WO2012037678A1

WO2012037678A1 - Process for sterilization of air spaces and surfaces using advanced oxidation stress

Info

Publication number: WO2012037678A1
Application number: PCT/CA2011/050576
Authority: WO
Inventors: Michael Edward Shannon; Arkady Mandel; Dick Eric Zoutman
Original assignee: Medizone International Inc
Current assignee: Medizone International Inc
Priority date: 2010-09-20
Filing date: 2011-09-20
Publication date: 2012-03-29
Anticipated expiration: 2013-03-20

Abstract

Bacteria, for example the superbugs, Clostridium difficile (C. difficile); E coli; Pseudomonas aeruginosa; methicillin-resistant Staphylococcus aureus (MSRA); and vanomycin-resistant Enterococcus (VRE); food-infecting bacteria such as Listeria monocytogenes or Salmonella enteridis; Bacillus bacteria; or bed bugs, their eggs and larvae, are deactivated or killed by a process which includes exposing them to the oxidative action of a highly reactive polyoxide such as trioxidane or the oxidative free radicals and species derived from it. The process can involve treating a bacterially contaminated room such as a hospital ward with a polyoxide or oxidative free radicals which can be formed by mixing ozone and hydrogen peroxide. Physical agitation of carpet and drapery surfaces can also be carried out in the room subjected to the atmosphere.

Description

PROCESS FOR STERILIZATION OF AIR SPACES AND SURFACES

USING ADVANCED OXIDATION STRESS

FIELD OF THE INVENTION

This invention relates to disinfecting systems for use in healthcare facilities, public health facilities, food handling facilities, sports facilities and the like, to eliminate or at least to reduce to acceptable levels, microbial residues and bed bug infestations which are resistant to conventional disinfectant and sterilization systems.

BACKGROUND OF THE INVENTION

Despite intensive preventive efforts over the past several years in hospital and other healthcare facilities, the incidence of life threatening infections caused by a growing array of antibiotic resistant bacteria (sometimes referred to as "superbugs") has grown significantly and is now posing a serious problem for medical staff world wide. According to an editorial in the journal "Science" (July 2008), the number of deaths in 2006 attributable to bacterial infections in healthcare facilities in the United States exceeded the U.S. death toll attributed to HIV/AIDS in the same year, and probably result in as many as 70,000 deaths per year in the United States. This is despite the best efforts of healthcare personnel properly to clean their facilities and the equipment contained therein.

The major causative agents (bacteria) for hospital-based infections (nocosomial infections) are Clostridium difficile (C. difficile); E. coli; Pseudomonas aeruginosa; methicillin-resistant Staphylococcus aureus (MRSA); and vancomycin-resistant Enterococcus (VRE).

Approximately 5% of all acute care hospitalizations in the U.S. develop a nosocomial infection with an incidence rate of five infections per thousand patient days, and an added expenditure in excess of $4.5 billion (Wentzel R, Edmond M D, "The Impact of Hospital Acquired Blood Stream Infections," Emerg. Inf. Dis. Mar-April 2001 :7(174)). When this rate is applied to the 35 million patients admitted to 7,000 acute-care institutions in the U.S., it is estimated that there are more than 2 million cases per year. Nocosomial infections are estimated to double, at least, the mortality and morbidity risks of any admitted patient.

The significant, and growing, incidence of antibiotic resistant bacteria in healthcare facilities has been termed by some as a "Silent Epidemic". On the international scene, a World Health Organization survey of 55 hospitals in 14 countries representing four WHO regions (Europe, Eastern Mediterranean, South-East Asia and Western Pacific) reported that an average of 8.7% of hospital patients had nocosomial infections. The WHO estimates that, at any time, over 1 .4 million people worldwide suffer from infection acquired in hospital.

Of particular concern in this context are the bacteria C difficile and

MRSA. Until recently, C difficile was relatively uncommon, but has now become epidemic in many regions of the world. Indeed, it is now recognized by a growing number of public health officials as a worldwide epidemic (pandemic) with incalculable financial and health implications. MRSA has been identified by the American Academy of Orthopaedic Surgeons as the single biggest concern for surgical procedures, and concurs with recent journal articles that it constitutes a "silent epidemic." Under current healthcare facility cleaning and sterilizing procedures, both C difficile and MRSA, as well as the aforementioned E. coli; Pseudomonas aeruginosa; and vancomycin-resistant Enterococcus (VRE), are ineffectively treated and subsequently removed, so that colonies of these pathogens accumulate in healthcare facilities, especially on porous surfaces such as carpets and drapes.

This invention also relates to bacterial disinfection treatments for food handling premises such as food processing rooms, meat packing plants, food packaging rooms, kitchens and the like, for disinfecting food handling premises of human-harmful, food poisoning-causing bacteria including Listeria species bacteria such as Listeria monocytogenes and Salmonella species such as S.typhium, causative agents of food poisoning in humans and animals.

