Microorganisms and Resistance

Gerald McDonnell

Microbiology is the study of microorganisms. Microorganisms can be classified under eight major types based on their essential structures: prions, viruses, bacteria, archaea, fungi, algae, protozoa, and the helminths. They present a wide variety of structures and sizes, with known resistance profiles to inactivation (Table 3.1 and Figure 3.1).¹ Because these are the targets of the many types of preservation, disinfection, and sterilization, a basic understanding of their concerns to public health and commerce and their resistance to inactivation is a useful introduction. Prions and viruses are noncellular structures that depend on host cells for survival and replication. They are some of the smallest types of microorganisms, typically <0.4 µm, and for viruses, they consist of an essential nucleic acid (DNA or RNA structure) contained within a protein-(or lipid and protein) based structure. The essential protein-only structure (or infectivity component) of prions is even simpler than this and is still a matter of some investigation as to their true nature. Bacteria, archaea, fungi, algae, and protozoa are all unicellular organisms, but themselves range greatly in structure (from prokaryote to eukaryotic cell types) and size, typically from 0.1 µm to 200 µm (see Table 3.1). Our knowledge of many of these continues to develop, particularly in our understanding of archaea with remarkable abilities to survive extreme environments that would traditionally be considered adverse to life. Equally, it is often wrong to consider these unicellular microorganisms just as individual, free-living cells because they are often closely associated or even coexisting together in various environments. Communities of bacteria and fungi can grow to develop into colonial structures that are visible. But the advantageous and/or detrimental roles of these populations of microorganisms in the human body, animals, plants, or in the environment is an area of active research, such as in the study of biofilms (defined as a community of microorganisms; see chapter 67) and microbiomes (the combined genetic material of the microorganisms in an environment). The much larger, multicellular helminths (or microscopic worms) can be >1000 µm and are often directly visible to the human eye.

This diversity of life presents varying resistance profiles to the effects of the different types of physical and chemical antimicrobial processes used for disinfection, sterilization, and preservation. This chapter briefly considers the various types of microorganisms and particularly their resistance profiles to inactivation by physical and chemical biocidal mechanisms. Traditionally, certain types of microorganisms have been well studied in this area, such as bacteria and, to a much lesser effect, fungi. This is not only due to their ease of laboratory identification and manipulation (for many but not necessarily all of these) but also because they are common environmental contaminants and are frequently associated with negative impacts such as product spoilage, infection, or toxicity. Viruses have also been the subject of more recent investigations in the last 30 years, primarily due to their importance as pathogens and contaminants of mammalian biotechnology processes used for manufacturing protein therapeutics. But there has also been a greater focus on the investigation of other types of microorganisms such as prions, protozoa, and helminths, with the development of laboratory-based methodology to study their viability and a greater understanding of their impact on health and safety. Finally, a brief consideration will be given to the understanding on various toxins than can be produced from microorganisms, including when the vegetative organism is itself inactivated and its presence can continue to have potential negative health and product impacts.

GENERAL HIERARCHY OF RESISTANCE

Microorganisms can be classified based on their innate resistance profiles to inactivation by chemical and physical microbicidal mechanisms.¹ A summary of this hierarchy

and the relationship to the expectations of antimicrobial claims made with disinfectants is given in Figure 3.1.

