WO2003045446A1

WO2003045446A1 - Method for the management and/or treatment of microbially contaminated environments and the use of a new class of microbicidal reagents in such management

Info

Publication number: WO2003045446A1
Application number: PCT/ZA2002/000156
Authority: WO
Inventors: Suha Rawhani; Robin Duncan Kirkpatrick
Original assignee: Radical Waters (Ip) (Pty) Limited
Priority date: 2001-10-12
Filing date: 2002-10-14
Publication date: 2003-06-05
Also published as: AU2002342361A1

Abstract

According to the invention there is provided a method for the management and/or treatment of microbes characterised in having acquired microbial resistance to conventional microcidal and/or bactericidal preparations, the method including the step of electrochemically activating a dilute aqueous saline solution such that the solution includes separable and both of an aqueous, mixed oxidant, predominantly anion-containing solution and an aqueous, mixed reductant, predominantly cation-containing solution; and applying the electrochemically activated, bactericidal aqueous solution to the specific environment where the microbes are present and where microbial decontamination is required. The invention also extends to the use of an electrochemically activated, dilute aqueous saline solution in the management and/or treatment of microbes, and the electrochemically activated, dilute aqueous saline solution for such use.

Description

Method for the management and/or treatment of microbiallv contaminated

environments and the use of a new class of microbicidal reagents in such

management

Technical field

This invention relates to a method for the management and reduction of the incidence and intensity of microbial resistance as currently evidenced with conventional chemical and antimicrobial treatments. More particularly, the invention concerns alleviating the difficulties associated with such microbial resistance, which often render traditional and current antimicrobial remedies ineffective and largely obsolete.

Background art

It is a scientifically proven fact that repeated interventions with chemical substances, which are theoretically designed to exert an antimicrobial effect against bacterial growth, progressively fail to exert the full extent of the desired antimicrobial efficacy. A variety of mechanisms associated with a microbe facilitate the development of resistance to these chemical interventions.

Factors affecting efficacy of antimicrobial treatment programs

The antibacterial activity of bactericides is determined by their chemical reactivity with certain organic groups. Bactericides do not select between free and cell-bound groups and oxidising bactericides therefore react with any readily oxidisable organic compound, and not only with live cells. Bactericide activity is influenced by the chemistry of the surroundings where it is employed. Factors affecting bactericide efficacy include inter alia pH, temperature, water hardness, the presence of organic compounds such as proteins or saccharides, and the presence of additives such as anti-scaling agents or corrosion inhibitors.

These factors affect different bactericides to different degrees. Some bactericides are not very stable in concentrated form and undergo changes. So, for example, formaldehyde polymerises when exposed to polar compounds (acids or alkalis) or high temperature and oxidises to formic acid when exposed to air. Isothiazolones are unstable at temperatures above 40 °C and chlorhexidine is unstable above 70 °C. A decrease in the efficacy of a bactericide treatment programme can be due to a decrease in bactericide activity, or due to inactivation by adverse conditions, and does not always indicate bacterial resistance.

Chemistry of the water

Chemicals inhibiting scaling and corrosion are often added to industrial water systems and some of these chemicals interact with certain biocides. So, for example, chromates are used to inhibit corrosion, but also suppress microbial growth, acting synergistically with the biocide used. Glycolic acid secreted by algae can, however, reduce chromate, rendering it inactive. Dithiocarbamates reduce chromate, so the two substances are incompatible. QAC's form insoluble chromate precipitates at high concentrations, so the two should not be added simultaneously to water. Careless application of chloride lowers the pH to a point where the protective chromate film is solubilised. Na-2-mercaptobenzothiazole is a corrosion inhibitor, which is oxidised by chlorine dioxide. Methylene bis-thiocyanate is hydrolysed under slightly alkaline conditions (pH 7,5). Where the chlorine demand of the water is high, the large quantity of chlorine added leads to a high chloride level, which increases the corrosion potential of the water. Chlorine and quaternary ammonium compounds increase the corrosion rate of copper alloys. Bacterial resistance

Bactericide treatment regimes for cooling water systems often fail, posing the question of bacterial resistance to the bactericide. Certain authors have argued that failure of treatment programs is due to selection for resistant strains. The applicant believes, however, that susceptible bacterial isolates acquire increased tolerance to bactericides following serial transfer in sub-inhibitory concentrations.

Microbial resistance is often defined as the temporary or permanent ability of an organism and its progeny to remain viable and/or multiply under conditions that would destroy or inhibit other members of the strain. Bacteria may be defined as resistant when they were not susceptible to a concentration of antibacterial agent used in practice. Traditionally, resistance refers to instances where the basis of increased tolerance is a genetic change and where the biochemical basis is known. Whereas the basis of bacterial resistance to antibiotics is well known, that of resistance to antiseptics, disinfectants and food preservatives is less well understood, and of the basis of bacterial development of resistance to water treatment bactericides is very little known.

