Principles of Antimicrobial Activity

Principles of Antimicrobial Activity

Gerald McDonnell

Joyce M. Hansen

This chapter provides a general introduction to the central principles of antimicrobial activity and application as they apply to the different preservation, disinfection, and sterilization objectives discussed in this book. It starts with a general overview of the important balance between antimicrobial activity and safety in any application, with greater emphasis on environmental safety in the last 20 years. This chapter further considers central requirements in the development and practical application of preservation, disinfection, and sterilization technologies as well as an introduction into the antimicrobial mechanisms of action.


Antimicrobial technologies employed for preservation, disinfection, and sterilization are widely used for industrial and medical purposes in reducing or eliminating microorganisms. But in the development and application of these technologies, there are at least two major considerations: the desired antimicrobial effects (the most obvious reason behind employing these technologies) and safety requirements. Antimicrobial targets can range from preventing the growth of certain types of microorganisms such as bacteria and fungi (as in the goal in many types of preservation systems) to inactivating microorganisms, to either reduce the levels and types present (as is the goal in disinfection) or to completely eliminate all microorganisms to reach a sterile level (as with sterilization processes). Therefore, the objective of the desired end point with an antimicrobial process is important in its choice and application. This can range from the unique control of an individual type of microorganism (eg, in a biosafety or research laboratory that may only be used for a certain type of microorganism), to the control of a range of potential pathogenic or spoilage microorganisms (eg, in environmental disinfection requirements in food production, research laboratory, general microbiology detection laboratories, and health care facilities), and to the complete eradication of all types of harmful or product degrading microorganisms in higher risk situation (as during the administration of injectable drugs or surgical intervention with devices). Safety requirements will also vary depending on the use of the antimicrobial technology. These can include material compatibility, safety to those using the technology, safety to the end consumer or patient, and safety for the environment. They will also vary depending on the application such as in the preparation of food/water for consumption, treatment of environmental surfaces, use on the skin, mucous membranes or in wounds, products to be placed or injected into the body or blood stream, or in other industrial situations such as during microelectronic manufacturing. Equally, the definition of these requirements and the risk-benefit balance in each situation are important to consider.


The desired antimicrobial end points will depend on a risk-benefit equation. If we consider preservation requirements, the goal is to prevent the multiplication of microorganisms. In many cases, the concern is the presence of environmental microorganisms in raw materials; during manufacturing or distribution; or from the use of the product, surface, or material over time. The greatest risks are bacteria and fungi because they can multiply under the right conditions (eg, water availability, presence of nutrients, temperature, time, etc.). Bacteria, particularly gram-negative bacteria, are equally a concern in water or water-based product systems, but other risks can come from the presence of alga and protozoa. These are often associated with the intrinsic microbiological quality of an environmental source (including the potential for cross-contamination with water used for hand washing
or food preparation) or only seen as a concern under situations where they have the ability to survive and grow (eg, in contaminated drinking or recreational water with a ready supply of organic and inorganic nutrients). In many of these situations, preservation strategies may be appropriate to consider not only for during manufacturing or initial treatment but also for the intended use of the product. During product use it may be needed to ensure that any microorganisms it may contact from other sources (such as from water, air or human contact) are not allowed to proliferate to levels that can cause harm or product damage. An example was an outbreak of Acanthamoeba keratitis associated with use of a multipurpose contact lenses solution, which on investigation was not found to be due to microorganisms found during product manufacturing but was suggested to be due to the inability of the preservative system to control the presence of Acanthamoeba introduced during the use (or repeated use) of the product.1 Equally important are measures taken to provide water for recreational use. Protozoal outbreaks are increasingly reported in recreational water, such as with Cryptosporidium and Naegleria fowleri. The Centers for Disease Control and Prevention2 reported a 13% rise per year in reports of cryptosporidiosis in the United States from 2009 to 2017; although rarer, N fowleri outbreaks have been linked to nasal contamination from contaminated natural water sources (eg, hot springs, lakes, and rivers).3 In other cases, microorganisms such as viruses and certain types of obligate intracellular bacteria can be present at low levels but may not have the opportunity to proliferate unless they are under unique environments such as in cell culture applications. Overall, the focus on preservation systems and applicable test methodologies to test preservative effectiveness predominantly focus on bacterial and fungal challenges in preserved products such as cosmetics and/or detection technologies (eg, in drinking water monitoring).4

For many other types of applications, it is not enough to prevent the growth of microorganisms, and their inactivation is a requirement or a specified goal, and this is the purpose of disinfection and sterilization. As already defined in chapter 2, disinfection is required to inactivate viable microorganisms to a level previously specified as being appropriate for a defined purpose, and sterilization is essentially the ultimate process that renders a surface or product free from viable microorganisms. In most practical cases, the objective is not limited to the inactivation of a specific, targeted microorganism, although this may be the case in certain situations such as in a research laboratory that may only be investigating a certain microbial strain or in a specific outbreak or contamination event (eg, with a known virus). It is more common that an attempt is made to reduce or control a range of microorganisms that may be present or are of a concern. In many cases, the exact types or strains of microorganisms present will not be known. Examples include environmental disinfection of surfaces in a food-handling facility or antiseptic hand washing or hand rinsing of staff that are employed in patient care. It would be impossible and impractical to test every type (genus, species, or even strain) of microorganisms that can be a concern; therefore, antimicrobial efficacy is usually demonstrated using indicator microorganisms that represent the different kinds of microbial life. These primarily include viruses, vegetative bacteria, fungi (molds and yeasts), mycobacteria, and bacterial spores as the major classes but may also include indicator protozoa, helminths, alga, and prions depending on the specific requirements (Table 5.1). If a disinfection application is shown to be effective against certain types of indicator organisms, this will imply activity against a wider range of microorganisms with similar structures or sensitivity to inactivation. This premise has led to the development and continued optimization of standardized antimicrobial test methods (see chapters 61, 62, 63). These test methods include requirements to meet regulatory claims and support product labeling associated with disinfection and/or sterilization as well as for testing specific antimicrobial applications such as the treatment of human/animal tissues (see chapter 50) or in the control of biofilms (see chapter 67). The differences in the intended use of disinfectants can lead to some confusion from users when considering the use of terms like bactericidal, viricidal, and germicidal (see chapter 2). It is important to remember that the suffix “-cidal” correctly implies the ability to kill a certain class or type of microorganisms. Therefore, “bactericidal” may be interpreted as killing all types of bacteria, but this may not be the case and is generally limited to many types of common vegetative bacteria; for example, this term may not apply to the inactivation of mycobacteria and spore forms of bacteria that are intrinsically more resistant (see chapter 3). “Viricidal” can be used to describe the inactivation of easier to kill enveloped viruses but not nonenveloped viruses, depending on the product, application, or associated labeled claims/validated test methods. Finally, “germicidal” may imply the ability to inactivate all germs (generally used to describe all microscopic microorganisms), but this is not the case such as in the United States, where this term is widely used but is only intended to indicate the ability to inactivate certain types of common vegetative bacteria (eg, Staphylococcus aureus and Escherichia coli). The original use of the term germ (from the 1600s, having its original from the Latin germin or French germe for the verb to beget), implies a small living substance capable of developing into some larger, which was essentially the basis of the germ theory in the 1800s and clearly focused on what was known at the time as the major types of germs, being types of vegetative bacteria. Although our knowledge of microbiology has certainly changed since that time, the use of these label claims needs to be closely considered to prevent misinterpretation.

Overall, the antimicrobial claims against indicator microorganisms in standardized test systems are the basis of
labeling requirements for disinfectants and disinfection processes internationally. The claims can range in their meaning and therefore specific label claims should be reviewed carefully to ensure expected efficacy. As we consider antimicrobial properties, it is important to remember that microorganisms range in their intrinsic resistance patterns to antimicrobial processes (see chapters 3 and 4). These can include types of pathogens that are relatively easier to inactivate by many different types of physical and/or chemical microbicidal technologies such as heat, radiation, and chemicals like aldehydes and oxidizing agents. Enveloped viruses and many (but not all types) of vegetative bacteria and fungi have been well described as being relatively susceptible to inactivation by many antimicrobial technologies (see chapter 3). But equally, types of mycobacteria (due to their unique tolerance profile to chemical disinfectants), nonenveloped viruses, protozoal (oo) cysts, and fungal and bacterial spores have much higher levels of tolerance and therefore create a more significant challenge (see chapters 3 and 4).5 This known hierarchy of resistance is also the basis for the generalization of antimicrobial claims such as low-, intermediate-, or high-levels of disinfection that are widely used in many countries (see chapters 2 and 3).