TABLE 3.1 The eight major types of microorganisms

Microorganism Type	Description	Examples
Prions	Transmissible agents composed of protein and lacking any specific nucleic acid. They are associated with types of neurologic diseases known as transmissible spongiform encephalopathies.	PrP^Sc or PrP^res forms of proteins implicated in diseases such as scrapie, Creutzfeldt-Jakob disease, and chronic wasting disease
Viruses	Consist of a virus-specific nucleic acid (DNA or RNA), surrounded by an external protein-based capsid. Some viruses are further surrounded by a lipid-based envelope. Enveloped viruses are relatively sensitive to inactivation, whereas nonenveloped viruses demonstrate greater resistance. They depend on host cells for multiplication, such as animal, plant, or bacterial cells depending on the virus type.	Enveloped viruses: human immunodeficiency virus, hepatitis B virus, influenza virus, and herpes viruses. Nonenveloped viruses: parvoviruses, papillomaviruses, and poliovirus
Bacteria	Bacteria (or eubacteria) are prokaryotic, single-celled microorganisms that can range from cell wall-free forms (mycoplasmas) and cell wall-containing types (traditionally classified based on the Gram stain and microscopic morphology). Some bacteria, such as Bacillus species and Clostridium species, can morph between vegetative cell and spore forms, the latter being particularly of high resistance to inactivation. They are ubiquitous and can multiply in various environments under desired growth conditions. Some forms are obligate intracellular bacteria, such as Chlamydia species and Rickettsia species.	Cell wall free: Mycoplasma pneumoniae Gram-positive: Staphylococcus, Bacillus, Streptococcus Acid-fast: Mycobacterium Gram-negative: Escherichia, Klebsiella, Pseudomonas
Archaea	Prokaryotic, single-celled microorganisms that are genetically distinct from bacteria. Often identified from “extreme” environments, so are often studied for their unique resistance to such conditions, such as heat and high salt; generally, not associated with being pathogenic or as routine environmental contaminants	Thermococcus, Halobacterium, Methanococcus
Fungi	Eukaryotic, single-celled microorganisms. The two major types include the molds (or filamentous fungi) that can grow as longer lines of cells and yeasts (unicellular growth forms). Fungi can also develop from vegetative forms of growth to dormant spores of various types, depending on the fungus type. These spores can also present varying resistance profiles to inactivation due to their structures. Fungi can be ubiquitous in the general environmental.	Molds: Aspergillus, Penicillium, Trichophyton Yeasts: Candida, Saccharomyces
Algae	Eukaryotic cells that can grow as filaments or single cells. Photosynthetic and frequently associated with water habitats, particularly when contaminated; can be a source of toxins that can lead to health risks	Chlamydomonas, Gonyaulax
Protozoa	Eukaryotic, cell wall-free cells that can develop into multiple forms during their life cycles, including dormant forms of cysts or oocysts. These latter structures can be highly resistant to chemical disinfectants; often associated with natural water supplies and as human or animal parasites	Giardia, Cryptosporidium, Acanthamoeba
Helminths	Multicellular eukaryotes that are often free-living (eg, in water) or as parasites in humans or animals (known as various types of microscopic “worms”). Many grow to such sizes as to be visible to the human eye. During their respective life cycles, they can form ova (or eggs) that present with high resistance to inactivation.	Enterobius, Ascaris, Schistosoma
Abbreviations: PrP^res, protease-resistant PrP; PrP^Sc, scrapie-type PrP.

Prions are well cited as being highly resistant to inactivation, and this should not be a surprise considering their nature as protein-only transmissible agents. It would be wrong to assume that the many types of physical and chemical antimicrobial methods would be effective against these proteins.² For example, methods such as those like radiation are known to target nucleic acids as their mechanisms of action and would not be expected,
or have been shown, to have significant activity against prions due to the lack of specific DNA or RNA molecule in these agents. But equally, methods have been described as effective at neutralizing prion infectivity but may not be effective against other types of microorganisms (eg, proteases). Due to their unusual properties and unique risks of transmission, it is important to consider them as the most resistant forms of microorganisms to both physical and chemical methods.²

FIGURE 3.1 Hierarchy of the various types of microorganisms and their resistance profiles to inactivation. The various expectations from claims made with disinfectants are also shown (see chapter 2). The resistance profiles can vary depending on the specific antimicrobial method under investigation, and this profile is given as a guide. Specific claims of effectiveness against algae and helminths are less well defined and are given as estimates based on limited studies conducted with these microorganisms.

It is well established that many types of dormant forms of vegetative microorganisms such as bacterial endospores, protozoal oocysts, helminths, and, to a lesser extent, protozoal cyst forms and fungal spores classically demonstrate high resistance to inactivation.¹ By far the most studied in this group are bacterial spores such as those formed by Bacillus atrophaeus and Geobacillus stearothermophilus. They have been widely used to evaluate and test various types of sterilization processes, and the various stages of the development of spores from vegetative cells (sporulation) have been well described as well as detailed investigations at which stage the developing spores demonstrate increased resistance to various types of biocidal processes (eg, earlier in the sporulation to ultraviolet [UV] and formaldehyde, whereas later to heat and glutaraldehyde). Much less yet more recent investigations have described the resistance profiles of protozoal cysts, or particularly oocysts, as well as helminth eggs. The dormant forms of protozoa have been a concern due to their unexpected resistance profiles to various types of antimicrobial chemistries used to treat water (eg, chlorine or bromine), thereby having the ability to survive water disinfection mechanisms and their association with waterborne illness (eg, dysentery). Fungal spores have been typically shown to be much less resistant than bacterial spores, but they should not be underestimated because they can be found within dried, tightly-packed, and protected bodies that resist the penetration of disinfectants and sterilants (eg, Aspergillus species ascospores and Pyronema species). Overall, the term sporicidal is generally confirmed using bacterial spores as being the most resistant to this group, and when demonstrated to be effective against these, they are often assumed to be effective against other types of dormant and vegetative forms of microorganisms, including fungal spores.

