Published in Disinfection Practice magazine, №1, 2003
V. M. Bakhir, V.I.Vtorenko, B.I.Leonov, S.A.Panicheva, V.I.Prilutsky, N.Y.Shomovskaya
The Russian Scientific & Research Institute for Medical Engineering (VNIIIMT MZ RF)
Nowadays a range of disinfectants is essentially increased. There are over four hundred biocidal agents for pre-sterilizing treatment, sanitation and all-levels disinfection used in Russia. However, appreciating results of man’s fight against microbes it is easy to see that the odds are not in man’s favor. An amount of cultures of pathogens resistant to liquid chemical germicides is increased. Recurrent planned replacements of one biocidal agent to another do not solve a problem of nosocomial infection control, but only keep wavering balance that could be upset by an accidental factor.
Official program documents that determined the strategy for nosocomial infection control and the way of disinfection business development are based on analysis results of statistical data from application one or another biocidal agent and reflect offers of chemical’s market practically exerting nothing influence on it.
Absence of united scientific conception of fight against microbes based upon fundamental laws of biology, modern progress in physics, chemistry and other sciences does not live any hope for some considerable progress in sanitary epidemiological protection of population at least at present time.
It can be corroborate by following examples. In conception of preventive measures on nosocomial infection control  approved by First Deputy Surgeon General of Russian Federation that the most perspective class of liquid chemical germicides for different surfaces disinfection in clinics and hospitals are cationic surfactants – quarternary ammonium compounds and some other organic substances. It is claimed there that substances may be used in presence of patients because these substances are not dangerous. Besides cationic surfactants it is also recommended to use aldehydes and alcohols for disinfection and pre-sterilization.
However, three years later it was stressed in the report  that all disinfectants’ disparity to modern requirements for biocidal efficacy and hygienic safety. Cationic surfactants are stable at good detergent power at the same time are inactive or have low-activity to such unsusceptible forms of pathogens as mycobacteria Tuberculosis, funguses, spores and bacillus. One of cationic surfactants disadvantage is fast and frequent adaptation of microbes to their effect. Liquid concentrated cationic surfactants possess resorptive and irritating influence on skin and eyes mucous membrane and frequently act as allergens. A high level of toxicity and sorptive power of aldehydes prevents from a wide recommendation of its use for surface treatment, linens, and food utensils treatment.
A high toxicity of glutaral aldehyde is well known . That is why its application has been forbidden in England since May, 2002 . However, disinfectants based on it are still advertised and on sale in Russia.
Ideal (perspective) disinfectants should posses high bactericidal activity, long shelf life, be ready for use without any preliminary activation and utilized after use without negative effect to the environment. Long storing of stable chemicals is available, but their utilization requires equivalent spending of another agent or energy. Therefore, combination of stability and easy utilization is impossible.
As for the demand to exclude the preliminary activation stage before usage of “ideal” liquid chemical germicide, it ought to take into consideration that all variety of biocidal agents are belong to few classes of chemicals well-known for tens of years. So appearance of new class of chemicals, which will meet the requirement, is unlikely. Modern tendency in developing new disinfectants is in search for activation means of known disinfectants, and not the creation of new ones. For example, up till recently, 6% hydrogen peroxide had been used for purposes of sterilization and high-level disinfection. Now the technology of sterilization/high-level disinfection with hydrogen peroxide plasma is created to decrease its corrosion activity and increase its biocidal activity . Thus, liquid chemical germicides activation is directed to development of conditions under which minimal concentration of active ingredients provides a high biocidal effect at minimal corrosion, destructive activity and toxicity. Exposure time, concentration, temperature are the most important characteristics of disinfection process and are major parameters of any practical methodic.
Addition of activators, synergists, using an extra physical influence, i.e. creating conditions converting active ingredients into metastable state at the moment of disinfection, is one of main directions for the disinfection efficacy improvement in Russia .
Let us consider the processes accompanying application of stable organic solution for room disinfection’s treatment on the example of compounds having membrane-attacking mechanism of pathogens. Cationic surfactants, phenols, iodophors and some others are related to mentioned compounds.
Due to complexity and multi-functionality of microorganisms’ membrane specific interaction between membrane’s biopolymers and abovementioned chemicals is hardly studied at all.