As biocides are selective in their action, application of any one could result in selection for resistant bacteria. As cells in biofilms and planktonic communities are in continuous exchange, death of cells in the planktonic phase would influence the equilibrium and shifts would occur in both the planktonic and the sessile populations. Biocides attack targets of cell function, placing the bacterium under stress. It is well recognized that communities under stress have lower species diversity and select for fitter species. Therefore, a more resistant community could develop. The concentration of a biocide is not related linearly to its activity; a concentration exponent is involved in the relationship. In many cases a small decrease in concentration will result in a notable decrease in activity.

In an in situ biocide evaluation study it was found that the dominant planktonic survivor after 48 hours was a species most effectively killed by the relevant biocide under laboratory pure culture conditions. An example is dichlorophen, which killed 99.94% of Pseudomonas stutreri at 50 ppm and yet left this species the dominant isolate after 48 hours (43%) in the cooling system. Also, thiocarbamate killed 99.87% of P. stutzeri at I74ppm and left it the dominant planktonic survivor (62.5%) in the system treated. QAC-tin killed 100% of Acinetobacter calcoaceticus and lef c/r/efoi acterspp. dominant (40%) in the system. Although the surviving strains could be different ones, the correlation is striking.

Two reasons exist why the efficacy of bactericide treatment programmes can decrease at times. The one is a decrease in the activity of the bactericide, and the other is a decrease in the bacterial susceptibility towards the bactericide. Three mechanisms of resistance have been reported in the field of antibiotic study: inaccessibility of the antimicrobial agent to its site of action,

*Absence of the susceptible site, or alteration to an insusceptible form, and

^*lnactivation of the antibacterial agent.

Bactericides are less specific in their action than some antibiotics, so that the alteration of a reactive site or the substitution of an amino acid in a protein will not render bacterial cells resistant. Therefore, inaccessibility and inactivation are the two classes of possible mechanisms of resistance. Additionally, active removal of biocide is a third class of resistance. Decreased permeability The initial stage of bactericide action is binding to the bacterial cell surface. Then it must traverse the cell wall (gram-positive) or outer membrane (gram-negative) to reach its site of action at the cytoplasmic membrane or cytoplasm. In gram-positive bacteria there are no specific receptor molecules or permeases to assist or block bactericide penetration. (The cell wall of Bacillus megaterium is permeable to molecules up to 30 kl). Intrinsic

resistance of gram-positive bacteria to bactericides is therefore low. The gram-negative cell envelope has, however, evolved to regulate the passage of substances into and out of the cell to a remarkable degree of specificity.

All the components of the cell envelope except peptidoglycan play a role in the barrier mechanisms because peptidoglycan is spongy and therefore permeable. P. aeruginosa is the most resistant non-spore forming bacteria to most bactericides, due to the superior barrier properties of its outer membrane. In a recent study the antimicrobial activity of a series of new 2-arylthio-N-alkylmaleimides were compared and many were found active against Staphylococcus aureus, Bacillus subtilis and E. coli. Only one of the 51 derivatives tested was marginally active against P. aeruginosa.

The physiological state of cells and the nature of the habitat can lead to considerable variation in the susceptibility of bacteria to bactericides. The composition of the bacterial cell envelope does change as a response to available or limiting nutrients, so that the barrier properties of the envelope are affected. Exposure to sub-inhibitory concentrations of bactericides can lead to phenotypic adaptation, resulting in a resistant cell population. In E. coli certain proteins induced by heat or starvation stress also confer resistance to UV light. Most bactericide-resistance is due to adaptation and the resistant phenotype is mostly lost upon removal of the bactericide.

Efflux systems

Bacteria can actively pump compounds out of the cell via membrane efflux systems. Only one type of bactericide-efflux system has been described to date, namely the QAC efflux system of Staphylococcus aureus. This efflux system is coded by two gene systems: the genes qacA and qacB encode for a high level of resistance, and qacC and qacD encode for a low level of resistance. qacC and qacD are further identical to the ebrgene encoding for resistance to ethidium bromide in Staphylococcus aureus, explaining why resistance to QAC is often concurrent with resistance to ethidium bromide. The qacA gene codes for a 50 kD protein which mediates energy-dependant efflux of both benzalkonium chloride and ethidium bromide. The qacC gene also mediates energy-dependant efflux of benzalkonium chloride and ethidium bromide. Two different but isofunctional gene systems appear to have evolved in Staphylococcus aureus.