Listeria is a genus of Gram-positive bacteria of the bacilli class. It contains six species, typified by L. monocytogenes, the causative agent of listeriosis, an uncommon but potentially lethal food-borne infection. L. monocytogenes is one of the most virulent food-borne pathogens. Listeriosis has been reported to be the leading cause of death among food-borne bacterial pathogens, responsible for about 2,500 illnesses and 500 deaths annually in the United States.

L. monocytogenes is commonly found in soil, stream water, sewage, plants and food. Vegetables can become contaminated with L. monocytogenes from the soil. Uncooked meats, unpasteurized milk, products made from unpasteurized milk such as certain cheeses, and processed foods commonly contain Listeria. Sufficient heating and cooking will kill Listeria, but contamination of food products can occur after cooking and before packaging. Meat processing plants, for example, producing ready-to-eat products such as deli meats and hot dogs, follow extensive sanitation policies to guard against listeria contamination.

Outbreaks of Listeria have reportedly been caused by hot dogs, deli meats, raw milk, soft-ripened cheeses, raw and cooked poultry, raw meats, ice cream, raw vegetables and raw and smoked fish. Pregnant women, the elderly and those with compromised immune systems are the most vulnerable patients. In its early stages Listeria infection is effectively treated with antibiotics such as ampicillin, ciprofloxacin and azithromycin, but it is commonly not recognized until a more advanced stage is reached. Prevention of such infections is accordingly of high importance.

Salmonella is a large genus of bacteria, many species of which can cause disease if ingested by humans. Salmonella bacteria infections are commonly termed "Salmonellosis" and are manifested by diarrhea, vomiting, fever and abdominal cramps (food poisoning). Among the human harmful Salmonella species are S. enteridis and its sub-species, S. bongori and S. typhi, the human pathogen of typhoid fever.

Attempts to combat and kill nosocomial infections caused by bacteria such as Pseudomonas aeroginosa and Staphylococcus aureus, and food handling facility infections caused by Listeria and Stapphylococcus infections are hampered by the fact that the bacteria grow within biofilms that protect them from adverse environmental factors. A biofilm is an aggregate of microorganisms in which cells adhere to each other and/or to a surface. They are frequently embedded in a self produced matrix of extracellular polymeric substance (EPS), a polymeric conglomeration generally composed of extracellular DNA, proteins and polysaccharides. Biofilms form on surfaces in the presence of water vapour.

Free floating microorganisms in planktonic (single cell) mode attach to a surface, and if not immediately removed, will anchor themselves more permanently to the surface. These first colonists provide more diverse adhesion sites for the arrival of other cells, thus beginning to build a matrix that holds the biofilm together and provides additional anchoring sites for arriving cells. The biofilm grows through a combination of cell division and recruitment. When the biofilm is established, the aggregate cell colonies are apparently increasingly antibiotic resistant. It has also been reported that biofilm bacteria used in bioterrorism can defend themselves against disinfectants and antibiotics (see "Biofilm Bacteria Protect Themselves With Chemical Weapons", Dr. Carsten Matz et al., Helmholtz Cetre for Infection Research, Brauschweig, reported on lnforniac.com, July 23, 2008).

Bacteria living in a biofilm have significantly differently properties from the planktonic form of the same species, as the dense and protected environment of the film allows them to co-operate and interact in various ways. Traditional antibiotic therapy is usually not sufficient to eradicate chronic infections, and one major reason for their persistence seems to be the capability of the bacteria to grow within biofilms that protect them from adverse environmental factors.

Also of growing concern are threatened bioterrorist and warfare attacks using potentially lethal bacteria. Some of the deadliest bacteria, for example anthrax, are highly resistant to conventional sterilization agents and treatments. Contamination of public facilities with such bacteria constitutes a significant threat to human life with residual amounts of such bacteria being almost impossible to remove using current methods. This invention also relates to methods and systems for inactivating spores of the bacterium Bacillus anthracis, the cause of the anthrax disease.

Bacillus anthracis bacteria spores have a very long lifetime. At animal burial sites, they have been known to re-infect animals over 70 years after burial of an anthrax- killed animal. There are many (c. 90) known strains of anthrax. The Vollum strain was developed but never used as a biological weapon during World War II, and is a particularly virulent strain. Another virulent strain is the Ames strain, which was used in 2001 anthrax bioterrorism attacks in the United States.

Anthrax can enter the human body by ingestion through the intestines, by inhalation through the lungs, or cutaneously through the skin. All three infection routes are of concern in the context of bioterrorism attacks with bacteria such as anthrax, but inhalation of an anthrax-contaminated environment in an enclosed room, and skin contact with anthrax contaminated objects, are of special concern since either or both can result from receipt of anthrax contaminated objects into an enclosed space such as an office. Respiratory infection with anthrax in humans initially manifests itself as cold or flu-like symptoms for several days, but then progresses quite suddenly to severe and commonly fatal respiratory collapse. If diagnosed early, anthrax infections can be treated with appropriate antibiotics, but not after the disease has progressed.

