Sterilization and Disinfection

Sterilization and Disinfection G McDonnell, Steris Ltd., Basingstoke, UK ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Introduction General Considerations Physical Disinfection and Sterilization

Glossary antimicrobial The ability of a process or product to be effective at killing microorganisms. This can vary depending on the process/product and the target microorganism (e.g., antibacterial or antiviral). Antimicrobial agents can include physical and/or chemical methods. antisepsis Destruction or inhibition of microorganisms in or on living tissue, for example, on the skin. An antiseptic is a disinfectant product used on the skin. biocide A chemical or physical agent, usually broad spectrum that inactivates microorganisms. Chemical biocides include hydrogen peroxide and phenols, whereas physical biocides include heat or radiation. -cidal/-static The combining form ‘-cidal’ refers to lethal activity against a group of microorganism (e.g., sporicidal, refers to activity to kill bacterial spores and bactericidal, refers to the ability to kill bacteria). Similarly, ‘-static’ refers to the ability to inhibit the multiplication of an organism (e.g., bacteriostatic). cleaning Removal of contamination from a surface to the extent necessary for further processing, or for intended use. decontamination Physical and/or chemical means to render a surface or item safe for handling, use, or disposal. Decontamination is generally a combination of cleaning and disinfection/sterilization. disinfection The antimicrobial reduction of the number of viable microorganisms on a product or surface to a level previously specified as appropriate for its intended further handling or use. Disinfectants are often subdivided into high level, intermediate, or low level (depending on the product claims and country-specific registrations). High-level disinfectants are considered

Abbreviations CHG ClO2 EO EPA

chlorhexidine gluconate chlorine dioxide ethylene oxide US Environmental Protection Agency

Chemical Disinfection and Sterilization An Introduction to Biocide Resistance Concluding Remarks Further Reading

effective against all microbial pathogens, with the exception of large numbers of bacterial spores (that are generally considered the most resistant to inactivation). Intermediate-level disinfectants are effective against mycobacteria, vegetative bacteria, most viruses and fungi, but not necessarily bacterial or some fungal spores. Low-level disinfectants are generally effective against most bacteria, some (in particular enveloped) viruses and some fungi, but not mycobacteria and bacterial spores. formulation Combination of ingredients, including active (e.g., biocide) and inert ingredients (e.g., chelating agents, surfactants), into a product for its intended use (e.g., cosmetics, antiseptics, and disinfectants). fumigation Delivery of a disinfectant/disinfection process (gas or liquid) indirectly to the internal surface of an isolator/enclosure. Fogging is a form of fumigation, which refers to the indirect application of a liquid product to a given area for the purpose of antimicrobial activity. pasteurization Destruction of microorganisms that can be harmful or cause product spoilage. preservation The prevention of multiplication of microorganisms in products. sanitization The removal or inactivation of microorganisms that pose a threat to public health. sterilization Validated process used to render a product free from viable organisms, including bacterial spores. ‘Sterile’ is defined as being free from viable organisms. tolerance Decreased effect of an anti-infective or biocide against a target microorganism and requiring increased concentration or other effects to be effective.

FDA H2O2 IR LTSF MIC

US Food and Drug Administration hydrogen peroxide infrared low-temperature steam formaldehyde minimal inhibitory concentration

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O3 PAA PVPI

ozone peracetic acid povidone-iodine

Defining Statement Disinfection and sterilization methods are described under various physical and chemical types. They are widely used for various antimicrobial applications in infection prevention and contamination control, including food/water treatment and surface treatments. Their mechanisms of action and resistance are briefly discussed.

Introduction The control of microorganisms and microbial growth is an important consideration in many laboratory, medical, veterinary, industrial, and environmental situations. Disinfection and sterilization processes play an important part in achieving this goal. They are essential for health and safety, if one considers the range of disinfection and sterilization processes that affects our lives, including medical device sterilization, food sanitization and pasteurization, water treatment, and product preservation. Yet, in more recent years we have come to expect these infection or contamination prevention strategies as being standard, with their effect on human and animal health generally only highlighted when the various products or processes have failed or been compromised. In this article, the various types of disinfection and sterilization applications are considered briefly. Sterilization is the ultimate process, which is required to ensure that all microorganisms (or indeed all that are a concern for a given purpose/use) have been removed or inactivated from a given surface, within the air or in a liquid. Disinfection is alternatively considered only to reduce the microbial load to a level that is considered safe for subsequent use (which will vary depending on the application) and incorporates a variety of antimicrobial processes and products. For the purpose of this article, disinfection can include a wide range of other antimicrobial processes including pasteurization, sanitization, antisepsis, and fumigation. Preservation may also be considered as a disinfection process, as the act of reducing and/or preventing the growth of microorganisms to prevent spoilage and extend shelf life. It should be noted that the use of these terms and their respective definitions may vary from country to country, application to application, and are often times misused (in particular the term ‘sterilization’); however, care has been taken in this article to use each term as universally accepted or defined in international standards.

QAC UV

quaternary ammonium compound ultraviolet

General Considerations Microbiology Microorganisms play an important part of our lives, both for our benefit as well as for our detriment. The goal of any disinfection or sterilization application is to remove or inactivate microorganisms that are a specific concern. These include the inactivation of pathogens (to prevent subsequent transfer, say, to the skin, via a contaminated surface or released from a biosafety laboratory), product protection (to prevent spoilage), remediation (to clean up a contamination event), and surface or material sterilization (to prevent complications or severe infections following say the use of a device during a surgical procedure). Therefore, the risks associated with a given situation or product to be treated should be the first consideration in the choice of any antimicrobial application. These primarily include any risks of disease transmission or contamination (e.g., in product spoilage). The types and potential numbers of microorganisms should be the next consideration. Obviously, higher populations of microorganisms can provide a greater challenge to disinfection and sterilization, to ensure adequate contact times, concentrations of chemical, and so on, and also to ensure that the contamination levels are indeed reduced to a safe level or completely inactivated. The types of microorganisms require further discussion. For example, a biosafety laboratory that handles pathogens such as the enveloped viruses HIV and Hepatitis B will have a wider choice of disinfection options available than a laboratory that handles large concentrations of mycobacteria. This is due to the natural sensitivity of the enveloped viruses to disinfection and sterilization, in contrast to the higher level of resistance observed with Mycobacterium and related species. For most applications, it is convenient to consider microorganisms as ranging in known sensitivity from those that are relatively sensitive (e.g., lipid, enveloped viruses) to those that are very resistant (e.g., bacterial spores; Figure 1). Biocide resistance depends on natural and, to a lesser extent, acquired mechanisms. Natural mechanisms include the external structure of the microorganism that can afford a penetration challenge to a given biocide. For example, spore-forming bacteria (including Bacillus and Clostridium species) are relatively sensitive in their vegetative form, but have the ability to form spores that are remarkably resistant to chemical and physical processes. Bacterial endospores have a unique structure, protected from the

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Microorganism More resistant

Prions Bacterial spores Protozoal oocysts Helminth eggs Mycobacteria Small, nonenveloped viruses

Scrapie, creutzfeld-jakob disease, chronic wasting disease Bacillus, geobacillus, clostridium Cryptosporidium Ascaris, enterobius Mycobacterium tuberculosis, M. terrae, M. chelonae Poliovirus, parvoviruses, papilloma viruses Giardia, acanthamoeba

Fungal spores

Aspergillus, penicillium

Gram-negative bacteria

Vegetative helminths and protozoa Large, nonenveloped viruses Gram-positive bacteria Less resistant

Examples

Protozoal cysts

Vegetative fungi and algae

Enveloped viruses

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Pseudomonas, providencia, escherichia Aspergillus, trichophyton, candida, chlamydomonas Ascaris, cryptosporidium, giardia Adenoviruses, rotaviruses Staphylococcus, streptococcus, enterococcus HIV, hepatitis B virus, herpes simplex virus

Figure 1 The ascending order of microbial resistance to disinfectants and sterilants. This representation is given only as a guide and can vary dramatically depending on the disinfection or sterilization process used. Prion resistance, for example, remains under debate but these unusual infectious agents are generally considered highly resistant to typical inactivation mechanisms.

environment by multiple, external layers. Similar intrinsic mechanisms are responsible for the environmental survival of many fungi (such as Aspergillus ascospores that have known high levels of resistance to biocides) and parasitic dormant forms, including eggs and various forms of cysts, although to a lesser extent than some bacterial endospores. In contrast, the lipid, enveloped viruses (including influenza, Newcastle virus, HIV, and Hepatitis B) are very sensitive to environmental factors (such as drying alone) and various types of disinfectants. Acquired resistance has also been described, in particular in bacteria, with particular significance for certain classes of biocides. The mechanisms of resistance, both intrinsic and acquired, are discussed in more detail later in this article. If we consider biosafety laboratory situations, the types and levels of pathogens are generally well defined and controlled. The primary role of disinfection and sterilization processes within these facilities will be prevention of pathogens to escape into the general environment, but these facilities will also be concerned about cross-contamination and the potential of biofouling (e.g., in water or air). Therefore, microbiology laboratories generally use a variety of processes, such as feed-water disinfection, waste disinfection and/or sterilization, continuous filtration of the air, periodic surface disinfection, and fumigation. In contrast, within a hospital, where a variety of devices are

repeatedly used for diagnostic and surgical procedures or environmental surfaces are found to have a range of pathogens present, the types and levels of microorganisms are less defined and more than likely unknown on contaminated instruments or surfaces; blood-borne pathogens are a particular concern, but other pathogens will cause a problem if introduced inadvertently into the right environment. In these specific clinical examples, a series of standard precautions are applied that are considered to reduce the levels of contamination to a safe level. In medical, veterinary, and dental use, devices that directly contact the patient’s bloodstream or remain within the patient are considered to be of much higher risk (referred to as ‘critical’ devices) and are generally recommended to be sterilized before use. It is important to note that many methods can be considered ‘sporicidal’, referring to their ability to kill bacterial spores, which are the most resistant microorganisms to various physical and chemical biocidal methods, but may not necessarily provide ‘sterilization’. Sterilization is an absolute term that describes the complete destruction or removal of all viable microorganisms in a validated process. In comparison, ‘semicritical’ devices that contact only intact mucous membranes or in some cases broken skin are recommended to be ‘high-level’ disinfected, to be able to inactivate most pathogens with the exception of a high concentration of bacterial spores (based on a widely used, simplified version

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of Figure 1). Finally, ‘noncritical’ devices or even various high-risk surfaces in hospitals, generally contact only intact skin and as they present the lowest risk to health are reprocessed or disinfected by cleaning or with ‘low’ or ‘intermediate’ disinfectants. As a final consideration, in addition to the type(s) and concentrations of microorganisms, the effectiveness of these processes and products can be dramatically affected by the presence of interfering materials that can adsorb, react/neutralize, or protect microorganisms from adequate treatment. These materials can be organic (e.g., protein, lipids) and/or inorganic (e.g., salts). Examples are patient materials such as blood remaining on a surgical device and the presence of extraneous carbohydrates and proteins in water-line biofilms. All disinfectants and sterilants can be compromised by these effects and therefore should be considered during their practical use.