Viruses can also range dramatically in resistance profiles based on their structures.¹ They are typically classified into two major types—nonenveloped and enveloped—referring to the presence or absence of an external lipophilic envelope. The external envelope is derived from the modified cell membrane structure sourced from the host cell during infection and release of virus particles, consisting of host cell phospholipids and proteins as well some virus protein such as glycoproteins. Because the envelope structure is typically required to allow the virus to successfully infect target cells, damage to this relatively sensitive structure is often sufficient to render the virus nonviable and therefore particularly sensitive to biocidal physical and chemical effects. Notable enveloped viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), and the flu (or influenza) viruses are therefore considered relatively sensitive to inactivation to include drying, heat, and chemical biocidal treatments. Nonenveloped viruses are dramatically more resistant. They can be further subdivided into larger and smaller nonenveloped viruses, where larger viruses (such as adenoviruses) are considered more resistant than enveloped viruses but less resistant than smaller, nonenveloped viruses. Traditionally, the most studied virus in the smaller, nonenveloped class was poliovirus, which was widely used as the test virus for testing disinfectant efficacy (see chapters 62 and 63). But in recent years, there has been a greater understanding of the extremes of resistance in nonenveloped viruses from studies with pathogens such as noroviruses, coxsackieviruses, and parvoviruses. Parvoviruses particularly demonstrate higher levels of resistance to physical and chemical inactivation methods due to their unique, tightly packed protein-capsid structures.³ Therefore, it is important to carefully inspect “virucidal” claims because efficacy against HIV or HBV will not be good indicators of efficacy against more resistant forms of viruses, and even then, the efficacy in certain types of nonenveloped viruses like poliovirus may not necessarily confirm efficacy against all nonenveloped viruses depending on the antimicrobial method under investigation.

Bacteria also range in resistance profiles. At a gross level, there has been three types of bacteria described as having increased levels of resistance to inactivation, from lower to higher being gram-positive bacteria, gram-negative bacteria, and mycobacteria. Such resistance profiles are due to their intrinsic cell wall structure differences, with gram-positive bacteria having a larger quantity of peptidoglycan in its cell wall, gram-negative bacteria with less peptidoglycan but containing an additional outer lipid-based membrane, and mycobacteria consisting of a tripartite structure of inner peptidoglycan, arabinogalactan, and outer lipid layer of mycolic acids. Mycobacteria have been traditionally found to have the highest level of resistance in the vegetative bacteria group to inactivation. Therefore, it is important to note that label claims or efficacy studies that often claim inactivation of bacteria (bactericidal; see chapter 2) are typically restricted to testing representative gram-negative and gram-positive bacteria but not mycobacteria (claims against which are specifically referred to as mycobactericidal). Added to this complexity can be the ability of certain bacterial genera to develop into bacterial spores, as described earlier. But overall, such general statements of efficacy against bacteria can be an oversimplification due to the diversity of bacteria types described to date and their ability to adapt to various situations. Consider, for example, that mycoplasmas (eg, the pathogens Mycoplasma pneumoniae and Mycoplasma genitalium) are known as cell wall-free bacteria and for this reason were often assumed to be very sensitive to inactivation, but this may not always be the case due to the unique lipids in their cell membrane structures that provide greater rigidity and tolerance to microbicides.⁴ Bacterial genera and species can vary in their tolerance levels to microbicides due to their structures, often depending on the chemical or physical antimicrobial method under investigation. For example, Salmonella, Enterococcus, and Legionella species have been described to have higher levels of tolerance to heat.⁵,⁶ Bacteria can vary due to their ability to present various resistance mechanisms that can be both a natural ability in the bacteria type or due to their ability to mutate or acquire resistance mechanisms to survive adverse conditions, including the presence of preservatives, antiseptics, and disinfectants. Examples include innate stress responses, biofilm development, cell wall structural changes, and efflux of chemical microbicides out of the cell (see chapters 4 and 67). Acquired mechanisms can be due to mutations and the acquisition of transmissible genetic material that can encode resistance mechanisms (see chapter 4). These mechanisms of intrinsic and acquired tolerance to antimicrobials can allow bacteria to persist in harsh or hostile environments.