Because of the dangers of experimenting with the highly toxic anthrax bacterium and its spores, it is common to experiment with another, less dangerous Bacillus species as a surrogate for anthrax, for example Bacillus subtilis. Results of deactivation treatment of B. subtilis are generally accepted as reliable indicators of effectiveness against Bacillus anthracis.

This invention also relates to processes and systems for disinfecting sports apparel such as athletes' clothing and protective equipment, and sports premises such as locker rooms, change rooms and gymnasiums, of bacteria such as the superbug Methicillin Resistant Staphylococcus Aureus, MRSA.

As reported in a recent article entitled "Assessment of Athletic Facility Surfaces for MRSA in the Secondary School Setting" (Journal of Environmental Health, Feb., 2010), the authors stated that "Methicillin-Resistant Staphylococcus Aureus (MRSA) was once largely a hospital-acquired infection, but increasingly, community-associated MRSA (CA-MRSA) is causing outbreaks among otherwise healthy people in athletic settings. Secondary school athletic trainers, student athletes, and the general student population may be at elevated risk of MRSA infection." This invention also relates to methods and systems for attacking and destroying infestations of "bed bugs" and similar insect pests, lodged in interior room spaces, upholstery, bedding, draperies, fabrics and other fibrous locations.

Bed bugs are parasitic insects of the family Cimicidae. They feed preferentially on human blood and the blood of other warm-blooded animals. They are mainly active at night. They grow to a length of 4 -5 mm and a width of 1 .5 - 3 mm. Best adapted to human environments is the common bedbug Cimex lectularius, found in temperate climates throughout the world. Other species include Cimex hemipterus, found in tropical regions, and Leptocimex boueti, in tropical regions of South America and West Africa.

Bites from bed bugs often go undetected at the time, and in many instances there is no visible sign of the bite. However, they cause a skin condition known as cimicosis which is accompanied by serious skin itching which can lead to anxiety, stress and insomnia, as well as secondary infection as a result of scratching. Largely because of their nocturnal habits, bed bugs are hard to detect and eradicate. They are not an easily identifiable problem. Their eggs and larvae present a particularly difficult eradication problem, since at least the eggs can remain viable, and hatch, several days after all adult insects have been eradicated from the vicinity.

U.S. Patent 7,404,624 Cumberland et al., issued August 5, 2008, describes methods for abating allergens, pathogens, odours and volatile organic compounds in air, using an atmosphere having specific combinations of ozone concentration, hydrogen peroxide concentrations, temperature and humidity delivered over a specified period of time. The patent contains an experimental account of treating rooms of a residence, effectively treating cladosporium mold spores and penicillium/aspergillus molds in the room air. No details of the precise conditions used are given. The general disclosure of the patent states that selected conditions of ozone concentration, hydrogen peroxide, humidity and temperature are effective in killing various pathogens, including dust mite allergens, at ozone concentrations below 6 - 9 ppm, but the precise conditions used are not disclosed. In general, the patent teaches use in an atmosphere of 2 - 1 0 ppm ozone, hydrogen peroxide which is 75% - 150% by weight of the atmospheric ozone concentration, at a temperature of 15 - 27 °C and time 0.5 - 3 hours. Insect infestations are not disclosed as a target for the process described. SUMMARY OF THE INVENTION

According to one aspect of the present invention, there is provided a process of combating an ex vivo biological target which comprises exposing the target to a lethal amount of a highly reactive polyoxide or oxidative free radicals derived therefrom, said target being at least one of bacteria, bed bugs, bed bug eggs and bed bug larvae.

The biological target may comprise bacteria simultaneously exposed to ultraviolet radiation. Preferably the ultraviolet radiation is UVC, further preferably having a wavelength 250 - 260 nm.

The bacteria may simultaneously be exposed to hydrogen peroxide, preferably in an atmosphere containing from 0.2 - 20% hydrogen peroxide at a relative humidity of at least 60%. Soft and porous surfaces carrying bacteria may also be subjected to physical agitation while exposed to said atmosphere.

Preferably, the polyoxide is trioxidane.

The biological target preferably comprises bacteria including at least one of the superbugs, Clostridium difficile (C. difficile); E. coli; Pseudomonas aeruginosa; methicillin-resistant Staphylococcus aureus (MRSA); and vancomycin-resistant Enterococcus (VRE).

The biological target may also comprise bacteria, namely Listeria monocytogenes or Salmonella enteridis, or both.

The biological target may also comprise bacteria, namely Bacillus subtilis or Bacillus anthraci.

Preferably, the bacteria are simultaneously exposed, in a closed room, to an effective amount of additional reactive oxidative species, to enhance the oxidative power, for a period of time which substantially reduces levels of bacteria on the surfaces in the room, the process including the subsequent step of reducing the residual hydrogen peroxide in the room's atmosphere to a safe level of 0.04 ppm or less. Preferably, the bacteria are simultaneously exposed, in a closed room, to an effective amount of ozone and additional reactive oxidative species, to enhance the oxidative power, for a period of time which substantially reduces levels of bacteria on the surfaces in the room, the process including the subsequent step of reducing the residual hydrogen peroxide and ozone in the room's atmosphere to a safe level of 0.04 ppm or less.