Enzymatic degradation of biocides

Resistance to antimicrobial agents can be due to enzymes transforming the bactericide to non-toxic form. The phenomenon is usually investigated from the biodegradation point of view, i.e. the biodegradation of toxic pollutants. A host of aromatic, phenolic and other compounds toxic to many bacteria (some of which are employed as bactericides) can be degraded by certain bacteria. The topic has been reviewed by various authors and the current literature reports extensively on biodegradative pathways.

Two types of enzyme-mediated resistance mechanisms have been documented, i.e. heavy metal resistance and formaldehyde resistance. Resistance to heavy metals includes resistance to the following: mercury, antimony, nickel, cadmium, arsenate, cobalt, zinc, lead, tellurite, copper, chromate and silver. Detoxification is usually by enzymatic reduction of the cation to the metal. Where some heavy metal resistance genes are carried on plasmids, others are chromosomal. The resistant phenotype is usually inducible by the presence of the heavy metal. Some heavy metals induce resistance to a broader spectrum of heavy metals. Arsenate, arsenite and antimony, for example, induce resistance to each other in E. coli.

The detoxification of formaldehyde by P. aeruginosa and P. putida has been studied extensively. Formaldehyde is reduced by an NAD + - gluthathione-dependant dehydrogenase, formaldehyde NAD + oxidoreductase. This enzyme is probably plasmid- encoded and appears to be constitutively expressed. Resistance to most formaldehyde- releasing formaldehyde condensates is also due to formaldehyde dehydrogenase activity, as the antibacterial mechanism of these condensates appears to be via formaldehyde.

Biofilm-associated resistance

Bacteria in biofilms are much more protected from bactericidal action than are planktonic bacteria. In a recent study, biofilm bacteria were found 150 to 3000-fold more resistant to hypochlorous acid and 2 to 100 times more to monochloramine than were unattached cells. Pseudomonas aeruginosa growing as a biofilm has been found 20 times as resistant to tobramycin as are planktonic cells. Three reasons for the increased biocide resistance of biofilm bacteria have been put forward. These do not adequately explain the phenomenon of biofilm resistance, but they are listed below:

1. The EPS material is a polyanionic polymer and acts as an exchange resin. It quantitatively adsorbs biocide, protecting the bacterial cell from biocide action. Biofilms contain large amounts of EPS which would protect resident bacteria from biocides.

2. Gram-negative bacteria growing in biofilms have a higher ratio of unsaturated to saturated fatty acids and a higher ratio of Ciβto Cιβ fatty acids. Resistant bacteria show similar changes in membrane-lipid profiles.

3. Surface hydrophobicity of QAC and amphoteric resistant cells were higher than that of unadapted cells. Biofilm bacteria often have a higher surface hydrophobicity due to attachment structures.

The first reason is questionable because the reason why bacteria grow in biofilms is because the EPS acts as a nutrient sink, attracting organic compounds from the surrounding water. Recent work has supported the theory that organic material is somehow attracted to the EPS and associates favourably with it. At least some of these molecules must diffuse to and into the microorganisms embedded in the EPS to facilitate the observed growth. Non-oxidising biocides, being organic molecules of small to intermediate size would also associate favourably with the EPS. At least some would diffuse to and into the microorganisms embedded in the EPS and exert their antibacterial activity. The mechanism of increased resistance must be related to altered surface properties of cells growing in the biofilm environment. Reasons 2 and 3 are possibly correct. Object of the invention

It is an object of this invention to provide a method for the effective management and/or treatment of microbial resistance to conventional chemical and antimicrobial agents used in the control of microbes.

Disclosure of the invention

According to the invention there is provided a method for the management and/or treatment of microbially contaminated environments, the method including the step of applying electrolytically activated, aqueous solution to the specific environment where microbes are present and where microbial decontamination is required.

Properties of Electrolytically Activated Solutions

The properties of electrolytically activated solutions are dependent upon a number of factors. These factors comprise the solution flow rate through the cell, type of salt, the voltage and current being applied, temperature, inter-flow dynamics of the solutions between the anode and cathode chambers, such as the degree of feedback of catholyte into the anolyte chamber, the design and geometry of the cell and the degree of mineralisation of the water.