Product/Process Application In the practical consideration of any disinfection or sterilization product or process, at least three factors should be considered (Figure 2): antimicrobial efficacy (including the desired outcome), compatibility with the material being treated, and any potential safety concerns. Microorganisms, efficacy, and some of the potential risks associated with contamination were considered earlier. Regionally, antimicrobial claims on products and processes can vary considerably, in particular for liquid disinfectants and antiseptics. Attention should be paid to review these claims, including the test methods used and recommended contact times for minimal activity claims; for example, suspension tests (in which test organisms are directly inoculated into a test liquid) are only an indication of the efficacy of a product when applied to a contaminated surface (e.g., on the skin or surfaces such as food or metals). Antimicrobial claims should be supported by standard, recognized test methods (e.g., the AOAC and ASTM antimicrobial test methods); in the United States, for example, environmental surface disinfectant claims need to be registered with the US Environmental Protection Agency (EPA) Antimicrobial efficacy

Biocidal product/process

Safety

Compatibility

Figure 2 The basic requirements in consideration of disinfection and sterilization processes or products.

and likewise for medical/dental device applications with the US Food and Drug Administration (FDA). The European Union has also developed and published a series of antiseptic and disinfection test methods, depending on the use of the product and specific antimicrobial claim (e.g., bactericidal, virucidal). In some cases, efficacy may need to be directly tested and verified for particular applications in a facility (e.g., in some manufacturing and critical biosafety laboratory facilities). When a given product or process has been chosen, a successful outcome further depends on its correct application. Variables such as biocide concentration, correct dilution, temperature, stability, humidity, application method, and contact time can dramatically affect the efficacy achieved, depending on the process or product used; again, close attention should be paid to the label or associated instructions provided. A unique consideration in liquid biocides, such as those containing chemicals such as hydrogen peroxide (H2O2), phenolics, and quaternary ammonium compounds (QACs), is formulation. Unlike most antimicrobial drugs, chemical biocides are often formulated to optimize their activity. A ‘formulation’ is therefore a combination of ingredients, including not only the biocide but also other inert ingredients. These ‘inerts’ can include, for example, chelating agents, surfactants, buffer, and dispersants to optimize the biocide activity, stability, and aesthetics for its required application. It is quite common to observe formulated products, such as hard-surface disinfectants and skin antiseptics, with similar concentrations of biocides but dramatically different efficacy profiles. Therefore, the active ingredient is the biocide but the formulation is the disinfectant. In other cases, the active ingredient alone is required and effective but only when controlled under certain conditions; for example, the low-temperature sterilizing agent gaseous ethylene oxide (EO) is dependent on the presence of humidity (generally in the 40–80% range) and temperature (30–70  C) for optimal and controlled antimicrobial efficacy. Similarly, EO is therefore the biocide but the overall process affords sterilization. The efficacy of decontamination can also vary depending on the contact surface. Porous surface materials (such as certain types of plastics and paper) are a particular concern, in comparison to nonporous surfaces such as stainless steel. The biocide can be adsorbed and even degraded by the surface material, thereby affecting the overall concentration available for antimicrobial effects. This can also affect physical sterilization methods such as steam where air can become trapped in materials, thereby limiting steam penetration, and large sterilization may also take longer to reach the desired sterilization temperature. A further requirement is that the biocidal method used is compatible or has limited, acceptable damage to a surface or product treated. Examples are electrical equipment, in which liquids should not be used because of safety and damage risks.

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Temperature-sensitive devices as a further example cannot be sterilized using heat and require the use of low-temperature, biocidal methods as alternatives. For this same reason, only a limited number of biocides are used on the skin or mucous membranes. The optimum processes for any given application should be nondestructive to surfaces over repeated use. Finally, there can be a variety of safety considerations, including environmental and personnel/user safety. The optimum biocidal process should be nontoxic, nonallergenic, and nonirritating. This is a particular consideration with chemical biocides. As these chemicals are designed to be effective against a wide range of microorganisms, they invariably will affect human and plant cells to different effects. Some biocides can be particularly toxic even at low concentrations. In further cases, reactions of the biocide with certain surfaces or on natural breakdown can produce toxic by-products, which should also be considered. Environmental effects on the use of many biocides are an increasing concern with particular restrictions being placed in various geographic areas regarding their use and disposal. Furthermore, in some applications (e.g., in disinfecting reusable surfaces such as surgical devices and production vessels) if chemical residues remain on the surface they may lead to toxicity issues on subsequent use (in particular with patient contact). Monitoring Effectiveness The efficacy of various disinfection and sterilization methods can be verified for specific applications and routinely monitored. In certain industrial, laboratory, and clinical situations, this is mandatory and can be achieved through various ways. For physical methods, parametric monitoring of process variables is often considered sufficient for confirming process effectiveness. These include measuring temperature, concentration, humidity, air removal, and other process variables. Heat-based systems are widely validated using these methods. Similarly, gaseous and liquid chemical methods can also be validated if important variables have been defined and can be monitored, such as humidity and biocide concentration. Various different types of chemical indicators, which change color on exposure to the chemical or physical process, can be used to simply indicate the qualitative presence of the biocide, but can also be designed to indicate multiple process conditions quantitatively. These are usually in the form of small strips with a color pad that changes on exposure. Finally, various microbiological methods can be used. Direct methods of microbial sampling include contact plates and air samplers. Indirect methods use swabs, cloths, or surface extractions that are then transferred onto culture media. There are no universal microbial cultivation methods that can be used for detecting the growth of all microorganisms. For example, although PCR (or other nucleic acid)-based methods may

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be used to detect microorganisms, they would not necessarily be capable of differentiation between live and dead states. In general, the monitoring of surfaces for aerobic (grow in the presence of oxygen), mesophilic (grow at ambient temperatures) bacterial, or fungal contaminants is the most convenient, although specific culture media and incubation conditions may be selected in cases where certain microbial contaminants are suspected or for particular environmental concerns. Virus analysis is much less used, except when specific viruses are suspected or known to be present. Similar to chemical indicators, biological indicators are also available for monitoring the effectiveness of processes, particularly sterilization. These are usually in the form of paper or stainless steel coupons that are inoculated with bacterial spores (as they are generally considered the most resistant microorganisms to sterilization processes), but other test organism can be used. These are simply exposed to a process and then incubated/cultured to determine the presence or absence of growth.

Physical Disinfection and Sterilization Physical disinfection and sterilization methods include heat, radiation, and filtration. Filtration methods are nonbiocidal in nature; physically removing microorganisms from liquids or air and are therefore considered only briefly. Heat Rapid surface disinfection on materials can be achieved by immersion in hot, preferably boiling, water or treatment with steam at atmospheric pressure (e.g., in steam cleaning applications). These methods are also used for the treatment of wastes, water, and other liquids. In contrast, low or freezing temperatures are generally not biocidal but are widely used as preservative methods. In heat-based methods, disinfection time will depend on the temperature, and is typically recommended to be >65  C for most pathogenic and food-spoiling microorganisms. Moist heat disinfection and sterilization is therefore one of the most effective and used methods for a variety of applications. There are, however, notable reports of microorganisms that present extreme resistance to heat disinfection. For example, some strains of the nonenveloped parvoviruses demonstrate higher levels of resistance to heat disinfection (with some reports up to 100  C, or boiling conditions). Various types of bacterial spores require much higher temperatures for inactivation, notably Geobacillus stearothermophilus spores at temperatures in excess of 115  C, as well as various vegetative, hyperthermophiles such as Thermococcus, Pyrococcus, and Pyrolobus species (the latter actually requiring temperatures in excess of 85–90  C to allow growth!). These

534 Applied Microbiology: Industrial | Sterilization and Disinfection

organisms may require specific consideration in certain situations, but are generally not considered to be of pathogenic concern. Examples of typical disinfection conditions using moist heat (e.g., various devices or materials that could be used for medical and dental procedures) include 100 min at 70  C, 10 min at 80  C, 1 min at 90  C, and 0.1 min at 100  C. A commonly used form of heat disinfection is pasteurization, being widely used in the liquid/ solid food industry for both safety (to remove pathogens) and shelf-life (to extend storage time) reasons. Pasteurization was first introduced to control mycobacteria (in particular slow-growing Mycobacterium bovis and Mycobacterium tuberculosis) and other pathogens in milk. The process can vary from application to application, but essentially consists of rapid heating to 65–80  C, holding for the required disinfection time, and rapidly cooling. In this way, heat-sensitive foods and materials can be treated with minimal effects on the taste, structure, or other characteristics. In general, moist or wet heat can be a very predicable form of both disinfection and sterilization. As the temperature rises the efficacy of heat increases, to include various types of microorganisms with known resistance to heat (in particular bacterial spores). To reach temperatures in excess of 100  C, in particular for sterilization applications, steam under pressure is used; steam, as a gas will follow the gas laws where the temperature is a function of the pressure and volume (of a given area where the steam is supplied). Therefore, at atmospheric pressure (101.35 kPa at sea level), water will boil to produce steam at 100  C, but if the pressure is increased, the boiling temperature of steam will also increase and high sterilization temperatures can be achieved. ‘Saturated’ steam is the most optimal and refers to holding as much water as possible in the steam phase, without allowing excessive condensation to occur. For example, if the steam is too ‘dry’ (‘superheated’ is more like a dry-heat sterilization process, see below) or if it is too ‘wet’ (oversaturated), there is excess condensation (or moisture) on the various surfaces, which can be inefficient as a sterilization process; in some applications, condensation also adds the requirement of drying afterward (as a ‘wet’ load can be recontaminated and cannot be stored long term). Steam sterilization is therefore performed in specially design pressure vessels (commonly referred to as autoclaves; Figure 3), which expose a given load to saturated steam at a specified temperature, pressure, and exposure time. In these situations, materials can be packaged in various barrier systems for terminal sterilization in an autoclave and then stored sterile until subsequent use. Recommended steam sterilization cycles do vary from country to country and also depending on the specific application. Typical cycles used include 121  C for 15 min (this is often a reference cycle) and 134  C for

Figure 3 A typical steam sterilizer (also known as an autoclave).