Preferably, the bacteria are simultaneously exposed, in a closed room, to an effective amount of ozone and additional reactive oxidative species, to enhance the oxidative power, for a period of time which substantially reduces levels of bacteria on the surfaces in the room, the process including the subsequent step of reducing residual ozone in the room's atmosphere to a safe level of 0.04 ppm or less.

Preferably, surfaces in the room are subjected to physical dislodgement while exposed to the oxidative species.

The biological target may comprise one or more of bed bugs, bed bug eggs or bed bug larvae. Preferably, the process further comprises exposing the bed bugs, bed bug eggs or bed bug larvae to the lethal amount of polyoxide, the polyoxide formed from mixing ozone at a concentration of 2 - 350 ppm by weight and hydrogen peroxide at an amount of 0.2 - 10 wt. %, at a relative humidity of at least 30%. The bed bugs, bed bug eggs or bed bug larvae may be exposed to the lethal amount of polyoxide for a period of from 3 to 48 hours, preferably from 6 to 48 hours, more preferably from 24 to 48 hours, most preferably for a period of from 24 to 36 hours. The ozone concentration may be from 10 - 350 ppm, preferably from 20 - 200 ppm, more preferably from 20 - 100 ppm, most preferably from 35 - 90ppm. The hydrogen peroxide amount may be from 0.5 - 1 0%, more preferably from 0.5 - 7%, most preferably from 1 - 5%.

THE PREFERRED EMBODIMENTS

"Highly reactive polyoxides" useful in the present invention include compounds corresponding to the formula HO_nH, where n is 3 or higher. In effect they are higher homologues of hydrogen peroxide HOOH. The simplest of these compounds, and a preferred compound for use in the present invention, is dihydrogen trioxide, of formula HOOOH, also known as peroxone or trioxidane. The higher homologues such as dihydrogen tetraoxide are also useful. These compounds are relatively unstable, and tend to decompose spontaneously and/or under the effects of incident radiation (e.g. UV), and/or under the effects of accelerated catalytic reaction (e.g. singlet oxygen, triplet oxygen, superoxide ^"0₂, ozonide^"0₃, hydroxyl radical OH, hydroperoxide radical HO₂, etc) to produce reactive oxidative species, such as singlet oxygen, HO, HOO and HOOO radicals. The use of these and their precursor trioxidane or peroxone characterize what can be termed advanced oxidation processes which form the basis of the present invention, as a means of enhancing the efficiency of lethal action against bacteria and bed bugs.

The mechanism of peroxidone (trioxidane) formation has been the subject of a thorough theoretical study - see Xin Xu and William A. Goddard III, Proceedings of the National Academy of Sciences (PNAS), vol. 99, no. 24, pp 15308 - 15312 (Nov. 26, 2002), the contents of which are incorporated herein by reference.

Because of the instability of these highly reactive polyoxides, it is preferred to generate them in situ, in the vicinity of the bacterial targets. There are several methods by which this can be done.

One of these methods is a reduction of hydrogen peroxide (e.g. dry mist or vapor) or ozone, in ambient room atmosphere or a room charged with moist air (to create the desirable relative humidity) with the highly reactive species, such as singlet oxygen (atomic or molecular), triplet oxygen, superoxide ^"O₂, ozonide ^"O₃ , hydroxyl radical OH or hydroperoxide radical HO₂.

The reactive species can be generated by various means including photolyic methods (see for example Carrigan J. Hayes et.al., "The Chemistry of Reactive RadicL Intermediates in Combustion and the Atmosphere," Advances in Physical Organic Chemistry, vol. 43, 2009, pages 79 - 134) or catalytic methods (see for example M. Christina Yeber et.al., "Advanced Oxidation of a PulpMill Bleaching Wastewater," Chemosphere vol. 39 issue 10, October 1999, pages 1 679 - 1688).

Thus according to this embodiment, a process for disinfecting a room and surfaces therein comprises exposing the room and surfaces therein to an atmosphere which includes: 1 ) an effective amount of hydrogen peroxide and an effective amount of additional reactive oxygen species; or 2) an effective amount of ozone and an effective amount of additional reactive oxygen species; or 3) an effective amount of hydrogen peroxide with an effective amount of ozone and an effective amount of additional reactive oxidative species to achieve highly effective oxidative power for a period of time which substantially reduces levels of bacteria on the surfaces, and subsequently removing the residual hydrogen peroxide and/or ozone in the room atmosphere down to a safe level.

