate ions and combining them with one or more agents from the group comprising chelators, phosphates, soaps, detergents, and/or surfactants.

These compositions have a molar ratio of chlorite ions to chlorate ions in the range from about 2:1 to about 1000:1, a molar ratio of chlorite ions to chloride ions in the range from about 0.1:1 to about 1000:1 and a molar ratio of chloride ions to chlorate ions in the range from about 0.1:1 to about 1000:1.

The chlorite ion source is present in amounts from about 40 grams to about 0.04 milligrams per thousand grams of water. The above ion components are combined in a ratio range from 0.1:1 to about 1000:1 with one or more agents from the group consisting of chelators, phosphates, soaps, detergents and/or surfactants including but not limited to disodium EDTA, mono- and dibasic sodium phosphates, trisodium phosphate, and sodium lauryl sulfate which are present in amounts from about 140 grams to about 0.04 milligrams per thousand grams of water.

The latter agent(s) act in concert with the oxy-halogen, non-chlorinedioxide-generating disinfectants to produce antimetabolites which are very effective biocides and have selective biocidal action, based on concentration and exposure time, against aggressively growing cells and microorganisms including Gram negative and Gram positive bacteria and other pathogens which have a high infectivity.

Such a composition can then be applied to various articles or administered to infected hosts to kill microorganisms which have a high infectivity. The stability of the composition can be improved by adding an appropriate pH-adjusting material to adjust the resulting composition to a buffered pH range from about 6.6 to 13.

In preparing the compositions of this invention, various commercially available materials are utilized as the starting materials. For example, the source for the chlorite ions can include materials such as alkali metal chlorites and the like.

Sodium chlorite is especially useful in preparing the compositions of this invention because of its availability and its solubility in water. Other suitable sources for the chlorite ions include the alkali metal chlorites and the alkaline earth metal chlorites, as well as ammonium chlorite.

Suitable sources for the chlorate ion include various commercially available chlorates with alkali metal chlorates being preferred. It has been found that sodium chlorate and potassium chlorate are especially useful in producing the compositions of this invention because of their solubility and availability. Other sources of the chlorate ions include the alkaline earth metal chlorates and ammonium chlorate.

Suitable sources of the chloride ion include various commercially available chlorides, with alkali metal chlorides being preferred. Sodium chloride and potassium chloride are especially useful because of their cost and solubility.

Alkaline earth metal chlorides and ammonium chlorides can also be used.

Suitable sources of the chelators, phosphates, soaps, detergents and/or surfactants include various commercially available sodium derivative compounds and are especially useful because of their cost and solubility.

In preparing the compositions of this invention, sufficient water should be available to dissolve the starting materials.

While water is an essential ingredient, it should be understood that other solvents can also be present such as various alcohols, glycols and the like. It has been found that water should be present in an amount of at least 0.1 gram molars per liter.

In order for the composition to have good inhibitory biocidal activity against virulent microorganisms, the amount of the chlorite ion source should be sufficient to provide at least 104 gram mols per liter of chlorite ion.

The molar ratio of chlorite ions to chlorate ions added to the water-containing solvent is in a range from about 2:1 up to about 1000:1. The preferred ratio of chlorite ion material to chlorate ion material that is added to the water-containing solvent is in the range from about 3:1 to about 500:1.

While the broad ratio of chlorite ions to chloride ions is from about 0.1:1 to about 1000:1, the preferred range is from about 1:1 to about 50:1.

The broad ratio range of chloride ions to chlorate ions is from about 0.1:1 to about 1000:1, and the preferred range is from about 3:1 to about 10:1.

The molar ratio of the one or more chelators, phosphates, soaps, detergents and/or surfactants including but not limited to, disodium EDTA, sodium lauryl sulfate, mono- and disodium phosphates, and trisodium phosphate that are added to the watercontaining solvent should be in a range from about 2:1 up to about 1000:1. The preferred range is from about 3:1 to about 500:1.

