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New and Emerging Technologies

Peter A. Burke1 and Gerald McDonnell2

1 STERIS Corporation, Mentor, OH, USA2 Research and Technical Affairs, STERIS Ltd, Basingstoke, UK

16

Introduction

Microbicidal practices are hundreds of years old, including the preservation and disinfection of foods/water for consumption, which developed over time to the disinfection/sterilization of reusable medical/dental devices and many other types of materi-als in healthcare, industrial and research settings. For example, Lister in 1867 discovered that simple carbonic acid (phenol), in its own right significantly reduced surgical site infections. Although phenol itself is not practically used for such applica-tions (the limitations of phenol were described by Lister himself), different types of phenolic compounds remain in wide use today as antiseptics and disinfectants [1, 2]. Many types of physical, chemical and combination technologies are in use today as microbicidal agents. Our objective here is to explore the new and emerging technologies in this area. It is worth noting here that many of the basic microbicidal methods in use have not changed much except for certain improvements in their microbicidal activity, delivery of the active, in surface compatibility as well as in enhanced safety for patients, staff and the environment. This applies to physical agents such as heat and also to a wide variety of microbicidal chemicals.

A microbicide can be described as any single chemical or mixture of chemicals used for the purpose of preservation, disin-fection, antisepsis or sterilization. Properly formulating such a chemical or mixture of chemicals with other inert ingredients (sometimes referred to as “excipients”) is the key to the success of a given product for its intended use. The microbicide can either be placed directly in/on the target or released into the air for space decontamination. The main types of chemicals with broad-spectrum microbicidal activity are listed in Table 16.1 [1].

Since the 1950s the introduction of truly new microbicidal agents has been limited mainly due to ever-escalating develop-mental costs coupled with increasingly strict regulations on human safety and ecotoxicity. However, examples of relatively recently introduced microbicides include triclosan, octenidine, ortho-phthaldehyde and the newer generations of phenolics and quaternary ammonium compounds (QACs).

A microbicidal agent must have a wide spectrum of activity against harmful organisms (dependent on its desired applica-tion), be rapid in its action with high materials compatibility while being as safe as possible for humans and the environment. Though these key criteria are obvious, the ability of such chemi-cals to be properly formulated is very crucial and merits further comment. Most microbicidal agents (with the exception of gases

Russell, Hugo & Ayliffe’s: Principles and Practice of Disinfection, Preservation and Sterilization, Fifth Edition. Edited by Adam P. Fraise, Jean-Yves Maillard,

and Syed A. Sattar.

© 2013 Blackwell Publishing Ltd. Published 2013 by Blackwell Publishing Ltd.

Introduction, 371General considerations, 372Ultrahigh pressure and supercritical fluids,

373High-voltage electric pulses, 375Other physical processes, 376Gas plasma, 377Vapor-phase oxidants, 378

Nitric oxide and nitrogen dioxide, 380Bacteriophages and other biological

substances, 380Glucoprotamines, 381Microbicidal surfaces, 381Conclusions, 383References, 383

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with the result that soon the use of many existing microbicides will no longer be allowed in the EU. Also, the inclusion of any new chemicals in the list will be a lot more demanding, in par-ticular, for gases.

General considerations

Process optimizationThe most widely described and used method for killing microor-ganisms is heat, both “dry” and “wet”. Heat can also enhance the microbicidal activity of chemicals. Examples of improvements in applying direct heat include the methods of heat generation (e.g. the use of microwaves), heat distribution (alternatives to fans and optimized pressure/vacuum combinations in steam sterilization processes) and, for food or other liquid applications, other factors such as osmotic pressure, surface tension modifiers and oxygen levels in combination with heat treatment. Other advances in this area relate to the development of alternatives to heat due to the growing use of heat-sensitive materials and devices requiring reprocessing, and to improve organoleptic perceptions in the food and beverage industry. Such physical methods include E-beam radiation, hydrostatic pressure, high-voltage electric dis-charges, high-intensity lasers, high magnetic field pulses and nano-thermosonication, some of which are further discussed in this chapter. Optimization of process conditions has also been an important development in the use of chemical microbicides. The control of variables such as temperature, pH, concentration, etc., has been successful in the optimization of disinfection and steri-lization applications with microbicides. This has been shown with formulated chemistries such as those based on peracetic acid [7] or peracid generational systems [8], as well as for chemical gas sterilization methods such as EtO [9], formaldehyde [10] and ozone [11] where defined temperature ranges, humidity levels

used for space decontamination and some halogens used for dis-infection of water) are formulated with various excipients for specific applications. For example, most liquid disinfectants or sterilants contain surfactants, chelating agents, anticorrosive agents and other ingredients to enhance stability and to optimize activity in the presence of soils.

It should be noted, for example, that formulation effects can actually increase or decrease efficacy as well as toxic effects [3, 4]. In rare cases, synergistic effects between chemical microbicides and/or formulation excipients can be observed, where the micro-bicidal agent(s) alone has less activity than when mixed together in formulation. This is not only true with liquid chemicals, but also important in optimizing the effects of gas or physical micro-bicides. An important example is the effect of water (humidity) content and temperature in the microbicidal activities of ethylene oxide (EtO), ozone and chlorine dioxide. Clinical or other field testing may often have to meet defined criteria for microbial reduction (e.g. the US Food and Drug Administration (FDA) [5], and as proposed by the European Union (EU) EN Phase 3 micro-bicidal tests [6]).

The development and introduction of a new microbicidal formulation can be quite complex and costly, often taking a decade or more for safety/compatibility testing and regulatory approval. In the USA, human-use antiseptic drugs and most medical device disinfectants/sterilants come under the purview of the US FDA, while the US Environmental Protection Agency (EPA) reviews and approves for sale microbicidal products to be used on environmental surfaces and for aerial decontamination of indoor spaces. The ultimate environmental fate of such chemi-cals is reviewed not only by federal government but also by those states that have their own environmental requirements. This is also true in the EU under the Biocides Directive (98/8/EC); in this case, an initial review of the safety and efficacy of various micro-bicides in use today has been made for inclusion in the directive,

Table 16.1 Commonly used microbicidal agents.

Microbicidal agents Microbicidal use Date of introduction

Alcohols (ethanol, isopropanol, n-propanol) Antisepsis, disinfection Alcohols have been described and used for millennia, though not necessarily for their disinfectant qualities. In modern times the first true chemical description was in 865 AD and in 1763 was reportedly used as an antiseptic

Chlorhexidine digluconate Antisepsis 1954Ethylene oxide Disinfection, sterilization 1936Glutaraldehyde Disinfection, sterilization 1957Halogens (iodine, iodophors, chlorine or chlorine-releasing agents such as hypochlorites, related compounds)

Antisepsis, disinfection, sterilization Chlorine discovered in 1744; iodine in the 1800s

Hydrogen peroxide Antisepsis, disinfection, sterilization Discovered in 1818, used in medicine since 1891Mercury, mercuric chloride Antisepsis Middle AgesPeracetic acid Disinfection, sterilization 1955Phenols Antisepsis, disinfection Mid-1800sQuaternary ammonium compounds Antisepsis, disinfection 1916Silver, silver complexes, silver sulfadiazine Antisepsis, disinfection Use for water applications as far back as 450 BC

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hydrogen peroxide itself is of interest due to its latent microbi-cidal activity; complex stabilized formulations known as “accel-erated” hydrogen peroxide (AHP) have been developed as hard surface disinfectants (Virox Corporation). The formulations can range in peroxide concentration (e.g. 0.5–7%), but have much greater microbicidal activity when mixed with peracetic acid than comparable concentrations of peroxide alone. Simi-larly, synergistic reactions between hydrogen peroxide gas and ozone have been proposed, although little evidence of the true effects of each active alone and/or in combination has been published to date.