The archaea are notably missing from the resistance hierarchy in Figure 3.1. Due to their similarities (essentially as single-celled prokaryotes), they were traditionally classified as bacteria but are now considered to be phylogenetically distinct. But despite our expanding knowledge of archaea types, they are considered nonpathogenic, are not known to be associated with spoilage, and are typically associated with “extreme environments” such as extremes of heat. Unlike the other microorganisms described, the archaea are not typically targets for preservative, disinfection, and sterilization processes. But they are of interest to microbiologists due to their various unique structures and metabolism that allow them to survive extreme environments as vegetative microorganisms.

Although less studied, fungi and protozoa can present similar variability in their resistance profiles to biocidal mechanisms. Vegetative fungi (molds and yeast) and protozoa are traditionally considered midway in resistance between gram-positive and gram-negative bacteria, with their dormant forms of spores and cysts demonstrating higher resistance. But resistance profiles can vary
significantly depending on their individual structures. As already noted earlier, Aspergillus and Pyronema species can demonstrate higher levels of resistance to some biocidal treatment methods. The ability of fungus to grow as masses of hyphal growth incorporating various types of spores can be a penetration barrier particularly to chemical microbicides. Protozoa, being more free-living, are considered more likely to be inactivated (such as in water) but can circumnavigate biocidal treatments by their association with bacterial biofilms. As described previously, the formation of dormant cyst forms can demonstrate resistance to chemical microbicides but generally not to physical methods such as heat inactivation. Helminths are much less studied, but their egg forms are known to be more resistant to inactivation than their adult worm forms.¹

TABLE 3.2 Examples of atypical resistance levels with microorganisms against biocidal mechanisms

Microorganism^a	Biocidal Method(s)	Examples of Mechanisms of Resistance
Deinococcus radiodurans⁷	Ionizing and nonionizing radiation, including γ radiation in the 5-10 kGy range; can include cross-resistance to oxidizing agents and dehydration	Gram-positive, vegetative bacteria that can have multiple mechanisms of tolerance including DNA damage repair mechanisms, multiple copies of their genomes, presence of pigments that act as free-radical scavengers, and unusual (thicker) cell wall structure in comparison to other gram-positive bacteria. Other bacteria with high-level resistance include Deinobacter, Rubrobacter, and Methylobacterium species.
Pyronema domesticum⁸	Ethylene oxide	Fungi (molds) originally reported from cotton-containing products that survived typical ethylene oxide sterilization process, due to hyphal mass protection, tightly packed ascospore-containing fruiting bodies (“apothecia”), and desiccated ascospore protoplasm
Mycobacterium chelonae and Mycobacterium massiliense⁹,¹⁰	Glutaraldehyde and ortho-phthalaldehyde (OPA)	Certain strains of these acid-fast, nontuberculosis bacteria have demonstrated unique resistance to aldehyde disinfectants. Mechanisms may be related to the lack of available protein targets at the surface of their lipophilic cell wall surface.
Pseudomonas species¹¹	Cross-resistance with antibiotics and certain types of microbicides such as triclosan, quaternary ammonium compounds, chlorhexidine, and metals (eg, copper)	Multiple efflux mechanisms that pump the antimicrobial out of the target cell and control of porin synthesis to allow for a greater level of tolerance to the microbicides, with consequences at preservative or lower levels. These mechanisms have been described in other gram-negative and gram-positive bacteria.
Staphylococcus species¹²	Quaternary ammonium compounds and cross-resistance with other microbicides and antibiotics	Efflux mechanisms that are hosted on plasmids and are known to be widespread in staphylococci
Escherichia coli¹,¹³	Triclosan and cross-resistance to some antibiotics such as isoniazid	Mutations in genes expressing enoyl-(acyl-carrier-protein) reductases that prevent binding of the microbicide. These enzymes are involved in cellular fatty acid synthesis and have been described as important targets for triclosan; also identified in other bacteria. Efflux-associated mechanisms have also been described.
Cryptosporidium oocysts¹⁴	A wide variety of chemical disinfectants and sterilant depending on their formulation/exposure conditions	Thick-walled oocyst structure, consisting of a four-layered matrix of protein/carbohydrate/lipid. These structures were initially considered unique in their resistance profiles, but subsequent investigations of other protozoa (eg, Acanthamoeba¹⁵) have also demonstrated high-level resistance.
^aNote that higher resistance levels have been described in some of the species listed but may not be a property of all strains in that species.