TABLE 5.1 Examples of microorganism types, commonly used indicator organisms of each group, and associated efficacy claimsa

Microorganism Type

Indicator Organism

Example Associated Claim

Vegetative bacteria

Staphylococcus aureus

Pseudomonas aeruginosa

Salmonella choleraesuis

Salmonella enterica

Escherichia coli

Klebsiella pneumoniae

Germicidal, bactericidal


Pseudomonas aeruginosa

Staphylococcus aureus



Trichophyton interdigitale (or mentagrophytes)

Candida albicans

Aspergillus niger

Fungicidal, yeasticidal, mildewicidal

Enveloped viruses

Hepatitis B virus


Herpes simplex



Nonenveloped viruses



Feline calicivirus



Mycobacterium bovis

Mycobacterium terra




Cryptosporidium parvum

Giardia lamblia

Acanthamoeba castellanii

Protozocidal, cysticidal





Ascaris lumbricoides

Enterobius vermicularis


Bacterial spores

Bacillus subtilis

Bacillus cereus

Clostridium sporogenes

Clostridium difficile

Geobacillus stearothermophilus




Not applicableb

HIV, human immunodeficiency virus.

aIndicator microorganisms and claim structures can vary internationally and/or on the associated product or product application.

b No accepted claim to date but can be referred to as “reducing the risk of prion infectivity,” “prion decontamination,” or “prionocidal” as examples.

So overall, with these claims and a close inspection of the hierarchy structure, we can expect the antimicrobial effectiveness
against a wide range of microorganisms. For example, a low-level disinfection claim implies effectiveness against vegetative forms of bacteria (but not necessarily mycobacteria), some fungi, and enveloped (or lipid) viruses, whereas high-level disinfection can be considered effective against all of these, including a wider range of fungi and mycobacteria but also some activity against bacterial spores. Overall, this hierarchy can be challenged depending on the antimicrobial process or product under investigation or the intrinsic resistance of certain types of microorganisms that would be expected to be within these claims5,6,7 (see chapters 3 and 4), but in general they apply. These terms are well established based on years of experience from industry and academic derived data proving antimicrobial efficacy against a wide range of microorganisms and, in most cases, the verification of such a claim based on regulatory requirements. Similarly, other disinfection requirements can be applied to specific applications such as for skin antisepsis (see chapters 42 and 43) and in the parametric control of moist heat treatments, as in thermal disinfection or pasteurization processes (see chapters 11 and 28). In the latter case, the ability of heat to reliably inactivate microorganisms above a given temperature (eg, 65°C-70°C) and then follow classical predictable profiles for kill in a temperature-time relationship allows for the demonstration of disinfection (and indeed sterilization) by simply confirming that the actual temperature is achieved over a given time. Disinfection can also be demonstrated by more traditional means, such as by applying the process/product and demonstrating that no viable microorganisms are detected or demonstrating that the quality of microorganisms have been reduced to a given level. This may seem to be a simple approach but can be laborious and difficult to control. Analysis is often hindered by the fact that only certain types of microorganisms (such as many bacteria and fungi) can be easily handled under laboratory conditions. Further, the risk of introduction of other microorganisms as contaminants can create misleading (false positive) results. This approach is still used to demonstrate the effectiveness of disinfection methods in high-risk or regulated environments or applications but is overall not considered practical or reliable if not performed with close attention to detail and best microbiological practices.

As noted previously, it is not always necessary to ensure the complete removal or inactivation of all microorganisms or essentially that a sterile condition is established and/or maintained. In situations like food or water consumption, industrial applications, or in the antimicrobial treatment of the skin or mucous membrane, many types of sterilization treatments (see Part IV) would not be appropriate due to limitations in application, costs, or deleterious effects on the application of these technologies such as changes in organoleptic properties on consumption, the presence of toxic by-products that cannot be tolerated, and damage to tissues or products. This is important to consider in the risk-benefit equation in considering requirements for antimicrobial applications. For these reasons, many gentler technologies may be practically used to reduce the presence of certain types of high-risk pathogens or spoilage microorganisms, such as vegetative bacteria and viruses in food, water or skin/mucous membranes, or many types of industrial products. These will be able to achieve relatively safe levels for human, animal, or plant situations but not at the expense of unacceptable safety risks or damage. There is also the growing consideration that the constant overexposure of antimicrobial technologies may themselves lead to unwanted effects, such as lack of fitness of the human or animal immune system and chemical-associated health risks linked to the direct or indirect use of antimicrobials.

But in certain situations, and more critical applications, sterilization is the expectation and can be readily achieved. Sterilization is essentially the ultimate disinfection process, being a process used to render product free from viable microorganisms. This definition is important in two aspects. First, it implies that the process should be well established or verified to inactivate all microorganisms that can be present (known or unknown) to render the target sterile and therefore free of living microorganisms. Therefore, all the requirements for disinfection discussed earlier will apply but will also need to encompass all types of microorganisms. Different methods to demonstrate and maintain these requirements are introduced later in this chapter as well as the antimicrobial properties of the limited types of technologies widely used for this purpose (see Part IV). But second, the definition also highlights that this is a controlled process. This will not only be in the act of applying the sterilization process itself but also will include the preparation before sterilization and the maintenance of sterility after the point of use. This concept is best considered as an “end-to-end” supply chain process to ensure microbiological quality, not just a focus on the terminal act of sterilizing (discussed later in this chapter). Both criteria and their ramifications essentially differentiate disinfection from sterilization, but although the end points and level of scrutiny may be different, the impact of both concepts to achieve the desired goal is essentially the same. For these reasons, in comparison to disinfection modalities (see Part III), there is a more restrictive list of technologies that are practically used to achieve sterilization such as heat, ionizing radiation, and certain types of chemical processes (see Part IV). To these we may also include filtration methods (although these generally do not provide antimicrobial processes but physically remove microorganisms) that can be used for liquids or gases (including air; see chapter 30) and the use of aseptic processing, based on the concept of ensuring the sterilization of various product components and keeping them under aseptic conditions during the manufacturing process to ensure a sterile product presentation (see chapter 58). In this latter case, the final product is not terminally sterilized (or subjected to a sterilization process in its final, manufactured state) but is essentially sterile based on best aseptic practices during the product manufacturing. The
combined benefits of aseptic processing techniques and terminal sterilization can allow for unique applications for delivering new sterile products to continue to innovate rather than being considered as separate approaches.8

The requirements for the first or antimicrobial considerations for preservation, disinfection, and sterilization are further discussed in this chapter. These can be broadly considered based on the requirements for achieving desired end points. Similarly, the second requirement for safety will also be considered. Other chapters in this book discuss the different types of physical and chemical technologies used for microbial control and public health in further detail.


Preservation, disinfection, and sterilization technologies are used to control microorganisms, thereby reducing the risks of adverse impacts such as infection, toxicity, and spoilage. But there are other safety requirements that may need to be considered in using these technologies. These can be considered as relating to impacts on the target application (eg, surface, product, functionality, or material compatibility), safety to those applying the technology for a particular purpose, safety to the end consumer or patient, and safety for the environment. As an example of surface or material compatibility, types of construction metals and plastics can be damaged by heat, thereby restricting the types of antimicrobial processes that can be used in certain applications. As an example of an exception to this is with incineration or other treatments for waste disposal because materials in these cases are typically discarded (see chapter 54). An even more restrictive example is seen in the antimicrobial applications to living (eg, skin) or nonliving tissues and many pharmaceutical products. Food and water applications will also be affected by more subtle criteria such as organoleptic properties (eg, taste, smell) and consumer perception that may limit product acceptance (eg, visual appearance). So overall, the balance lies between obtaining the desired level of antimicrobial activity with the minimum acceptable level of deterioration or damage to the target application. For sterilization, this can be important in the choice of and application of sterilization processes that may damage certain types of materials of impede product functionality depending on the technology used.9

The next consideration is the risks associated with those using the technology, such as from burns (eg, due to handling and application of heat) to side effects from the use of various antimicrobial chemicals that are hazardous or toxic. It is important to remember that technologies that are designed to affect the growth or multiplication of microorganisms are likely to have similar effects on human, plant, or animal cells based on their more general mechanisms of action (see discussion later in this chapter). These requirements are especially important in understanding short- and long-term health impacts, which can range from those that may resolve quickly (eg, minor burns) to those that can be more dangerous to health over time (eg, carcinogenicity, mutagenicity, and, at an extreme, instant death). Many of these can be considered as occupational risks that should be minimized, such as those defined by regulatory agencies such as Occupational Safety and Health Administration in the United States, Control of Substances Hazardous to Health in the United Kingdom, and the International Labor Office.10 Occupational risks may not only come from direct exposure to the antimicrobial process itself or by-products from that process but also due to accidents in the use or insufficient controls in using some of these technologies (eg, explosion accidents with ethylene oxide [EO] gas11). It is also important to consider that many widely used chemicals have been considered “safe” in the past but are now known to be of a higher risk, such as in the case of the historic use of mercury, types of organic solvents, and more recently with the ubiquitous use of microbicides such as hexachlorophene and triclosan.12 Essentially, it may be simple to conclude that all microbicides by their nature will be expected to do harm as much as we expect them to have their effects on microbial targets, but also some technologies will be more of a concern than others.