During the process of electrolytic activation in the electrolytic cell utilised by the authors, three broad classes of products are believed to be produced, namely:

(i) Stable products: These are acids (in the anolyte) and bases (in the catholyte) that influence the pH of the solution in question, as well as other active species; (ii) Highly active unstable products: These include free radicals and other active ion species with a half-life of typically less than 48 hours. Included here are electrically and chemically active micro bubbles of electrolytic gas, 0.2 to 0. 5 micrometer in diameter and with concentrations of up to 10⁷ ml^"1, distributed uniformly through the j solution. All these species serve to enhance the ORP of the anolyte and catholyte;

(iii) Quasi-stable structures: These are structures formed at or near the electrode surface as a consequence of the very high voltage gradient (10⁶ V cm^"1) in those regions. These are free structural complexes of hydrated membranes around ions, ) molecules, radicals and atoms. The size of these water clusters is reduced from about 13-18 to approximately 5-6 molecules per cluster. All these features enhance the diffusion, catalytic and biocatalytic properties of the water.

It is important to note that the level of mineralisation of input water required to generate ) optimally metastable solutions is insignificantly different from the composition of potable water. However, the heightened electrical activity and altered physico-chemical attributes of the solutions differ significantly from the inactivated state, yet they remain non-toxic to mammalian tissue and the environment. Without maintenance of the activated state, these diverse products degrade to the relaxed state of benign water and the anomalous ⁾ attributes of the activated solutions such as altered conductivity and surface tension similarly revert to pre-activation status.

Biocidal properties of anolyte and mixed anolyte and catholyte Most of the earlier technologies that have employed electrolytic activation to generate > biocidal solutions have not been capable of separating the anolyte and catholyte solutions during generation in the cell. In these earlier technologies, the two opposing solutions have greatly neutralised each other with regard to potential electrical activity.

One of the advantages of the more modern ECA systems is that the biocidal activity of hypochlorous acid generated in these systems is up to 300 times more effective than the sodium hypochlorite generated by earlier systems. Additionally, comparison of neutral anolyte (pH=7) with alkaline gluteraldehyde (pH=8.5) showed that the latter required a concentration of 2% versus 0.05% of the former, in order to achieve the same biocidal efficacy. Similarly, it has been shown that a 5% solution of sodium hypochlorite ("Jik") can only be used for purposes of disinfection, whilst a 0.03% solution of neutral anolyte has both disinfecting and sterilising properties. In general, the biocidal activity of non-activated neutral anolyte (only stable products and no electrical charge) is 80 times the potential activity of the hypochlorite solution, but still exhibits only one third of the full biocidal potential of the optimally activated ECA solution.

Thus, using non-toxic salts, these activated solutions have been shown conclusively to exceed chemically derived "equivalents" both in low dosage effectiveness as well as physico-chemical properties. This heightened biocidal capacity relative to traditional chemical solutions permits the incorporation of activated solutions at substantially lower dose rates, eliminating the risk of toxicity and adverse environmental impact, while providing cost effective resolutions.

The electrochemically activated, aqueous solution may be selected from a group consisting of an anion-containing solution; a cation-containing solution; and a mixture of an anion-containing solution and a cation-containing solution. The anion-containing solution may have been prepared from an anion-containing solution, a cation-containing solution or a mixture of an anion-containing solution and a cation-containing solution. The cation- containing solution may have been prepared from an anion-containing solution, a cation- containing solution or a mixture of an anion-containing solution and a cation-containing solution.

The anion-containing solution is referred to hereinafter for brevity as the "anolyte solution" or "anolyte" and the cation-containing solution is referred to hereinafter for brevity as the "catholyte solution" or "catholyte. The electro-chemically activated, aqueous solution may be prepared by means of electrolysis of an aqueous solution of a salt. The salt may be selected from the group consisting of alkali and alkali earth metal chlorides; -carbonates; - bi-carbonates; -phosphates; -sulphates, or -nitrates; for example, sodium chloride; non- iodated sodium chloride; potassium chloride; calcium chloride; sodium carbonate; - bicarbonate; and - phosphate; potassium carbonate; - bicarbonate; and - phosphate; calcium phosphate; sodium-; -potassium; and calcium nitrate; and a mixture of at least two of the aforementioned salts. The electro-chemically activated, aqueous solution may be produced from a relatively low concentration aqueous salt solution. Depending on the particular process being performed, and the method for producing the electrochemically activated solution (with or without pre-dilution of the feed solutions), the aqueous salt solution concentration may be between 0.0001 % to 10%, and, for some specific systems, without pre-dilution of the feed preferably between about 0.05 to 5% aqueous salt solution.

The anolyte and the catholyte may be produced by an electrochemical reactor or so-called electrolysis device, having a through flow electro-chemical cell with two co-axial cylindrical electrodes, with a tubular ceramic diaphragm located co-axially between the two electrodes so as to separate an annular inter-electrode space into a co-axial, annular catholytic and an annular anolytic chamber arrangement. The electro-chemical cell may have predetermined design and geometrical relationships, ensuring optimum fluid flow and re-circulation patterns. The cell may have a relatively small, annular, cross-sectional total i open area for fluid flow, thus causing turbulent fluid flow there through so as to ensure maximum exposure of the solutions to the electric field.