Cytoplasmic membrane is extremely vitally important structure of any cells including microbes. Organic compounds are part of it and have many reactive groups that cause a high sensitiveness of membrane to damaging factors of different nature. It is known that high concentration of membrane-attacking agents destroy biopolymers of membrane, resulting in damaging lysis of microbe’s cell. The same chemicals in small doses affect membrane functions – change osmotic pressure, permeability, transport processes of molecules and ions through membrane, inhibit metabolic processes, bio-oxidation and cell divisions.
Cationic surfactants (quarternary ammonium compounds) are concentrated at membrane and bind with phosphatidic groups of its lipids; anionic surfactants such as alkaline detergents, alkyl- and arylsulfones, iodophors react with membrane lipids. Phenols and alcohols dissolve lipid’s fragments of membrane.
After disinfection treatment is completed, the moist surfaces get dry, so organic compounds are concentrated in a volume of porous material and turn into superfine and invisible to the eye film. Then it evaporates by sublimation with less intensity than under evaporation during wet treatment. Formed aerosol frequently has no smell that creates illusions of its harmlessness. One should take into account that in accordance with known physical laws each liter of the air in the room contains about some milliards of molecules of matter vaporized with natural course or due to sublimation even if its concentration could be hardly measured and does not exceed hundreds or thousands parts from maximum permissible concentration (MPC). During breathing as well as through the skin and mucous membrane such molecules penetrate to human organism (patients or medical staff) and each one of it keeps realizing its main function – suppression of vital function of cells, but this time in a human body. Stability of liquid chemical germicides creates their accumulation in organism followed by migration through digestive cycle.
Colonies of microorganisms form resistance to dry inefficient disinfectant and start using it as a nutrient medium. Processes as described above have recently become an object of attention; so it is in a stage of study now [7, 8].
It is quite evident that the development of new liquid chemical germicides which allow bacteria to develop resistance in a short period of time, creates conditions for improvement of mutability mechanism of pathogens and initiates appearance of new isolates of microorganisms. It is particularly dangerous because systematical investigations are seldom carried out in hospitals, therefore it is impossible in the most cases to determine during what time after beginning of use new disinfectant, microorganisms form resistance and adaptive reactions.
Today by efficacy of disinfectants is implied its spectrum of biocidal activity. Efficacy also relates to exposure time required for disinfection. However, taking a broad view on the subject we should say that disinfectant is effective only in the case it has a broad spectrum of biocidal activity and does not stimulate microorganism’s adaptation during a long-term use. In other words, effective disinfectant must be used for years with certainty that microorganisms could not form adaptation to it for principal reasons.
Different means and technologies of disinfection that meet requirements mentioned above are known and widely used. For instance, these are penetration of electromagnetic radiation (X-rays, gamma rays), ultraviolet irradiation, treatment by ionized plasma, and, in conclusion, thermal methods of microorganism’s destruction. Case of cold high-level disinfection/sterilization by liquid, it is caused by metastable state of dissolved disinfectant.
Let us consider a mechanism of antibacterial defense created by nature and functions in internal environment of life organisms – from unicellular organisms up to human – over million of years and without any fail.
It is proved  that leading role in bactericidal effect of neutrophils belongs to hypochlorous acid (HClO) made by phagocytes. Under respiratory burst about 28% of oxygen used by neutrophils is spent for formation of HClO. HClO is generated from hydrogen peroxide and chloride-ions in neutrophils. Catalyst of this reaction is myeloperoxidase (MPO):
H2O2 + Cl [Cat (МPО)] HClO + OH [9, 10].
Hypochlorous acid dissociates in aqueous media with formation of hypochlorite-anion and hydrogen-ion:
HClO ClO + Н+.
Concentrations of HClO and hypochlorite-anions ClO are almost equal at neutral pH. A decrease in pH shifts reaction balance towards to HClO, and an increase of pH raises concentration of hypochlorite-anions.
A formation of H2O2 and HClO in a short time (fractions of a second) in a little volume of aqueous media (parts of microliter, in a volume of active zone of phagocytosis) – inevitably must be followed by reactions of spontaneous decomposition and interaction of reaction products with formation of active particles similar to once formed by radiolysis or electrolysis of water.
Spontaneous decomposition of hydrogen peroxide in aqueous media is followed by formation of highly active biocides (in parenthesis appropriate reactions are presented):
HO2 – hydroperoxide-anion (H2O2 + OH HO2 + H2O);
О22 – peroxide-anion (OH + HO2 O22 + H2O);
О2 – superoxide-anion (O22 + H2O2 O2 + OH + OH );
НО2 – hydrogen peroxide radical (НO + H2O2 H2O + HO2);
HO2 – hydrogen super-oxide (O2 + H2O HO2 + OH).