The third consideration is an extension of the second, being safe to the patient or consumer. The associated risks can come from the presence of residual by-products from the use or application of the microbicidal technology. Obvious examples include the presence of residual levels of chemical disinfectants or sterilants. These will include low levels of chemical sterilants such as EO and hydrogen peroxide (see chapters 31 and 32). The EO, for example, is classified as a carcinogen and mutagen; therefore, it is prudent to ensure that the use of EO is controlled and even restricted in its use under some situations. As an antimicrobial, its most widespread use is in the sterilization of medical devices, although it is also used for other applications such as with some foodstuffs (see chapter 31). For medical device applications, regulatory and standard requirements restrict the levels of acceptable EO residues that can remain on devices prior to patient exposure.13 These requirements include not only residuals of EO itself but also by-products on reaction with chlorine radicals (ethylene chlorohydrin) and water (ethylene glycol). As an example of a benefit-risk approach, the levels of acceptable residual levels can then be defined based on the expected patient contact, such as permanent contact, prolonged exposure, or limited exposure.13 Another example of the health risks associated with chemicals is the use of chlorine for water disinfection (see chapter 15). Chlorine was introduced in the early 1900s as a widespread drinking water disinfectant with clear benefits in reducing the infection risk with water and foodborne pathogens (see chapters 37 and 56). But by 1974, it was reported that the chemical reaction of chlorine with organic and inorganic compounds in water can produce halogenated compounds associated with health risks (eg, chloroform and other trihalomethanes).14 Disinfection
by-products are associated with all types of chemical microbicides widely used for water disinfection, with health risks at different levels including genotoxicity and carcinogenicity; estimates of at least 600 by-products were reported from the use of chlorine in drinking water, with the trihalomethanes widely used as indicators for drinking water safety.4,15 There is also a growing debate, often associated with conflicting data, on the effects of preservatives used in many consumer products that are used on the skin or mucous membrane, such as shampoos and cosmetics, or directly used in food. These can include effects on the immune system, complications in those with asthma, and at the extreme to include carcinogenicity risks.16,17,18

Central to the consideration of toxicity, particularly with chemical antimicrobials, is the concept of selective activity. Selective activity may be defined as injury to one kind of living organism without harming another that is intimately associated with it.19 This principle is used in agriculture, pharmacology, and diagnostic microbiology, but its most dramatic application is the systemic chemotherapy of infectious disease (eg, antibiotic use in the prevention and treatment of bacterial infections). Selective action against microorganisms is based on differences in the cell physiology of the microorganism and the mammalian or other host. Despite the general similarity of nutritional requirements, enzyme composition, and nucleic acid structure among all forms of life, there are many differences between microbes and humans in structure and metabolism, especially in the processes of cell synthesis. Energy-yielding processes do not offer the same possibilities for selective toxicity. Chemicals that inhibit a specific step in a metabolic pathway that is vital to the microorganism, but that does not occur or is not accessible in the cells of the host, exhibit selective toxicity. The treatment of bacterial infections has traditionally been more successful than that of viral disease because viruses depend on many enzymes of the host cell for their replication. Overall, chemotherapeutic agents are therefore often limited in their target antimicrobial activity and, equally, due to the ability of microorganisms to adapt or mutate to their presence, are at a high risk of becoming less effective over time. This has been well established since the widespread introduction of antibiotics but with more alarming consequences recently with the lack of development of new anti-infectives and parallel emergence of multidrug-resistant bacteria such as carbapenem-resistant gram-negative bacteria.20 The limitation for selective toxicity is different for surface disinfectants or sterilants that are used on inanimate surfaces or skin/wound antiseptics in comparison to systemic chemotherapeutic agents. A more limited range of chemicals can be practically used on the skin in comparison to a wider range used for work surface or product disinfection or sterilization. But in general, many of these chemicals demonstrate a broader range of antimicrobial effects with less specific, wider toxicity. Examples include the oxidizing agents that will target any microbial (or host) structure that can be oxidized to culminate in inactivation or the effects of increased heat in disrupting the structure and function of the various macromolecules that make up life. It is also less likely for resistance to develop to such broad-spectrum antimicrobials over time in comparison to the chemotherapy agents with a high degree of selective toxicity that are associated with a narrow antimicrobial spectrum and the emergence of drug-resistant microorganisms. But in between these types of extremes, there are antimicrobials such as the bisphenol, quaternary ammonium compounds (QACs), and chlorhexidine that are in the midrange, with limited spectra of activity against microorganisms and variable degrees of selective toxicity between the microbial targets and hosts.

The final point is the impact to the environment. There has been a greater focus on the use of chemicals as microbicides and their impact on the environment, especially those that may not readily break down in the environment (or as highlighted earlier in this chapter, may breakdown or react with other chemicals to form by-products that can impact the environment and its associated life). An example is the harmonized data requirements for biocides under the European Union’s Biocidal Products Directive (Directive 98/8/EC of the European Parliament21), which considers not only the toxicity risks on primary exposure to biocides (discussed earlier) but also the secondary risks through the environment.22 The regulation defined four categories of biocides (disinfectants, preservatives, pest control, and other products, such as embalming fluids) and 23 product types (eg, for disinfectants based on their use for human or veterinary hygiene as well as food, public health, or water use). Registration requirements include toxicological studies, ecotoxicological studies, and data assessment. Similar requirements are also in place regarding other chemicals that can impact the formulation of antimicrobial products (see chapter 5) as defined under the European Union regulation Registration, Evaluation, Authorisation and Restriction of Chemicals.23 The impact of these, and similar regulations in other countries and regional areas, has reduced the availability of many types of microbicides from practical use, particularly many chemicals known to be carcinogens, mutagens, and reprotoxic substances; endocrine disruptors; and those that are persistent and bioaccumulative.24 Other examples of environmental safety considerations include the following:

  • The safe disposal, including neutralization, of chemical disinfectants (eg, aldehydes) following their health care or industrial use into septic systems25,26

  • The EO27,28,29 and other gases released into the environment

  • Incineration controls, often due to the release of toxic byproducts from the incineration process30 (see chapter 54)

  • Handling and disposal of radioactive materials (see chapter 29)

  • Energy sources for heat generation

It may be expected that further regulatory controls in different countries as well as international mandates regarding the short- and long-term risks to the environment
will continue to impact the use of technologies for preservation, disinfection, and sterilization.

Overall, safety requirements can vary depending on the application. It is important to consider the risks and benefits in these situations to determine the best antimicrobial solution for a given situation, to include its safe, effective, and optimal use.


The use of preservation, disinfection, and sterilization technologies will often be associated with legal requirements including international; regional; national; and area-specific (eg, state or province) regulations, standards, and guidelines (see chapter 70). These can include specific requirements in order to legally commercialize or use different technologies for various applications as well as determining the continued safety and effective use of antimicrobial methods under routine use in laboratory, medical, veterinary, food production/handling, public health, and industrial environments. These requirements can have different legal consequences with regulations having the greatest impact, being defined as a rule or directive made and maintained by an authority. Examples include the Biocides Directive21 and Registration, Evaluation, Authorisation and Restriction of Chemicals23 requirements in the European Union; US Food and Drug Administration (FDA) regulations regarding food,31 medical devices32 (FDA CFR 21-Part 807), and pharmaceuticals33,34 (CFR 21 Parts 210 and 211); National Medical Products Administration (formerly China Food and Drug Administration or CFDA) regulations on medical devices35 (Supervision and Regulation of Medical Devices, No. 680) and drugs (Drug Administration Law 2019)36; Indian regulations on medical devices37; and many other specific country regulations that are periodically subject to amendments or revisions that may impact antimicrobial applications.