The anolyte and the catholyte may be produced in the electro-chemical cell under predetermined operational parameters, including a relatively low current, preferably of i about 5 to 7A, and a relatively high voltage, preferably of about 6 to 48 V, and more preferably between 8V and 24 V, thus providing a relatively high voltage gradient or electric field intensity at the interface between the electrode surface and electrolyte, estimated to be about 10⁶ V/cm.

i The salt solution may be electrolysed to produce the anolyte and the catholyte with mixed oxidant and mixed reductant species. These species may be labile and after about 96 hours, the concentration and activity of the various activated species may reduce substantially with relatively little, alternatively, no active residues remaining.

i The anolyte solution has a suitable redox potential, preferably of about between +300 mV and +1 200 mV. The anolyte solution may have a suitable pH, preferably of between 2 and 8, and more preferably 7. Depending on the salt(s) in the feed stream and used in the activation process, the anolyte solution may include oxidant species such as CIO; CIO^"; HCIO; OH^"; HO₂ ^"; H₂0₂; O₃; S₂O₈ ^2" and CI₂O₆ ^2". The catholyte solution generally may have a suitable pH, preferably of between 7 and 13, and more preferably about 11.5, and a suitable redox potential, preferably of between -200 mV and -1100 mV and more preferably about -800 mV. Depending on the salt(s) in the feed stream and used in the activation process, the catholyte solution may include i reductant species such as OH^"; H₃ ^"; O₂; H₂; HO₂'; HO₂ ^" and O₂ ^".

Depending on the source of the water and the salt used, anolyte may contain organic radicals and other components such as Cl₂, HCIO, CIO^", CIO^', CT, HO₂, HO₂\ O₂, HO^', O₃,

0₂\ 3O₂, 0\ H₃O⁺, Cl', H-, H₂O₂, CI₂O, CIO₂ ^", HCI, CI₂O₇, S₂O₈ ^2" C₂O₆ ^2', HCIO, H₂SO₄, and

) HSO_gCI. Catholyte may contain HO^", H₃O₂ ^", O₂ ^", HO₂ ^", H₂O₂, H₂, HO, H₂ ^", NaOH, KOH,

Ca(OH)₂ and Mg(OH)₂. Analysis of the inorganic components of these solutions has

shown varying quantities of aluminium, calcium, magnesium, manganese, potassium, sodium, molybdenum, ammonium, orthophosphate, silica and chloride.

i The varying levels of saline concentration and the mineral content of the feed water as well as the operational variables of the electrochemical reactor, preferably the different flow rates, flow regimes, -paths, and -rates of recycle, currents and potential differences, may be adjustable so as to produce anolyte and catholyte with suitable physical and chemical characteristics, with specific conductivity, redox potential and pH, concentration i of "activated species" and other characteristics, for particular applications.

According to a second aspect of the invention there is provided an electro-chemically activated oxidant aqueous product, characterised in being produced by a method substantially as hereinbefore defined. Specific embodiment of the invention

The invention will now be further described and exemplified in light of the following.

Experiments were conducted using laboratory and field isolates of proven antimicrobial J resistant microbial organisms. The resistant microbes were exposed to the aqueous electro-chemically activated oxidant solution in accordance with internationally standardised and accepted antimicrobial assessment protocols.

Anolyte production for antibacterial effect I Anolyte was produced at 5.0 Amps. The pH was adjusted to 7.0 (within a 0.02 limitation) so that the pH itself would not be responsible for any bactericidal effect. The ORP or oxdation-reduction potential readings (since not all tests could be done on the same day) ranged between + 770 and + 810 mV. The anolyte was used within 2 hours.

i Anolyte was decanted into sterile disposable plastic test tubes in 1.8 ml (1800 μl) quantities and 200μl of culture (1 + 9 = 1/10 dilution) was added. A baseline control tube was included with saline in the place of anolyte to determine the actual number of organisms being exposed to the anolyte. All tests were done in triplicate.

Anolyte, which is basically the positively-charged portion of a saline solution that has been split into its ionic components and which remains stable for several days, fulfils all of the above criteria. Because of its small molecular size, it enters the cell regardless of changes in the outer membrane. It acts so rapidly that there is no time for the cell to excrete it or degrade it before the lethal effect occurs. There is also no subsequent division of the bacterial cell, so that selective growth of resistant mutants cannot occurr. The advantage of keeping the bacterial load (and consequently the amount of DNA) low reduces the chance of mutations eg due to transformation.