At the same time it is possible the formation of extremely reactive singlet oxygen 1О2 : (ClO + H2O2 1О2 + H2O + Cl ). Participation of molecular oxygen ion-radical О2 in reactions of phagocytosis is determined experimentally [10,11]. One of the described above could be the way of its formation.
Formation of free radicals СlO, Сl, НО is possible in aqueous media in presence of НСlО and СlO
HClO + ClO ClO + Cl + НO.
By modern theory of catalytic processes, a formation of interim activated complex with myeloperoxidase as a catalyst seems also to be most possivle. A dissociation of this complex is followed by formation of О , and medium acidification:
HClO + ClO [HClO Cat (МПО) ClO ] 2Сl + 2O + Н+
Active hypochlorite radical СlO can participate in reactions of atomic oxygen (O ) and hydroxyl radical (НO ) formation:
СlO + СlO + ОН Сl + 2O + ОН.
Followed by formation of chlorine radicals:
OH + Cl Cl + OH.
Formed radicals and atomic oxygen take part in microbe’s destruction, oxidizing biopolymers, for example, by the following:
RH2 + OH RH + H2O;
A metastable mixture of compounds formed during phagocytosis is a very effective mean for microbe’s destruction due to many spontaneous realized possibilities of changing (irreversible damage) of essential functions of microorganism’s biopolymers at a level of electron transmission. Metastable particles with different values of electrochemical potential possess universal spectrum of action, i.e. they are able to damage all large systematic groups of microorganisms (bacteria, mycobacteria, viruses, funguses, spores) and without damaging of human tissues and other multicellular system organisms.
That can be explained by texture and living activities of cells of that living organisms. Cells of multicellular organisms during their life process, for example, in oxygenase’s reactions of cytochrome P-450, during phagocytosis under microbe’s adhesion and cidal action produce a range of highly efficient oxidants. These cells have a strong chemical system of antioxidant protection with preventing a toxic effect of such compounds on vitally important cellular structures. Antioxidant properties of somatic cells are related to a presence of a strong three-layered lipoprotein’s shell that contains diene conjugates (–С=С–) possessing electron-donor properties and sulfhydric groups (SH). Microorganisms do not have strong mechanisms of antioxidant protection due to absence of mentioned chemical groups.
All somatic cells of living organisms are heterotrophs: their trophism depends on availability of nutritive materials in extracellular medium – glucose, amino acids, fatty acids. Though biological well-being of any somatic cell is up to place it keeps in a process of dispensing of trophic functions of all elements of multicellular system (cell is supported by cell).
Trophic functions of multicellular organisms cells are obeyed to interchangeability law. If a trophism of single cell is disturbed, then this disturbance can be corrected by neurotrophic regulation, functions of adjoining cells, reparative processes, nutritive function of blood and so on.
All microbe’s cells are autotrophs, so their nutrition depends on their own activity, in other words if enzymatic processes in microbe’s cell are depressed, it dies since there is no compensatory mechanism. Microbial cell gets all its trophic functions by enzymatic reactions only. An interaction between microbial cells in their habitat is not a compensatory one, that is to say susceptibility of microbe is in its autonomy.
Maximum use of fundamental difference between living organisms of micro- and macro-biological life is an ideological basis of electrochemical activated biocidal solutions .
As physicochemical process electrochemical activation is an electrophysical and electrochemical influence on water that contains ions and molecules of dissolved substances in it. It takes place under conditions of minimal heat release in the area of dimensional charge at the electrode surface (anode or cathode) of electrochemical system at nonequilibrium charge transfer through the interface “electrode – electrolyte” by electrons [13, 14].
As a result of electrochemical activation water converts into metastable (activated) condition showing increased reactivity in different physical-chemical processes during some tens hours. Electrochemical activation allows directly change a composition of dissolved gases, acid-base and redox characteristics of water within the bigger scale then under the equivalent chemical regulation. Chemical reagents (oxidants or reducing agents) in metastable condition can be generated from water and dissolved substances. It is used in processes of water purification and disinfection as well as for water or diluted electrolyte solution transformation into ecologically friendly biocidal (disinfecting/sterilizing solution), cleaning, extractive and other functionally useful solutions.
Flow through electrolytic modular (FEM) is used for electrochemical transformation of water and dissolved substances. A distinctive feature of FEM is in combination of properties of ideal displacement reactor and ideal mixing reactor in one element as well as high technical and economic characteristics at processing of fresh water and low-mineralized solutions.