An alternative method is by dissociation of water vapour by means of an electrical discharge - see "Chemistry of Dissociated Water Vapour and Related Systems", by M. Venugopalan and R. A. Jones, Wiley, New York 1968, Chapter 7, the contents of which are incorporated herein by reference. Another such method is irradiation of hydrogen peroxide vapour by ultraviolet light, especially UVC, in the presence of oxygen and water vapour. Depending on the wavelength of the UV employed, this can be accompanied by the generation of ozone. This is a preferred method for use in the process of the present invention, since it allows in situ generation of trioxidane in a room contaminated with bacteria, and also takes advantage of the known germicidal properties of UV radiation. The room can be charged with moist air to create the desirable relative humidity, and with hydrogen peroxide to the desired extent (0.2 - 20%, preferably 0.2 to 1 0%, or 0.5 to 1 0%, or 0.5 to 7%, more preferably 0.5 to 5% or 0.5 to 3%, or 1 to 3%, most preferably 0.5 to 1 .5%), for example, and then irradiated with UVC to create the trioxidane and other reactive species which participate in the advanced oxidation process of the invention, in the room atmosphere.

An additional method of creating trioxidane is by pulse radiolysis of acidified air-saturated perchloric acid solutions - see B.H.J. Bielski and H.A.Schwarz, J. Phys. Chem., 1968, 72, 3836 - 3841 , and B.H.J. Bielski, J. Phys. Chem. 1970, 74, 3213 - 3216, incorporated herein by reference. .

Solutions of trioxidane in organic solvents (for example methyl acetate, t- butyl methyl ether or acetone) can be prepared by reduction of ozone with 1 ,2- diphenylhydrazine, 2-ethylanthrohydroquinone, isopropyl alcohol, isopropyl methyl ether or cumene at low temperatures - see for example J. Cerkovnik, B.

Plesnicar, J. Am. Chem. Soc 1993, 1 15, 12169 - 12170; B. Plesnicar, T. Tuttle,

J. Cerkovnik, D Cremer, J. Am. Chem. Soc. 2003, 125, 1 1553 - 1 1564; and B.

Plesnicar, J Cerkovnik, T. Tekavec, J. Koller, J. Am.Chem. Soc, 1998, 120, 8005 - 8006. Such solutions may be useful in contact disinfection of surfaces and objects, with the trioxidane formation taking place in situ.

Another method is the decomposition of hydrotrioxides of 1 ,3-dioxolanes

(see T. Tuttle et.al., J. Am.Chem. Soc, 2004, 126, 16093 - 16104).

Trioxidane HOOOH can take a variety of forms, and use in all its various forms is encompassed in the present invention. For example, there is the dimeric form [(HOO)(HOOO)-7r], a head-to-tail seven member ring complex.

Under irradiation, or by thermal unimolecular rearrangement, H₂0₃ forms from this complex, as well as [(H0₂)(HO)] radicals. Other ring structures have been postulated as forming on the decomposition of trioxidane. It can also form complexes with one or two molecules of water. Trioxidane HOOOH is a linear molecule, and can adopt cis or trans configurations. The free radical HOOO derived from trioxidane normally has a planar cis configuration.

Reactive, oxidative species derived from trioxidane and its dimeric form include the free radicals H0₃, H0₂, and OH, singlet oxygen, and the complex [(H0₂)(HO)]. Use or these as advanced oxidation agents is contemplated in the present invention.

A preferred embodiment provides a process for disinfecting a room and surfaces therein to combat at least one of the microorganisms bacteria Clostridium difficile (C. difficile); E. coli; Pseudomonas aeruginosa; methicillin- resistant Staphylococcus aureus (MRSA); vancomycin-resistant Enterococcus (VRE); Bacillus subtilus, anthrax, Listerium monocytogenes and/or Salmonella enteridis which comprises exposing the room and surfaces therein to a gaseous atmosphere which includes an effective amount of a highly reactive polyoxide or oxidative free radicals derived therefrom, an effective amount of hydrogen peroxide, and ultraviolet radiation, for a period of time which substantially reduces levels of bacteria on the surfaces. Other food-borne pathogens which are susceptible to the bactericidal treatment of the process of the present invention include Shigella, Campylobacter and Yersinia sp., as well as S. Aureus and Bacillus cereus.

The process is particularly effective with or without physical agitation, in disinfection of stainless steel surfaces which abound in medical treatment facilities, food handling facilities and sports facilities such as locker rooms, and on which bacteria are tenacious and difficult to destroy, due at least in part to their generation of a biofilm on such surfaces.

The physical agitation can be done by provision of a discharge system through which one or more of the reactive gases are applied to a surface, the system having a discharge end equipped with a dislodgement system. The dislodgement system causes physical agitation of the surface and allows the highly oxidative species to penetrate deep into the carpet, drape and similar surfaces in the room, thereby gaining better access to concealed/sequestered spores and/or colonies of bacteria. The dislodgement system is preferably manually operated from within the room, with operators protected by hazard suit and a mask. It may take the form of one or more outlet jets, with associated manually operated jet pressure controls. It may take the form of a revolving fixed brush with bristles of appropriate stiffness, alone or in combination with an outlet jet. Any form of dislodgement system which sufficiently disturbs the fibrous surfaces to allow the gases access to the spores and colonies concealed therein can be adopted. This includes non-physical applications such as air jets, ultrasonic waves, and electromagnetic energy, for example.