Ultrahigh pressure and supercritical fluids

The inactivation of vegetative bacteria by ultrahigh pressure (also known as ultrahigh pressure homogenization (UHPH)) is not new, with initial reports demonstrating some effects at or above 100 MPa for over 100 years. As a reference, atmospheric pressure is 101.35 kPa (14.7 psi, 1 bar or 760 mmHg (or Torr) at sea level) and a typical steam sterilization process operates at 204–308 kPa. Therefore, these pressures are quite significant and are proposed for various types of foods and vaccines. The extent of microbi-cidal activity was found to be limited, with bacterial spores, for example, being shown to be much more resistant, surviving pressures higher than 1200 MPa [19]. Practical limitations of high-pressure technology prevented commercial use for food preservation until recently, but applications now include the treatment of jams, fruit juices, meat and an expanding variety of other foodstuffs [20–22]. The effects of high pressure (and there-fore changes in volume) are essentially derived from LeChatelier’s principle on any system in equilibrium, where a change in pres-sure (as well as other variables such as concentration, tempera-ture, etc.) will cause a shift to counteract the change and establish a new equilibrium. Such dramatic changes in biological vegetative systems that are essentially in equilibrium will lead to a variety of detrimental effects, such as the unfolding of protein and lipid structures, increases in the ionization of dissociable molecules due to “electrostriction”, etc. [23, 24]. Such changes may be expected to culminate in cellular death or at least inhibition of growth [25]. Effects on gene expression, protein synthesis, nucleic acids, ribosomes, specific proteins, maintenance of cytoplasmic pH and tran-membrane pH gradients have all been observed [26], although none are expected to be key targets during pressure-induced inactivation. Of interest to the food industry, low molec-ular weight flavor and odor compounds in foods tend to survive pressure treatment unchanged, with quality advantages in some types of products in comparison with heat treatment. Overall, these effects may be limited from a microbicidal point of view as kinetic studies have shown some examples of exponential inacti-vation of cells held at constant pressure (e.g. Escherichia coli [27]), but the majority of the studies have reported “tails” on survivor curves, that is a decreasing death rate with increasing treatment time (Figure 16.1) [28].

and exposure times need to be tightly controlled for optimal microbicidal effects.

Particular advances over the past decade have been in methods of ozone generation ([11] and associated patents), the use of UV light [12] and various proposed synergistic methods (such as UV and ozone and/or hydrogen peroxide gas).

Formulation optimization, including synergismIt is outside the scope of this chapter to describe the various types of formulation effects that have or could be used to enhance the activity of a microbicide. In short, a variety of effects can be combined to demonstrate optimized activity, stability, tolerance of variable water quality (with dilutable products), and synergism between chemicals; many of these effects have been successfully patented. A novel, recent example is the description of a liquid formulation including 0.2% sodium dodecyl sulfaate (SDS) and 0.3% NaOH in 20% n-propanol that was shown to be effective against surface prion contamination [13]. In addition, such for-mulations also showed significant inactivation of viruses (polio-virus, hepatitis A virus and caliciviruses), bacteria (Enterococcus faecium, Mycobacterium avium) and fungi (Aspergillus brasilien-sis). Alkali are known to be effective microbicidal and anti-prion agents, but generally at relatively higher concentrations (e.g. 1 m NaOH is widely used for both purposes), although recent evi-dence would suggest that such high concentrations are not required for activity against prions and that formulation concen-trations can have a dramatic impact even against these agents [14]. Similarly, concentrations of alcohols are known to enhance the activity of aldehydes such as glutaraldehyde in high-level dis-infectant applications [15].

Direct synergism between microbicides is often a debatable point [16]. Synergism in this context is defined as two or more microbicides that work together so the combined microbicidal effect is greater than the sum of the individual microbicides alone. With this in mind, many have claimed to observe synergistic benefits with combinations of microbicides but have not excluded the cumulative effects of the individual components [16, 17]. Despite this, many synergistic effects can be observed. A common example is in the use of chelating agents such as ethylenediamine tetraacetic acid (EDTA; a weak microbicide but some preservative qualities) with triclosan, other chelating agents and some QACs. These effects are limited to certain types of bacteria and appear to be from destabilization of the cell wall/membrane, allowing the microbicide access to its target site(s) [18]. Another example is the acidification of hydrogen peroxide for increased mycobacte-ricidal effects, possibly due to the disruption of the highly lipophilic cell wall structure of these bacteria.

The reported synergistic effects between hydrogen peroxide and peracetic acid in formulation may be primarily due to preservation of the peracetic acid concentration (known to be a much more potent microbicidal at lower concentration than peroxide) as such solutions are always in equilibrium. Although these are also known microbicides, their contribution to the activity in formulation is debatable. The formulation of

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pressure can cause spores to initiate germination and further pressure increases can then inactivate the vegetative forms. This has led to further studies on the synergistic effects with factors such as temperature [37] and low-dose irradiation [38] to achieve a higher level of spore inactivation. Such effects have been con-firmed for a wide range of spore types, although the effectiveness of the combination varies greatly in magnitude for different spores [39, 40]. Bacillus cereus spores, for example, are considered relatively sensitive to pressure, while those of others such as Geo-bacillus stearothermophilus and Clostridium botulinum are quite resistant [21]. This may change with the development of presses that operate at higher temperature–pressure combinations, or the development of other effective combination techniques. The fact that high-pressure application can directly lead to temperature increases, for example by about 3°C per 100 MPa for water, and as an opposite effect of cooling, allows pressure to be used to heat/cool various products very rapidly. For example, the application of pressure at 800 MPa may raise the temperature of products, without any need for time/energy-consuming conduction or con-vection, very quickly by about 25°C and, most importantly, reduce the temperature by the same amount as the pressure is reduced at the end or treatment, perhaps within a few seconds. Such rapid temperature changes could be very useful in gaining much tighter control over thermal processes, with reduced heat-induced damage, and consequent benefits in product quality [41]. Opti-mization of such processes will have benefits to certain applica-tions, particularly in food production.

A further application of high pressure is in the use of “super-critical” (or dense) liquids as disinfecting/sterilizing agents for surfaces and/or materials. Supercritical fluids are defined physi-cally as any substance that is above its critical point, where the phase boundary between a solid, liquid and gas no longer exists. Essentially, any matter can exist as a solid, liquid or gas depend-ing on the pressure and temperature. A typical example is with water that forms a solid (ice) below 0°C, when heated assumes its liquid phase (water) and at higher temperatures (>100°C) forms a gas (steam). Equally, as the pressure is changed, the phase can change (due to the relationship between temperature and pressure described in the gas laws), where, for example, water can be maintained in a liquid form at temperatures above 100°C by changing the pressure. Therefore, at increased tem-peratures and pressures, typical examples such as water and carbon dioxide cross the phase boundaries of being a liquid/gas to become supercritical fluids. By controlling very high pressure levels, the temperatures can be maintained at low levels for more sensitive applications.