It is important to note that exceptions to these profiles are frequently cited in the literature. In considering these reports, it is important to look carefully at the test methods being use. Close inspection of many reports can show significant differences in the growth and isolation of test organisms and their presentation to an antimicrobial process for comparing their resistance profiles to inactivation. Important variables can include the ability of cells, dormant forms, or viruses to clump together; the presence of interfering factors (such as organic or inorganic materials that can compete or even neutralize the biocidal activity); and the presence of mixed populations of microorganisms or various forms. Despite these test methodology variances, there are reports of unique and dramatic resistance profiles to inactivation methods with specific types of microorganisms (Table 3.2).

Prions

Although often considered a controversial topic, prions are well established as unique transmissible agents that are composed of protein and lacking any specific nucleic acid.¹⁶ The specific source of the prion protein is PrP (or in its normal cellular form is known as PrP^c), a glycoprotein associated the cell membranes of human or animal cells and particularly in the neurons of neural tissues such as the brain and spinal cord. The protein is not considered essential for life, but it has been associated with various higher order functions including transport of copper, stress responses, central nervous system structure, and memory.¹⁷ Like other proteins, PrP^c assumes a normal folded secondary and tertiary structure that is produced and broken down by normal protein metabolism processes. But in the disease process, a conformational change in its structure can occur to render the protein resistant to proteases and therefore to cellular degradation. This form of the protein, known as a prion, is referred to in the literature under a variety of similar names such as PrP^res (referring to “resistant”), PrP^Sc (denoting “scrapie”—a sheep prion disease), or PrP^TSE (for transmissible spongiform encephalopathies [TSEs] or prion diseases). The prion form acts to promote a conformational change in other PrP^c proteins. Because the prion form cannot be completely broken down by normal cellular processes, it accumulates in affected cells, can lead to cell damage/death, and can transfer to other cells. Over time, this cascade reaction has a significant impact on neural tissues, leading to gross damage and the pathologic implications typified in prion diseases such as loss of brain function and eventually death. It is firmly established that the PrP plays a central role in these diseases and is devoid of any unique nucleic acid typical for other transmissible diseases such as with viruses (see chapter 68). But there is some debate on the role of other cofactors in these diseases that may also be linked with disease infectivity, the prion conversion process, and “strain” diversity. These include various types of lipids and RNA fragments.¹⁸,¹⁹

The TSEs or prion diseases are fatal degenerative brain diseases of animals and humans.²⁰ Human diseases include Creutzfeldt-Jakob disease (CJD), kuru, and Gerstmann-Sträussler-Scheinker syndrome and are considered rare. For example, CJD is the most widely described and is reported to occur in about 1 to 3 cases in 1 million populations. Specific cases of disease transmission between humans has been reported with tissue transplantation, reusable medical devices, and, if often speculated (but not confirmed), blood transmission.² Zoonotic transmission has also been reported, most notably with the link between bovine spongiform encephalopathy (BSE or often referred to as mad cow disease) and a variant form of CJD (vCJD) in humans due to contaminated meat products.²¹ Certain animal diseases are considered more widespread for reasons that are unknown, including scrapie (in sheep) and chronic wasting disease (CWD, in deer/elk).²⁰ In addition to these specific TSEs, there is much speculation about the existence of prion-like mechanisms in other neurodegenerative diseases such as Parkinson disease and Alzheimer disease.²⁰ It is interesting to note that Alzheimer disease has been shown to be induced (by a protein-seeding mechanism) in certain animal models in mechanisms similar to those understood for prion diseases and suggesting the potential transmissibility of these diseases in contact with contaminated tissues.