>2 min. These indicate the minimum time and temperature for the load to be held within the chamber for sterilization and do not take into consideration the requirement to prepare or ‘condition’ the load for sterilization and any drying or cooling after sterilization. Conditioning of the load is important for at least one major reason, which is air (or other noncondensable gas) removal. Air can inhibit penetration and therefore effectiveness of steam during the sterilization process. Autoclave designs can therefore be classified based on the mechanisms of air removal to include upward displacement, downward displacement, and prevacuum designs. Upward displacement designs are traditional, small, laboratory autoclaves. Water is placed at the base of the vessel and boiled to produce steam. Air is then expelled from the vessel under pressure, through a safety valve on the lid, for a given time and closed. Heating continues during the sterilization time, and then the autoclave is allowed to cool. Downward (or ‘gravity’) displacement autoclaves can vary in size from small, bench-top to larger-scale autoclaves. In a typical model, steam is introduced at the top of a pressure vessel and, because it is heavier than air, forces the air out through a valve at the base of the chamber. When the required temperatures and pressures are achieved, the valve is closed and the load is held for a given sterilization time. At the end of the cycle, pressure is released and the contents are allowed to cool (generally to below 80  C). Finally, prevacuum autoclaves are more reliable for use

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with various kinds of porous and nonporous materials, from which air removal may be difficult. These sterilizers use a vacuum pump that removes the air from the chamber. Steam may be also pulsed into the chamber, to aid in the removal of air and allow for even penetration of steam (and heating) through the load. After sterilization, the steam is evacuated and the contents are allowed to cool (which can be assisted using vacuum again). Dry-heat methods may also be used for disinfection and sterilization purposes. Dry heat is an effective antimicrobial but, in general, requires exposure to extended periods at elevated temperature or extremely high temperatures to ensure sterilization. For these reasons, these methods can be limited in their application because of their destructive nature. Incineration (combustion or burning to ashes) is an example and is essentially a destructive method used for the disposal of contaminated wastes. Incinerators typically operate from 800 to 1300  C. A simple example at a microbiology laboratory level is the use of flaming (or passing a small device through a flame, usually via a Bunsen burner). Hot-air disinfectors and sterilizers are often used for various laboratory and industrial applications. Temperatureresistant surfaces (such as some metals and glass) and materials (such as powders and some oils) can be sterilized by dry heat, but longer times are required as air is not effective at heat transfer. Typical dry sterilization cycles used (minimum) include 160  C for 120 min, 170  C for 60 min, and 80  C for 30 min. An important industrial use of dry heat is in the inactivation of bacterial endotoxins and other pyrogens (depyrogenation), which have known highlevel resistance to moist-heat sterilization. In the cases of both dry and wet heat, the mechanisms of action are associated with the loss of structure and function of various microbial macromolecules because of the transfer to heat. Radiation Radiation is energy in the form of particles or electromagnetic waves. Biocidal applications may be considered under nonionizing methods (generally used for disinfection purposes) and ionizing methods (more restrictive and used in sterilization processes). Nonionizing methods include sources that emit energy at the lower end of the electromagnetic spectrum, notably ultraviolet (UV; 5  10 19 2  10 17 J of energy), infrared (IR; 2  10 22 3  10 19 J), and to a lesser extent microwaves (2  10 24 2  10 22 J). Both IR and microwaves are considered to be effective only due to the rise in heat associated with their applications and may therefore be predominantly considered as heat-based methods (although this is debated in the literature). UV light has greater associated energy to elicit a direct antimicrobial effect in its own right. UV light is produced from various forms of

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lamps (e.g., mercury-vapor lamps) and has maximum antimicrobial efficacy in the 200 to 280 nm (‘UV-C’ or ‘short’) wavelength range. Due to a lack of penetration, the effect of UV light, in comparison to other forms of radiation, is dramatically reduced the further a surface is from the light source and from shielding, and in the presence of soils or other interfering substances. UV light can be optimized for bactericidal, cysticidal, and virucidal efficacy, but requires longer exposure times or higher radiation levels for fungicidal and sporicidal activity. UV light is widely used for water disinfection (by passing water across a UV light) and small surface area decontamination (e.g., in microbiology handling laminar flow cabinets when not in use). Ionizing radiation methods can be used as very effective disinfection and sterilization processes. They include the use of -radiation, X-rays, and electron (or ‘E’) beams that have much higher energy (e.g., -rays are >210 14 J) and therefore greater penetration and biocidal activity.

-Radiation and X-rays are electromagnetic radiation, whereas E-beams are sources of particle radiation consisting of beams of electrons (or -radiation) that are accelerated to improve their penetration. -Radiation is sourced from the delay of isotopes (or radionucleotides) such as 60Cobalt and 137Caesium, whereas X-rays and E-beams are generated through specially designed generators. These methods are some of the most potent biocidal processes used, but subsequently have limited widespread applications because of the safety risks associated with their use. There are generally used for industrial purposes, in the sterilization of manufactured products or to reduce the microbial load in materials, and require specific facility designs. For example, in a typical irradiator using a -radiation source, the source is usually stored under water and raised only during exposure to a load on a conveyor system for only a few minutes. A radiation dose of 25 kGy (or 2.5 MRad) of absorbed energy is generally considered sufficient for sterilization, but this will be load specific. The exposure area needs to be shielded by concrete (typically up to ten foot thick) to prevent worker exposure. The application of any disinfection or sterilization process is always a balance of safety, compatibility, and biocidal efficacy. Therefore, despite the potent antimicrobial activity of ionizing radiation methods, negative effects can include material damage (in particular on plastics), unpleasant odors, changes in taste (in food applications), and accelerated aging of materials (in particular drugs). Also, despite the antimicrobial activity, various strains of the nonsporulating bacteria Deinococcus and Deinobacter have dramatic intrinsic resistance to ionizing radiation, even in comparison to bacterial spores. They are found to be widely distributed in the environment, having been isolated from soil, dust, and, not surprisingly in this case, nuclear waste. These vegetative bacteria present with various

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novel mechanisms that accumulate to afford extreme resistance to radiation. The most widely studied is Deinococcus radiodurans and resistance mechanisms include efficient DNA and other macromolecular repair, production of protective pigments and defense enzymes, and a unique cell wall structure. The major mechanism of action of UV light and ionizing radiation methods is through the transfer of energy to microorganisms, with particular dramatic effects on structure and function of nucleic acids. In addition to the direct mutational effects in DNA and other direct structural damage to other macromolecules, radiation has been shown to produce free radicals and other reactive species on reaction with water and oxygen within and associated with microorganisms, which react with and disrupt the function of macromolecules and their associated structures.

Filtration Filtration can be an effective method in the physical removal of microorganisms, in particular in the treatment of liquids and gases (such as in air-handling systems). In these cases, various types of filtration media are used for the retention of microorganisms based on their size (Figure 4). Applications can range from the use of course filtration (to 5 mm size exclusion, depending on the filter specification) to nanofiltration or reverse osmosis methods being used to remove contaminants at <1 nm in size. Filters can be very efficient in reducing or completely removing microbial contamination, but an important variable is ensuring that the retentive properties of the filter are intact, because of the risks of structural damage or bacterial grow-through over time of use. In certain applications, biocides may be integrated onto the material surface of the filter to allow for some antimicrobial activity during the filtration process. An interesting application of this is the use for a class of biocides known as the N-halamines. These are nitrogencontaining compounds with anchored and available chlorine or bromine groups, which can react with and inactivate microorganisms (see ‘Halogens’); they are

Parvovirus

10 nm

Endotoxin

unique in that on exhaustion of available antimicrobial activity, they can be reactivated by treatment with a bromine- or chlorine-based liquid.

Chemical Disinfection and Sterilization Various chemical biocides are used for disinfection purposes, although only a subset has the ability to be used in sterilization processes. In the cases of chemical sterilization, the biocide must demonstrate the prerequisite to be effective against a wide range of microorganisms and in particular bacterial endospores. Further, the method of application, including various process conditions (depending on the defined process) to all areas/surfaces, needs to be controlled in order to confirm that sterilization can be reliably and reproducibly achieved. A much wider range of biocides and methods of delivery can be used for disinfection and preservation. For general surfaces, this can include liquids (including the biocide itself alone or in combination with other formulation effects), foams, or impregnated wipe disinfectants that can be used for localized or area decontamination. It is important that these products are prepared (if provided as concentrates) and used according to the manufacturer’s instructions (including correct dilution, restrictions on reuse, and safety precautions). Chemical preservatives can be generally used at relatively low concentrations to reduce or control the microbial levels within a given product; the actual activity of the biocide or biocides used in these applications will vary depending on the biocide type, its concentration over time, and the product it is present in/on (not just added but uniformly available to illicit its antimicrobial effect). A further subset of biocides is used for true antiseptic applications, that is, for use directly on the skin or mucous membranes. Overall, the most widely used chemical biocides are further discussed. It is reemphasized that the activity of any biocide or biocidal product/application depends on its formulation, application, and proper use; the discussion below is meant only as a guide for each biocide type and may not represent the true antimicrobial activity of labeled products and applications.