It should be understood that the amount of the chlorite ion materials, the chlorate ion materials, and the chelators, phosphates, soaps, detergents and/or surfactants that are added to the water-containing solvent can go up to a point where the solvent material is completely saturated. In some instances, it may be desirable to heat the mixture gently to assist in dissolution.

The stability of the composition of this invention can be improved by adding a pH-adjusting material to the composition to adjust the pH of the resulting mixture to above 6.6. The concentration of the buffer can range from 0.001 molar up to the saturation level of the solution. The preferred buffering materials contain borate or phosphate salts. The preferred buffer concentration is in the range of 0.001 molar to 0.5 molar. It has been found that if the pH is adjusted to about 13, the compositions are very stable and will retain their biocidal properties over long periods of storage. Various pHadjusting materials such as alkali metal hydroxides and the like are preferred. Other pH-adjusting materials that can be utilized in this invention include buffers containing inorganic anions such as phosphates, sulfates and borates.

It has been found that various other materials can be added to the compositions of this invention to improve their efficacy.

For example, it has been found that the addition of materials such as hydrogen peroxide will inhibit the production of chlorine dioxide. Various other materials such as borates, perborates and percarbonates can also be utilized to retard the formation of chlorine dioxide. Such materials include borax, and various peroxides such as hydrogen peroxide, peroxysulfate, peroxyborate and peroxydisulfate.

In preparing the compositions of this invention, a very simple procedure can be followed whereby the chlorite ion source, the chlorate ion source, and the chelator, phosphate, soap, detergent and/or surfactant source(s) are mixed with the water-containing solvent and allowed to dissolve therein. It has been found that the mixing can take place at any desirable temperature and pressure as long as the starting materials are dissolved in the water-containing solvent. Following the mixing, in most instances a precipitate will form from heavy metal impurities in the starting materials. In order to improve the stability of the compositions of this invention, the clear liquid should be decanted and pressure-filtered to remove such precipitates.

Following this procedure, the various other materials including a membrane-altering agent and hydrogen peroxide or a similar stabilizing agent can be added to the resulting solution. The unadjusted pH of the solution ranges from 10.8 to 11. This is adjusted to pH 8.5.

At this point the solution is further stabilized by cationexchange removal of impurities. This can be accomplished either by passage of the solution through a cation-exchange column or by introducing the cation-exchange resin as a slurry into the solution followed by pressure filtration to remove said resin.

Once the compositions of this invention have been formulated, they can be utilized to treat infected hosts or to disinfect various articles on or in which there are microorganisms with a high infectivity. This can be achieved by administering the compound to the host or by contacting the article with the composition.

Although it is not intended that the inventive compositions be limited to any particular theory of operation or mechanism, it is believed that the biocidal compositions formed by mixing the aforementioned ingredients contain some type of reaction product or intermediate product that is highly effective in killing various types of virulent organisms. As will be appreciated by those skilled in the art, such intermediate products of course can be "pushed" to produce final reaction products. In this instance, it is possible that the various materials that are combined can be "pushed" to produce final reaction products that include significant amounts of chlorine dioxide.

However, it has been found that if one controls and limits the amount of chlorine dioxide that is actually formed, the resultant intermediate products are indeed more effective than chlorine dioxide itself in killing organisms that are aggressively growing.

Therefore, the theory or mechanism, it is now believed, centers on the combination of the aforementioned materials to form highly active, biocidal intermediate products (with the addition of chelators, phosphates, soaps, detergents and/or surfactants) without requiring production of chlorine dioxide.

Thus, the instant invention differs from the prior art by controlling or preventing the formation of significant amounts of chlorine dioxide while providing a composition that has outstanding biocidal properties which demonstrate a clear improvement over the prior art teachings. This improvement arises as a consequence of adding one or more surface-reactant and/or viscosity-altering compounds to act in concert with oxyhalogen, non-chlorine-dioxide-generating disinfectants to produce antimetabolites which are very effective biocides and have selective biocidal action, based on concentration and exposure time, against aggressively growing cells and microorganisms including Gram negative and Gram positive bacteria and other pathogens which have a high infectivity.