The next level down from these are more specific standards that can also be international (International Organization for Standardization [ISO]), regional (European Standard [EN]), or country specific (eg, American National Standards Institute-Association for the Advancement of Medical Instrumentation, American Society for Testing and Materials International, or Association of Official Analytical Chemists [AOAC] in the United States; Canadian Standards Association in Canada; British Standards in the United Kingdom; Deutsches Institut für Normung in Germany; Australia/New Zealand standards in Australia-New Zealand; and the Guobiao (GB) standards in China). For pharmaceutical applications, including drug manufacturing or dispensing, many of these are published in various pharmacopeia such as the US pharmacopeia, European pharmacopoeia, and Japanese pharmacopoeia, with international efforts to ensure the standardization of these requirements. Standards can be harmonized internationally or regionally, where the same standard is applied (such as those developed by ISO or International Electrotechnical Commission [IEC]) or modified with exceptions for a specific country. They can also be considered as extensions of certain regulations when deemed appropriate or best practice. Many standards are more specific in their requirements, but some can be more general such as in the case of ISO 9001 quality management systems,38 ISO 13485 for quality management systems in the manufacturing of medical devices,39 and ISO/IEC 17025 for the competency of test and calibration laboratories.40 Examples of specific standards in the area of disinfection and sterilization include those for washer disinfectors (ISO 15883 series), biological indicators (BIs) (ISO 11138 series), general sterilization processes (ISO 14937), specific sterilization processes such as steam (or moist heat sterilization under the ISO 17665 series), and radiation (ISO 11137 series).

Finally, there are different guidances published by many organizations that provide further information for specific applications such as in different health care situations, industrial manufacturing, food handling or manufacturing, and public health. Some are specific to those providing greater guidance of specific standards (eg, ISO/TS 11137-3 provides specific guidance on the parent ISO 11137-1 radiation sterilization standard), whereas others provide more general guidance on options for specific applications such as medical device reprocessing,41,42 clinical practice (eg, surgical best practices43,44,45), biological safety,46 water disinfection and preservation,4 pharmaceutical manufacturing requirements,47 and food safety.48 Some of these guidances are based on detailed literature reviews (referred to as “evidenced based”), consensus building by expert committees, or developed by key opinion leaders for publication by industrial or public health organizations. Guidance documents can be useful in the development or application of best practices for many situations and in some cases may be expected to be employed as requirements by regulatory or auditing organizations. It is important to note that regulatory expectations are not only required, when defined, to allow for the safe and effective use of antimicrobials or antimicrobial processes in various situations but are also often subject to periodic verification of these requirements such as during announced and unannounced audits to ensure compliance to those requirements.


Antimicrobial agents may be subdivided into either physical (eg, heat, radiation) or chemical (eg, halogens, oxidizing agents) types and in many cases are used in combination. Table 5.2 depicts some of the most common agents that are available and in widespread use. Chemotherapeutic agents are those that are used orally or systemically for the treatment of microbial infections of humans and animals.

The most important agents are antibiotics, together with synthetic compounds that may be used for their antibacterial, antifungal, or antiviral properties. These are not considered further in this chapter.

TABLE 5.2 Major types of antimicrobial processes used for preservation, disinfection, and sterilization

Type of Process





Dry heat (≥160°C)

Disinfection, sterilization

Less efficient than moist heat

Moist heat (≥115°C)


Use of steam

Moist heat (<100°C)


Inactivation of most pathogens over 70°C



Repeated freeze-thaw cycles can inactivate microbial cells.

Ionizing radiation


Includes multiple sources (γ sources, x-ray, E-beam)

Ultraviolet radiation


Requires direct light contact for optimal activity; may provide sterilization under some applications

Other nonionizing radiation


Used as sources of heat

Cell lysis methods such as those based on hydrostatic pressure changes or pulsed electrical field


Can inactivate microbial cells, with activity dependent on the application method, temperature, and in the presence of chemicals

Chemical (vapor phase)

Ethylene oxide


Most widely used chemical in sterilization processes


Disinfection, sterilization

Laboratory room disinfection and limited use for sterilization

Hydrogen peroxide, peracetic acid, chlorine dioxide, ozone

Disinfection, sterilization

Oxidizing agents for low-temperature disinfection and sterilization processes

Gas plasma

Disinfection, sterilization

Can be generated from inert or antimicrobial gas(es)

Chemical (liquid phase)

Acids, alkali, and derivatives

Preservation, disinfection

Widely used for preservation, including foods


Preservation, antisepsis, disinfection

Widely used in antiseptics and for surface cleaning/disinfection

Aldehydes (glutaraldehyde, OPA)


Glutaraldehyde is used as a chemical sterilant.

Halogens (including sources of chlorine, iodine and bromine)

Preservation, disinfection, sterilization

Widely used for surface disinfection and food applications

Oxidizing agents (hydrogen peroxide, peracetic acid, chlorine dioxide, ozone)

Antisepsis, disinfection, sterilization

Increasing used as alternatives to aldehydes

Antimicrobial metals including silver and copper

Antisepsis, disinfection

Often used in or as impregnated surfaces. Many other metals have been in traditional use such as mercury and tin.


Antisepsis, disinfection

Skin applications and surface disinfectants

Quaternary ammonium compounds

Antisepsis, disinfection

As surfactants, can be combined for cleaning applications


Preservatives, antisepsis

Chlorhexidine is the most widely used, particularly as an antiseptic.


Preservatives, antisepsis

Examples include acridines and crystal violet.

Essential oils

Preservatives, antisepsis, disinfection

Plant extracts, often consisting of multiple antimicrobials such as phenolics and aldehydes (eg, pine or tea tree oils)


Peptides, proteins, and enzymes Bacteriophages

Preservatives, antisepsis, disinfection

Often associated with limit spectrum of activity in comparison to other microbicides above

OPA, ortho-phthalaldehyde.

a Some of these methods can be applied for both disinfection and sterilization depending on their applications, but others are limited to disinfection due to their lack of practical antimicrobial activity against more resistant forms of microorganisms such as nonenveloped viruses, protozoal (oo)cysts, and bacterial spores (see chapters 3 and 4).

b Filtration methodologies used for disinfection and sterilization are generally excluded from this definition as they may be used for the same end points by physical removal but are not typically associated with antimicrobial activity (see chapter 30).

Several physical processes such as moist heat, dry heat, and ionizing radiation under defined conditions can be relied on to kill all types of microorganisms, including more resistant forms such as bacterial spores, and thus are expected to achieve sterilization when correctly validated and maintained (see Part IV). These processes can be controlled even under sometimes difficult situations (such as in complex loads to be sterilized with features or materials that are difficult to ensure access of the antimicrobial process), and the assurance of such penetration capabilities has seen their widespread adoption as the major types of sterilization processes used today. Moist heat is not only a practical and relatively straight forward sterilization process to apply to heat-stable materials for traditional infectious agents but has also been shown to be effective against nontraditional infectious agents such as prions that are not readily inactivated by other methods such as radiation or dry heat (see chapter 68). Temperatures below 100°C (eg, as low as 50°C-60°C) can be of value in various thermal disinfection applications, including pasteurization (a widely used disinfection method in the preparation of and risk reduction in foods and many other materials or surfaces) and in the preparation of certain bacterial vaccines when the aim is to inactivate all the cells without affecting their antigenic identity. Moist heat is preferred over dry heat due to being more efficient as an antimicrobial process (a concept that is considered in more detail in chapters 11 and 28). At the opposite end of the heat spectrum, cold or even freezing temperatures are widely used as preservative methods. These are not generally appreciated to cause microbial death, although in some applications, freeze-thawing cycling can be associated with cell death and presumably due to the disruption of the membrane structure/function of cells. Ultraviolet (UV) radiation, as a nonionizing radiation source with less associated energy in comparison to ionizing radiation, is considered less effective as antimicrobial agent but still can have the ability to inactivate all microorganisms including bacterial and fungal spores; but due to limits in penetration capabilities and ensuring that all surfaces in a target applications have direct contact with UV light to enable antimicrobial activity, this technology has been more widely used for liquid, gas/air, and surface disinfection. Despite this, certain applications, such as in space with UV exposure, can be sustained, and high use of this technology for surface sterilization may be a practical reality.49 Other physical methods have been identified for niche applications such as those based on hydrostatic pressure and pulsed electrical fields that target lysis of vegetative cells and may otherwise be limited in antimicrobial effectiveness (eg, against spores or viruses) and scope of applications (eg, in certain food or other industrial uses). Hydrostatic pressure, for example, is a method that uses the high pressures exerted by liquids on microorganisms, whereas pulsed electrical fields use an electric charge to cause disruption of cell walls in bacterial and fungi. Outside the scope of this section are physical methods used to preserve, disinfect, or sterilize by filtration mechanisms such as liquid or air filtration (see chapter 30) and the use of packaging materials for preservation or sterile presentation (see chapter 41) because these do not generally provide any intrinsic antimicrobial activity; their mechanism of action is simply by physical removal or exclusion of microorganisms.