The ultraviolet radiation which is used in the preferred embodiments of the invention is preferably UVC, which has wavelength in the range 10 - 280 nm. UVC has germicidal properties of its own, so that its presence helps in the bactericidal action. It also interacts efficiently with the atmospheric oxygen and the hydrogen peroxide which is used, to produce the required highly reactive polyoxide trioxidane, particularly if UV of wavelength about 250 - 260 nm is used. UVB, of wavelength 280 - 350 can also be used. It has approximately the same germicidal effects as UVC, but is less efficient than UVC in generating trioxidane. Thus the process of the present invention is preferably conducted in a closed room, which contains air humidified to at least 60% relative humidity, hydrogen peroxide in an approximate amount of 0.2 - 20%, preferably 0.2 - 10% and even more preferably 0.5 - 1 .5% (or as mentioned above), with UVC generation within the room to impinge on the air and hydrogen peroxide within the room and generate polyoxide such as trioxidane therein. The UV dose should be at least 1 0mJ/cm², preferably 10 - 200 mJ/cm ². Contact of bacteria- carrying surfaces in the room with such an atmosphere for a period of 20 - 90 minutes is sufficient for an effective destruction of live bacteria and spores thereof, even those contained in biofilm. For contaminated soft, porous surfaces such as draperies, carpets, textiles and upholstery, it is preferred to subject them to physical agitation whilst exposed to the humid, hydrogen peroxide, UVC atmosphere, using pressure jets directed at the soft porous surfaces, physical scrubbing brushes, fans, etc., worked by a protected operator in the room or remotely.

An embodiment of the invention is further described, for illustrative purposes, in the following specific example.

EXAMPLE

An empty laboratory room of dimensions 20 x 1 6 feet, with a 9 foot ceiling was sealed with polypropylene tape to render it air tight. Six ultraviolet light emitters, producing UVC of wavelength c. 253 nm were placed in the room, along with a high volume humidifier. Sterile hydrogen peroxide solution 1 % v/v was added to the humidification reservoir. Stainless steel discs carrying MRSA were placed in the room approximately three feet above the ground on small tables 6 - 8 feet from the UV source which was located in the centre of the room. The discs were exposed to the UV radiation and 1 % hydrogen peroxide for periods of 30 or 60 minutes, at a relative humidity of 80%.

Under these conditions, the UVC radiation interacts with the atmospheric oxygen present to produce ozone, which in turn reacts with the hydrogen peroxide supplied to the atmosphere in the room, to form trixidane HOOOH in one of its isomeric forms. These forms include the dimeric, 7-member ring complex form [(HOO)(HOOO)-7r] mentioned above, believed to be formed first (Xin Xu et.al., op.cit.) and subsequently to decompose to form other reactive oxidative species. The cyclic complex precursor is essentially a "capped" hydroxyl radical, which has a much longer lifetime than the OH radical itself. Detection of HOOOH can be done by 170 NMR spectroscopy - see Nyffeler et al., op.cit.

Other test samples were tested in a chamber supplied with ozone prepared by corona discharge and supplied to the chamber along with hydrogen peroxide. Trioxidane forms under these conditions also. In preparation of the test plates, a single pure colony of methicillin-resistant Staphylococcus aureus (MRSA) was inoculated to a Columbia agar plate with 5% sheep's blood. They were incubated at 35 ^ in room air for 18 - 24 hours. From the plate, 4-5 isolated colonies were selected, and suspended in tryptic soy broth to achieve a 0.5 McFarland turbidity standard (1 .5 x10 ⁸ cfu/ml) measured using a spectrophotometer. Inoculum was prepared by performing a series of serial dilutions of 0.9 ml 0.85 NaCI broth with 0.1 ml of original 0.5 McFarland inoculum (6 x 10 fold) to give solutions of 10^~1 , 10^~2, 10^~3, 1 0^~4, 10^~5, 10^"6 and 1 0^"7 cfu/mL.

Brushed stainless steel discs of 10mm diameter and 0.7 mm thickness were used as the test surface medium. The steel discs were cleaned and then sterilized in a steam autoclave. 20 μΙ of the freshly prepared bacterial inoculums in tryptic soy broth was applied to the steel discs and allowed to dry at room temperature while resting in a sterile petri dish without a lid for approximately 45 - 60 minutes in a biological safety cabinet. Three steel discs were placed per petri dish and all exposure conditions of inoculated discs were done in triplicate. Once dry, the lid of the petri dish was placed over the discs and they were carefully placed in the test chamber where the lid was removed and the discs were exposed to the test conditions. Control discs in triplicate were left covered in the biological safety cabinet and were not exposed to the test conditions.

Immediately after the exposure to the test conditions, and similarly for the control discs, the steel discs were vigorously mixed in 10 ml sterile saline using a vortex mixer at high speed for 60 seconds to elute off surviving viable bacteria or spores. The eluted suspension was serially diluted 10 fold in sterile 0.85% saline and the diluted bacteria were quantitatively plated onto Columbia sheep's blood agar plates, incubated under appropriate conditions, in duplicate so as to determine the original inoculums concentration.