The addition of various chelators, phosphates, soaps, detergents and/or surfactants to the composition assists in translocation across the cellular membrane of the formed biocidal intermediates and results in the selection and formation of new intermediates, which in turn improves the composition's activity against Gram negative and Gram positive bacteria and other microorganisms which have a high infectivity.

The improvement of the instant compositions does not merely lie in their enhanced potency in terms of quantity and time of killing. The inventive compositions provide a vehicle for enhancing the transport of an active ingredient into the target site of a cell or microorganism. The cooperative effects of the oxy-halogen, non-chloride-dioxide-forming intermediates and the membrane-altering agent(s) result in a biocidal composition which is more effective the more rapidly the pathogen population is metabolizing/growing. One of ordinary skill in the art acquainted with the prior art would not be led to the concept of formulating a biocidal composition whose mechanism of killing action is directly based on the rate of metabolism of the target cells.

The successful, preferred embodiments of the invention are compositions and uses of said compositions which are independent of the genus and species of the target microorganism. The compositions are especially effective against particularly virulent organisms whether they be Gram negative bacteria, Gram positive bacteria or other pathogens. The compositions according to the invention also have superior stability over those previously known in the art.

Based on the properties of the inventive compositions, one would expect said compositions also to be effective against such aggressively growing cells as cancer cells and against virulent pathogenic microorganisms such as viruses.

The unexpected, enhanced potency of the instant compositions is such that very low concentrations of the components which make up the composition are necessary to achieve complete and rapid killing of the target cells; the risk to the host is thus significantly lowered if not rendered insignificant. The lower required concentrations of components also means that the instant compositions are more cost effective and less wasteful than those of the prior art.

In order to demonstrate the properties of the biocidal compositions of the instant invention, the following examples are offered. It should be appreciated that these are merely examples to show the utility and effectiveness of some of the compositions of the instant invention. The inclusion of these examples should not be interpreted in any manner as limiting the scope of the present invention to the conditions set forth in the examples.

EXAMPLE 1
A biocidal composition was prepared by dissolving 950 grams of sodium chlorite in 12 liters of water. The mixture was stirred well until all of the solid dissolved. 300 grams of sodium chlorate and 350 grams of sodium chloride were then added to the aqueous mixture and it was stirred for approximately 10 minutes until all of the solids had dissolved. After the solution was mixed, it was allowed to stand for 20 minutes and 25 grams of sodium borate, 25 grams of sodium sulfate and then 20 grams of hydrogen peroxide were added. Finally, surfacereactant/viscosity-altering agents in the form of mono- and dibasic sodium phosphate were added to the mixture.

The composition was agitated for approximately 60 minutes.

The composition was filtered to remove catalase sources. The filtrate was also passed through a cation exchange column to remove trace mineral cations. These measures were taken to prevent the decomposition of hydrogen peroxide.

The composition of the final supernatant material had a density of 1.08. The chlorite ion was present in an amount of 0.54 mols per liter. The chlorate ion was present in the amount of 0.15 mols per liter and the chloride ion was present in an amount of 0.33 mols per liter.

EXAMPLE 2
A composition that was prepared as in Example 1 was utilized to demonstrate its effectiveness in killing Salmonella enteritidis (wild type). The composition was used in an undiluted form as well as diluted (tenfold by the addition of water) form. In both of the tests, multiple colonies of the test organism grown on Mueller-Hinton agar for 24 hours at 350 C were inoculated into a Mueller-Hinton broth. A mid-log-phase broth culture was prepared by incubating the inoculated Mueller
Hinton broth at 350 C and at 200 rpm (shaking incubator) for 3-4 hours until the culture turbidity equaled that of a number one
MacFarland st inoculated agar plates were incubated for 350 C for 48 hours.