As an example, supercritical carbon dioxide can be generated above the critical temperature (c. 31°C) and pressure (c. 7.4 MPa); under such conditions it assumes the properties of both a gas and a liquid. Supercritical fluids are used industrially for a variety of purposes, such as water for power generation and carbon dioxide for dry cleaning, decaffeination and oil extraction (often used for their intrinsic microbicidal properties). Supercritical carbon dioxide itself has been described for its direct microbicidal

It has been proposed that at higher temperatures (e.g. in the case of E. coli, 40°C and above at 250 MPa) inactivation is near first order, whereas at lower temperatures (e.g. 30°C for E. coli) it is nearer second order, and that membrane lipid changes may account for these differences [29]. Temperature may play an important role in these effects, with such pressures shown to be more effective in inactivating vegetative bacteria at the lower (<0°C) than the higher (>10°C) temperatures [30]. Indirect effects from pressure changes may be important; for example, pressure increases ionization that can lead to a reduction in pH. Many vegetative bacteria are known to be quite sensitive and others resistant to acidic pH [1]. pH effects may also have benefits in combination with various microbicides, for example in the activity of sorbic acid against Saccharomyces bailii [31]. Further, such effects may be specific to the material being treated, as shown with foods. Salmonella enterica serotype Typhimurium was inactivated more efficiently in pork in 10 min at 300 MPa, than in baby food at 350 MPa [32]. The spectrum of antibacterial activity may also be limited in such cases, as strain-to-strain vari-ability in sensitivity to pressure is greater than with other inacti-vation techniques, such as heat [28]. Exponential-phase bacteria are more pressure sensitive than when in the stationary phase [33] and Gram-positive bacteria are more pressure tolerant than Gram-negatives [34]; overall, these generalizations will vary depending on the target/resident bacteria and the food product under investigation. Despite these variations, a number of suc-cessful applications have been published, such as against a wide range of bacteria and fungi in fruit juices at 100, 200 and 300 MPa, and at 2 and 4°C [35], with similar results in milk [36].

Although bacterial spores are known to be pressure tolerant, under certain conditions inactivation of the spores proceeded more successfully at lower pressures [37]. This may be explained by the inactivation of spores in two stages. First, increased

Figure 16.1 Hydrostatic pressure–survivor curves of Yersinia enterocolitica in pH 7 phosphate buffered saline at 20°C showing the effect of increasing pressure and the non-linearity of survivor[28].

0

0

–1

–2

–3

–4

–5

–6

–7

–85 10 15

Time (min)

log

(N

/No

)

20 25 30

300 MPa275 MPa

250 MPa

225 MPa

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from about 10 to 100 kV/cm, with very short pulses (micro- or millisecond ranges) in repeated delivery to obtain the desired inactivation. The rate and duration of electric pulsing can prevent excessive increases in the temperature created [49]. In some appli-cations, limiting any unwanted thermal effects are important (e.g. food processing) but in others this may be less of a concern. While the effects of high-voltage fields on microorganisms are not understood at the molecular level, the gross effects and the mech-anisms that cause them are well described. Significant effects result from the permeabilization of the cell wall/membrane [50]. Gross structural effects occur when the potential difference across the membrane exceeds about 1 V – not surprisingly considering the electrical balances across bacterial cell membranes that are important for cell metabolism and outer cell structures. This was rapidly followed by massive leakage of the cytoplasm, culminat-ing in cell death. Microscopical examination of electrically-treated cells has shown an initial thinning of the membrane, followed by hole formation and leaking of the cytoplasm.

Electric pulse inactivation has been reported to have a limited effect on vegetative bacteria, yeasts and molds, including E. coli, Salmonella spp., Staphylococcus aureus, Listeria monocytogenes, Saccharoomyces cerevisiae and Candida albicans [49, 51]. Consid-ering their structures and dormancy, bacterial spores [52] and yeast ascospores [53] are a greater challenge and appear to be resistant, even at very high-voltage gradients (i.e. above 30 kV/cm). There may be some benefits in applying low concentrations of chemical microbicides in combination with such electric pulses to enhance sporicidal activity. Indeed a number of intrinsic and extrinsic factors have already been shown to influence the effec-tiveness of the electrical treatments. Inactivation increased greatly with elevations in temperature, pH variations and osmotic pres-sure of the surrounding media [54]. For example, in a skimmed milk application, a reduction in pH (from 6.8 to 5.7) led to a doubling for observed E. coli reduction. It may be expected from these results that the growth phase of the target organism will also be important, a phenomenon not usual in disinfection studies or in genetic electroporation experimentation.

Some other potentially useful synergistic reactions have been described. For example, electroporated cells of E. coli, L. monocy-togenes and S. enterica serovar Typhimurium were more sensitive than untreated cells to microbicidal peptides such as nisin and pediocin [55]. These results suggest that the greater peptide pen-etration is achieved through electrically-treated cells to access internal cell targets of these actives.

Electric pulse treatments have been applied to a number of liquid foods as an alternative to cold pasteurization methods against vegetative bacteria and yeasts. For example, treatment of apple juice at temperatures below 30°C, with fewer than 10 pulses in a continuous treatment chamber, gave a greater than 106 reduc-tion in S. cerevisiae populations at a voltage gradient of 35 kV/cm; lower voltage pulses (22 kV/cm) gave a respectively lower reduc-tion of about 102 [54]. Applications for the use of pulsed electric field technology to treat liquid foods have included fruit juices, milk and liquid egg [49]. Field intensities employed range from

properties. The microbicidal effects were first described in the 1950s, but have been particularly studied and applied to various industrial applications in the last 20 years [42]. Typical conditions apply to carbon dioxide at 20–70 MPa, 40–100°C for 30 min to 6 h of contact; for example disinfection (pasteurization) activity can be demonstrated at 20 MPa, 40°C for 15 min [42, 43]. Overall, there are mixed reports in the literature regarding sporicidal activity; however, recent advances have been made to enhance sporicidal activity as the basis for sterilization process develop-ment. For example. little to no activity against G. stearother-mophilus and Bacillus atrophaeus spores was observed at low temperatures and cycle times [44]. At higher temperatures (c. 100°C) and pressures (20 MPa) a 6-log reduction in the viability of the spores was observed within 25 min. Similar enhancements of sporicidal activity at elevated but traditionally non-sporicidal temperatures have been described by others [45]. It has also been reported that the addition of low concentrations of water or hydrogen peroxide had some synergistic effects, with 100 ppm of hydrogen peroxide being particularly effective at 40°C for 1 h [44]. The addition of various chemical modifiers at low concentrations appears to enhance sporicidal activity. Bacillus pumilus spores were inactivated at 45–50°C and 10 MPa of supercritical carbon dioxide in the presence of hydrogen peroxide, formic acid, Triton X-100 and mixtures thereof [46]. Similar effects have been reported with low concentrations of peracetic acid, water and surfactants [43]. It may be expected that the microbicidal effects of supercritical carbon dioxide are due to the same effects on vegetative microorganisms as described above, with a variety of direct (e.g. oxidative agent) and indirect (permeabilization) action on the outer and inner structures of bacterial spores that culminate in spore death. Initial diffusion of carbon dioxide (and other chemicals) into the cell may be expected to have dramatic effects on metabolism, with changes in intracellular pH and further effects on microbial surface layers. Microbicidal applica-tions for supercritical carbon dioxide to date have included various disinfection/sterilization-sensitive materials such as human/animal tissues, protein preparations and drug–device combinations.

High-voltage electric pulses

While the direct application of electricity to heat materials has become well established (e.g. in food applications [22]), the use of electric pulses for non-thermal inactivation of certain vegeta-tive microorganisms has been investigated [47]. Such techniques using lower, non-lethal voltage gradients are widely used for the introduction of genetic material into recipient cells (predomi-nantly bacteria) in a process known as electroporation; presum-ably, this process allows for the temporary permeabilization of the cell wall/membrane to permit uptake but without bacterial inac-tivation. Studies have demonstrated the inactivation of bacteria and yeasts by a variety of electrical parameters [48]. For bacteri-cidal activity, field strengths shown to be effective have ranged

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and most often including part of the UV wavelength spectrum) with pulse durations from 10−6 to10−1 s and with energy densities from about 0.1 to 50 J/cm2. Different spectral distributions and energies are selected for different applications. For example UV-rich light, in which about 30% of the energy is at wavelengths shorter than 300 nm, is recommended for treatment of packaging materials, water or other transparent fluids. In contrast, for food surfaces, where high intensities of UV may accelerate lipid oxida-tion or cause color loss, etc., the shorter wavelengths are filtered out and the killing effects are largely considered thermal. The advantage of delivering heat in this manner is that a large amount of thermal energy is transferred to a very thin layer of product surface very quickly, while the temperature rise within the bulk of the product can be very small [61]. Significant effects of pulsed UV have been reported [62], including activity against a broad range of viruses [63] and protozoan oocysts [64]. Some synergistic reactions have been reported in combination with other chemicals, such as the microbicidal peptide nisin [56], although these and other true synergistic reactions remain to be confirmed.