After cultivation for 24 hours in an incubator, the plates were stained, examined through a microscope, and the number of colony forming units on each plate was counted.

The control plates had colonies too numerous to count, at dilutions 10^~1 and 10^~2. Test plates which had received 30 minutes exposure (6 in total) or 60 minutes exposure (3 in total), with relative humidity 80% and hydrogen peroxide 1 %, all showed zero viable colonies, at both the aforementioned dilutions.

Detailed results showing the effectiveness of polyoxide on MRSA can be seen in a series of experiments conducted as described above and summarized in the "Table - MRSA" below. Results obtained for treatments with combinations of relatively lower amounts of hydrogen peroxide and ozone are surprisingly effective given the results obtained for treatments with each of these alone.

Table MRSA

A B C D E F G H

1 MRSA - ATCC 33952

Log 10

2

Material 03(PPM) H202 % Humidity % Exp. PEEP Cham./Room RED

3 Steel 400 0 80 90 90 Chamber 1 .223

4 Steel 80 0 80 90 90 Chamber 0.83

5 Steel 80 0 65 90 90 Chamber 1 .44

6 Steel 130 0 65 90 90 Chamber 0.22

7 Steel 130 0 65 90 90 Chamber 1 .08

8 Agar >1000 0 80 90 90 Chamber 5.15

9 Agar 80 0 80 90 90 Chamber 4.899

10 Agar 130 0 ? 90 90 Chamber 4.695

11 Steel 80 0 60-70 90 90 Chamber 0.49

12 Steel 180 0 80 90 90 Chamber 0.66

13 Steel 500 0 80 90 90 Chamber 6.73

14 Steel 180 0 80-85 90 0 Chamber 0.99

Further results showing the effectiveness of polyoxide in the context of treating bed bug infestations are also shown herein.

In the "Table - Bed Bugs" below, are the results of testing the present inventive method on bed bugs provided and verified by the Public Health Unit of the city of Kingston, Ontario, Canada. Each data point represents the effects of ozone and/or H₂O₂ on four such bed bugs in a test chamber at room temperature. Eggs laid by such bed bugs during their captivity were also exposed at the same time in the same test chamber as the adults. The number of eggs varied from one group of bed bugs to the next ranging from three to ten. Following exposure, these eggs were kept at room temperature and observed for one week for viability (i.e. hatching).

TABLE - Bed Bugs

*this and all other data points in chart indicate: percentage of bed bugs killed / percentage of bed bug eggs not viable

The foregoing shows that for a polyoxide forming combination of a relatively low level of hydrogen peroxide (1 %) and a relatively low level of ozone (80 ppm), a surprising 100% of bed bugs were killed between 24 and 36 hours, and that a surprising 1 00% of eggs were rendered non-viable after 36 hours of exposure time. Much higher levels of hydrogen peroxide alone, or ozone alone, were completely ineffective in rendering eggs non-viable. While it is apparent that neither ozone alone, nor hydrogen peroxide alone, provided useful results, there is also nothing to indicate (apart from the inventive combination) that a combination of ozone or hydrogen peroxide, even at higher levels, would have any effect on eggs. Thus, the polyoxide forming combination of such low levels of ozone and hydrogen peroxide were surprisingly efficacious.

Preferred ozone amounts for mixing with hydrogen peroxide so as to form a lethal amount of polyoxide in a disinfecting atmosphere for treatment against bed bugs, bed bug eggs and bed bug larvae according to the invention, are from about 10 - 350, preferably 10 - 200 parts per million of the disinfecting atmosphere. More preferably ozone is present at from 20 to 350, or from 20 to 200, or from 20 to 100, or from 35 to 1 00 parts per million, and most preferably from 35 to 90 ppm ozone. Preferred amounts of hydrogen peroxide are the amounts supplied to the disinfecting atmosphere using an aqueous solution containing 0.2 - 10%, more preferably .5 - 7%, more preferably again 1 - 5% and most preferably 1 - 3% hydrogen peroxide. The peroxide percentages used are sometimes expressed in terms of these solution percentages. The amounts are chosen so that no serious deleterious effects are suffered by other equipment in the treatment room or the fabrics (including carpets and drapes) to which the disinfecting atmosphere is supplied. The amount of hydrogen peroxide in the disinfecting atmosphere can be calculated from the volume of aqueous hydrogen peroxide evaporated into the disinfecting atmosphere, the volume of the room being disinfected and the concentration of hydrogen peroxide in the starting solution. Times of exposure of the room and its surfaces to the disinfecting atmosphere are suitably from 3 to 48 hours for combinations of hydrogen peroxide amounts (supplied as described above) of from 0.5 to 7% and ozone amounts of from 10 to 200 ppm. More preferably, such times of exposure are from about 6 to 48 hours, or from 12 to 48 hours, or 12 to 36 hours and most preferably about from 24 to 36 hours. These times are constrained to some extent by the need to clear the room of ozone (down to a maximum of 0.04 ppm) following the disinfection phase, and return the room to normal use within a reasonable period of time, with the entire start-to-finish time not exceeding 60 hours. The ozone removal is an extremely rapid and fully effective process. The hydrogen peroxide, ozone and polyoxide products of interaction between them should be removed before the room is put back into normal use.