Aims and objectives of the study Anolyte has been shown to be rapidly bactericidal to a large number of organisms including Mycobacterium spp. No resistance to the product is able to develop due to the rapid death of the organism. In other words, selective growth of resistant mutants cannot occur as no futher division of the cell takes place, lysis being so rapid due to outpouring of the internal contents of the cell as a result of efflux because of ion imbalances. This phenomenon has been shown in time lapse photography by a group in Birmingham, UK (unpublished data).

Materials and Methods

Source of bacterial strains

Strains of multiresistant organisms were obtained mainly from the Microbiology Laboratory of the NHLS, at the Chris Hani Baragwanath Hospital in Gauteng. As we were unable to obtain a vancomycin-resistant Enterococcus from CHB Hospital during the study, we obtained VRE strains from the ATCC (American Type Culture Collection). A ward isolate of a P. aeruginosa which was not especially resistant but which was of an antibiogram type frequently isolated in the wards was included.

The organisms tested included: of 5 strains of Acinetobacter baumanii, 4 strains of Escherichia coli, 1 strain of P. aeruginosa, 1 strain of Enterobacter species, 2 serotypes of Salmonellas (1 unspeciated, 1 S.isangi), 5 strains of Klebsiella species, 6 strains of S. aureus, 2 strains of Ent faecium and 1 strain of Ent faecalis. These are all types frequently encountered in the wards and considered to be of particular relevance in the paediatric ward. Most isolates were obtained from specimens received from respiratory ICU patients.

The Gram negative bacilli included extended spectrum B-lactamase producers, which simultaneously had aminoglycoside resistance. Salmonellas with ESBL's have been isolated from this hospital, often belonging to the S.isangi serotype. MRSA's were also included, mainly because Staphylococci produce catalase and SOD (superoxide dismutase) in a relatively large quantity, compared to certain other organisms, which may have affected the anolyte as it contains some hydrogen peroxide radicles.

)

Halogen-based disinfectants such as hypochlorite are known to have poor performance in the presence of organic soiling. The effect of adding 1.0 % horse serum to anolyte was therefore assessed. Jik household bleach, a well-known hypochlorite product, was made up to the manufacturer's designated working solution strength and used as a "like product" J control.

Test conditions, Exposure time and Neutralisation

Anolyte was diluted in standard hard water obtained from the NHLS (National Health Laboratory Services). Cultures (which had been maintained in semisolid medium and i subcultured just prior to testing) were prepared to give an approximate 10 million orgs. per ml concentration by observing the opacity and comparing it with prior test work. A 0.5 ml

(500 :l) aliquot was added to 4.5 ml (4,500 :l) of disinfectant solution- ie a 1/10 dilution,

giving an effective 1 million orgs/ml. Jik (hypochorite) was used as "like product" control. A viability count control was included, by omitting the disinfectant and the actual number of organisms/ml determined from this. The test organisms were exposed for 5 minutes to the solutions. Where 1.0 % horse serum was included, the serum was added first and rapidly dispersed evenly, just prior to the organisms being placed in the test solutions. This is because the Anolyte acts within seconds and would have caused an error in estimating the biocidal effect if the serum had i been added afterwards.

On completion of the exposure period, the tubes were inverted 3 times to ensure homogenous distribution of the organism. Thereafter, 0.5 ml was removed and added to 1.0 % sodium thiosulphate to neutralise the disinfectant. After mixing, 1.0 ml aliquots were removed and then spread on the surface of Mueller-Hinton agar plates with a sterile metal spreader. The plates were then allowed to dry for 1 hour.

All plates were incubated at 37°C for 24 and 48 hours. Plates with discrete colonies (i.e. the counts were not so high as to be inaccurate) were counted. All tests were carried out in triplicate and the mean of the colony counts recorded.

Results

The results of the various tests are given in Tables 1 - 3.

As can be seen in Table 1 below, when using anolyte in unsoiled conditions, within 5 minutes all organisms were eradicated. Providing the anolyte is undiluted, even the presence of 1.0 % horse serum did not decrease efficacy. This fact is borne out by the ORP study where undiluted anolyte did not show a marked effect on exposure to the bio- soiling. (See Table 2) The SABS Standard Disinfection Test method advocates an efficiency limit of 99.9% (9) or a 3-log reduction. Even the diluted anolyte (1/10) with added 1.0 % horse serum achieved this in all but one of the cultures tested, with an Entfaecalis just missing the limit. However, only 1 isolate was tested of this organism. The results may be more favourable in an extended study.

Regarding the ORP study, the addition of 1.0 % horse serum did not rapidly decrease the ORP reading. However 10 % horse serum caused a marked drop (Table 2). Rotary agitation of the beaker, in comparison to stationary anolyte, caused a rapid drop in the ORP in levels of bio-soiling above 1.0 %. This was apparent when only 1.5% horse serum was used. For this reason 10 % horse serum was not tested as it was obvious that the ORP drop would be rapid.