Pulsed light is very sensitive to and has significantly reduced activity in the presence of any interfering factors such as surfaces not directly exposed to the light source and any porous surface (due to restricted penetration). Despite these initial limitations, some applications have been shown to be successful in reducing foodborne pathogens such as Salmonella [57]. The benefits include no process residuals, rapid cycle times and generally good compatibility (particularly with limited temperature increases). The mechanisms of action appear similar to other non-ionizing radiation sources, including direct effects on DNA, proteins and lipids [1]. Overall, high-intensity light treatments appear to be effective due to localized heat and/or similar effects to UV treat-ments, with some advantages for industrial and clinical applica-tions [53].

Magnetic fieldsExposure to oscillating magnetic fields has been reported to have a variety of effects on vegetative cells, ranging from selec-tive inactivation of malignant eukaryotic cells [65] to the inac-tivation of prokaryotes (bacteria) on packaging materials and in foods [66]. Treatment times tested have been relatively short, ranging from 2 to 25 ms and high field strengths (typically from 0.26 to 13.3 kPa (2–100 Torr) at frequencies between about 5 and 500 kHz). However, the findings of the limited number of studies conducted on magnetic fields so far show the overall microbicidal effects to be limited. For example a 102 reduction of vegetative bacteria, yeasts and molds inoculated into milk (Streptococcus thermophilus), orange juice (Saccharomyces) or bread rolls (mold spores) was observed in one study, but no effects were observed with bacterial spores [66]. Based on these results, the practical potential for the technique on its own, as it has been developed so far, appears to be limited although it may deserve further investigation [53, 67]. It has been suggested that the mechanism of action could involve alteration of ion

12 to 25 kV/cm, with treatment times from 1 to 100 ms over multiple pulsing cycles depending on the level of disinfection desired. Electric fields may be delivered in a variety of ways, but the most economic method to date involves raising the field strength as high as possible, while reducing the duration of the pulses, without reducing pulse energy.

On the other hand, the use of very high field strengths demands more complex and expensive engineering. As a result of these competing requirements, most modern pulse-field devices employ field strengths from about 20 up to about 70 kV/cm, with pulse durations between 1 and about 5 ms. Repetition rates are typically from 1 up to 30 s or so at the higher voltages in order to minimize rises in temperature. While applications to date have focused on the use of electric pulses for pasteurization applications in foods, further applications can be envisioned in other liquid prepara-tions and may be further enhanced (in particular with the addi-tion of other microbicides) to increase observed microbial reduction rates and spectrum of activity.

Other physical processes

High-intensity lightVarious non-ionizing (ultraviolet wavelength range) and ioniz-ing (γ) radiations are traditionally used for disinfection and sterilization [1]. High-intensity laser and light in the visual wavelength range (“white light”) are also known to inactivate microorganisms under certain conditions [53]. The wavelengths of light generally applied range in wavelengths from near UV (c. 200 nm) to the infrared (c. 1500 nm) range. White light (sun-light) is not considered a microbicide per se, but when the intensity of the light is increased (e.g. in the energy range of 0.01–50 J/cm2) microbicidal effects have been reported. Although test results vary depending on the lamps used and doses applied, the technology has been shown to be bactericidal, fungicidal (against yeasts and molds), virucidal, sporicidal and cysticidal. Similar to that described for electrical and high-pressure systems, during high-intensity light treatment the light source is pulsed to limit any unwanted heat generation; the number, intensity and extent of pulses will depend on the application and desired microbicidal level. The exact microbicidal effects and extent may vary depending on the choice of lamp used and light wavelengths/intensities produced for a given application. For these reasons, the degree of microbicidal activity can vary considerably from low-level disinfection to sterilization. The delivery of light to packaging materials, food surfaces and transparent liquid prod-ucts, in short pulses of high intensity, has been shown to inac-tivate vegetative and spore forms of microorganisms [56, 57]. Further, applications have included medical and dental instru-ments [58, 59] and antiseptic applications for the control of acne [60].

Commercially available systems for treating exposed surfaces of foods, materials and medical devices have been described [61]. Many use broad-spectrum light (across the visible light range

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MicrowavesMicrowave energy is a source of non-ionizing radiation within the wavelength range of 0.1–0.001 mm and an equivalent energy level of 2 × 10−24 to 2 × 10−22 J. It is generally accepted that micro-waves have little to no direct microbicidal effects from studies to date, in comparison to other irradiation sources at higher energy levels [1]. Despite this, significant microbicidal activity has been attributed to microwave energy, predominantly due to direct heating effects of water molecules associated with target surfaces/solutions. Activity has been described against bacteria, fungi and viruses, with little to no activity against thermoresistant bacterial spores (e.g. Geobacillus stearothermophilus spores). Sporicidal activity can be achieved in some applications; for example 2.0 kW microwave applications led to breakdown of B. licheniformis spore coat and inner membrane structures, as indicated by the leakage of internal spore proteins/DNA [79]. Because the primary effects are due to localized heating of water, microbial contamina-tion on wet surfaces is more rapidly inactivated than on dry surfaces (e.g. as shown with yeast inactivation studies [80]).

Microwaves have the benefit of being conveniently produced in widely available and electrically-operated ovens. The microbi-cidal activity observed is essentially equivalent to the tempera-tures produced; although some investigations have reported enhanced activity with microwaves this may be due to localized heat production in the test medium rather than direct effects of the wavelength on microbial components [81]. Microwaves have been primarily used as flash disinfection methods for a variety of applications including tissues (e.g. heart valves [82]), medical devices [83], catheters [84] and other materials. A typical applica-tion includes treatment of biomedical waste that is shredded, sprayed with water, heated by microwave to c. 95°C and held for the desired decontamination time [85].

Gas plasma

The applications of gas plasmas for microbicidal, particularly sterilization, purposes are detailed in Chapter 15.4 and are, there-fore, only briefly touched upon in this section. Plasma is essen-tially an excited or energized liquid or gas. In consideration of the gas laws, matter can assume any of the three essential states (solid, liquid, gas) depending on the temperature and pressure applied. Energy can be applied, for example in the case of water and tem-perature as described earlier in this chapter, to form a gas but when the gas is further energized it can form the so-called fourth state of matter, plasma. In a true plasma generation model, the gas molecules become ionized to form a variety of active and potentially microbicidal species such as ions, radicals, etc. In such forms, potent microbicidal activity has been reported [86]. Plasmas naturally occur in space, but for microbicidal purposes artificial plasmas can be created by the application of a strong energy source (such as electromagnetic fields and/or tempera-tures) to a variety of gases or liquids such as oxygen, nitrogen, helium, water and hydrogen peroxide.

fluxes across the cell membrane in target microorganisms, but this has not been confirmed. Potential synergistic effects may be explored in combination with chemical microbicides to test for greater uptake or direct effects to enhance microbicidal activity. To date this has not been widely reported, although similar potentiation effects have been described in biofilm disruption and in improving the activity of antibiotics against biofilm-resident bacteria [68].