At the end of the incubation period, complete kill was observed.

Agar plates inoculated with only the organism suspension showed vigorous cell growth.

EXAMPLE 3
The composition of Example 1 was tested to determine its effectiveness in killing Campvlobactor fetus ssp. nefuni (wild type strain). In this procedure, the composition of Example I and a tenfold dilution of the composition were utilized. In the series of tests, the composition and the diluted composition were mixed in volumes of 0.3 ml each with 2.7 ml of thioglycollate broth. Multiple colonies of the test organism grown on chocolate agar for 48 hours at 420 C in microaerophilic bags (5% oxygen) were suspended in the broth. The suspension was ready to inoculate in the test composition when the culture turbidity equalled that of a number one MacFarland standard. A 0.1 ml volume of the organism suspension was added to a 1.9 ml volume of the test compounds.

An aliquot of 0.2 of the organism suspension was removed and serially diluted (tenfold) in the broth, and aliquots of 0.1 ml of the dilution were subcultured and spread onto chocolate agar for enumeration. After 10 minutes of incubation at 250 C, 0.2 ml aliquots were removed from the compound-supplemented broth and by use of calibrated micropipette droppers, 0.05 ml of the dilutions were subcultured on chocolate agar for enumeration. All of the inoculated agar plates were incubated at 420 C for 48 hours in microaerophilic bags. In both tests, complete kill of the organisms was observed. Agar plates inoculated with only the organism suspension showed vigorous cell growth.

The following in vitro and in vivo microbiological studies were performed to demonstrate a clear improvement over the teachings of Gordon as a consequence of adding one or more surface-reactant and/or viscosity-altering compounds to act in concert with disinfectants containing oxy-halogen, non-chlorinedioxide-generating intermediates to form antimetabolite oxyhalogen intermediates which are very effective biocides with selective biocidal action, based on concentration and exposure time, against living cells including Gram negative and Gram positive bacteria and other pathogens having a high infectivities.

In vitro microbiological studies # 1, # 2 and # 3, described in Examples 4, 5 and 6, respectively, were conducted to validate that the composition has an unexpected and unique biocidal selectivity for Gram negative and Gram positive bacteria and other pathogens, based on exposure time and the microorganisms' infectivity.

EXAMPLE 4
The composition of Example 1 was diluted to a concentration of 0.093 Molar and was added for 60 minutes to aqueous cultures, using standard AOAC microbiological testing procedures, inoculated with the following actively growing Gram negative and
Gram positive bacteria (108 CFU/ml). The biocidal activity was monitored at 15-minute intervals.

Gram negative bacteria:
Salmonella typhimurium NAR
Escherichia coli 0157.H7 2018
Gram positive bacteria:
Listeria monocytoqenes ATTC 19111
Figure 1 clearly shows that each of the Gram negative and
Gram positive bacterial strains has a unique biocidal sensitivity to the compound based on exposure time.

colony forming units
EXAMPLE 5
The composition of Example 1 was diluted to a concentration of 0.092 Molar and was added for 60 minutes to aqueous cultures, using standard AOAC microbiological testing procedures, inoculated with the following actively growing Gram negative and
Gram positive bacteria (108 CFU/ml). The biocidal activity was monitored at 15-minute intervals.

Gram negative bacteria:
Salmonella tvphimurium NAR
Salmonella choleraesuis ATTC 10708
Pseudomonas aeruqinosa ATTC 15442
Gram positive bacteria:
Listeria monocytopenes ATTC 19111
Staphvlococcus epidermidis ATTC 12228 Staphvlococcus aureus ATTC 12600
Figure 2 also shows that each of the bacterial strains has an unexpected and unique biocidal sensitivity to the composition based on exposure time. Studies # 1 and &num; 2 clearly show that the composition demonstrates an unexpected and unique selective biocidal activity against both Gram negative and Gram positive bacteria based on exposure time and the microorganisms' infectivity.