TABLE I:

AVERAGES OF ALL ISOLATES OF EACH GROUP OF ORGANISMS TESTED: PERCENTAGE KILL AFTER VARIOUS 5 MINUTE TREATMENTS OF MDR ORGANISMS

NOTE: ANOLYTE WOULD NORMALLY BE USED UNDILUTED. THE DILUTIONS ASSESSED HERE WERE DONE TO TESTS LIMITS OF EFFICACY ONLY.

MDR (MULTI-DRUG RESISTANT) ORGANISMS LISTS TABLE 2: GRAM NEGATIVES

MDR (MULTI-DRUG RESISTANT) ORGANISMS LIST- all CHB /BARA unless stated.

TABLE 3:

GRAM POSITIVES

Pre- and Post bio-load addition on biocidal effect:

1. The E.coli count dropped from 7.5 x 10⁵ orgs./ml to 0 .0 orgs. /ml whether undiluted Anolyte was added before or after addition of 1.0 % serum + bacteria to the beaker.

2. The E.coli count dropped from 7.5 x 10⁵ orgs./ml to 6.08 x 10³ orgs./ml when addition of 1.0 % serum + bacteria to the beaker was followed by adding 1/10 Anolyte 3. The E.coli count dropped from 7.5 x 10⁵ orgs./ml to 7.2 x 10³ orgs./ml when 1.0 % serum + bacteria was added to the 1/10 Anolyte already in the beaker. Thus, the biocidal effect was a 2-log drop with the 1 /10 Anolyte and a 5-log minimum drop with the undiluted product.

Discussion i Organisms such as VRE's (vancomycin resistant enterococci) are opportunistic pathogens that have become problematic in the USA (2) and elsewhere. In the USA, during the 1990's Gram-positive MDR infections with VRE's and MRSAs increased in prevalence as agents of nosocomial infections (3). It is believed that this resistance may have evolved due to cattle being treated with vancomycin analogues prophylactically.

)

The use of expensive antibiotics, not to mention the cost of prior failed therapy, has focussed more attention on infection contro of nosocomial pathogens. Methicillin resistant S. aureus has been around two decades in South Africa (4) and our strains of Klebsiella (especially pneumoniae) are resistant to most antibiotics, producing a variety of aminoglycoside-modifying enzymes and extended spectrum beta-lactamases.

These results show that anolyte is a comparatively safe, effective alternative to routinely used disinfectants. The product requires a small device for production of limited quantities of anolyte, and even the larger volumes required to supply a moderately sized hospital need a device that is less than 3 cubic meters. The raw materials (salt and water) are cheap and obtainable anywhere. There is no costly biohazdardous waste to dispose of.

It will be appreciated that many variations in detail are possible without departing from the scope or spirit of the invention as defined in the claims.

Claims

1. A method for the management and/or treatment of microbes characterised in having acquired microbial resistance to conventional microcidal and/or bactericidal preparations, the method including the step of electrochemically activating a dilute aqueous saline solution such that the solution includes separable and both of an aqueous, mixed oxidant, predominantly anion-containing solution and an aqueous, mixed reductant, predominantly cation-containing solution; and applying the electrochemically activated, bactericidal aqueous solution to the specific environment where the microbes are present and where microbial decontamination is required.

2. The method as claimed in claim 1 wherein the aqueous saline solution is electrochemically activated so as to produce three categories of products, namely relatively stable products; highly active unstable products, including free radicals and other active ion species; and quasi-stable structures formed at or near an electrode surface and including free structural complexes of hydrated membranes around ions, molecules, radicals and atoms.

3. The method as claimed in claims 1 and 2 wherein the three categories of products are present in both the aqueous, mixed oxidant, predominantly anion-containing solution and the aqueous, mixed reductant, predominantly cation-containing solution produced by the process.

4. The method as claimed in claim 1 wherein the electrochemically activated, aqueous solution is selected from a group consisting of an anion-containing solution, a cation-containing solution, and a mixture of an anion-containing solution and a cation-containing solution.

5. The method as claimed in claim 4 wherein the anion-containing solution is prepared from an anion-containing solution, a cation-containing solution or a mixture of an anion-containing solution and a cation-containing solution; and wherein the cation- containing solution is prepared from an anion-containing solution, a cation- containing solution or a mixture of an anion-containing solution and a cation- containing solution.

6. The method as claimed in claim 1 wherein the electrochemically activated, bactericidal aqueous solution is prepared by means of electrolysis of an aqueous solution of a salt, electrolysed to produce mixed reductant and mixed oxidant species.