SonicationSonication is a method widely used for a variety of cleaning applications, but has also been reported to have some microbi-cidal effects. The use of sonication (or the generation of ultra-sound waves in a liquid) to inactivate microorganisms was first reported in the 1920s. The mechanism of action derives from the rapidly alternating compression and decompression zones gener-ated in a liquid (for surface application or for direct effects within the liquid itself) leading to cavitations. Cavitations involve the formation and collapse of small bubbles, generating shock waves often associated with very high temperatures and pressures, which can be sufficiently intense to catalyze chemical reactions and disrupt animal, plant and microbial cells [69, 70]. Liquid products are easily sonicated, but in solids the structure and high viscosity severely impede efficacy. Generally, the larger the cells, the more susceptible they are to such cavitation effects. The cell structure also seems to affect efficacy, with rod-shaped bacteria being reportedly more sensitive than cocci [71] and Gram-positive bacteria more sensitive than Gram-negative [72]. Bacte-rial spores and many fungal spores are essentially resistant in studies to date [72].

Although the direct use of sonication may have limited micro-bicidal effects alone, potential synergistic effects have been reported. Combinations with heat and pressure have shown advantages for the inactivation of bacterial spores (B. cereus and Bacillus licheniformis [73]), thermoresistant streptococci [74], S. aureus and other vegetative microorganisms [74–76]. In studies with spores, as the temperature was raised, the potentiating effect of ultrasound became less and less, and had essentially no effect near the boiling point of water. Increased pressure–temperature effects may have further optimization potential [77]. The combi-nation procedure was reported to generally have the effect of reducing the apparent heat resistance of microorganisms by about 5–20°C or so, depending on the temperature, the organism and its z-value. Since sonication can generate, locally, very high temperatures, it has been difficult to disentangle the influence of heat and specific ultrasonic effects. Nevertheless, nano-thermosonication has been claimed to operate in some liquid foods [77], and requires further investigation.

Observed indirect effects of sonication have been shown in wastewater applications to be simply due to the disruption of larger microbial particles (clumped microorganisms in the presence/absence of soils) into smaller particles and improve-ments in efficacy in the presence of UV light [78] and presumably other chemical microbicides.

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unlike those described for “activated” or electrolysed water pro-cesses that are primarily microbicidal due to the production of hypochlorous ions [1]. Similar effects may be observed in plasma generation of air (containing a significant proportion of nitrogen and oxygen) for various applications. Such processes could be used for a variety of microbicidal purposes, including food, skin, wound care, air and general hard surface applications [90, 91].

A notable indirect use of plasma is in hydrogen peroxide plasma sterilization processes, such as the Sterrad® sterilizers. Initial patents around this process described the distribution of hydrogen peroxide gas in an evacuated chamber and the subse-quent generation of the plasma for sterilization [87], as well as the use of plasma to remove residual levels of hydrogen peroxide liquid/gas remaining following sterilization with peroxide gas [92]. Hydrogen peroxide gas is a powerful microbicidal agent in its own right [93] and the Sterrad processes developed so far have used peroxide liquid/gas exclusively for sterilization, while only using plasma generation either as a heating/drying mechanism prior to gas introduction or following gas evacuation to remove any remaining residuals of liquid/gas (see Chapter 15.4). Essen-tially, gas plasma is not used for any true microbicidal benefit in these cases. Although the plasma may have a theoretical effect on microbicidal activity in such processes, studies have confirmed that they are insignificant from a microbicidal point of view [94], but necessary in the design of these particular processes for safe residual removal/aeration.

Vapor-phase oxidants

Oxidizing agents such as hydrogen peroxide, peracetic acid and chlorine dioxide have been widely used as antiseptics, disinfect-ants and sterilants. Until recently, the major uses of these micro-bicides were primarily in liquid form, either on their own (as in the case of chlorine dioxide for water treatment or hydrogen peroxide on skin/wounds [1]) or in formulation with other excip-ients. Over the last 15 years, further research and applications have been described in the use of these microbicides in vapor (or gas phase). The most widely and successfully used to date is hydrogen peroxide gas (often referred to under its trademarked name as VHP®, for vaporized hydrogen peroxide). The terms “gas” and “vapor” can be alternatively used in this case; chemi-cally, a vapor is a gas but can also exist in the liquid (or solid) form below the critical temperature of the substance. Essentially, depending on the concentration of peroxide and its temperature/pressure it may be readily present as a gas, a liquid or liquid/gas (“fog”) mixture. This point is often underestimated in considera-tion of vapor-phase oxidants.

Hydrogen peroxide in its liquid form is a powerful microbicide, but has limitations in sporicidal, cysticidal and even virucidal activity at relatively high concentrations. Its microbicidal (par-ticularly sporidical/cysticidal) activity is dramatically increased and different to liquid activity in the gaseous phase. As an example, 1 mg/l gaseous peroxide demonstrates equivalent spori-

There is often confusion in the literature regarding the use of the term “plasma”. Many patents and publications erroneously describe certain disinfection/sterilization processes as being plasmas, and direct applications of plasmas have been limited so far to laboratory investigations for their microbicidal purposes. Optimal activities of such experiments have been shown with argon/oxygen, nitrogen/oxygen and hydrogen peroxide plasmas (as discussed in Chapter 15.4) [86]. For example, various types of microbicides including hydrogen peroxide, peracetic acids, nitric oxide and aldehydes such as formaldehyde and glutaralde-hyde have been investigated to try and increase their activity. Initial patents focused on the use of oxidizing gases such as hydrogen peroxide [87] and peracetic acid [88]; however, these studies have already highlighted limitations in the practical application of plasma technologies, in particular limited pene-tration for packaged materials. The optimum use, in these cases, was to allow the gas to distribute first within the load and then apply the required energy to generate the plasma locally at the required sites of activity. However, most of these investigations included the use of gaseous oxidizing agents with intrinsic microbicidal activity and it was therefore difficult to differentiate the true contribution of plasma generation to microbicidal activity. Subsequent investigations have focused on the use of what may be referred to as inert gases, such as oxygen, nitrogen, argon, helium, xeon and neon, both on their own and particu-larly in various mixtures. Unlike previous investigations, these gases have little to no intrinsic microbicidal activity until acti-vated into the plasma form. Particular success has been reported with N/O and Ar/O gas mixtures in various proportions (see Chapter 15.4). In addition to traditional microbicidal effects (against bacterial spores, fungi, viruses, etc.), some studies have shown activity in neutralizing endotoxin and infectious proteins (prions) (see Chapter 10).

Further developments are required to bring these technologies, including cleaning, disinfection and sterilization applications, to commercial use. It is important to not only focus on the optimiza-tion of the desired microbicidal effects but also on limiting any negative effects such as overheating and damage to materials and on ensuring uniformity in applying the plasma process to a target load [89].

To date, most plasma processes that have seen commercial success are actually indirect. For example, the plasma may be generated in a gas or liquid at a remote site and then transferred to the point of use. In this case, the true plasma will only be maintained when the energy applied is constant and it may be expected that the initial plasma activation allows for reassociation/generation of other microbicides that are actually responsible for the observed microbicidal effects. A typical example is found in processes using nitrogen and oxygen, where both gases are essen-tially inert but when activated by plasma formation and then allowed to reassociate will theoretically lead to the generation of a variety of microbicidal molecules such as ozone and nitric oxide. A further example is the activation of water, where a variety of microbicidal chemicals can be generated on energization, not

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parameters for microbicidal activity are peroxide concentration, temperature and distribution of the peroxide to ensure contact with all target surfaces. Humidity levels can be important at close to saturation concentrations (increasing the risk of peroxide/water condensation), although the direct impact on the activity of peroxide gas deserves further study.