Anolytes A, AN, ANK are types of electrochemically solutions generated by STEL-devices. They are different from each other by physicochemical properties and biocidal activity due to different technological processes used for their generation. At present time anolyte ANK generated by STEL devices is the most perfect solution by its functional and technological properties among other electrochemically activated solutions. There are following stages of ANK generation: cathode treatment of pre-mixed brine, during which pH value rises and electrolyte is saturated with dissolved hydrogen; removing hydrogen and hydroxides of heavy metals formed by cations and hydroxide anions; generation of hydro- peroxide and oxygen- chlorine- released type oxidants by anode treatment. Some reactions of anolyte ANK generating process are presented below.
Reactions of the first stage:
2H2O + 2Na+ + 2e 2NaOH + H2 (sodium hydroxide formation);
2H2O + 2e H2 + 2OH (formation of free anions of hydroxyl at current density over 500 А/m2; reaction rate raises when concentration of sodium chloride decrease);
О2 + Н2О + 2е НО2 + ОН- (formation of peroxide anion by dissolved in water oxygen);
О2 + 2Н2 + 2е Н2О2 + 2ОН (formation of hydrogen peroxide by dissolved in water oxygen).
Reactions of the last stage of anolyte ANK generation:
2Cl - 2e Cl2 (formation of molecular chlorine which immediately reacts with components of near-electrode medium):
Cl2 + H2O HClO + HCl;
HCl + NaOH NaCl + H2O;
2H2O - 4e 4H+ + O2 (formation of oxygen and ions of oxony);
OH + Cl - 2e HClO (direct synthesis of hypochlorous acid);
Cl + 2OH - 2e ClO + H2O (direct synthesis of hypochlorite anion);
3OH - 2e HO2 + H2O (formation of peroxide anion); HO2- " e ® HO2 (formation of hydrogen superoxide);
HO2 - e HO2 (formation of hydroxyl radical);
Cl + 4 OH - 5 e ClO2 + 2H2O (formation of chlorine dioxide);
O2 + 2 OH - 3 e O3 + H2O (ozone formation);
H2O - 2e 2 H+ + O (atomic oxygen formation).
Besides, in a volume of anolyte ANK during processes of relaxation reactions proceed; as a result of it other biocide substances are generated, in particular, singlet molecular oxygen (1О2), molecular anion-radical of ozone (O3 ), hypochlorite-radical (ClO ), atomic chlorine (Cl ), chlorite-anion (СlO2 ) and others. A comparison of bactericidal substances being in phagocytosis and contained in anolyte ANK practically recognizes their clear identity.
Environmentally friendly electrochemically-activated anolyte ANK has “life time” that is necessary for procedure of disinfection. After its use it spontaneously degrades without formation of toxic xenobiotics and does not require any neutralization before discharging to sewerage. Anolyte ANK is activated during a period of relaxation, i.e. the time during which spontaneous change of its physicochemical characteristics, catalytic and biocatalytic activity takes place. The spontaneous change of these parameters is evidence of dissipation of excess internal energy due to dissipative processes.
A mixture of metastable active agents eliminates microbes’ ability for adaptation to bactericidal effect of anolyte ANK. A small total concentration of active oxygen and chlorine compounds guarantees for absolute safety for man and environment under long-term use of anolyte.
A chemical potential of molecules and ions in anolyte ANK is much higher than in hypochlorite solutions. A low mineralization of anolyte ANK and its hydration ability helps penetration through cell membrane, creates conditions for intensive osmotic and electroosmotic oxidant’s transfer into intracellular media. The osmotic transfer of oxidants through shells and membranes of microbe’s cells is more intensive than through membranes of somatic cells due to inherent difference in osmotic gradient of these types of cells. Electrically charged cluster structures formed by dissolved gas molecules in water and electron-active components of medium promote high-speed electroosmotic carry of oxidants into bacterial cell, because this clusters produce strong local electric fields with high heterogeneity in zones of contact with biopolymers.
Activated anolyte ANK produced by STEL-devices kills microorganisms of bacterial, viral and fungous etiology (Staphylococcus aureus, Pseudumonas aeruginosa, Escherichia coli, hepatitis B virus, poliomyelitis virus, HIV, adenovirus, pathogens of tuberculosis, salmonellosis, dermatomycosis and others). By its efficacy anolyte greatly exceeds chloramines, sodium hypochlorite and overwhelming majority of other disinfectants and sterilizing agents.