7 A method as claimed in claim 6 wherein the mixed oxidant and reductant species are labile and, after about 96 hours, disappear with relatively no residue being produced.

8. The method as claimed in claim 6 wherein the salt is selected from a group consisting of alkali and alkali earth metal chlorides; -carbonates; -bi-carbonates; - phosphates; -sulphates or -nitrates, for example sodium chloride; non-iodated sodium chloride; potassium chloride; calcium chloride; sodium carbonate; - bicarbonate and -phosphate; potassium carbonate; -bicarbonate and - phosphate; calcium phosphate; sodium-; -potassium; and calcium nitrate; and a mixture of at least two of the aforementioned salts.

9. The method as claimed in claim 6 wherein the aqueous salt solution has a concentration of between 0.0001% to 10% and, for specific systems without pre- dilution of the feed, preferably a concentration of between about 0.05 to 5%.

10. The method as claimed in claim 1 wherein the anion-containing solution and the cation-containing solution are produced by an electrochemical reactor or so-called electrolysis device, having a through flow electrochemical cell with two co-axial cylindrical electrodes, with a tubular diaphragm located co-axially between the two electrodes so as to separate an annular inter-electrode space into a co-axial, annular catholytic and an annular anolytic chamber arrangement.

11. The method as claimed in claim 10 wherein the electrochemical cell has a relatively small, annular, cross-sectional total open area for fluid flow so as to cause turbulent fluid flow there through for ensuring maximum exposure of the solutions to the electric field.

12. The method as claimed in claim 10 wherein the anolyte and catholyte are produced in the electrochemical cell under influence of a relatively low current, preferably of about 5A to 7A, and a relatively high voltage, preferably of about 6V to 48V, and more preferably between 8V and 24V, so as to provide a relatively high voltage gradient or electric field intensity at the interface between the electrode surface and the electrolyte.

13. The method as claimed in claim 1 wherein the anion-containing solution has a redox potential preferably of between +300mV and +1 200mV, and a pH preferably of between 2 and 8, and more preferably 7; and wherein the cation-containing solution has a pH preferably of between 7 and 13, and more preferably about 11.5, and a redox potential preferably of between -200mV and -1100mV, and more preferably about -800mV.

14. The method as claimed in claim 1 wherein the anion-containing solution includes mixed oxidant species selected from a group including CIO; CIO^"; HCIO; OH^"; H0₂ ^"; H₂0₂; 0₃; S₂0₈ ^2" and Cl₂0₆ ^2".

15. The method as claimed in claim 1 wherein the cation-containing solution includes mixed reductant species selected from a group including OH^"; H₃ ^"; 0₂; H₂; H0₂ ^'; H0₂ ^"and 0₂ ^".

16. The method as claimed in claim 1 wherein the anion-containing solution includes organic radicals and other components selected from a group including Cl₂, HCIO,

CIO^", CIO^', Cϊ, H0₂, H0₂\ 0₂, HO^', 0₃, 0₂\ 30₂, 0\ H₃0⁺, Cl , H\ H₂0₂, Cl₂0, CI0₂ ^",

HCI, Cl₂0₇, S₂0₈ ^2" C₂0₆ ^2", HCIO, H₂S0₄, and HS0₃CI.

17. The method as claimed in claim 1 wherein the cation-containing solution includes organic radicals and other components selected from a group including HO^", H₃0₂ ^",

0₂ ^", H0₂ ^", H₂0₂, H₂, HO, H₂ ^", NaOH, KOH, Ca(OH)₂ and Mg(OH)₂.

18. The method as claimed in claim 1 wherein the physical and chemical characteristics, including specific conductivity, redox potential and pH, concentration of activated species and other characteristics of the anolyte and catholyte are adjusted for particular applications by varying levels of saline concentration and the mineral content of the feed water, as well as the operational variables of the electrochemical reactor, including flow rates, flow regimes, -paths and -rates of recycle, currents and potential differences.

19. The use of an electrochemically activated, dilute aqueous saline solution in the management and/or treatment of microbes characterised in having acquired microbial resistance to conventional microcidal and/or bactericidal preparations, wherein the electrochemically activated dilute aqueous saline solution includes separable and both of an aqueous, mixed oxidant, predominantly anion-containing solution and an aqueous, mixed reductant, predominantly cation-containing solution.

20. An electrochemically activated dilute aqueous saline solution including separable and both of an aqueous, mixed oxidant, predominantly anion-containing solution and an aqueous, mixed reductant, predominantly cation-containing solution, and suitable for use in the management and/or treatment of microbes characterised in having acquired microbial resistance to conventional microcidal and/or bactericidal preparations .