A similar range of chemical agents and applications has been described. Like physical agents, they can range from those that are used to prevent the growth of or inactivate microorganisms. Chemicals can be limited not only in their antimicrobial activity but also in their ability to be safe for various applications. Preservatives, for example, need to be able to prevent the growth of many types of bacteria and fungi, and without reacting with or affecting the use of the product, they are used in either from a toxicity, effectiveness or, in many cases, aesthetic point of view. As discussed earlier, disinfection can be achieved with a wider range of chemical and physical methods. At the lower end are those microbicides that can be safely used on the skin or mucous membranes such as the bisphenol (eg, triclosan), chlorhexidine and other biguanides, alcohols, silver, and
iodine, depending on their in-use concentration. Thermal methods are more restrictive as typically pain tolerance is in the 50°C to 55°C range, which only demonstrates limited antimicrobial activity. But these requirements become less of a concern with inanimate surfaces, applications to food and water, manufacturing equipment, etc. Therefore, in these cases, a wider variety of chemical (aldehydes, oxidizing agents, QACs, and phenolics) and physical (radiation sources and heat) methods can be used.

As a greater expectation of microbial inactivation is required, this list of choices becomes more limited to meet the needs for sterilization. A traditional and minimal requirement (or prerequisite) for this is often sporicidal activity due to their resistance profile to inactivation (see chapter 3). Physical methods (heat and radiation) are widely used but equally are certain types of chemicals (eg, aldehydes, oxidizing agents, and EO). Many types of chemical agents can also act as sporicides, such as glutaraldehyde (pentanedial), formaldehyde (methanal), hypochlorites, and EO gas. But their activity can often be slower against bacterial spores; however, this can be influenced by many factors such as the condition of the spores (present in liquid suspension or dried onto test objects), the presence of organic matter, the concentration and pH of the antimicrobial agent, presence of humidity, temperature, formulation, and state in which the chemical is in (eg, liquid or gas).5,50,51 For example, optimal antimicrobial activity has been described under controlled humidity levels or, perhaps more accurately, the microenvironmental water content, for many sporicidal gases such as EO, formaldehyde, chlorine dioxide, and ozone. Methods of potentiating the activity of an agent are obviously important in antimicrobial product or process optimization. Formulation effects, defined as the combination of chemical ingredients such as microbicidal agents and antimicrobial agents and other ingredients (eg, stabilizers, buffers, solvents, chelating agents, etc), can have significant impacts in effectiveness (see chapter 6). pH can be important in optimizing antimicrobial activity and stability of the microbicide over time, with examples being in the use of chlorine (see chapter 15) and aldehydes (see chapter 23). Acids and bases can have intrinsic antimicrobial activity, but there can also be combined effects in increasing the activity of formulated microbicides. The activity of most compounds increases with an increase in temperature (see chapter 7),50 but this will reach a limited dependent on the microbicide and may also increase the degradation of the antimicrobial to decrease its availability over time. The impact of other physical factors in the activity of microbicides is considered in further detail in chapter 7.

Combinations of antimicrobial agents may be used in disinfectant formulations. They offer the following advantages:

  • Gaps in the range of action of a certain antimicrobial agent can be filled by a second agent. This helps to widen the range or have a broader spectrum of action. Some agents act preferentially against gram-positive bacteria, and others against gram-negative types. A third group may have preferable activity against molds and yeasts. Instead of increasing the concentration of an agent to get a broader spectrum of activity, it is often better to add a second agent of complementary activity. Only a small amount of the additional agent may be necessary.

  • Lower toxic and ecologic risk. If a combination of two or more antimicrobial agents is used in a formulation, the concentration of each agent can be lower than in a formulation with only one of these agents. By reducing the concentration of an agent, the toxic effects are also reduced, if possible, below the threshold value (eg, for sensitization), which is becoming increasingly desirable.

  • Solubility. If a saturated solution of a given agent of low solubility does not show sufficient efficiency, the required efficiency might be achieved by the addition of a second agent.

  • Product or regulatory requirements. The choice of microbicides may be limited in some applications such as in the case of preservatives in cosmetics (see chapter 40) and on the skin (see chapters 42 and 43), where combinations are required to show product expectations. Commercial antiseptics and disinfectants may be subjected to official testing for registration purposes, and a variety of test method requirements may need to be fulfilled. A good example for this is the requirement of many test method or efficacy requirements in skin hygiene to show a substantiate (or residual) antimicrobial effect, which will be less likely with some microbicides (eg, alcohols) in comparison to others (see chapter 43), but the combination of these actives may provide some overall benefit.

  • The risk of tolerance or resistance development against the antimicrobial(s) is lower when two or more antimicrobial agents are used.

In selecting antimicrobial agents, mutual effects on the action of the substances are of significance. Some substances have an inhibitory effect on each other, such as active chlorine compounds with an oxidative effect on aldehydes or phenols. Other combinations of active substances mutually enhance their actions, a fact well known from the patent literature. It is therefore the aim of those developing disinfectants (especially in liquid formulations) and sterilization processes to satisfy the demands on the product as far as possible by selecting suitable combinations of antimicrobial agents. Thus, the content of active substances in disinfectants may be reduced, which may even help to make production more economical.52,53 Many studies have been published on the combined effects of two or more active substances, but it is important to consider that these effects may be considered as additive (when both microbicides contribute together to the antimicrobial effects), in synergism (where the they act together to give greater efficacy than either alone), or in antagonism (and therefore a negative overall impact).
These concepts not only apply to the various types of microbicides considered in this book but also to other chemotherapeutic agents. The effects can be best described graphically. In Figure 5.1, the full antimicrobial effect of two different substances, A and B, is displayed on the abscissa and the ordinate of a coordinate system at points M and N, respectively. This effect is achieved by substance A at concentration M and by substance B at concentration N, respectively. Assuming an increase up to the full effect has a linear character, the subdivision of the effect is supposed to be equivalent to the corresponding partial concentrations. There must be a series of partial concentrations of A and B giving the same effect in combination as the effective concentration of each substance alone. The simplest case would be the addition of the two partial effects corresponding to the respective partial concentrations. This may be shown graphically by drawing a straight line between N and M. All points on this line represent combinations giving identical effects (Figure 5.1A). The points M and N represent the inhibitory effects caused by the minimum inhibitory concentrations (MICs), M and N, of the two agents A and B. All points on the line between M and N represent the inhibitory effects caused by the different combinations of agents A and B. The combinations lying within the triangle of 0 (intersection of the X and Y axis), M and N (except those on the line MN) fail to give an inhibitory reaction. All the other combinations give positive results. This typical pattern is characteristic for additive effects. Synergism of antimicrobial combinations can be defined as the supra-additive effects of combined antimicrobials. Figure 5.1B shows an example of the synergistic action of two agents. Combinations of parts of the inhibitory concentrations of the two agents A and B reveal a higher activity than that expected on the basis of a purely additive effect. The curved line in Figure 5.1B indicates the limit of effectiveness.

FIGURE 5.1 Combined effect of two antimicrobial agents A and B in the case of an additive behavior (A) and a synergistic behavior (B).

A mathematical formula to describe these relationships can be given as:

where x and y refer to the actual concentrations of the combination of the agents A and B, which exert the same antimicrobial effect as the MICs, M and N, of the two agents alone.54 If the left side of this equation totals 1, we have a combination showing an additive effect. A result smaller than 1 indicates a synergistic effect of the combination.