Prions have noteworthy higher resistance profiles to most physical and chemical antimicrobial methods. A summary of processes known to be effective or ineffective against prions are given in Table 3.3. It is important to note that efficacy reports can vary widely and do depend on the methods used for evaluation (see chapter 68).² For example, many studies have focused on the investigation of tissues highly contaminated with prions under which conditions full efficacy against the target agents would not be expected, particularly if compared to investigations with other types of microorganisms. This is due to the interference of the tissue material in such investigations and the lack of penetration to the target agents. Despite this, some clear conclusions can be made from these studies. First, antimicrobial methods that specifically target nucleic acids as their primary modes of action are not effective. Despite this conclusion, there are some exceptions, such as in the case of certain unique phenolic-disinfectant formulations that were verified to be effective against prions.²² Second, steam sterilization is considered effective, but dry heat methods are not. The effectiveness of steam can range depending on the material being inactivated (eg, whole tissue or contaminated surfaces with a cleaning step prior to steam treatment).²,²² Incineration is considered effective, but even in this case, viable prions could be detected in some studies.²³ Third, processes that are effective at degrading protein structures are generally effective, especially aggressive chemical treatment based on alkalis (especially sodium hydroxide), some oxidizing agents (eg, sodium hypochlorite, gaseous hydrogen peroxide), and (in certain situations) with extended protease digestion. The reports of effectiveness can also range depending on the test method, product formulation or test conditions, and so forth.² For example, proteases may often appear effective in in vitro tests, but in infectivity (in vivo) tests, many of these methods were not found to be effective and this may be due to partial protein degradation.² Combinations of heat and chemical (eg, alkali) processes have been shown to be particularly effective, including for whole carcass degradation.

So overall, prions present a unique challenge for traditional disinfection and sterilization procedures. In many cases, control of prions and prion contamination, such as in the handling of animal tissues, is most often a risk reduction exercise. Examples include the sourcing of tissues used for human introduction from animals or human known to be at a low risk of prion disease, the
use of treatment that is known to inactivate or remove prion contamination (particularly alkaline and oxidizing agents), or the complete removal of prion risk by exclusion (eg, removing all materials that are of animal origin or disposal of any materials that contact tissues known to be contaminated). Despite this, specific inactivation methods for prions have been defined and this continues to be an area for further research and development. Further consideration of the unique nature and inactivation of prions is given in chapter 68.

TABLE 3.3 A summary of antimicrobial processes known to be effective, potentially effective but requiring further investigation, and ineffective against prions^a

Effective	Potential Effectiveness	Ineffective
Steam sterilization but typically under extended exposure time (eg, 121°C for 1 h and 134°C for 18 min) or, for surface contamination, when combined with a cleaning step prion to steam treatment	Oxidizing agents (such as liquid hydrogen peroxide, peracetic acid, or ozone) under certain formulation and/or process conditions	Aldehydes such as formaldehyde and glutaraldehyde
1 N sodium hydroxide for 1 h or with other alkaline-based chemistries or formulations	Some acids, depending on concentration, formulation, and temperature exposure	Alcohols
2% available chlorine for 1 h		Hot or boiling water
Incineration		Radiation (ionizing or nonionizing)
Hydrogen peroxide gas		Dry heat
Certain types of gas plasmas (eg, oxygen)		Ethylene oxide
Some phenolic formulations		Quaternary ammonium compounds
4 M guanidine hydrochloride (≥1 h)
Prolonged protease digestion
^a Reprinted from McDonnell.²

Viruses

Viruses are a major group of pathogens and are therefore important targets for disinfection and sterilization applications. Unlike bacteria and fungi, they cannot grow or multiply in the environment without a specific host. Therefore, they are generally not targeted for preservative applications but are key targets for many purposes such as environmental disinfection (particularly in disease outbreak situations), treatment of foods or liquids for consumption, skin or mucous membrane infection treatments, mammalian biotechnology process systems, wastewater or material treatment, and device/tissue sterilization. Once viruses are in a host, they can lead to infections that are often serious and are responsible for significant rates of morbidity and mortality. Influenza outbreaks alone continue to be a significant cause of death during yearly flu seasons, but the different variants of influenza viruses do vary in virulence and have been associated with pandemics. The most noteworthy was the Spanish influenza pandemic that is estimated to have led to about 50 million deaths in 1918 worldwide.²⁴ Previous editions of this book focused attention on viruses such as HIV and HBV due to serious concerns at that time on their risks of transmission and particularly in health care facilities.²⁵,²⁶ In recent years, concerns with rotavirus, noroviruses, Zika virus, dengue, Ebola virus, and other emerging virus outbreaks, many with threats of pandemics, continue to highlight the risks of viruses as serious pathogens.²⁷ Even preventable diseases in first-world countries, such as measles, have reemerged as concerns and causes of death due to unfounded concerns with vaccinations.²⁸