Gas-based systems have also been described for chlorine dioxide, a newer generation of ozone processes and peracetic acid [1]. In contrast to hydrogen peroxide gas, all of these systems are dependent on high humidity conditions for optimal microbicidal activity. Gaseous peracetic acid has not been widely utilized, primarily due to compatibility concerns; disinfection and sterilization (in combination with plasma [105]) have been described but have seen little new development. On the other hand, gaseous chlorine dioxide and ozone processes have seen some recent developments. Chlorine dioxide is a water-soluble gas that has been widely used for liquid disinfection (e.g. water and surface disinfection) and can be generated by a variety of chemical reactions such as acidification of sodium chlorate (NaClO3) [1, 106]. Examples include the reaction of sodium chloride and citric acid to form chlorine dioxide or by passing chlorine gas over sodium chloride under high humidity condi-tions [107–109]. To be more specific, the humidity needs to be between 85% and 90%, which is thought to be a prerequisite for optimal microbicidal action. Gaseous applications have been described for sterilization and disinfection (fumigation) use, although its primary use today is for laminar flow cabinet, room and building fumigation [109]. Fumigation applications as of yet are limited, in comparison to formaldehyde or hydrogen perox-ide gas. Chlorine dioxide has some reported negative attributes such as being highly toxic via inhalation and causing ocular damage at low concentrations and dermal irritation, as well as being very corrosive to steel and aluminum. Despite this, it has been successfully used for various fumigation applications. The anthrax attack of the Washington, DC Senate mail building system was remediated with multiple rounds of chlorine dioxide gas, which after reaching a minimum of 90% relative humidity has also proved effective as a sporicidal agent. Chlorine dioxide gas is more effective at lower concentrations (with typical use concentrations varying from 0.5 to 30 mg/l), but is more depend-ent on high humidity levels for activity (>65% relative humid-ity), with broad-spectrum (including sporicidal) microbicidal activity. Mobile and fixed generator systems that can control the humidification, gas generation and aeration process are com-mercially available. Applications have also been described directly on foods, to prevent fungal spoilage, at 10 ppm of ClO2 at 95% relative humidity, with fungicidal activity reported against Candida, Saccharomycetes and Penicillin [110]. Although sterili-zation processes have been patented [109], such systems have yet to be successfully commercialized.

From an antiseptic perspective, a blend of 0.1% sodium chlo-ride and 0.5% mandelic acid has been used as a preoperative preparation [110, 111]. A triple approach was theorized as the mechanism of microbicidal action via the formation of chlorous

cidal activity to c. 400 mg/l liquid peroxide [95, 96]; the most resistant organism to peroxide gas is G. stearothermophilus spores, in comparison to liquid peroxide preparations where the most resistant organism is B. atrophaeus spores [93, 97]. Concentra-tions as low as 0.1 mg/l gas peroxide retain appreciable sporicidal activity, with estimated D-valves (time for a log10 reduction of bacterial spore populations) decreasing exponentially from about 10 min at 0.1 mg/l to 1 min at 1 mg/l and <0.1 min at 10 mg/l. For these reasons, gas peroxide has been successfully used for disinfec-tion [93] and sterilization [98] purposes in industrial, pharma-ceutical, research and healthcare applications. Examples include fumigation of rooms/buildings (as an alternative to formalde-hyde) and for sterilization of reusable, thermosensitive devices (as more rapid alternatives to ethylene oxide and formaldehyde steri-lizers). In addition to potent microbicidal effects, peroxide gas has also been shown to be effective in the inactivation of prions [99, 100], endotoxins and cytotoxic drugs [101]. Recent studies on the mechanisms of action of gas and liquid peroxide have shown that, at least for activity against proteins and peptides, gas peroxide targets the peptide bonds that hold together the amino acids in protein structures, while liquid peroxide targets amino acid side-chains [102]. Unlike older reports in the literature, the gas has been found to be reactive with but can tolerate high levels of soiling (depending on the application [93]), but also has impor-tant advantages in materials compatibility and a reasonable safety profile (e.g. breaking down rapidly into water and oxygen in the environment).

Gas peroxide is generated by heating, typically by flash vapori-zation of liquid peroxide applied directly onto a hot surface [103]. However, the commercial applications of gas peroxide can vary significantly with various terms being used including VHP, hydrogen peroxide vapor (HPV), dry mists, etc. [104]. These systems can all use hydrogen peroxide as a microbicide but in different ways; some may generate and apply the gas in an unsatu-rated (gas) form, others in a saturated (liquid/gas or condensed gas) form and, finally, simply dispensing a liquid peroxide formu-lation in a given area over surfaces to be treated. The safety and efficacy of such processes can vary [103, 104]. To date, not much has been published on the impact of various process conditions such as humidity, temperature and concentration on the micro-bicidal activity of peroxide gas. From some studies, peroxide does not require high humidity levels to be effective in comparison with other gaseous microbicides, including formaldehyde, ethyl-ene and chlorine dioxide. Typically, gas is generated from perox-ide solutions in water (35–60% v/v in water); therefore, a humidity level is always present and can affect the saturation level of per-oxide at a given temperature. For 35% peroxide, humidity levels are typically in the range of 40% although this level will increase as the peroxide degrades into oxygen and water vapor. For this reason, regeneration (closed) systems have been developed that constantly release peroxide gas into a given area but also remove peroxide/water vapor from a treated area. Other systems release an initial bolus of peroxide/water vapor into a given area and allow this to naturally degrade over time. Overall, the key

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Sterilization systems that describe the use of NO and NO2 have been patented, with the emphasis on NO2 as the major microbi-cide [117]. It provides many advantages as a sterilant, including few aeration requirements following exposure (unlike ethylene oxide), it is not believed to generate toxic residues, it is non-explosive and has a reasonable materials compatibility profile. Typical sterilization conditions proposed include 0.25–3% NO2 at 70–90% humidity, 18–30°C and 30–60 min of exposure. Similar to other such processes, air removal is important but various processes based on vacuum, pressurized and atmospheric pres-sure conditions can be envisioned. A typical sterilization process could include conditioning (for humidity/air removal), introduc-tion and holding of gas/humidity (for the required number of pulsing phases to achieved sterilization) and aeration to release the load for safe handling/use. Other applications have included use in antiseptic creams [118] and as a general disinfectant, including food applications [119].

In addition to these forms, nitrous oxide (N2O; more com-monly known as laughing gas) may also be proposed as a micro-bicide as it is a known oxidizing agent, but it has yet to be investigated for this purpose. N2O is a non-flammable, colorless gas with a slightly sweet odor. Its primary use today is as an anesthetic/analgesic.

Bacteriophages and other biological substances

The idea of using bacteriophages and microbicidal peptides as alternatives to conventional antimicrobials is not new. Bacteri-ophages (“phages”), ubiquitous viruses that prey upon bacteria, are experiencing a renewed interest as therapeutic alternatives to antibiotics, especially to counter multidrug-resistant bacterial pathogens [120–122]. The highly bacterial host-specific nature of phages, their ability to multiply in situ and safety for humans are added advantages over antibiotics and conventional microbicides. However, and unlike many microbicidal chemicals, the need for phages to infect and multiply in their respective hosts inevitably produces a lag of several hours between application and the desired level of activity. This is not necessarily an impediment in wound care where a single type of phage or a group of carefully selected phages can be applied as a preventive or therapeutic measure without the need for instantaneous action.

Microbicidal peptides have been identified from a wide variety of eukaryotes and prokaryotes [123, 124]. From a microbicidal perspective, and similar to bacteriophages, their microbicidal applications are generally limited as alternatives to antibiotics for specific antiseptic, disinfectant and/or preservative uses. Such peptides, which are produced naturally for host defense often in response to injury [125], show not only microbicidal effects but may also be effective in wound care, reducing inflammation reac-tions, and even in cancer therapy [124, 126]. As compared with antibiotics, they are considered broad spectrum with antimicro-bial activity against a wide range of bacteria (including mycobac-teria), fungi, protozoa and some enveloped viruses, depending on

acid and chlorine dioxide as well as residual mandelic acid [112]. Periodontal preparations have been described for gingivitis treat-ment using chlorine dioxide as the active microbicidal agent.