These effects can also be demonstrated experimentally based on both microbiostatic and microbicidal test methods.52,53 Examples include bacteriostatic tests such as MICs that are often employed in the testing of preservative considerations and bactericidal tests such a time-kill studies in the development and optimization of disinfectants (see chapter 61). The first step is the determination of the MIC of the two antimicrobial agents A and B alone. A series of successive dilutions of each agent is prepared in a liquid nutrient medium and then inoculated with the test bacteria (or fungi, if applicable) to be tested to give a known cell number (eg, 103-107/mL). After an incubation period (eg, for bacteria, 24-48 h), the tubes are examined for growth or no growth, where the lowest concentration observed for no growth is determined to indicate the MIC of each test agent. In a second step, the combination of A + B is tested in an analogous manner by using the MIC of the two agents and some higher and lower concentrations of this mixture. After inoculation and incubation, the MIC of the mixture is again determined. With the help of the formula in the earlier equation, it can be calculated whether the two components give an additive, a synergistic, or an antagonistic reaction. It is important to note in these cases that interference factors (eg, growth media) are present that can influence the activity of the microbicide(s).
A good example would be attempting to demonstrate a true MIC with oxidizing agents (eg, chlorine, iodine, hydrogen peroxide) that will readily react with media components will clearly underestimate their true efficacy. In bactericidal tests, dilutions of the two disinfecting agents to be tested are also prepared separately and in combination, but in these cases, it is recommended for the test to be performed in water (or a similar noninterfering diluent) without any nutrient compounds. The dilutions are again inoculated with a known cell concentration (eg, 106-107/mL). After exposures that can range from a few seconds to many hours (depending on the test microorganism and product requirements), samples are removed and analyzed for surviving cells (see chapter 61; note the importance of neutralization to ensure the antimicrobials under tests are immediately halted in their activity to give a true account of the extent of kill at each exposure time point). Microbial quantification can be done by two different methods, direct analysis of cell numbers remaining or end point analysis. In direct enumeration, the number of viable cells is determined by plating, indication, and counting, with the results plotted against time logarithmically (Figure 5.2). Synergism is shown by the combination of two agents showing a faster rate of killing than twice the concentration of either agent alone. Addition effects will show the rate of killing of the agents in combination is approximately that expected from simple algebraic summation of a single agent alone. Finally, antagonism will show a rate of killing of the combination lower than that of one or both of its components. In end point analysis, the samples are transferred into tubes containing liquid medium to allow the surviving cells to grow. After the desired incubation time, the tubes demonstrating growth/no growth are examined, and those with no growth indicate complete killing of the test population (end point). For each dilution, the time needed for complete inactivation can be determined, and again, addition effects will be indicated by the killing time of the combination being nearly the same as that needed by twice the concentration of either agent alone (ie, a mean value of the two killing times), and synergism where the combination is shorter, preferably by one-half or less, than that needed by twice the concentration of either agent alone. Many different iterations of these essential tests are useful in optimizing the benefits of formulation effects as well as combinations of actives (see chapters 6 and 61).

But it is worthwhile to have a note of caution at this stage in the identification and use of synergistic effects of two or more substances in disinfectants due to the potential inconsistency in the increase of effectiveness against different microorganisms. Many of the examples of synergism of two substances described in the literature and particularly in the patent literature concern a limited selection of test organisms. In some cases, improvement of action on one group of microorganisms may be accompanied by reduced effects on another group. For example, a combination of a QAC with the anionic agent undecylenic acid resulted in an antagonistic effect when S aureus was used as the test organism but not with the fungus Trichophyton mentagrophytes.55 Thus, some additive or synergistic combinations can be used only to compensate for a specific weakness against a single microbial species but may not apply to others.

FIGURE 5.2 Interaction between two disinfecting agents A and B in a quantitative bactericidal test.53 A, Synergism. B, Additivity. C, Antagonism.

It is outside of the scope of this chapter to provide further details of the many difficult combination of actives (including chemical-physical interactions) that can provide positive impact in antimicrobial activity, but some examples include the following53:

  • Chelating agents (eg, ethylenediaminetetraacetic acid [EDTA]) with overall poor activity against bacteria is used at low concentrations to demonstrate
    greater bactericidal activity of bisphenol such as triclosan against gram-negative bacteria (where intrinsically gram-negative bacteria are more resistant than gram-positive bacteria).

  • UV radiation (and indeed other energy sources such as temperature) potentiates the activity of hydrogen peroxide.56

  • Hydrogen peroxide and peracetic acid have been shown to have synergistic activities in their subtly different modes of action against bacterial spores (see chapter 18).

  • Combinations of QACs are widely used as broad-spectrum (against gram-positive and gram-negative bacteria, including in some situations mycobacteria) surface disinfectant formulations (see chapter 21).

  • Reaction of two chemicals or microbicides that result in a more active product. For example, sodium iodide, a chemical without appreciable antimicrobial potency, may be oxidized by a reagent such as 2,2-dibromo-2-cyanoacetamide, a highly active disinfectant, giving free iodine, which provides an even higher antimicrobial activity than that of 2,2-dibromo-2-cyanoacetamide.52 The microbicidal activity of peroxy compounds, such as hydrogen peroxide or sodium perborate, is enhanced by combination with a suitable catalyst, which accelerates the formation of oxygen radicals as the active principle of these compounds. Because of their high chemical reactivity, they have to be mixed just before application, giving a disinfectant solution with a short life span. This may be an advantage if only a short active period of disinfection is required, but similar and more stable combination products have also been described with greater benefits from a generational, stability, and safety points of view.

  • Surfactants are often important constituents of disinfectants. They are used to achieve both uniform wetting of the surface to be treated and frequently for the additional cleaning effect they provide (see chapter 21). Particular attention should be given to this group of substances when formulating a disinfectant because there are many ways in which the two groups of compounds can interact (see chapter 6). For example, anionic surfactants promote the inactivation of cationic QACs, and nonionics (eg, Tween 80 or polyoxyethylene sorbitan monooleate) are capable of binding microbicides such as cetylpyridinium chloride, some phenols, and chlorhexidine (hence its use in neutralizing solutions52,53). In contrast, low surfactant concentrations of 0.1% or less are often accompanied by an improvement in the action of antimicrobial agents such as p-hydroxybenzoates preservatives.57 Alkyl glucosides have shown synergistic activity in combination with biguanides, alcohols, and organic acids.52 As another example, nisin is an antimicrobial peptide with antimicrobial properties that is produced by the gram-positive bacterium Lactococcus lactis subsp lactis particularly against gram-positive bacteria and has limited antimicrobial efficacy against gram-negative bacteria, yeasts, and mold; this can be significantly increased in formulation with 30 to 300 ppm nisin, 20 mmol/L EDTA, and 0.1% to 1.0% Triton X-100.58

Overall, despite the individual or combinations of microbicides used, the response of microorganisms to adverse agents will depend on the type of target organism(s), the biocidal agent(s), and the intensity (eg, concentration of a chemical, temperature of exposure, radiation dose) and duration of exposure. In bacteriostasis or fungistasis, reversible inhibition of growth and multiplication can be achieved by restoring the organism to favorable conditions. Microorganisms injured but not killed by a particular process may be able to repair the damage inflicted on them, and such damaged cells should be considered in the design of procedures used for the validation of sterilization and disinfection processes, in their detection and enumeration, and as a health hazard in processed foods.59 It may also be necessary to use an appropriate antagonist or neutralizing agent (inactivator, inactivating agent, neutralizer, antidote) to distinguish between a lethal effect and mere inhibition in cells exposed to disinfectants, antiseptics, or preservatives. The theoretic principles in using neutralizing agents is further discussed in chapter

61. Table 5.3 summarizes the uses of neutralizing agents. Moreover, in some instances (eg, mercury, silver, and thiol compounds), the chemical nature of the neutralizing agent may provide a clue as to the mechanism of action.


The main goal of preservation is to prevent the growth of microorganisms in (or in the case of packaging systems, the ingress of microorganisms into) various products or applications. There are two main methods to achieve this goal: the physical exclusion of microorganisms and the use of antimicrobials or antimicrobial processes.

Physical exclusion as a concept for preservation was elegantly confirmed by Louis Pasteur in 1862 in the demonstration that a torturous path using a swan-neck shaped flask was sufficient to prevent the passage of microorganism to a culture media (see chapter 2, Figure 2.2). Similar designs (including food canning methods) and innovations in microbial barrier materials since that time are the basis of modern sterile barrier or exclusion systems used today (see chapter 41). Similarly, microbial retentive materials and methods are the basis for disinfection and sterile filtration technologies for air and liquid applications for products and environmental controls (see chapters 30 and 58, 59, 60). These are essentially based on the same exclusion processes such as providing a complex, torturous path to microorganisms or limiting their passage by size exclusion.

Historic methods of preservation were widely used in the past prior to any modern understanding of microbiology, including salting, drying, and use of heat. Salting and drying of food was found to allow foods to last for longer time periods without degradation by various bacteria and
fungi now known to be present. The use of other chemicals such as vinegar (as a mild acid) in pickling is another common example in more modern times, although the use of variety of chemicals was only directly shown to have effects on microorganisms (or “animalcules”) by Antonie van Leeuwenhoek in the 1670s with the development of microscopy. Plants and plant extracts have a long traditional history of use in the treatment or even prevention of illness with examples from herbal remedies used in diverse worldwide cultures.60 Although often controversial in modern scrutiny of therapeutic requirements, these extracts are known to have direct antibacterial requirements due to the presence of various natural antimicrobials such as aldehydes and phenolics. High heat mechanisms, including boiling and incineration have been historically appreciated for disinfection and preservation but only became fully understood since the 1800s and again with the development of methods such as pasteurization. Pasteurization is a disinfection method, but it is used to extend the shelf life of foods and liquids as a preservation method by reducing the numbers and therefore growth of microorganisms that lead to spoilage. Similar methods may be used to reduce the levels or types of microorganisms in raw materials used in a variety of manufacturing environments. Maintaining foods or tissues at cold or even freezing temperatures has the same impact as effective preservation methods. Similarly, product dehydration or drying is a useful way of preventing the growth of bacteria and fungi due to the reduced levels or even lack of available water for metabolism.