Pox virus Staphylococcus

100 nm

1 µm

Pseudomonas Giardia cysts

Pollen

10 µm

Yeast

100 µm

1 mm

Sand

Figure 4 A representation of the various sizes of microorganisms (note: the scale is a log range of size).

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Acids and Bases In general, acids and bases are widely used at low concentrations for their preservative activity, and to a much lesser extend as disinfectants. Acid examples include weak acids such as acetic, benzoic, citric, sorbic, and salicylic acid. Salicylic acid is widely used in antiseptics, such as for wart treatments (particularly for its exfoliant activity) or in many common acne or skin infection treatment products. Esters (formed by reacting acids with alcohols) are also used as preservatives, for example, in p-hydroxybenzoic ester derivatives (‘parabens’). Strong acids such as hydrochloric acid are not generally used because of safety and compatibility concerns (e.g., surface damage), except in specific industrial applications. The ‘weak’ acids vary in antimicrobial activity, but are generally particularly effective microstatic agents. A smaller range of bases (or alkalis) is used in similar formulation and some direct antimicrobial applications. These include sodium hydroxide, potassium hydroxide, and the acridines (which are considered relatively weak bases). Strong alkalis are used for specific microbicidal applications in industrial and laboratory settings, including for their virucidal activities at high concentrations. It is important to note that high concentrations of bases or formulated lower pH alternatives are effective against prions, which as atypical pathogens are considered highly resistant to disinfectants. Similar to the acids, bases are useful as preservatives at low concentrations and are required to be used in higher concentrations (e.g., direct use of 0.5–2 N NaOH) for virucidal and priocidal activity. The mechanism of action of acids and bases is similar in that they inhibit and/or inactivate normal metabolic functions and disrupt microbial structures. Acidophilic (e.g., many fungi and Helicobacter) and in contrast alkaliphilic (e.g., Enterococcus) bacteria and fungi can survive specific extremes of pH growth conditions. Investigations carried out in these organisms have shown that they achieve this by maintaining the internal cytoplasm at pH neutral conditions by active efflux mechanisms that can pump hydrogen ions in either direction across the cell membrane. Other mechanisms of tolerance can include exclusion due to various microbial surface structures (e.g., that reduce ion permeability). Many acids and bases are also used in combination with other biocides to improve their efficacy in formulation and to increase their efficacy or stability at the desired pH level.

Alcohols The most widely used alcohols for biocidal processes are isopropanol (isopropyl alcohol, propan-2-ol, ‘rubbing alcohol’), ethanol (‘alcohol’, also the major component in methylated spirits), and n-propanol (propan-1-ol). The

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typical concentrations used range from 50 to 90% (in water), where lower and higher concentrations outside this range demonstrate significantly less antimicrobial activity. Alcohol-based products can be rapidly bactericidal, mycobactericidal, fungicidal, and viricidal (varying depending on the type of alcohol used), but have no activity on bacterial spores (apart from preventing germination of spores, as sporistatic activity). They are particularly used as general surface disinfectants, widely used as skin antiseptics and to aid drying applications due to dispersion of water and rapid evaporation. Efficacy can be affected by the rate of alcohol evaporation following application; therefore, it is possible to improve the efficacy of much lower concentrations of alcohol by preventing its evaporation, which is particularly important in skin disinfection (to improve efficacy and reduce damage to the skin on repeated application). Alcohols are often combined with other biocides for various applications, including chlorhexidine or triclosan in antiseptics (which remain on the skin or mucous membranes following application for sustained antimicrobial activity or preservation). Care should be taken when using high concentrations, in particular, because of flammability risks. The mode of action is considered to be particularly due to protein denaturation and coagulation, as well as various generalized effects on lipids, lipid structures, and cellular metabolism. Aldehydes Glutaraldehyde and formaldehyde are both widely used as biocides with broad-spectrum activity, although longer exposure times are generally required for sporicidal activity. In general, aldehydes are mycobactericidal, but specific resistant strains to glutaraldehyde have been reported (e.g., Mycobacterium chelonae strains). Glutaraldehyde is usually provided in liquid mixtures at 0.1–2.5% and it is particularly active under alkaline conditions (pH 8), although acidic formulations (e.g., pH 4) are also used because of their greater stability; activity can be enhanced at higher contact temperatures. It is widely used as a surface or device disinfectant. For example, glutaraldehyde is one of the most common biocides used for disinfection of thermosensitive medical and dental devices. An alternative aldehyde in these applications is orthophthaldehyde (e.g., supplied at 0.55% and at a neutral pH), which has more potent mycobactericidal activity but has little practical sporicidal activity. Formaldehyde is used in both liquid and gas form. It is usually provided in liquid suspension (34–40%, ‘formalin’) or in solid, polymer form (paraformaldehyde). Formaldehyde-releasing agents (e.g., taurolin and hexamine) are used for some antiseptic applications and, more widely, as preservatives. Although formaldehyde in alcohol solutions was widely used in the past for surface disinfection, this is now rarely used because of safety considerations

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(as formaldehyde is considered toxic, mutagenic, and carcinogenic). Formaldehyde gas is used in biosafety laboratory facilities for area/room fumigation, and in a limited number of countries in various sterilization processes. In both cases antimicrobial efficacy depends on the presence of least 70– 80% humidity. In laboratory fumigation applications, processes include heating paraformaldehyde or formalin solutions in water, to form the gas/humidity mix, although specific practices do vary widely. A typical application in rooms would be to introduce and hold the formaldehyde gas for 7 h at 400–600 mg l 1 (and at >70% humidity), followed by adequate aeration to allow reentry into the treated area. The most widely used sterilization processes are referred to as low-temperature steam formaldehyde (LTSF) systems, which use steam (under vacuum) to heat and humidify a sterilizer vessel (generally an airtight chamber) and its load, followed by introduction and holding of the formaldehyde gas for a given time and then aeration using steam/vacuum pulses to purge the formaldehyde from the chamber. Although all biocides should be used with caution, aldehydes have been reported to be particularly toxic, sensitizing, and irritating to mucous membranes; therefore specific attention should be paid to any local guidelines and standards regarding their safe use. In some geographic areas their use may be no longer be recommended and even restricted. Aldehydes are particularly effective because of their cross-linking activity, in particular, with proteins and nucleic acids. Some amino acids are known to be particularly sensitive to these cross-linking reactions (e.g., lysine and arginine). Glutaraldehyde is one of the most widely studied biocides from a mode of action point of view, being shown to be particularly active on the various microbial surface structures, such as the bacterial cell wall, the effects of which alone are sufficient to inactivate bacteria and other vegetative microorganisms. Formaldehyde and orthophthaldehyde acid (OPA) are considered to be capable of penetrating further into microbial structures, with formaldehyde known to generally react with available carboxyl, sulfhydryl, and hydroxyl groups. Unlike formaldehyde, OPA does not seem to be capable of penetrating bacterial spores but does have some sporidical activity over much longer exposure times, although it is its effects are rapid against vegetative bacteria, including and particularly mycobacteria. Halogens Halogens and halogen-releasing agents are widely used for antiseptic, preservative, and disinfectant purposes. These include chlorine and iodine, although bromine is also widely used industrially. The chemical equilibria of the halogens in solution can be rather complicated. For example, ‘chlorine’ is one of the most widely used disinfectants for surface and liquid (e.g., water) applications.

The true activity of chlorine in water is actually due to a combination of three actives namely Cl2 (chlorine gas), OCl (the hypochlorite ion), and predominantly HOCl (hypochlorous acid). Most households, for example, keep a ready supply of ‘bleach’, a stabilized dilution of sodium hypochlorite (typically at 5%), which is one of the most widely used disinfectants. A one in ten dilutions of freshly prepared bleach (or more correctly 0.5% hypochlorite, as bleach is provided at many different concentrations) is widely recommended in hospitals, laboratories, and other institutions because of its demonstrated rapid virucidal, bactericidal, fungicidal, and somewhat slower sporicidal activity. However, its antimicrobial activity will vary and is often taken for granted, with little data provided to confirm its efficacy under the conditions of use. In dilution it is not only important to consider concentration and time, but also pH, where a neutral pH is optimal and activity is reduced by a factor of 10 for every pH unit above 7. Also, active chlorine solutions are reactive and short-lived biocides. For this reason, only freshly prepared dilutions should be used and reuse overtime should not be considered. For some applications, the amount of active (free) chlorine measured in a given preparation should be monitored to ensure activity, which can be easily done using a variety of commercially available methods. Water (and water system) disinfection concentrations also vary, with a typical recommendation at 0.5 to 5 mg l 1 of free (active) chlorine. Chlorine and chlorine-releasing agents (such as the chloramines) are widely used for surface and water disinfection; lower concentrations can provide adequate preservation, as widely used in drinking and swimming water. Because of its reactive nature, repeated use on surfaces can be damaging, in particular at the concentrations required for true disinfection. Chlorine is generally considered safe for use and demonstrates low toxicity, but can be irritating at higher concentrations (particularly when heated or present in gas form). Hypersensitivity is a common side effect with chlorine exposure and disinfection by products such as trihalomethanes can pose health risks if not routinely monitored. Iodine, or the more widely used iodophors (iodinereleasing agents), also demonstrate broad-spectrum activity, but require higher concentrations and exposure times to show sporicidal activity against bacterial endospores. Similar to chlorine, activity depends on the amount of ‘free’ iodine and water and the true antimicrobial activity is because of the presence of molecular iodine (I2) and hypoiodous acid (HOI). Although simple solutions of iodine or iodine in alcohol still have some use (in particular from an antiseptic or wound-treatment point of view), the various iodophors or other iodine-releasing agents are more commonly used. An example is PVPI (povidone-iodine), which releases iodine over time for antimicrobial activity and is used in many hard-surface