Ozone is well described as one of the most effective oxidizing microbicides, but recent advances in the generation and mainte-nance of ozone concentrations allow for further practical uses of the technology, and in particular for medical device sterilization applications [1, 11, 113]. Ozone can be readily generated by applying energy (e.g. UV light or an electric charge) to oxygen (pure gas or as present in air), but close control of humidity levels is required for optimal microbicidal activity. Typical processes (fumigation or sterilization) consist of three phases: humidi-fication (to 70–80% relative humidity), ozone concentration/humidification maintenance for a defined time, and aeration to remove any residual levels to below a safe level (generally reported as 0.1 ppm). Newer ozone-based sterilization systems have been developed for medical device applications including the TSO3 sterilizer [11, 113]. This vacuum-based process humidifies the sterilizer load during preconditioning, followed by two identical stages of humidification (85–100%) and ozone diffusion (85 mg/l) at 30–36°C followed by ventilation, for a cycle time in the range of 4–6 h. Similar, ozone disinfection technologies have been described for laundry reprocessing as alternatives to heat-based methods. Humidified ozone is a powerful microbicidal, but can be limited in use with certain types of plastics and metal surfaces, including aluminum, brass, polyurethanes and rubber materials. Similar to other gaseous oxidants, specific process conditions may be defined or used with ozone for the disinfection of liquids and cellulose-based materials (due to their reactive and absorbing nature to the microbicide). There has been a recent resurgence of interest in the use of ozone for environmental space decontami-nation, with a variety of generation systems commercially avail-able for miscellaneous applications.

Nitric oxide and nitrogen dioxide

Nitric oxide (NO, nitrogen monoxide) and nitrogen dioxide (NO2) gases have both been investigated for their microbicidal activities. NO is an important, naturally occurring, reactive bio-logical molecule expressed in cells as a signaling molecule, for protection of tissue damage, particularly during inflammation, and as part of the immune response; over- or continuous expres-sion can lead to cell damage and a variety of tissue toxicity effects [114]. It is rapidly oxidized in air to form NO2, a brown-colored gas that is also cytotoxic. Both molecules are considered effective microbicides and have been particularly studied for their effects against bacteria. Mechanisms of action are primarily referred to as being fragmentation of DNA, although investigations have shown a variety of cellular effects (from investigations with bac-teria that can also be extrapolated to proposed effects on viruses and other microorganisms) such as enzyme inactivation, lipid modifications (affects cell membrane structures) and protein nitrification as well as DNA fragmentation [115, 116].

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therefore, target peptidoglycan as their site of action. Synergistic reactions have also been described with other cationic, amphiphilic peptides [130].

While it is envisioned that phages and antimicrobial peptides may offer certain benefits over conventional microbicidal chemi-cals, their particular promise to date (with the notable exception of nisin) is as alternatives to antibiotics.

Glucoprotamines

Glucoprotamines are a group of chemicals that can be generated by condensation of N-substituted propylene diamines with 2- aminoglutaric compounds [134]. The first described microbicide was produced from reacting l-glutamic acid and cocopropylene-1,3-diamine to give a wax-like substance that can be dissolved in water [135]. In initial investigations, these non-sensitizing and readily biodegradable chemicals showed broad-spectrum activity against vegetative bacteria (including mycobacteria), fungi and viruses [135]. In addition, clinical investigations with soiled medical instruments demonstrated good bactericidal activity [136]. Longer contact times (15 min at 2500 ppm glucopro-tamine) were required to inactivate 4-log10 of atypical mycobac-teria, although a glutaraldehyde-resistant strain of Mycobacterium chelonae was also cross-resistant to glucoprotamine [137]. In these studies, up to 2200 ppm for 60 min or 5000 ppm for 15 min were required to be effective against that strain, suggesting that available surface proteins may be a key target for the initial myco-bactericidal activity of glucoprotamine [138]. The development of resistance may be unique to mycobacterial strains, as a further study of clinical and reference bacterial and fungal strains with two glucoprotamine-containing disinfectants demonstrated rapid activity [139].

Microbicidal surfaces

Recent years have seen much interest in producing microbicide-coated or impregnated materials (see Chapters 20.1 and 20.2) for environmental surfaces in healthcare settings and food-handling establishments, catheters and other device surfaces, textiles, and air and water filters. Common substances used to confer such microbicidal activity are triclosan, QACs and metals such as silver and copper. These and other novel means are reviewed below [140, 141].

Copper and silverThe metals silver and copper are well recognized for their micro-bicidal potential on their own and in formulation, and have been receiving increasing interest as microbicidal surfaces. While copper is widely used as a microbicide, particularly in agricul-ture, water treatment and as a preservative [1], copper-containing surfaces have been the focus of more recent investigations in hospitals and other environments in reducing contamination

the specific peptide under investigation. Many such peptides with specific antimicrobial properties are now known [127]. For example, a database updated in 2008 lists 1228 entries including 76 antiviral, 327 antifungal and 944 antibacterial peptides [128]. Structurally, most of them comprise of 1250 amino acid residues, although larger ones (>100 amino acids) also exist. Examples are shown in Table 16.2.

In general, their amino acid composition, hydrophobic nature, size and charge allow them to assume structures that can bind to and interact with the outer surface of microbes as their primary site of action. Others, such as microcidin J25, can interact with bacterial RNA polymerase as well as other intracellular targets [129]. A further example is a series of peptidoglycan recognition peptides (PRPs; [130]). For those cationic, amphiphilic peptides, cell membrane insertion is observed but the exact mechanisms leading to disruption of cell metabolism and death require further investigation [131]; it may be simply due to gross disruption of the structure/function of the cell membrane but could also be due to pore formulation, inhibition of specific membrane-associated metabolic functions or a variety of other effects. Interestingly, similar to antibiotics, the development of bacterial resistance to peptides has been described and a variety of mechanisms have been elucidated [130].

Nisin has been one of the most widely investigated antimi-crobial peptides and has been used for over 50 years as a food preservative [132]. Typical concentrations of it can range from 1 to 25 ppm. It is produced commercially from Lactococcus lactis as a 34-amino acid chain that contains some unusual amino acids such as lanthionine and methylanthionine. Nisin is par-ticularly active against Gram-positive bacteria including Staphy-lococcus spp. and is sporistatic against Bacillus and Clostridium spp. It has also been used in combination with other chemicals (e.g. chelating agents) to enhance activity against Gram-negative bacteria, predominantly to increase the penetration of the peptide into the cell membrane. The structure of nisin varies in aqueous solution, but assumes an amphiphilic and β-sheet structure when associated with lipid membranes and therefore in its active form [133].

Other peptides investigated for clinical applications such as pexiganan, iseganan, omiganan and the PGLYRP series have yielded mixed success [130]. Of note, the PGLYRP series have been described to be peptidoglycan-recognition peptides and,

Table 16.2 Examples of microbicidal peptides.

Structural subgroup

Example Primary (amino acid) sequence

α-Helical Magainin GIGKFLHSAKKFGKAFVGEIMNSβ-Sheet Lacteroferrin FKCRRWQWRMKKLGAPSITCVRRAF

α-Defensin ACYCRIPACIAGERRYGTCIYQGRLWAFCCLoop-structured Microcin J25 GGAGHVPEYFVGIGTPISFYGExtended Indolicidin ILPWKWPWWPWRR

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and Gram-negative strains of bacteria with intrinsic or acquired resistance to copper have been described; interestingly, such resistance was observed only under dry conditions and not when the inoculum was still wet [153]. Chromosomal and plasmid-mediated resistance mechanisms have been described in E. coli [1]. Plasmid-mediated tolerance to silver has already been well described in Salmonella and silver tolerance is emerging in methicillin-resistant S. aureus (MRSA) [154], although the clini-cal relevance of such levels of resistance is currently unknown.

N-halaminesN-halamines are a novel class of halogen-releasing agents (Figure 16.2). They are available as a range of monomeric and polymeric microbicides for both suspension and surface applications [123, 155, 156]. Such applications include the treatment of filters and textiles, and the disinfection of liquids, environmental surfaces and electron-spun fibers [157, 158].