TABLE 5.3 Examples of chemical agents that neutralize antimicrobial compoundsa


Neutralizing Agent



Dilution to subinhibitory level; glycine better

Sodium sulfite not recommended (may itself be toxic)


Dilution to subinhibitory level

Tween 80 is possible alternative.

Organic acids and their esters

Dilution to subinhibitory level

Tween 80 is possible alternative.

Hydrogen peroxide

Catalase, sodium thiosulfate

Rapid effect, but some bacteria can be inhibited by sodium thiosulfate

Peracetic acid

Sodium thiosulfate

Rapid effect, but some bacteria can be inhibited by sodium thiosulfate

Mercury compounds

Thiol compounds

Possible toxicity of sodium thioglycollate


Dilution to subinhibitory level

Quaternary ammonium compounds, biguanides

Letheen broth (or agar); lecithin + Lubrol W

Ethylenediaminetetraacetic acid and related chelating agents


Mg2+ useful

Hypochlorites, iodine

Sodium thiosulfate

Rapid effect, but some bacteria can be inhibited by sodium thiosulfate

Silver compounds

Thiol compounds

As for mercury

a Note that membrane filtration may be alternative method, where the microbicide does not bind to the filter material.

The earlier demonstration of the effects of chemicals as preservatives led to the development of modern chemical preservation methods that continue to evolve today to meet product and regulatory requirements (see Part V). Examples include the use of chemical preservatives of products to prevent product spoilage (as in the case of cosmetics, paints, and natural products like those based on wood), decreased product acceptance by consumers (eg, organoleptic, being acceptance of a product due to taste, color, odor), or the overgrowth of microorganisms (such as in water or foods) that can lead to an unacceptable infection or associated toxicity risk in consumers. In most of these cases, the presence of a chemical preservative can be used not only to initially reduce the presence of microorganisms but also to prevent their potential growth over time during the manufacturing, storage, shipping, and even use of the product. For this reason, there is an emphasis on controlling bacterial and fungal contaminants due to their ubiquitous nature, their ability to be able to multiply even under sometimes unfavorable conditions, and the historical implications of such microorganisms in reported cases of product failure leading to spoilage, infections, or associated toxicity. In food preservation applications, these will include notable foodborne bacterial pathogens such as Shigella, Escherichia, and Salmonella (see chapters 37 and 56). But similarly, there is an increasing emphasis on viral and protozoal pathogens, with a focus on noroviruses and rotaviruses that have become prevalent in food and waterborne infections and outbreaks.61,62 In cosmetic and non-sterile pharmaceutical applications, there is a much greater emphasis on certain pathogens that are known to be resilient or persistent in transmission situations such as those referred to as being objectionable
or opportunistic microorganisms.34,63,64 These terms has been become widely used in the United States in cosmetic and pharmaceutical applications. Microorganisms can be generally defined as being “objectionable” in view of the product’s intended use and in general in relation to products not required to be sterile. But overall, the term may also be generally applied to any situations where the detection of a specific type of microorganism is considered a higher risk due to its known resilience and virulence based on previous experience. Many of these have been widely described in the past such as Pseudomonas, Burkholderia, and Bacillus cereus that once detected in a manufacturing environment are cause of an alert due to their known persistence and association with potential patient impacts including infection and toxicity.63,64 Pseudomonads such as Pseudomonas and Burkholderia species are noteworthy given their propensity to persist in low nutrient availability conditions in even pure water conditions or the presence of low concentrations of biocides that are used for preservation conditions (eg, in cosmetics and drinking water situations); they are resilient due to many intrinsic and acquirement resistance mechanisms.5 But in addition to their resilience, they can be pathogenic, are often associated with water contamination and biofilm formation, and are associated with product-associated outbreaks.65,66 Other examples include bacteria such as Aeromonas, Enterobacter, Klebsiella, Clostridium, and Staphylococcus and fungi such as Aspergillus and Penicillium. As already stated, many of these microorganisms have been described as having known tolerance factors to antimicrobial processes such as spore formation, efflux mechanisms, cell surface alterations, and other innate and acquired tolerance of resistance factors that give them opportunities for survival in various industrial manufacturing and preservation technologies in comparison to other pathogens (see chapters 3 and 4). Therefore, these pathogens are often linked to product-associated outbreaks or contamination (eg, product spoilage) reports. It is important to note that microorganisms and their associated risks (eg, presence of toxin, enzymes, and chemicals) can lead to a variety of infection and toxic effects in consumers/patient depending on their quantitative levels, virulence, and host interactions.

Preservation requirements and technologies are further considered in chapters 38, 39 and 40 for industrial, pharmaceutical, and cosmetic applications. Consideration is also given to the preservative requirements in food and water (see chapter 37) and for packaging, particularly sterile packaging, to maintain product integrity (see chapter 41).


Disinfection, defined as a process to inactivate viable microorganisms to a level previously specified as being appropriate for a defined purpose, can encompass a wide range of applications and desired outcomes (see chapter 2). These include antiseptics, food or water disinfectants (including pasteurization), general or critical surface disinfection, area disinfection (fumigation), and medical/dental device applications. A variety of chemical and physical methods in their gaseous or liquid forms are used as summarized in Table 5.2. They vary in their antimicrobial effects and safety requirements. A good example of this is the restricted use of certain antimicrobials on the skin and mucous membranes due to their specific activity against certain microorganisms but also their minimal negative (including toxic) effects on those tissues. Other examples will include the direct use of antimicrobials on plants, foods, and in water. To these examples one can add the variety of filtration methods used to remove microorganisms depending on their sizes for a variety of liquids (including water) and gases (including air). The higher order of disinfection or filtration methods are associated with the complete inactivation or removal of microorganisms, which is further considered under the sterilization section in this chapter.

Before discussing the inactivation of microorganisms during disinfection, a brief consideration is given to filtration. Filtration is often considered separate to disinfection and sterilization methods as it generally does not encompass microbial inactivation, although in some cases, filtration can be combined with antimicrobial processes such as for filter disinfection/maintenance (eg, heat, UV light or periodic use of chemicals including chlorine, bromine, peracetic acid, and silver/copper) or where chemical antimicrobials are included into the structure of filter materials (eg, nanotechnology including silver or carbon, and natural products).67 Microorganisms vary in size from larger cells such as yeasts, molds, and protozoa in the approximately 10 µm size down to smaller viruses to approximately 10- to 20-nm diameter range.5 With this knowledge, filters can be designed to retain or prevent the passage of microorganisms by a knowledge of their exclusion size and demonstration of effectiveness by various test methods using physical tests (eg, the bubble-point test to determine the pressure at which a continuous stream of bubbles under pressure is observed passing through a wetted filter68), particulate tests (eg, testing the presence of particulates in air following filtration69,70), and indicator microorganisms (eg, the use of Brevundimonas diminuta or other challenge organisms71,72). Filters can be made of a variety of organic and inorganic materials including paper, glass, ceramics, metals, polysaccharides, and variety of polymers. For example, high-efficiency particulate air filters are widely used fiberglass filters that can be designed to remove particulates (including microorganisms) as low as 0.5 and 2.0 µm in diameter, such as those used in cleanrooms (see chapters 58 and 60) and microbiological cabinet designs (see chapter 59). Filters can be classified based on their size exclusion, with examples including course filtration methods (often used as prefilters to extend the lifetime of other more expensive, lower size
exclusion filter types), microfiltration (generally including most bacteria and fungi to 0.1- or 0.2-µm diameter), and other methods such as nanofiltration or reverse osmosis methods that can be designed to ensure the removal of the smallest known viruses (eg, in the 10-20 nm range) as well as chemical contaminants such as metal ions. The latter methods can be considered sterile-grade filters because they can be validated to remove microbial contaminants to provide sterile products (see chapter 30).73,74