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disinfectants and antiseptics (e.g., Betadine). In both cases, products are generally stabilized and release iodine on dilution in water, which should be done according to manufacturer’s instructions, and will vary considerably depending on their formulation. At typical used concentrations, iodine also has low toxicity making it a particularly useful antiseptic (being less reactive as a halogen than say chlorine), but can be undesirable because of staining of surfaces/materials. Although less widely used and referenced, bromine (released on dissolving NaBr or BrCl in water) and bromine-releasing agents (such as STABREX and Bronopol) are also used primarily as water-based or surfaceimpregnated disinfectants. The active agents in solution are hypobromous acid and the hypobromite ion, the antimicrobial activity of which will be dependent on their actual concentrations in a given application. Bromine can demonstrate broad-spectrum antimicrobial activity, including cysticidal, algicidal, and sporicidal activity at higher concentrations/exposure times. Although bromine is considered less corrosive than chlorine, reactions with other chemicals (in particular during water treatment) can lead to the production of toxic by-products. Overall, the halogens are useful biocides, but it is also important to note that in all cases they are not very stable and are particularly sensitive to the presence of contaminating organic molecules (such as proteins and carbohydrates) that are often associated with microbial growth and survival. This negative effect on halogen activity may be counteracted by using higher concentrations in such applications. Further, their relative instability has as opposite advantage environmentally as they do not accumulate. Clearly, these overall advantages need to be balanced and controlled with potential disadvantages of toxic by-product generation and damage to various surfaces treated. The primary mode of action of all the halogens is due to their potent oxidizing agent activity, thereby attaching the various molecules and structures that make up microbial structure and function. Specific reactions with proteins, lipids, and nucleic have been described, which appear to culminate in the observed antimicrobial activity. Epoxides Epoxides include propylene oxide (rarely used in some fumigation applications) and ethylene oxide (EO, also known as ETO), but only is widely used in recent times and in particular in sterilization processes. Prior or limited other uses include room or other area fumigation. EO is a stable, colorless gas with a slightly sweet, aromatic odor. It is generally provided in liquid form in compressed gas cylinders, being heated under vacuum to generate the gas. It is the most widely used method of low temperature sterilization (as an alternative to heat and radiation-based methods)

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Figure 5 An example of a small ethylene oxide (EO) sterilizer, showing a user inserting a gas cylinder into the chamber.

including materials such as medical devices (including reusable medical devices in hospitals), bandages, foods, paper, fabrics, and pharmaceutical products. This is particularly due to its broad spectrum efficacy, stability (for penetration), and compatibility with a variety of surface materials. EO is used as part of controlled sterilization processes in dedicated exposure chambers (sterilizers; Figure 5), with its typical activity based on efficient air removal from the load/chamber, temperature distribution (in the 30–70  C range), humidity (40–80%), EO gas distribution (400–1200 mg l 1), and finally exposure time. Air removal is particularly important, not only due to the inhibitory effects in gas/humidity distribution but also due to the explosive nature of EO in the presence of 3% air. EO demonstrates broad-spectrum (including sporicidal and cysticidal) stable biocidal activity. Despite its stability, it also can be significantly inhibited by the presence of soils and certain types of materials due to adsorption. Bacillus atrophaeus endospores are widely cited as being the most resistant organisms to EO and are routinely used to validate and monitor the various efficiencies of EO sterilization processes. Despite this, it is of interest to note that some forms of the fungus Pyronema domesticum (as ascospores and in hyphal clumps known as sclerotia) demonstrate unusually high resistance to EO processes and may be a particular concern in the lowtemperature sterilization of some cotton-based materials. Owing to its toxic nature at low concentrations (1 ppm), recorded as being carcinogenic and mutagenic, particular attention should be paid to occupational safety (e.g., adequate ventilation). This should be considered during the sterilization cycle itself and on any subsequent material aeration on storage, due to the absorption of the gas into device materials/packaging that is then released over time. Low-level gas monitors are available to detect toxic levels safely and are routinely used. A further issue

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can be various by-products that can form on reaction with EO (e.g., with chlorine to form ethylene chlorohydrin) that are also considered toxic. The toxic nature of the gas has recently restricted its widespread use in hospital or similar facilities, along with the development of quicker turnaround time liquid and gaseous alternatives, but it still remains a significant method of industrial sterilization. The mode of action of EO is attributed to its alkylating agent activity, specifically attacking hydrogen atoms on any available proteins, nucleic acids, and other organic compounds. EO has been shown to be particularly reactive with exposed sulfhydryl and other enzyme-reactive groups. These effects clearly culminate in microbial death. Metals As for other life forms, various metals are required at low levels for microbial survival (e.g., in a variety of structures and biochemical processes) but can be toxic at higher concentrations. Metals such as mercury and tin had traditional uses in disinfectant, preservative, and antiseptic applications, but are now less used as a result of toxicity and bioaccumulation concerns; indeed, it is now the other way around where microorganisms are used for environmental metal bioremediation. The metals that see widespread biocidal use today are silver and, currently to a less effect, copper. Historically, their use in urns and other vessels for food/liquid storage clearly had known beneficial preservative effects in ancient times. Sources of both metals in various applications include silver sulfate, silver sulfadiazine, copper sulfate and copper–silver ionization systems. Applications are wide and varied, including plant sprays (e.g., ‘Bordeaux mixture’ contains copper sulfate), water disinfection (e.g., Legionella control in water distribution systems), preservation and, in particular with silver, application directly in wounds or integration into wound dressings. Both metals demonstrate a broad range of biocidal and biostatic activities, in particular against bacteria, depending on the method of application and active concentration. In general, copper is reported to have broader antimicrobial activity (in particular against fungi, viruses, and algae) and silver to be bactericidal, but primarily inhibitory to other microorganisms; this may indeed reflect differences in the concentrations of both actives used, with silver investigations tending to focus on lower, antiseptic concentrations. In both cases, they may be considered as general cellular/ structural poisons. As positively charged, they have a high affinity for cellular structures leading to disruption of permeability and structure. Protein structures are particularly affected by direct binding to various amino acids, and particularly to proteins containing thiol (–SH) groups; direct interaction with nucleic acids, DNA in particular, inhibits its essential structure and functions.

Peroxygens and Other Oxidizing Agents The most widely used biocides in this group are H2O2, peracetic acid (PAA), chlorine dioxide (ClO2), and ozone (O3). These are potent, broad-spectrum oxidizing agents, but vary in sporicidal activity and material compatibility depending on their concentration and method of application. ClO2 is widely used as a water and, to a lesser extent surface (including medical device) disinfectant, and is distinct from chlorine as a biocide. Sterilization processes (using ClO2 under vacuum) have been described but are not generally in use. It has also been used, at low concentrations, as an antiseptic. Because of its reactive nature and rapid decomposition, it is generally generated at the site of use, by a variety of chemical reactions. A widely used method is by mixing sodium chlorite with chlorine (in gas form or from hypochlorite solutions). As a strong oxidizing agent, ClO2 is bactericidal, virucidal, fungicidal, and sporicidal, depending on the concentration. Typical working concentrations in liquids are in the 0.1–5 mg l 1 range, although for sporicidal activity higher concentrations (>11 mg l 1) are required depending on the contact time. Limited activity against some protozoal cysts and algae has also been reported. In addition to direct or liquid formulation use, ClO2 is also used in gaseous form for fumigation and sterilization applications. When in the gaseous form, ClO2 (in the presence of high humidity) is very reactive at low concentrations and demonstrates broad-spectrum efficacy. A typical fumigation cycle, controlled by ClO2 generators, consists of conditioning, gassing, and aeration phases (to nontoxic residues regarded to be <0.1 ppm). A minimum concentration of 500–550 ppm (at 65% humidity) for 12 h contact time has been recommended for broad-spectrum activity. High humidity (60–75%) levels are required for antimicrobial activity. Because of light sensitivity, fumigation should be conducted in the dark. Breakdown products include chlorine and ClO2 itself, which can be damaging to various different types of metals and plastics over time, depending on exposure concentrations. O3 is one of the more reactive oxidizing agents known, but also rapidly degrades in the environment, highlighting it as an attractive biocide for general use. It is produced at the site of use, by passing oxygen (or air) through a highenergy source (e.g., a UV light or corona discharge). Applications have included water disinfection (at pH 6–7 and 0.2–0.4 mg l 1) and for area deodorization (in the 0.5–3 mg l 1 range). O3 fumigation/deodorization applications also require high humidity levels (70–80%) to be effective under these concentrations, and following application need to be reduced to <0.1 ppm for safety reasons. O3, under these conditions, is an effective bactericide, virucide, and fungicide, but has limited sporicidal activity at typical use concentrations. Sporicidal concentrations have been

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reported at >10 mg l 1, depending on the exposure time and humidity/concentration maintained. Activity has also been shown against protozoal cysts in some liquid applications. O3 sterilization processes have also been developed, using O3 concentrations of 85 mg l 1 under deep vacuum (0.01 kPa) at 85–100% humidity and 30–36  C, for a typical sterilization time of 4–6 h. As one of the most reactive oxidizing agents, O3 can be damaging to surfaces at higher concentrations and is very sensitive to interfering substances (in particular organic materials) that deplete the activity concentration for antimicrobial activity. Liquid H2O2 is commercially available in a stable, clear, and colorless form at a range of concentrations (3–90%). Peroxide solutions may be used directly on the skin or in wounds at 3–6%, with lower concentrations being used for direct eye instillations. Higher concentrations may be used directly or in formulation for surface applications, sometimes in combination with other active agents such as peracetic acid and at lower pH levels. Liquid preparations will vary in antimicrobial activity depending on the concentration and combination with other biocides in formulation. In general, peroxide solutions show broad-spectrum antimicrobial activity, including sporicidal and cysticidal effects at higher concentrations (>20% for simple peroxide dilutions in water). Unlike lower concentrations, considering the direct use in wounds and the skin, higher concentrations can be damaging to surfaces and pose health risks. H2O2 has an excellent environmental safety profile, as it breaks down in the environment to water and oxygen. Similar to other oxidizing agents, it is also sensitive to the presence of soil or other interfering substances that react with and break down the available peroxide for antimicrobial activity; as previously discussed, this may be overcome by the use of higher concentrations of peroxide or over continued exposure time.