A sum of active oxygen and chlorine compounds in anolyte ANK (total oxidant content) is within 100 to 500 mg/l, that is dozens times less than in most solutions of currently used disinfectants. Anolyte ANK does not cause coagulation of protein that protects microorganisms and, due to its loosened structure, easily penetrate into pinholes of living and lifeless matter.
Anolyte ANK is generated by STEL-devices from dilute solution of sodium chloride in drinking water. Total mineralization of initial solution for anolyte ANK eneration is within 1,5 to 5,0 g/l. That prevents from anolyte active ingredients accumulation in porous materials after it used and dried up.
Useful properties of anolyte ANK such as its efficacy, safety and economy are confirmed in full measure by many hospitals. In some of them (for instance, in Moscow City Hospitals № 15 and № 52) anolyte ANK is practically the only disinfectant in last few years. A position of anolyte in disinfectants structure of the hospitals exceeds 97 %, and its annual consumption is about 1000 tons per hospital [15, 16].
Different disinfectants had been used in the hospitals before application of anolyte ANK. Many of that disinfectants corroded metals, dimnessed glass instruments, damaged synthetic materials, developed skin allergic reactions and headache to medical personnel. More over, it was necessary to train medical personnel to prepare of working solutions of each disinfectant and to provide them with personal protection (respirator, shoes, eye protectors, rubber gloves, apron).
Hospitals were spending considerable amount of money for buying disinfectants, detergents, antiseptics, tanks for their storage, special clothes and protection. It required periodical renewal of drains disabled because of aggressive solutions discharge. Taking into account limited efficiency of disinfectants, particularly for nosocomial infection control, and possibility of incorrect preparation of applied disinfectants, microbiological laboratories of these hospitals had to make primary and secondary bacteriological control on disinfection quality by taking 500 – 700 tests each month. It was spent a lot of test agents for control of parameters of prepared disinfectants.
Application of anolyte ANK generated by STEL devices eliminated mentioned above negative aspects from using traditional chemicals resulted in following beneficial effects: bacteriological contamination of treated objects was considerably reduced, a number of negative test results decreased on about 85 – 95 %, which allowed to cut down monthly bacteriological routine tests up to 200 – 300. Besides, secondary infecting rate by hepatitis “B” among patients have been reduced from 0,45 – 0,50% to 0,06 to 0,08 % which shows an improvement in quality of disinfection and nosocomial infection control.
Anolyte ANK is used in the following hospitals’ departments: surgery and purulent surgery departments, therapeutic department, department of transfusiology, gynecology department, department of proctology, pathology department, diagnostic centers, waiting rooms, rehabilitation centers, intensive care, traumatologic, clinical-diagnostic laboratory, opthalmology, vascular surgery, cardio-surgical, cardiovascular departments, neurology unit, operative, anesthetic departments, unit of endoscops, physiotherapy, dental departments, department of functional diagnostics, of ultrasonic and X-Ray diagnostics, and also in central sterilization department (Moscow City Hospital №15), in puerperal hospital with obstetric division, departments of pathology of pregnant, of newborns, operative and maternity rooms.
STEL devices in hospitals are compact, easy in operation, ecologically safe and could be installed in any room. They do not require special long training of medical personnel for work with. Their operation doesn’t require any special protective cloth. Amount of generated anolyte meets the required amount of departments and could be increased by request from medical personnel in compliance with epidemiological situation. It is possible to generate anolyte ANK with different concentration of oxidants . Testing of oxidants concentration in anolyte ANK is conducted by using test-strips for express analyses or by iodometric titration for more accuracy. Neutral anolyte ANK is easy-to-use and readily used in hospitals. Contrary to other disinfectants, it can be applied as a multifunctional solution for all-levels disinfection, as well as cleaning and sterilization simultaneously.
Resume. For effective sanitary-and-epidemiological protection of population, it is necessary to work out unified scientific conception of fight against microbes based on fundamental laws of biology, current achievements in physics, chemistry and another sciences that includes the following principals:
1. Besides data on antimicrobial activity and exposure time, an estimation on efficacy of liquid chemical germicides must also include data concerned with microorganisms ability to develop a resistance to the given disinfectant or sterilizing agent.
2. A creation of new effective and safe for human liquid chemical germicides must be based on fundamental differences between micro- and macro-organisms, by copying or modeling protection mechanisms used by cells of multicellular organisms.
3. The main criteria for safety of liquid chemical germicides must be defined whether active agents of given biocide are xenobiotics. Consumer should be informed by a special label with information on xenobiotic’s presence.