EXAMPLE 6
The compound of Example 1 was diluted to a concentration of 0.092 Molar and was added for 60 minutes to cultures (108
CFU/ml), using standard AOAC microbiological testing procedures, inoculated with actively growing Salmonella tvphimurium (NAR) in an organic cultured medium, brain-heart infusion broth. The biocidal activity was monitored at 15-minute intervals.

Figure 3 shows that the bacterial strain has a unique biocidal sensitivity to the composition based on exposure time, and that the composition has its greatest biocidal activity within 15 minutes of exposure.

Study # 3 clearly shows that the compound demonstrates an unexpected and unique selective biocidal activity against Gram negative bacteria based on exposure time and the microorganisms' infectivity.

EXAMPLE 7
In vitro microbiological study # 4 was conducted to demonstrate the composition's unexpected and unique selective biocidal activity, based on concentration, against living cells and microorganisms including Gram negative and Gram positive bacteria and other pathogens which have high infectivities.

The composition of Example 1 was diluted to 0.027 M, 0.054
M and 0.081 M concentrations and was added for 10 minutes to cultures, using standard AOAC microbiological testing procedures, inoculated with actively growing Gram negative
Salmonella tvDhimurium (NAR) bacteria (107 CFU/ml). The percent reduction (biocidal activity) at different concentrations was determined.

Figure 4 shows that the biocidal killing power is dependent on the composition's concentration. In vitro microbiological study # 4 validates the composition's unexpected and unique selective biocidal activity, based on concentration, against actively growing Gram negative Salmonella tvDhimurium (NAR) bacteria which have high infectivities.

Studies # 1 through &num; 4 support the premise that the composition acts either as a broad-spectrum biocide or as a selective biocide, based on concentration and exposure time, to microorganisms which have a high infectivity.

EXAMPLE 8
In vitro microbiological study # 5 was conducted to validate further that the composition has an unexpected and unique selective biocidal action, based on concentration and exposure time, against living cells and microorganisms including
Gram negative and Gram positive bacteria and other pathogens which have a high infectivity.

The composition of Example 1 was diluted to a 0.092 Molar concentration and was added for 60 minutes to aqueous cultures, using standard AOAC microbiological testing procedures, inoculated with the following actively growing Gram negative and
Gram positive bacteria (107 CFU/ml). The biocidal activity was monitored at 30- and 60-minute intervals.

Gram negative bacteria - Salmonella Species:
Salmonella tvhimurium NAR
Salmonella choleraesuis ATTC 10708
Salmonella worthington 206-4
Gram negative bacteria:
Pseudomonas aeruqinosa ATTC 15442
Escherichia coli 0157.H7 2018
Gram positive bacteria:
Listeria monocytoaenes ATTC 19111 Staphylococcus epidermidis ATTC 12228 StaPhylococcus aureus ATTC 12600
Lactobacillus acidoPhilus 0606 1-B (VPI)
Figure 5 shows biocidal selectivity at an exposure time of 30 minutes against living cells including Gram negative and Gram positive bacteria which have strong infectivities, and Figure 6 shows the results in 60 minutes.

The results of this study support further the findings in Studies # 1 through # 3.

EXAMPLE 9
The composition of Example 1 was diluted to 0.027 M, 0.054
M and 0.081 M concentrations and was combined with one or more chelators, phosphates, soaps, detergents, and/or surfactants including but not limited to disodium EDTA (1.34 mM), monobasic and dibasic sodium phosphates, trisodium phosphate (210 mM) and sodium lauryl sulfate (0.347 mM). The combined composition was added for 10 minutes to aqueous cultures, using standard AOAC microbiological testing procedures, inoculated with actively growing Salmonella tvPhimurium bacteria (107 CFU/ml).

The results were compared to the results from Study # 4 in which the composition was tested under the same conditions without the addition of one or more chelators, phosphates, soaps, detergents, and/or surfactants including but not limited to, disodium EDTA, monobasic and dibasic sodium phosphates, trisodium phosphate, and sodium lauryl sulfate. Figures 7, 8 and 9 show the unexpected and improved percent bacterial reduction by the combined composition.