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In contrast to liquid preparations, much lower concentrations of H2O2 are required in the gaseous form to be rapidly sporicidal and cysticidal. For this reason, gaseous applications have been used for area disinfection (fumigation) and device sterilization, with typical concentrations in the 0.1–10 mg l 1 range. H2O2 gas (also known as the trademark VHP, for vaporized H2O2) is an odorless, colorless gas that is produced by vaporization (flash heating) of liquid H2O2 (in the 30–59% range, depending on the process) to give a mixture of H2O2 and water vapor. H2O2 gas (as for steam) obeys the gas laws, and is thereby dependent on temperature, volume, pressure, and concentration to be maintained in the gas or liquid form. When the concentration is maintained below its dew point, and therefore in gas form, it demonstrates rapid, broad-spectrum activity and material compatibility. Gaseous peroxide is virucidal, bactericidal, fungicidal, cysticidal, and sporicidal; recent evidence has also confirmed effective activity against prions and protein toxins. Gas processes have the benefit of also being compatible and safe for use on a wide range of materials, including metals, plastics, and other materials (including artwork and electronics); further, the gas rapidly breaks down into water and oxygen. Delivery systems are available that control the decontamination process for enclosed areas, including isolators and rooms, as well as for the sterilization of materials and medical devices (Figure 6). Some sterilization systems use H2O2 gas alone (e.g., the V-Pro1 sterilization process) or in combination with plasma generation as part of the sterilization process (e.g., the range of STERRAD sterilizers; Figure 6). In these latter systems, the plasma is created by adding energy (an electromagnetic field) at the end of the sterilization cycle to assist in the breakdown of peroxide remaining in the chamber. Typical gas concentrations in room applications with peroxide gas are at the

Figure 6 Examples of various types of hydrogen peroxide (H2O2) gas delivery systems. On the left is a mobile gas generator for room/area fumigation, a peroxide gas sterilizer (middle), and on the right is a peroxide gas/plasma sterilizer.

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0.2–2 mg l 1 range and for equipment sterilization at 5–10 mg l 1, efficacy being a function of concentration exposure over time. From a safety point of view, concentrations of peroxide within a given area needs to be reduced to 1 ppm before entry or use. Peracetic acid (PAA; CH3COOH) is bactericidal, fungicidal, virucidal, and sporicidal at relatively low concentrations (0.3%), which can also be dramatically improved by increasing the exposure temperature. Cysticidal activity and endotoxin inactivation have also been described under certain conditions. It is available in liquid preparations in equilibrium with H2O2 and a low concentration of acetic acid. However, these low pH preparations are generally highly corrosive and are rarely used directly. Formulations of the biocide is important to optimize its activity and material compatibility, where increased temperatures can dramatically increase its biocidal activity but also its decomposition. Generational formulations have also been developed, by mixing inert, stable components to generate the working solution of PAA in situ; an example is using sodium perborate and acetylsalicylic acid to produce PAA, via the generation of H2O2 as an intermediate in water. PAA formulations are widely used for device disinfection/sterilization, waste treatment, and high-risk surface disinfection. Gas applications (also depending on high humidity levels), both for areas and as part if vacuum-based sterilization processes, have also be described but are rarely used because of corrosion and safety risks. PAA decomposes into safe, nontoxic products (water and a low concentration of acetic acid, or vinegar). PAA has been described as retaining greater activity in the presence of organic and inorganic soils; however this is primarily due to the lower concentrations being required for antimicrobial activity. Higher concentrations have a strong acetic acid odor and direct contact with the skin should be avoided at typical use conditions. Phenolics Phenol and phenolic derivatives are used as surface disinfectants and preservatives. These include biocides such as chlorophenol and o-phenylphenol, in which most disinfectant formulations usually contain two or more types of phenolics to provide a maximum range of antimicrobial activities. For this reason, products will vary considerably in activity but can include bactericidal, mycobactericidal, fungicidal, and both enveloped and nonenveloped virucidal activity. In general, only certain formulations have true activity against small, nonenveloped viruses (such as parvoviruses) and mycobacteria, highlighting the importance of formulation and the choice of phenolic derivatives used to optimize biocidal activity. Phenolics are not sporicidal, but they do inhibit spore germination and outgrowth. Formulations will also vary in toxicity,

can be corrosive, and have a strong ‘institutional’ odor. Their associated strong odors and stability of the biocides on surfaces may be limiting in some applications. They are widely used as general surface disinfectants, including walls, floors, and equipment. A certain number of phenolics have also been particularly used as antiseptics such as the bisphenols (triclosan and hexachlorophene), chloroxylenol (PCMX), and salicyclic acid (the latter was previously considered as an acid). Triclosan, for example, is widely used in various antiseptics such as antimicrobial soaps and toothpastes because of its very low irritation index in comparison to other biocides. Triclosan has a rather narrow spectrum of biocidal activity, being a particular effective bacteristatic and fungistatic agent. It is rapidly bactericidal against Gram-positive bacteria and virucidal against enveloped viruses, although activity against other bacteria and fungi can be enhanced in combination with other formulation ingredients; therapeutic applications have been described against some protozoa, such as Plasmodium. Salicyclic acid is widely used as a skin antiseptic, for example, in the treatment of acne and psoriasis, because of its antibacterial, antifungal, and keratinolytic activity. Although the phenolics range in activity, they are generally considered cellular poisons, reacting with a variety of macromolecules and microbial structures via their reactive hydroxyl groups. In some cases, in particular with the bisphenols triclosan and hexachlorophene, more specific mechanisms of action have been described in the inhibition of fatty acid synthesis (by inhibiting the activity of enoyl reductases) in some bacteria and other microorganisms. This was particularly a concern in that some antibiotics (such as isoniazid and ethionamide) have a similar cellular target and cross-resistance in Escherichia coli and other bacteria. However, unlike antibiotic resistance, ‘resistance’ to triclosan in these cases was only shown to cause an increase in minimal inhibitory concentration (MIC) of triclosan and the biocide has been shown to have multiple effects on these strains at higher (minimum bactericidal) concentrations. Despite this, the association between biocides such as triclosan and antibiotic resistance in bacteria has been the subject of some debate with certain biocides that have known, limited activity in comparison to other broad-spectrum biocides. Quaternary Ammonium Compounds Quaternary ammonium compounds (commonly known as quats or QACs) are cationic surfactants (surface active agents) that combine bactericidal and virucidal (generally only enveloped viruses) activity with good detergency and, therefore, cleaning ability. Although other surfactant types, such as anionic, nonionic and, amphoteric surfactants (referring to their overall charge) have some antimicrobial activity depending on the specific biocide,

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the cationic surfactants (and some of the amphoterics) have the greatest antimicrobial activity. Examples include hexadecyltrimethylammonium (‘cetrimide’), chlorhexidine, and benzalkonium chloride. As for other biocides, the activity of QAC-based formulations will vary significantly based on the types of biocides used and their respective formulations. Given that their primary mechanism of action is the structure/function disruption against cell membranes, they generally demonstrate bactericidal and fungicidal activity, with further activity observed against enveloped viruses. QACs are also potent microstatic (including sporistatic) agents, but only limited formulations have claimed activity against mycobacteria (presumably by combination of other formulation excipients that allow greater penetration of the mycobacterial cell wall structure) and are generally cited as being nonactive against nonenveloped viruses. Activity can be affected by the presence of water hardness (when used to dilute a concentrated product), fat-containing substances, and anionic surfactants. QACs have a pleasant odor, are not aggressive on surfaces, and have low toxicity. They are widely used as cleaners/disinfectants on general, noncritical surfaces, including the removal of gross soil. QACs and other surfactants are also used as preservatives (e.g., in paints and cosmetics). Some QACs and amphoterics are also used at low concentrations as antiseptics. The most widely used ones are the biguanides and in particular chlorhexidine (chlorhexidine gluconate, CHG) and polymeric biguanides (e.g., Vantocil). CHG is used in such products as antimicrobial soaps (e.g., Hibiclens), mouthwashes, wound dressings, and in contact lens storage solutions. In these applications, in addition to direct antimicrobial activity, CHG has the further benefits of low irritation and binding to, and remaining on, the skin and mucous membranes at low, bacteristatic concentrations following application (thereby providing longer term or ‘substantive’ antimicrobial protection). In addition to antiseptic applications, the polymeric bioguanides are also used as general disinfectants and for water sanitization (as chlorine alternatives). Overall, the antimicrobial activity of CHG and the polymerics are similar to other QACs, but have limited fungicidal activity in their own right that can be enhanced in formulation but are fungistatic and sporistatic at low concentrations. As for the QACs, the cell membrane is the main target for antimicrobial activity and the action of CHG in particular has been well studied. Being positively charged, they are rapidly attracted to the cell wall surface, with initial surface structure disruption, penetration to the cell membrane, and direct insertion to and interaction with the phospholipids, leading to structure/function disruption (including leakage of cytoplasmic components); these effects culminate in cell death and loss of viability of enveloped viruses.

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Other Miscellaneous Biocides For the purpose of this review, although it is not possible to describe all the various types of chemical biocides in detail, there are other miscellaneous classes of biocides that should be briefly considered. The anilides (such as triclocarban and tribromsalan) are used as preservatives and in antiseptics (cosmetics and antimicrobial soaps). They are generally bacteristatic and fungistatic, with little to no investigations on activity against viruses and other microorganisms. Their main target of activity is the cell membrane, by binding to and interfering with its structure and functions. A variety of dyes are also used for their biocidal activities, such as the acridines (in antiseptics, such as topical wound dressings as alternatives to antibiotic treatment) and crystal violet (e.g., in veterinary tinctures and as water preservatives). Although specific activity will vary depending on the biocide and their preparations, they are generally bactericidal and can be fungicidal (or at least fungistatic and algistatic), whereas their activity against viruses have not been studied much. The primary mode of action of acridines is intercalation into DNA structure, thereby disrupting its structure and functions. Further inhibitory effects include certain types of enzymes and other intracellular/extracellular interactions. The other dyes appear to have more generalized activity, with a variety of effects reported included intercellular radical formation and intercalation into cell wall peptidoglycan. The diamidines, such as propamidine, are primarily used as antiseptics because of their low toxicity. Applications include their use in creams/ointments for wounds and in eye drops, as well as their specific applications for antiparasitic chemotherapy. They have been described as being bactericidal, fungicidal, and active against the vegetative forms of parasites such as Leishmania and Pneumocystis. The cell membrane appears to be the main target for activity, with specific disruption of the structure and functions being described in Gram-positive and Gramnegative bacteria, as well as on the amoebael plasma membrane. In addition, interference with the activity of certain cytoplasmic enzymes and nucleic acids has been described. In addition to their uses as fragrances, various essential oils have been appreciated for their low level antimicrobial (in particular antibacterial) activities for many years, with their widespread use in apothecary applications; however, it is only recently that their true activity and analysis of the various chemicals responsible have been scientifically investigated. They are extracted as oils from various plants, such as tea tree, pine, and eucalyptus. Chemical analysis of these oils has found that a variety of biocides are present, including terpenes, terpenoids, and oxides that combine to give the overall antimicrobial activity. Overall, they range in bactericidal and fungicidal activity, but primarily are bacteristatic and fungistatic under typical use conditions.