The hierarchy of resistance of microorganisms to inactivation as well as labeling or testing requirements to demonstrate the extent or indeed limits of various disinfectants and disinfection processes were introduced earlier. Antimicrobial claims can be specific to certain types of microorganisms (with claims such as bactericidal and viricidal) and broader claims defined by regulatory requirements in various countries/areas (eg, low-, intermediate-, or high-level disinfection; see chapter 2). The associated test methods used to demonstrate efficacy are useful to ensure these products/processes can be effective under recommended use conditions and are required to be verified (and/or validated) in many specific applications (eg, in industrial manufacturing, high-risk microbiological laboratories, or other regulated environments; see chapter 61). Best practices in disinfection (and sterilization) science dictate not only to demonstrate that a given microbicide, disinfectant formulation, or disinfection process can inactivate certain types of microorganisms but also to study the effects of these under various test conditions, including time and the presence of interfering factors. Microbiological survival curves, which are generally graphed as semilog plots of the ratio of the concentration of surviving organisms to the initial number versus time, are commonly used to present inactivation data and to interpret the inactivation kinetics of microorganisms. These curves can be useful in studying the kinetics of microbial inactivation, determining the predictability of the product/process in microbial inactivation, comparing the resistance of microorganisms to antimicrobial processes (eg, in D-valve estimations), and providing a more detailed analysis of what is occurring during the microbicidal application (see further specific details on the studies and impacts of this analysis in chapters 7, 11, 28, and 61). Typical curves, as shown in Figure 5.3, may be linear but are frequently nonlinear. Nonlinear curves can include sigmoidal, concave-upward, or concave-downward configurations. Several concepts, based on theoretic and mechanistic approaches, have been developed to explain the configurations of these curves.

A linear microbial survival curve demonstrates a predicable antimicrobial process against a uniform population of microorganisms, when the inactivation curve is well established (see the discussion later in this chapter on “Sterilization”). This can allow for the extrapolation of the continued antimicrobial activity over a longer time (or dose in the case of a radiation process), which is an important concept in the demonstration of a sterility assurance level (SAL) for sterilization processes discussed later. But other curve types can equally provide information about the target microbial population and/or the antimicrobial process/product being applied. An example was originally proposed in 1918 based on the assumption that the resistance of different individual cells in a population of apparently similar microorganisms may be different, and a vitalistic concept was used to explain the sigmoidal or upward-concave survival curves.75 They suggested that organisms possessing an average degree of resistance to the antimicrobial process would be found in the majority of the population, whereas those possessing a maximum or minimum degree of resistance would be found in the minority. Consequently, the survival times were expected to be normally distributed, but no experimental evidence was available to verify this concept.76 Others demonstrated that the logarithm of survival time for some organisms was normally distributed, and this indicated that the microorganisms resisting in a minimum degree are present in the greatest number, in contrast to the vitalistic concept.77 In a comprehensive analysis of disinfection kinetics, several theoretic and practical drawbacks of the concept were discussed and indicated the need for further experimental evidence to substantiate the concept.78

FIGURE 5.3 Representation of the configurations of microbial survival curves (showing log10 microbial reduction over time) on disinfection and sterilization process/product exposures: concave upward (1), sigmoidal (2), linear (exponential) (3), and concave downward (4).

The initial shoulder effect associated with many microbial survival curves (eg, curve 1, Figure 5.3) has been analyzed in different ways. The expressions developed from the multitarget79 and multihit80 phenomena were able to generate survival curves that were concave upward. The multitarget approach assumes that the microorganisms
possess several vital sites, each of which must be hit once before inactivation. The multihit or series-event concept assumes that only one sensitive target requires several hits for inactivation. These approaches did not prove to be satisfactory in fitting data for some studies,81 but they were successful in fitting data for others.82 In another approach, it was considered that virus inactivation by iodine was first order and that the nonlinearity caused by the multihit effect was necessary to inactivate the organisms that remained as clumps.83 Moreover, it was deduced that a typical lagging curve occurs if all the virions form clumps of approximately equal numbers of infectious particles. In contrast, survival curves for the chlorine inactivation of single virion preparations of poliovirus type 1 at low pH also resembled typical lagging curves.84 Using the same organism and disinfectant at pH 6, others reported a similar curve configuration that was concave upward.85 These curves are a typical example of how antimicrobial activity can vary depending on the exact antimicrobial being applied, including concentration, temperature, time, pH, etc. The inactivation of a suspension of clump-free protozoan cysts with ozone produced typical lagging curves.86 Similar observations have been made in the inactivation of protozoan cysts by chlorine87 and chlorine dioxide.88 Thus, the existence of an initial lag in microbial inactivation may not be attributed solely to simple aggregation of particles.

The inactivation rate of a single-cell suspension of Naegleria gruberi cysts followed first-order kinetics when exposed to iodine, with a shoulder of survival curves proposed to be due to the aggregation of cysts.89 A multi-Poisson distribution model was developed to explain cyst inactivation kinetics, assuming that the destruction of each individual of the clump was first order of kinetics. Two additional assumptions were made in the derivation of the distribution model: First, the rate of inactivation of clumped organisms is directly proportional to the concentration of clumps of a specific size, and second, the destruction of a clump is sequential, with only one individual inactivated at a time. Although no experimental evidence substantiated the last two assumptions, the multi-Poisson distribution model appeared successful in fitting their data.

It is evident that none of the various hypotheses can be considered entirely correct because they have not been subjected to adequate independent verifications. A common kinetic theory is difficult to apply universally because different microorganisms behave differently under similar experimental conditions, and the same microorganism may behave differently under various experimental or growth conditions. But kinetics based on empiric or mechanistic approaches can be used with reasonable confidence for engineering applications, such as designing full-scale treatment units or predicting the extent of inactivation under different hydraulic and mixing conditions. For example, using the multitarget and series-event kinetic parameters generated from batch data, the response of microorganisms to UV was successfully predicted for inactivation in flow-through reactors.82

Depending on the type of organism, the kinetics of the inactivation of relevant subcellular components such as DNA and RNA may also need to be established experimentally if the inactivation of those components is deemed necessary. Inactivation kinetics can be established for different viability endpoints as needed. For example, kinetic data can be established for the inactivation of the microorganism as well as the denaturation of its genetic materials if such treatments are deemed necessary based on the risk associated with microbial survival (eg, with some viruses). Such applications may be required in the treatment of infectious wastes and materials from bioprocessing facilities that contain genetically engineered organisms. Similar to microorganisms, inactivation kinetics can also be established for genetic elements ex vivo (eg, by investigating nucleic acid fragmentation). Once the kinetics is established, the extent of inactivation of biologic agents can be predicted for a variety of conditions such as different biocide concentrations, pH, and temperature.

Selection of an appropriate disinfection or sterilization process is determined by a number of criteria including:

  • Effectiveness of the microbicide to inactivate microorganisms and, if necessary, the subcellular components

  • Applicability of the method to different media (eg, air, wastewater, sludge, or surfaces)

  • Availability of the antimicrobial process (eg, distribution of heat or chemical, presence of interfering factors, chemical demand, access to the microorganism, etc.)

  • Detoxification requirements and lack of toxic by-product formation

  • Hazards associated with the microbicide and treatment process

  • Ease of handling and application

  • Capital and operating costs, etc.

The effectiveness of different microbicides can also be evaluated by comparing the concentration-exposure time (C • t) data for the inactivation of microorganisms to a given level (eg, 99% inactivation). For a successful comparison, data should be generated under identical experimental conditions. For example, inactivation data need to be generated using the same suspension of organisms grown under similar conditions in a given medium (water or wastewater). Environmental conditions such as pH and temperature also should be the same. Finally, experiments should be conducted using appropriate reactors (eg, batch versus continuous flow) for which data will be applied. The C • t data can also be used to compare the relative resistance of different microorganisms to a given inactivation agent. This information is necessary, for example, when a waste stream contains several different microorganisms and subcellular components. In this case, inactivation data need to be obtained for each of the biologic agents of concern under identical environmental conditions.

A convenient way to evaluate C • t data is to compare the times required to inactivate 99% (2 log) of the organisms exposed to a certain concentration (eg, 1 ppm)
of each of the microbicides. A unique concentration for all the inactivation agents does not seem to be feasible because of the significant variations in the effectiveness of the decontaminant and resistance of microorganisms. For example, when E coli was exposed to 0.07 mg/L of ozone at pH 7.2 and 1°C in a batch reactor, the time required to achieve 99% inactivation was only approximately 5 seconds.90 In contrast, others reported that the time required to inactivate simian rotavirus by 10 mg/L chloramine at pH 8 and 5°C was more than 6 hours.91 As a result of these wide variations, the C • t products for 99% inactivation were compared by several groups for a range of decontaminant concentrations.92,93

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May 9, 2021 | Posted by in MICROBIOLOGY | Comments Off on Principles of Antimicrobial Activity

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