In such disease states, viruses have the ability to be disseminated, often at high levels, by many mechanisms such as through blood or other body fluids (eg, with HIV, HBV, and viral hemorrhagic fever viruses such as Ebola), diarrhea (eg, with noroviruses or rotaviruses), direct contact (eg, from warts such as with human papillomaviruses), and through the air or droplet routes (such as with smallpox, influenza, and rhinoviruses). Viruses are therefore often highly contagious or transmissible. Disinfection challenges can include the presentation of the virus (eg, often associated in blood or other body tissues/excretions), the viral load present (often high in cases such as active infections with Ebola or other hemorrhagic fever viruses estimated to be ≥10⁶ viruses per milliliter blood),²⁹ the ability of the virus to persist in the environment, and resistance of virus type to chemical and physical inactivation. Viruses can clump together, and this
has often been linked as a tolerance factor to allow virus survival following disinfection.¹ Furthermore, they are often associated with various organic materials (such as patient’s tissues or excretions that include proteins, carbohydrates, and lipids), which provide a penetration challenge to physical and chemical disinfectants. As with other microorganisms, the importance of cleaning (to remove extraneous materials) prior to disinfection or sterilization is important.³⁰ Even enveloped viruses that are considered relatively sensitive to environmental conditions, drying, and disinfection can show remarkable persistence profiles in the environment, depending on the specific virus protein structures³¹ and the presence of nonviral extraneous materials.³²

As introduced earlier in this chapter (see Table 3.1), viruses can present a wide resistance profile to disinfectants and disinfection processes due to their structures. The main determinants to resistance are the presence of an external envelope (therefore known as enveloped and nonenveloped viruses) and their size (large or small viruses). Traditionally, viruses were then classified into three types based on their innate resistance profiles to inactivation.¹,³³ These include small nonenveloped viruses, large nonenveloped viruses, and enveloped viruses. Enveloped viruses can range in size from approximately 40 to 400 nm, larger nonenveloped viruses are in the 50- to 100-nm range, and small nonenveloped viruses at 10 to 30 nm. Examples of pathogenic viruses in each of these types and their relationship to model or indicator viruses used for efficacy tests are given in Table 3.4.

TABLE 3.4 Examples of virus pathogens and surrogate viruses used to indicate disinfection effectiveness against viruses^a

Viruses		Diseases	Examples of Indicator Viruses^b
Group A: enveloped viruses			Duck hepatitis B virus Bovine viral diarrhea virus Influenza A virus Herpes simplex virus
	HIV	AIDS
	Ebola virus	Ebola virus disease, a viral hemorrhagic fever in humans and primates
	Influenza virus	Influenza or flu
Group B: large, nonenveloped viruses			Adenovirus
	Adenoviruses	A wide range of symptoms similar to having a cold, sore throat, pneumonia, sometimes diarrhea, and conjunctivitis (commonly called “pink eye”)	Adenovirus
Group C: small, nonenveloped viruses			Feline calicivirus Poliovirus Parvovirus Murine norovirus Bovine Enterovirus
	Poliovirus	Poliomyelitis or commonly called polio
	Human papilloma virus	Often asymptomatic but can lead to the development of skin or mucous membrane warts (eg, in the genital area) or precancerous lesions. Two types of viruses, HPV-16 and HPV-18, are associated with cervical cancer.
	Parvovirus	Human (eg, fifth disease) and animal disease, particularly associated with the gastrointestinal tract and lymphatic system. Symptoms can include vomiting, diarrhea, immunosuppression, and infertility.
^a From Klein and DeForest.³³ ^bUsed as surrogate viruses for disinfection testing and can vary depending on geographical area (see chapters 62 and 63).