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Tea tree oil is one of the most studied, with greater activity against Gram-positive bacteria, greater resistance observed against Gram-negatives, and fungicidal activity depending on the concentration used. Limited reports suggest some activity against enveloped viruses. Equally, the mode of action is difficult to determine based on the mixture of chemical present, but the major effects appear to be related to the cell wall/membrane structures and functions, as well as a variety of intercellular toxic effects. Finally, although they are not true ‘chemical’ biocides, various peptides and some enzymes have been shown to have biocidal activity. Lysozyme, for example, has limited activity against bacteria because of the enzymatic degradation of peptidoglycan, and is used in some applications as a preservative. Cationic peptides such as nisin (food preservation) and the magainins (skin infection treatments) have also been used. Nisin is particularly active against Gram-positive bacteria and has static activities against other microorganisms, whereas the magainins have more broad-spectrum activity with published bactericidal, fungicidal, and some protozocidal activity. Their main activity appears to be focused on the cell membrane, although other effects may include DNA and intracellular protein affinity. In addition to development of new biocides, new biocidal processes have been described and are the focus of further research. These include physiochemical processes such as plasmas. Plasma is essentially an excited or highly energized gas. Antimicrobial activity has been particularly associated with plasmas generated with nitrogen and oxygen mixtures or other oxygen-containing gases (e.g., H2O2). They can have rapid antimicrobial activity, associated with various oxidants or other reactive ions being produced on excitement of the gas, but are shortlived and nonpenetrating. Their antimicrobial activity is certainly related to the production of various oxidizing agents but conversely it can be damaging to surfaces at typical antimicrobial conditions.

An Introduction to Biocide Resistance In the study of microbiology, much attention is focused on the understanding of the mechanisms of action and subsequent resistance to the various types of anti-infective drugs (such as antibiotics and antiviral agents). In most of these cases, the development of resistance can often be due to minor changes in the specific structural or functional targets in normally sensitive microorganisms. Anti-infectives generally have a limited spectrum of activity and specific mechanisms of action, allowing for their safe use at very low concentrations within the infected host. In contrast, much less is known about biocides and biocidal processes, although this is of growing interest considering the widespread use of these physical

and chemical effects. Considering, in most cases, their broad-spectrum efficacy, their application methods, and more nonspecific mechanisms of action, it is often cited that microorganisms once susceptible are not expected to develop resistance to biocides. This is certainly not the case. Microorganisms are prolific survivors and have shown varied adaptability to circumvent biocides and biocidal processes. A simple example is in the study of extremophiles, or microorganisms with extreme resistance to ‘adverse’ environmental conditions. Various types of eubacteria and archaea have been identified that survive and grow at extremes of temperature, acidity and salt conditions; indeed, many of these do not grow under what we may consider ‘normal’ laboratory conditions and are adapted for their specific niche environments. Surprisingly, investigations into their various structures and survival mechanisms have shown many similarities to other well-studied bacteria (such as Bacillus, Pseudomonas, Escherichia, and Staphylococcus), and their resistance factors appear to be cumulative to allow for growth. This simple example, as well as others in the literature, reminds us not to be complacent in the study of microbiology and particularly in the use of various methods to control microbial growth. Indeed, in some examples, biocide resistance development has been shown to be dramatic with significant outcomes, including in some limited situations of cross-resistance to antiinfectives such as antibiotics. Mechanisms of microbial resistance to biocides and biocidal processes are only considered briefly in this section. They can be generally classified as being an intrinsic, innate, or natural ability within a given microorganism, or acquired by mechanisms not dissimilar to those particularly described in bacteria and fungi (i.e., mutation and acquisition of genetic material). Intrinsic Resistance A summary of some of the most notable mechanisms of microbial resistance to biocides are shown in Table 1. These mechanisms of resistance are in addition to the intrinsic mechanisms due to the various structures of microorganisms that afford them varied resistance to biocidal effects (see Figure 1 and associated discussion). For example, the cell wall structure of Gram-negative bacteria demonstrates greater resistance patterns to biocides than the Gram-positive cell wall, presumably due to biocide penetration. Similarly in viruses, the smaller nonenveloped viruses provide mechanisms of survival to drying and biocides than the relatively sensitive enveloped viruses; the associated envelop structures in the latter viruses are clearly more sensitive to damage and are known to be required for cellular infectivity. These mechanisms may be further considered as being passive or active. A typical passive example is the case

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Table 1 Examples of the mechanisms of intrinsic resistance to biocides and biocidal processes Mechanisms

Description

Examples

Stress responses

Various mechanisms of microbial survival under adverse environmental conditions (including stationary phase of growth)

Efflux

Pumping of specific biocides out of a target cell via the cell membrane; prevents toxic accumulation

Enzyme or chemical production

Production of enzymes and/or chemicals that neutralize the biocide

Aggregation

The ability to protect due to clumping or aggregation around a microorganism Communities of microorganisms (single or multiple species) that develop on or are associated with surfaces and interfaces The ability to dramatically change structure and metabolism into a dormant state of growth

Structural changes (dwarfism in Escherichia coli). Lower metabolic rate. Stringent response. SOS response. Enzyme production and biocide inactivation NorA expression in Staphylococcus aureus causes efflux of some biocides (dyes and QACs) and antibiotics (fluoroquinolones). MexAB-AprM efflux system in Pseudomonas aeruginosa can lead to biocide ( -lactams, fluoroquinolones) and antibiotic resistance (triclosan, QACs, and acriflavine) Catalase degrades hydrogen peroxide (H2O2) to water/oxygen. Formaldehyde dehydrogenase breaks down formaldehyde. Glutathione, as an antioxidant, neutralizes oxidizing agents Fungal hyphae production. Viral clumping

Biofilm development Dormancy

with mycelial development in mold growth and viral clumping, which can allow for some protection from the biocide by essentially sheltering the organism. In some cases, the attack of the biocide can actually protect a certain proportion of the microbial community, due to clumping or cross-linking of external materials/microorganisms and limited penetration to the remaining, inner survivors. This has been shown particularly with the coagulation/cross-linking reactions associated with some biocides (e.g., formaldehyde and glutaraldehyde) and with virus inactivation (e.g., as in the case of the famous Cutter incident in 1955, in which virus clumping protected live virus from formaldehyde inactivation, used in the preparation of polio vaccines). A further novel example of resistance in viruses is described as multiplicity reactivation, in which the viral structure can be broken apart to be considered noninfectious but by reassociation or cooperation between the various subcomponents that infectivity can proceed; indeed, this is highly possible with nonenveloped viruses in which the nucleic acid itself is only required for infectivity and therefore biocides that only break down the viral structure and do not inactivate the nucleic acid may not be considered to be completely effective. Although many of these types of mechanisms alone may not be sufficient to protect the microorganism against the assault of a given biocide, under minimal conditions or when combined together with other mechanisms they can allow for survival under normal use conditions. A typical example of these cumulative

Pseudomonas biofilm development in water systems. Staphylococci biofilm development on the skin or associated with medical devices Geobacillus, Bacillus, and Clostridium spore development. Fungal spores (e.g., Aspergillus ascospores). Helminth egg and protozoa cyst/ oocyst development

effects is with the active mechanism of biofilm development. Biofilms may be defined as communities of microorganisms, which can be single or multiple species that develop on or are associated with surfaces and interfaces. Certain bacteria and fungal types have been widely appreciated for their ability, and consequences of biofilm development such as Pseudomonas and other pseudomonads (in water distribution systems), Staphylococcus (various species in periodontal disease, skin, and wound infections, and device-related infections), Legionella (air/ water handling systems), and Candida (device-associated and endodontic infections). Biofilms can become very diverse in the types of microorganisms present within and associated with their structure; for example, in addition to actively dividing ‘base’ bacteria or fungi, various other microorganisms can be essentially trapped or can feed on its matrix, such as viruses and protozoa respectively. Biofilms can not only lead to infectious or contamination problems but can also lead to significant surface damage (e.g., biocorrosion) or product fouling. The resistance mechanisms involved include: attachment, in which microorganisms are gen• surface erally considered to be more difficult to inactivate in comparison to planktonic cells;

due to extrapolysaccharide and/or protein • protection matrix production, thereby limiting penetration into the biofilm and physical removal from the surface;

growth deeper within the biofilm, in compar• slower ison to the surface, with associated stationary phase

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•

phenomenon such as biocide-neutralizing enzymes/ chemicals, stress responses, and dormancy; various acquired mechanisms of resistance due to an excellent environment for exposure to sublethal concentrations of the biocide, close relationship between microorganisms and subsequent genetic exchange.