Claims

WHAT IS CLAIMED IS:

1 . A process of combating an ex vivo biological target which comprises exposing the target to a lethal amount of a highly reactive polyoxide or oxidative free radicals derived therefrom, said target being at least one of bacteria, bed bugs, bed bug eggs or bed bug larvae.

2. The process of claim 1 , wherein the biological target comprises bacteria, said bacteria being simultaneously exposed to ultraviolet radiation.

3. The process of claim 2, wherein the ultraviolet radiation is UVC.

4. The process of claim 3, wherein the ultraviolet radiation is of wavelength 250 - 260 nm.

5. The process of any one of claims 2 - 4, wherein the bacteria are simultaneously exposed to hydrogen peroxide.

6. The process of claim 5, wherein the bacteria are exposed to an atmosphere containing from 0.2 - 20% hydrogen peroxide at a relative humidity of at least 60%.

7. The process of claim 6 including subjecting soft and porous surfaces carrying bacteria to physical agitation while exposed to said atmosphere.

8. The process of any one of claims 1 - 7, wherein the polyoxide is trioxidane.

9. The process of any one of claims 1 - 8, wherein the biological target comprises bacteria, said bacteria are at least one of the superbugs, Clostridium difficile (C. difficile); E. coli; Pseudomonas aeruginosa; methicillin-resistant Staphylococcus aureus (MRSA); and vancomycin-resistant Enterococcus (VRE).

10. The process of any one of claims 1 - 8, wherein the biological target comprises bacteria, said bacteria are either, or both Listeria monocytogenes or Salmonella enteridis.

1 1 . The process of any one of claims 1 - 8, wherein the biological target comprises bacteria, said bacteria are Bacillus subtilis or Bacillus anthracis

12. The process of any one of claims 2 - 1 1 , wherein the bacteria are simultaneously exposed, in a closed room, to an effective amount of additional reactive oxidative species, to enhance the oxidative power, for a period of time which substantially reduces levels of bacteria on the surfaces in the room, the process including the subsequent step of reducing the residual hydrogen peroxide in the room's atmosphere to a safe level of 0.04 ppm or less.

13. The process of any one of claims 2 - 1 1 , wherein the bacteria are simultaneously exposed, in a closed room, to an effective amount of ozone and additional reactive oxidative species, to enhance the oxidative power, for a period of time which substantially reduces levels of bacteria on the surfaces in the room, the process including the subsequent step of reducing the residual hydrogen peroxide and ozone in the room's atmosphere to a safe level of 0.04 ppm or less.

14. The process of claim 1 wherein the bacteria are simultaneously exposed, in a closed room, to an effective amount of ozone and additional reactive oxidative species, to enhance the oxidative power, for a period of time which substantially reduces levels of bacteria on the surfaces in the room, the process including the subsequent step of reducing residual ozone in the room's atmosphere to a safe level of 0.04 ppm or less.

15. The process of claim 12, claim 13, or claim 14 further including subjecting surfaces in the room to physical dislodgement while exposed to the oxidative species.

16. The process of claim 1 wherein the biological target comprises bed bugs, bed bug eggs or bed bug larvae, said process further comprises exposing the bed bugs, bed bug eggs or bed bug larvae to said lethal amount of polyoxide, said polyoxide formed from mixing ozone at a concentration of 2 - 350 ppm by weight and hydrogen peroxide at an amount of 0.2 - 10 wt. %, at a relative humidity of at least 30%.

17. The process of claim 16 wherein the bed bugs, bed bug eggs or bed bug larvae are exposed to said lethal amount of polyoxide for a period of from 3 to 48 hours.

18. The process of claim 16 wherein the bed bugs, bed bug eggs or bed bug larvae are exposed to said lethal amount of polyoxide for a period of from 6 to 48 hours.

19. The process of claim 16 wherein the bed bugs, bed bug eggs or bed bug larvae are exposed to said lethal amount of polyoxide for a period of from 24 to 48 hours.

20. The process of claim 16 wherein the bed bugs, bed bug eggs or bed bug larvae are exposed to said lethal amount of polyoxide for a period of from 24 to 36 hours.

21 . The process of claim 1 6 wherein the ozone concentration is from 1 0 - 350 ppm.

22. The process of claim 21 wherein the ozone concentration is from 20 - 200 ppm.

23. The process of claim 22 wherein the ozone concentration is from 20 - 100 ppm.

24. The process of claim 23 wherein the ozone concentration is from 35 - 90ppm.

25. The process of any one of claim 16, wherein the hydrogen peroxide amount is from 0.5 - 10%.

26. The process of any one of claim 25, wherein the hydrogen peroxide amount is from 0.5 - 7%.

27. The process of any one of claim 26, wherein the hydrogen peroxide amount is from 1 - 5%.