The data from this study demonstrate that the combination of the oxy-halogen disinfectant composition with a chelator such as EDTA, a phosphate such as trisodium phosphate, or a surfactant such as sodium lauryl sulfate produces an antimetabolite composition of unexpected, unique and improved biocidal activity wherein the transport of the biocidal antimetabolites into actively growing cells and microorganisms is enhanced. Such cells and microorganisms include Gram negative and Gram positive bacteria and other pathogens having a high infectivity.

EXAMPLE 10
Study # 7 was conducted to evaluate the selective biocidal activity of the inventive composition of Example 1 in young chicks (7-14 days old) "stressed" with an inoculum of Salmonella typhimurium (10S CFU/ml). A series of concentrations of the composition was tested. Changes in protein absorption, growth and feed utilization of the chicks was determined upon chronic administration of the composition for 14 days in the drinking water.

Figures 10 through 16 clearly show that the bacterial strain has a unique biocidal sensitivity to the composition based on concentration, and that the composition has its greatest biocidal activity at a concentration of 0.01 Molar.

Furthermore, the results show that the composition has a unique selective biocidal activity against both Gram negative and Gram positive bacteria based on concentration, and the microorganisms' virulence. This premise is based on the results from Studies # 1 through &num; 5 which validated that Gram negative bacteria have a higher infectivity and have a greater biocidal sensitivity to the composition. The data of study #7 show that
Salmonella typhimurium were significantly reduced, but that the
Gram positive bacteria required for digestion and nutrient absorption were not destroyed. This conclusion is based on the improved protein absorption, growth and feed utilization of the chicks.

Study # 7 shows that the composition demonstrates a unique selective biocidal activity against both Gram negative and Gram positive bacteria in young chicks (7-14 days old) based on concentration, exposure time, and the microorganisms' infectivity. Furthermore, this study demonstrates that protein absorption, growth, and feed utilization are improved when the compound is administered in the drinking water of chicks inoculated with Salmonella tvphimurium.

EXAMPLE 11
Study # 8 was conducted to evaluate the selective biocidal activity of the composition of Example 10 in chickens (45 days old) when "stressed" with an inoculum of Salmonella typhimurium (10S CFU/ml). A series of concentrations of the composition was tested by administration in the chickens' drinking water for 8 hours prior to slaughter. The enteric infectious microorganisms in the intestinal tract of the chickens were stressed further by removing feed and water for a 4-hour period prior to the 8-hour administration of the composition.

This study was designed to validate that the composition is a very effective biocide, based on concentration and exposure time, against "stressed" living cells and microorganisms including Gram negative and Gram positive bacteria and other pathogens which are growing aggressively.

Figures 17 and 18 clearly show that the bacteria strain has a unique biocidal sensitivity to the composition based on concentration, and that the composition has its greatest biocidal activity at a concentration of 0.066 Molar.

Furthermore, the results show that the composition has a unique synergistic action against heavily stressed Gram negative bacteria based on concentration, and the microorganisms' virulence.

Figure 17 shows the biocidal activity in "moderately stressed" infectious environments and Figure 18 shows the biocidal activity in "heavily stressed" infectious environments.

Studies # 7 and &num; 8 show that the compound demonstrates a unique synergistic biocidal activity against Gram negative bacteria in young chicks (1-14 days old) and chickens (45 days old) based on concentration, exposure time, and the microorganisms' infectivity. Furthermore, this study clearly demonstrates that the biocidal activity of the composition in "heavily stressed" infectious environments is enhanced.

Claims

We claim:

1. A stable biocidal composition with selective biocidal action which comprises water, a source of chlorite ions, a source of chloride ions and a source of chlorate ions in combination with one or more components selected from the group consisting of surface-reactant and viscosity-altering agents and which includes a pH-adjusting material in an amount sufficient to adjust the pH of said composition to above 7.0.