The basis for the microbicidal activity of N-halamine is the anchoring of free (and therefore microbicidal) chlorine or bromine to nitrogen-containing compounds such as imidazolid-ins, oxazolidinones and modified polymers. On microbial contact, the halogens are released to elicit their microbicidal effects, but can also be constituted by the addition of halogen-containing liquids into the suspension or directly onto the surface. Due to the relatively low concentrations of the microbicide present, these preparations generally have a low odor, are considered stable and, being able to be reconstituted, have benefits over other impreg-nated microbicidal surfaces [157, 159–161]. The specific micro-bicidal activity depends on the N-halamine structure and its applications. Examples include the use of chlorine- and bromine-based N-halamines against bacteriophages (as a test model for other viruses) and in reducing toxins (with the bromine-based compounds showing superior activity [162]), fecal bacteria and coliphages in sewage-contaminated water [163]. A recent study with chlorinated, polymeric N-halamine in the form of N-chloro-2,2,6,6-tetramethyl-4-piperidinyl methacrylate showed persistent broad-spectrum activity over a year and rechargeable activity against bacteria (S. aureus, E. coli, Enterococcus), fungi (Candida, Stachybotrys) and a model phage (MS2) in microbicidal paint applications [164]. A recent patent has described methods for optimizing the generation of active polymers [165].

with nosocomial pathogens on frequently-contacted surfaces such as bed rails, door handles and bedside tables. One study examined the levels of bacterial contamination on copper and non-copper surfaces (toilet seats, tap handles, door push plates) and found the counts of viable bacteria to be statistically signifi-cantly lower on the copper ones [142]. Another study showed similar results and lower re-contamination rates over time [143]. Similar studies have shown benefits in other applications such as the control of E. coli on food-handling surfaces [144] and in controlling fungal contamination on polyester fibers [145]. Lab-oratory studies have also shown similar effects against the veg-etative form of Clostridium difficile over a 3 h contact time and in the presence/absence of soil in comparison with stainless steel [146]. The authors also suggested a reduction in the viability of the spores, although copper under these conditions is expected to have sporistatic attributes. More detailed studies showed that copper alloy surfaces with a copper content of >70% provided a significant reduction of both vegetative and spore forms of C. difficile [147].

In addition to direct copper alloy surfaces, other applications have been proposed using copper oxide nanoparticles that contain copper ions [148]. Such particles could be used in a variety of suspension and surface applications; in suspension studies bacte-ricidal activity could be further enhanced in combination with silver nanoparticles. It has often been assumed that copper and silver have similar mechanisms of action, but recent evidence suggests that this may not be so.

Silver is also being used to confer microbicidal attributes to environmental surfaces and, in particular, wound dressings and indwelling devices such as catheters [140]. Traditional sources for silver have been silver nitrate and silver sulfadiazine, but silver itself (or as silver nitrate or silver oxide) can be directly integrated into a variety of materials such as polymers. Similar to copper, further advanced methods of presentation have included nano-particles or crystalline silver [149, 150]. The benefits of these applications have been the subject of much debate and have been particularly described in situations requiring long contact times (such as larger wounds and long-term indwelling catheters). Despite these reports, a careful review of the literature shows little evidence to support the benefits of such applications in prevent-ing infections with short-term implantable devices [140]. In contrast, others have reported improved microbicidal efficacy of silver nanoparticle-based technologies, in particular as alterna-tives to antibiotic use [151].

The integration of surface microbicides, or indeed other mate-rials or surface modifications, may play an important role in preventing the initial attachment and subsequent colonization of surfaces, as the first stages in biofilm development are often asso-ciated with such devices [152]. The benefit of the widespread use of silver and copper for such microbicidal applications has been the subject of some debate, with many suggesting little role in reducing the rates of healthcare-acquired infections and the potential risks of microbial resistance development. These argu-ments should be considered further. For example Gram-positive

Figure 16.2 Examples of polymeric N-halamine chemical structures. The free chlorine (Cl) or bromine (Br) groups are responsible for microbicidal activity.

H

H

CH3 CH3

O

Br N

N N

N

Br CI

CIO O

O

n n

H

C

H

HC

H

CC

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Chapter 16 New and Emerging Technologies

implications for product safety, will be needed if application of the techniques continues to be considered. Third, with the excep-tion of hydrostatic pressure, which is now well established com-mercially, the efficacy of the other techniques is impaired by product structure and may, therefore, be limited to liquid prod-ucts or products containing small particulates, or (for light pulses) transparent products and surfaces, etc. At the same time, combination techniques, in which newer technologies are only one component of the total disinfection/preservation system, have already been described. If these are further developed and proved to be effective, the opportunities for use of the new tech-niques are very likely to grow in the future.

Changes in alternative chemical and biological processes for disinfection and sterilization, with some exceptions, have evolu-tionary and not revolutionary characteristics. For instance, the use of hydrogen peroxide for disinfection and sterilization involves new versions of an existing technology. Despite this, there are some novel microbicidals that have been developed recently. Applications of microbicides with nitrous oxide are in the early developmental phase but show promise in various dis-infection and terminal sterilization uses. However, significant research is required to materialize this development. The main factor limiting the use of new microbicidal compounds is the extreme cost of registration due to governmental regulation, which is retarding the consideration and development of new and innovative compounds. In the meantime, it is likely that further improvements will be made to the optimal use of microbicides in formulation and in synergistic mixtures.

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Titanium dioxideTitanium dioxide (TiO2; also known as titania), is a commonly-used white pigment for a variety of commercial applications (such as in paints, sunscreens, paper, etc.). It is also used for its photocatalytic activity under UV light, where surface radicals and other oxidizing agents are produced. This is used industrially for the oxidation of organic/inorganic contaminants as well as for its direct microbicidal purposes. Such applications include odor control, water disinfection, preservation, air treatment and on microbicidal (e.g. painted) surfaces. Reports of microbicidal activity vary in the literature and most investigated the activity under laboratory conditions. Recent studies have looked at more practical applications. For example, a continuous-flow water treatment pilot application has shown some success and stability using titania with solar activation [166] and silver/titanium dioxide photocatalysis showed particular use in the disinfection of air in medical settings [167]. Overall, such applications provide a low level (particularly bactericidal) of disinfection over time and may be further optimized depending on the specific applica-tion. For example, water disinfection studies showed activity against protozoan cysts (Giardia and Acanthamoeba) that generally show higher resistance to chemical microbicides [168]. Cysticidal activity was observed against Giardia cysts but not Acanthamoeba over a 30 min illumination time, being overall less effective than control UV studies. Other investigations have shown little to no appreciable sporicidal activity in recent surface studies [169]. Further, microbicidal activity is significantly affected by the presence of interfering organic or inorganic mate-rials [170]. However, many of these studies may be limited by the methods of preparation and illumination by UV light; for example nanometer films of titania may have some optimal benefits of bactericidal and cysticidal activity over conventional deposition methods [171, 172].

Conclusions

The use of physical techniques to inactivate microorganisms in some types of foods, pharmaceutical products and medical devices, without the application of heat, or with the use of less heat (or required time), is particularly attractive from the point of view of product quality. A wide range of methodologies such as high pressures, high-intensity light and sonication are potential alternatives for such applications. Three facts limit their wide-spread use and current usefulness for niche applications. First, bacterial endospores remain the organisms most tolerant to all the techniques, so that sterilization, as opposed to various levels of disinfection, is not yet possible. Further developments and specific applications (e.g. with supercritical fluids) may overcome this limitation with some of these technologies. Second, the kinet-ics of inactivation that result from some of the techniques are different from those resulting from heating. This means that a careful new approach, for example with respect to the potential for survival of low numbers of pathogens, and consequent

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