It is important to note that, in most cases, when the various bacteria or fungi associated with the biofilm are isolated, they are generally not found to be individually, or genotypically, resistant to biocides, although acquired resistance under these conditions has been described and should not be surprising. The most dramatic mechanisms of intrinsic resistance to biocides are associated with dormancy. Various types of microorganisms have the remarkable ability to respond to adverse environmental conditions and change from normal vegetative growth to produce dormant, resilient forms of themselves. Examples include endospore formation in certain bacterial species (with the notable examples of Bacillus, Geobacillus, and Clostridium species), fungi (yeasts and molds), helminths (eggs or cysts, as well as differences in adult and larval stages in their respective life cycles), and protozoa (cysts and oocysts, with the notable example of Cryptosporidium parvum oocysts that have significant resistance to biocides including chlorine and others used for drinking water disinfection). Indeed, the endospores of various bacterial species are used to test and confirm the efficacy of various sterilization processes against which they are known to be the most significantly resistant microorganisms, such as G. stearothermophilus (for steam, moistheat, or some oxidizing agent-based processes), B. atrophaeus (previously known as Bacillus subtilus, for EO and dry-heat sterilization), and Bacillus pumilus (in radiation processes). Of all these, bacterial endospore formation has been particularly well described (both biochemically and genetically) in the literature. Endospore formation involves a complex adaptation in the transcriptional and translational machinery of the mother cell that finally leads to the release of a single endospore. Endospores are essentially dormant biochemically and their specific structures can vary from species to species. They generally consist of an internal core structure (containing the nucleic acids, protective proteins, and chemicals, such as the small acid-soluble proteins and calcium dipicolinic acid, respectively) that is surrounded by various membranes, walls, and coats. It is the combination of these protective mechanisms that limit the penetration and inactivating effects of various physical and chemical biocides. If a typical endospore core structure alone is considered, it is essentially dehydrated (which limits penetration of chemicals/heat) and the DNA structure is stabilized/protected by the presence of the calcium dipicolinic acid and associated, stable proteins. External to the core, the various protective layers consist of lipid/ protein, peptidoglycan, and protein complexes, which

further limit chemical and heat penetration. Although the exact structures and mechanisms of development have been less described for fungi, helminths, and protozoa, their various forms also present greater resistance to chemical biocides but in general are not highly heat resistant. Various types of fungal asexual and sexual spores have been described, although in general they are consider less resistant that bacterial spores yet more resistant than their respective vegetative forms. A notable example is the sexual spores of Aspergillus species (ascospores), which are considered some of the more resistant fungal spores to biocidal processes and are therefore used in some geographical areas (e.g., Europe) for confirming fungicidal activity claims on products/processes. In some helminths, a further protective mechanism is the focus of recent research with emphasis on free-living amoeba such as Acanthameoba species. It has been known for some years that various types of bacteria and even viruses can survive within the vegetative, trophozoite stages of their life cycles; indeed, amoeba cell culture techniques have been developed as unique methods of fastidious microorganism culturing from various environments (including water). These include Legionella, Mycobacterium, and various Gram-negative bacteria, as well as newly described types of rickettsia and chlamydia-like microorganisms. These organisms not only survive in trophozoites, but are also found in respective cyst forms, thereby providing them a unique mechanism of surviving various biocidal effects. Amoeba are often associated with and feed on biofilms, thereby presenting a unique opportunity for survival and genetic exchange between microorganisms.

Acquired Resistance Acquired resistance to biocides has been described in bacteria, although similar mechanisms are expected in other prokaryotes and eukaryotes such as fungi and protozoa. Further, acquired mechanisms have been speculated to have effects in the resistance of viruses to disinfectants; for example, changes in the structure of proteins associated with viral capsid structures may give rise to greater heat- or chemical-tolerant structures. Acquired resistance may be defined as a genetic change, in which the microorganism acquires the ability to resist the action of the biocide due to mutation or the genetic acquisition of nucleic acids. Mutations are defined as specific, stable changes in the genetic material of a microorganism that result in a change in a given nucleotide sequence, whereas with acquisition the nucleic acid sequence defining the resistance mechanism is introduced into the host via plasmids or transposons. There are some notable examples of acquired biocide resistance in bacteria that have been particularly investigated, in some cases that have been linked with cross-resistance to antibiotics (Table 2).

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Table 2 Examples of the mechanisms of acquired resistance to biocides and biocidal processes Type

Mechanism

Examples

Mutation

Lipid metabolism

Cell wall/membrane fatty acid profile changes in Escherichia coli and Staphylococcus aureus leading to increased inhibitory concentration to triclosan Downregulation or mutations in various porin proteins associated with the outer membrane structure in E. coli (triclosan tolerance) Overproduction or upregulation for efflux systems in Gram-negative bacteria such as E. coli and Pseudomonas, leading to increased biocide (e.g., triclosan, QACs, and dyes) and antibiotic ( -lactams, tetracycline, and fluoroquinolones) tolerance Triclosan tolerance (increased inhibitory concentrations) and crossresistance to isoniazid (an antimycobacterial antibiotic)

Cell wall protein expression Efflux

Active site mutations in enoyl reductases involved in fatty acid biosynthesis Other cell wall structural changes

Plasmid/ transposon acquisition

Efflux

Decreased accumulation, including efflux and sequestration Alteration of cell wall structure Enzymatic degradation

Chlorhexidine and QAC resistance in Pseudomonas stutzeri Glutaraldehyde-resistant mycobacteria (presumably due to carbohydrate changes) Expression of plasmid-associated qac genes in Staphylococcus with increased tolerance profiles to cationic biocides (QACs and chlorhexidine) as well as some antibiotics (e.g., -lactams) Multiple mechanisms of silver and copper tolerance in Gram-negative bacteria Expression of pR124 in E. coli leads to outer membrane changes and increased tolerance to QACs Toluene and phenol degradation by TOM plasmids in Pseudomonas Mercury resistance in Gram-negative bacteria (mercuric reductase) Plasmids that express formaldehyde dehydrogenase in Serratia and E. coli

In most of these cases, the microorganism remains sensitive to the biocide at higher concentrations; therefore it is more correct to refer to these reports as evidence of increased ‘tolerance’ to the biocide. These include upregulation of efflux and degradative enzyme mechanisms, but under certain situations these may allow the microorganism to survive and grow in the presence of the biocide (e.g., under preservative levels). However, in some cases the single or multiple mechanisms lead to therapeutic failure of the biocide under normal use conditions and therefore similar to antibiotic or anti-infective resistance. An example is in the isolation of glutaraldehyde-resistance mycobacteria (e.g., M. chelonae) which can survive extended incubation in concentrations of the biocide and exposure times generally used for disinfection purposes; although the exact mechanisms of action require further understanding, initial reports suggest changes in carbohydrates and the exact (single or multiple) genetic changes are unknown. Interestingly, in the strains analyzed no cross-resistance to antibiotics or other biocides was observed, although the activity with a similar aldehyde (o-phthaldehyde) was somewhat delayed in comparison to tests with wild-type strains and required longer incubation times. This is a clear example that additional mechanisms remain to be identified and described. In most cases of resistance the mechanisms are simply not investigated. Further, the link between

some biocide and antibiotic cross-resistance is intriguing; it is true that the effect of biocide tolerance in these cases are not thought to be significant (as they are generally only associated with minor increases in MICs of the biocide and not bactericidal concentrations) but the associated changes in antibiotic resistance are clearly a concern (given their therapeutic use at lower concentrations). Most of these investigations have been conducted under laboratory conditions and any true effect in clinical use remains to be elucidated.

Concluding Remarks The many types of physical and chemical biocides available provide an extensive armamentarium for various preservation, disinfection, and sterilization applications. This review has only touched on the range of biocide types, their uses, activities, and mechanisms of action. They clearly play an important role in infection prevention, contamination control, and public health, in addition to their economic benefits. Indeed, their use is responsible for some of the remarkable advances we have made in modern living. An example is reducing the impact of water-, food-, and medical-related infections, at one time commonly associated with morbidity and mortality. Many of these biocides had been successfully used before

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the antibiotic and anti-infective revolution, but now with growing resistance mechanisms and restrictive recommendations around anti-infective use it may be time to renew our interest in the study and optimization of the use of biocides for various applications. Why use an antibiotic in some cases when a biocide could have an equal effect with less risk of adverse events? We have much to learn about the biocides themselves as well as extending their applications safely, and insisting on their prudent and correct use. A recent example is with the greater understanding on the mechanisms of action of and microbial resistance mechanisms to triclosan. This not only highlighted the importance of its correct use but also identified potential new applications (in antiprotozoal activity); further, we know that triclosan in a unique way not only targets key enzymes in bacterial fatty acid synthesis but also other major cellular metabolic processes (such as carbohydrate production) and from these investigations could lead to the development of less specific anti-infective molecules with less risks of resistance development. The emphasis in recent years is not on the development of new biocides, but on using biocides in different and better ways to optimize their benefits. However, we have also seen that the use of some chemical biocides add concerns of their own, in particular with resistance development, speculated cross-resistance to antibiotics, and environmental concerns (depending on the biocide type). These concerns remind us to be ever mindful of the risk–benefit scale that should be considered in the use of any biocidal/biostatic application. It is clear that certain biocide types will continue to be less

used because of safety and efficacy concerns, but these can be easily replaced with safer alternatives. In that way, we can keep all the benefits associated with disinfection and sterilization, but reduce any associated concerns. See also: Antibiotic Resistance; Biofilms, Microbial; Cell Structure, Organization, Bacteria and Archaea; Cosmetics Microbiology; Extremophiles:Cold Environments; Food Spoilage, Preservation and Quality Control; Genetics, Microbial (general); Global Burden of Infectious Diseases; Heavy Metals, Bacterial Resistance; Pesticides, Microbial; Water Treatment, Industrial; Water Treatment, Municipal; Water, Drinking

Further Reading Block SS (2001) Disinfection, Sterilization, and Preservation, 5th edn. Philadelphia, PA: Lippincott Williams & Wilkins. Gilbert P and McBain AJ (2003) Potential impact of increased use of biocides in consumer products on prevalence of antibiotic resistance. Clinical Microbiology Reviews 16: 189–208. McDonnell G (2007) Antisepsis, Disinfection and Sterilization: Types, Action and Resistance. Washington, DC: ASM Press. McDonnell G and Russell AD (1999) Antiseptics and disinfectants: Activity, action and resistance. Clinical Microbiology Reviews 12: 147–179. Russell AD, Hugo WB, and Ayliffe GAJ (1992) Principles and Practice of Disinfection, Preservation & Sterilization, 2nd edn. Cambridge, MA: Blackwell Science. Sattar SA, Tetro JA, and Springthorpe VS (2006) Effects of environmental chemicals and the host–pathogen relationship: Are there any negative consequences for human health? In: Zhu PC (ed.) Biocides Old and New: Where Chemistry and Microbiology Meet, ACS Symposium Series. New York: Oxford University Press.