2. The composition of claim 1 wherein the molar ratio of chlorite ions to chlorate ions is in the range from about 2:1 to about 1000:1, the molar ratio of chlorite ions to chloride ions is in the range from about 0.1:1 to about 1000:1, the molar ratio of chloride ions to chlorate ions is in the range from about 0.1:1 to about 1000:1, the chlorite ion source is present in amounts of from about 40 grams to about 0.04 milligrams per thousand grams of water, the collective surface-reactant and viscosity-altering agents are present in amounts of from about 40 grams to about 0.04 milligrams per thousand grams of water, and the ratio of the collective surface-reactant and viscosityaltering agents to the combined ion sources is in the range from about 0.1:1 to about 1000:1.

3. The composition of claim 2 wherein the molar ratio of chlorite ions to chlorate ions is in the range from about 3:1 to about 500:1, the molar ratio of chlorite ions to chloride ions is in the range from about 1:1 to about 50:1, the molar ratio of chloride ions to chlorate ions is in the range from about 3:1 to about 10:1 and the molar ratio of the added surface-reactant and viscosity-altering agents to the combined ion sources is in the range from about 3:1 to about 500:1.

4. The composition of claim 1 wherein the surface-reactant and viscosity-altering agents are selected from the group consisting of chelators, phosphates, soaps, detergents and surfactants.

5. The composition of claim 4 wherein the surface-reactant and viscosity-altering agents are selected from the group consisting of disodium EDTA, monobasic and dibasic sodium phosphates, trisodium phosphate and sodium lauryl sulfate.

6. The composition of claim 1 which additionally comprises a material which retards the formation of chlorine dioxide.

7. The composition of claim 6 wherein the material retarding the formation of chlorine dioxide is selected from the group consisting of peroxides, borate, perborates and percarbonates.

8. The composition of claim 7 comprising hydrogen peroxide.

9. The composition of claim 1 wherein water is present in an amount of at least 0.1 gram mols per liter.

10. The composition of claim l wherein sufficient water is available to dissolve the chlorite ion source, the chloride ion source and the chlorate ion source.

11. The composition according to claim 10 wherein the water can be combined with other solvents capable of dissolving the chlorite ion source, the chloride ion source and the chlorate ion source.

12. The composition according to claim 1 wherein said source of chlorite ions is an alkali metal chlorite, said source of chloride ions is an alkali metal chloride and said source of chlorate ions is an alkali metal chlorate.

13. The composition according to claim 12 wherein said alkali metal chlorite is sodium chlorite, said alkali metal chloride is sodium chloride and said alkali metal chlorate is sodium chlorate.

14. A stable biocidal composition which is particularly effective against aggressively growing populations of Gram negative and Gram positive bacteria and other pathogens, comprising water, a source of chlorite ions, a source of chloride ions and a source of chlorate ions in combination with one or more components selected from the group consisting of surface-reactant and viscosity-altering agents and which includes a pH-adjusting material in an amount sufficient to adjust the pH of said composition to above 7.0.

15. A stable biocidal composition which is particularly effective against aggressively growing populations of Gram negative and Gram positive bacteria and other pathogens which have been stressed prior to contact with said composition, comprising water, a source of chlorite ions, a source of chloride ions and a source of chlorate ions in combination with one or more components selected from the group consisting of surface-reactant and viscosity-altering agents and which includes a pH-adjusting material in an amount sufficient to adjust the pH of said composition to above 7.0.

16. A method of killing Gram negative and Gram positive bacteria and other pathogens, which comprises contacting said pathogens with an effective amount of the composition of any one of claims 1-6.

17. A method for achieving enhanced killing of aggressively growing Gram negative bacteria, Gram positive bacteria and other pathogens, comprising contacting said pathogens with an effective amount of the composition of any one of claim 1-6.

18. The method of claim 17 wherein the aggressively growing pathogens are cancer cells.

19. The method of claim 17 wherein the aggressively growing pathogens are viral.

20. A method for achieving enhanced killing of infectious, aggressively growing Gram negative bacteria, Gram positive bacteria and other pathogens which have been stressed within an infected host, comprising administering to said host an effective amount of the composition of any one of claims 1-6.

21. The method of claim 16 wherein the water can be combined with other solvents capable of dissolving the chlorite ion source, the chloride ion source and the chlorate ion source.

22. The method of claim 17 wherein the water can be combined with other solvents capable of dissolving the chlorite ion source, the chloride ion source and the chlorate ion source.

23. The method of claim 20 wherein the water can be combined with other solvents capable of dissolving the chlorite ion source, the chloride ion source and the chlorate ion source.

24. The method of claim 16 wherein said source of chlorite rons is an alkali metal chlorite, said source of chloride ions is an alkali metal chloride and said source of chlorate ions is an alkali metal chlorate.

25. The method of claim 17 wherein said source of chlorite ions is an alkali metal chlorite, said source of chloride ions is an alkali metal chloride and said source of chlorate ions is an alkali metal chlorate.

26. The method of claim 20 wherein said source of chlorite ions is an alkali metal chlorite, said source of chloride ions is an alkali metal chloride and said source of chlorate ions is an alkali metal chlorate.

27. The method of claim 16 wherein said alkali metal chlorite is sodium chlorite, said alkali metal chloride is sodium chloride and said alkali metal chlorate is sodium chlorate.

28. The method of claim 17 wherein said alkali metal chlorite is sodium chlorite, said alkali metal chloride is sodium chloride and said alkali metal chlorate is sodium chlorate.

29. The method of claim 20 wherein said alkali metal chlorite is sodium chlorite, said alkali metal chloride is sodium chloride and said alkali metal chlorate is sodium chlorate.

30. The method according to claim 16 wherein said composition additionally includes a material to retard the formation of chlorine dioxide, the material being selected from the group consisting of peroxides, borates, perborates and percarbonates.

31. The method according to claim 17 wherein said composition additionally includes a material to retard the formation of chlorine dioxide, the material being selected from the group consisting of peroxides, borates, perborates and percarbonates.

32. The method according to claim 20 wherein said composition additionally includes a material to retard the formation of chlorine dioxide, the material being selected from the group consisting of peroxides, borates, perborates and percarbonates.

33. The method of claim 30 wherein said material is hydrogen peroxide.

34. The method of claim 31 wherein said material is hydrogen peroxide.

35. The method of claim 32 wherein said material is hydrogen peroxide.

36. The method of claim 16 wherein the molar ratio of chlorite ion to chlorate ion is in the range from about 3:1 to about 500:1, the molar ratio of chlorite ion to chloride ion is in the range from about 1:1 to about 50:1, the molar ratio of chloride ion to chlorate ion is in the range from about 3:1 to about 10:1 and the ratio of the collective surface-reactant and viscosity-altering agents to the combined ion sources is in the range from about 0.1:1 to about 1000:1.

37. The method of claim 17 wherein the molar ratio of chlorite ion to chlorate ion is in the range from about 3:1 to about 500:1, the molar ratio of chlorite ion to chloride ion is in the range from about 1:1 to about 50:1, the molar ratio of chloride ion to chlorate ion is in the range from about 3:1 to about 10:1 and the ratio of the collective surface-reactant and viscosity-altering agents to the combined ion sources is in the range from about 0.1:1 to about 1000:1.

38. The method of claim 20 wherein the molar ratio of chlorite ion to chlorate ion is in the range from about 3:1 to about 500:1, the molar ratio of chlorite ion to chloride ion is in the range from about 1:1 to about 50:1, the molar ratio of chloride ion to chlorate ion is in the range from about 3:1 to about 10:1 and the ratio of the collective surface-reactant and viscosity-altering agents to the combined ion sources is in the range from about 0.1:1 to about 1000:1.