Mechanisms of action of microbicides commonly used in infection prevention and control

SUMMARY

Understanding how commonly used chemical microbicides affect pathogenic microorganisms is important for formulation of microbicides. This review focuses on the mechanism(s) of action of chemical microbicides commonly used in infection prevention and control. Contrary to the typical site-specific mode of action of antibiotics, microbicides often act via multiple targets, causing rapid and irreversible damage to microbes. In the case of viruses, the envelope or protein capsid is usually the primary structural target, resulting in loss of envelope integrity or denaturation of proteins in the capsid, causing loss of the receptor-binding domain for host cell receptors, and/or breakdown of other viral proteins or nucleic acids. However, for certain virucidal microbicides, the nucleic acid may be a significant site of action. The region of primary damage to the protein or nucleic acid is site-specific and may vary with the virus type. Due to their greater complexity and metabolism, bacteria and fungi offer more targets. The rapid and irreversible damage to microbes may result from solubilization of lipid components and denaturation of enzymes involved in the transport of nutrients. Formulation of microbicidal actives that attack multiple sites on microbes, or control of the pH, addition of preservatives or potentiators, and so on, can increase the spectrum of action against pathogens and reduce both the concentrations and times needed to achieve microbicidal activity against the target pathogens.

INTRODUCTION

While naturally occurring substances (e.g., alcohols, salts, sugars, and essential oils) have been used for dehydration and preservation of food, wood, and fabrics for many centuries (1, 2), formulated chemicals and disinfectants (microbicides) have only seen widespread use over the last 100 years (3). The mechanisms of action of microbicides have not been characterized to the extent of antibiotics and soaps (4). The application of microbicides to hand and surface hygiene has, and continues to have, a major impact on the control of environmental spread of infectious diseases (5). Their continued successful use has been due to their wide spectrum of action against multiple different pathogen types, and the ability of product developers to formulate the active agents to improve their performance (3).

Knowledge of the specific sites of action of these agents will aid in targeting formulations to meet the challenges of optimizing use and preparedness for emerging pathogens (6). This review focuses on the known or hypothesized mechanisms of action of the chemical microbicides (Fig. 1) most commonly used in infection prevention and control (IPAC). Physical inactivation of microbes is not addressed.

Fig 1.

Classes of active ingredients used in United States EPA-registered microbicidal formulations intended for (A) households or (B) industrial and institutional use [data from reference (7)].

CLASSES OF MICROBICIDAL CHEMICALS USED IN IPAC

Fu et al. (7) surveyed approximately 500 household disinfectant products registered with the United States Environmental Protection Agency (EPA) and reported that five classes of disinfectants represent the most commonly employed active ingredients (Fig. 1A). Among the microbicidal actives listed, quaternary ammonium compounds (QACs) are used most often, being used in 58% of the formulations as an active ingredient. Less commonly used active ingredients include pine oil, alcohol, phenolics, silver, and hypochlorite/hypochlorous acid. A survey of more than 2,300 EPA-registered disinfectant formulations intended for industrial and institutional use (Fig. 1B) also indicated QAC as the most commonly listed active ingredient. Quaternary ammonium compounds have been listed as the active ingredients in 36% of these formulated microbicides, while sodium hypochlorite (the active agent in bleach) is listed in over 18% of the formulations (7).

THE IMPORTANCE OF PROPER USE OF MICROBICIDES FOR IPAC

Direct contact of the target pathogen with the required concentration of microbicide for the required contact time is essential for the desired level of microbicidal action to occur. In contrast to antibiotics, microbicides used in IPAC are expected to kill microbes on contact (within seconds to minutes). How rapidly the microbicidal effect occurs depends on a combination of factors (listed in Table 1). The risks associated with improper or sub-optimal use of microbicides include microbe survival or sub-lethal effects on the targeted pathogens. This, in turn, may result in failure to interrupt pathogen transmission, but also may have the undesired effect of creating conditions favoring microbicide tolerance (8). Therefore, it is imperative to select the appropriate formulation for the intended purpose and to apply the formulation per the manufacturers’ instructions for an optimum microbicidal outcome.

TABLE 1.

Factors influencing the microbicidal activity of formulated microbicides^a

Factor	Comments
Factors related to the product formulation
Active ingredient concentration	The higher the concentration, the greater efficacy
Enhancing additives (potentiators, pH modulators, preservatives, synergistic compounds)	Enhance the microbicidal efficacy, and broaden the spectrum of activity
Factors related to the application
Duration of contact	Seconds to minutes is preferred
Temperature	More effective at higher temperatures
Humidity	Can affect the drying time of active ingredient and susceptibility of pathogen to agent
Presence of soil load (organic/inorganic) in the inactivation matrix	Reduces efficacy, but is more relevant to field conditions of microbicide use
Factors related to the target pathogens
Pathogen type, titer, location, and condition of infectious agents targeted	Growth phase or developmental state (e.g., vegetative vs spores or parasitic ova/(oo)cysts) of the pathogens, type and porosity of surface

^a See also reference (9).

HIERARCHY OF SUSCEPTIBILITY OF PATHOGENS TO MICROBICIDES

Although pathogens vary widely with regard to susceptibility to the microbicidal action of chemicals, a closer examination reveals a general microbial hierarchy of susceptibility (Fig. 2) (10, 11). For instance, the most susceptible among pathogenic agents are the enveloped viruses (such as SARS-CoV-2, other coronaviruses, influenza virus, rabies virus, Ebola virus, and monkeypox), while the least susceptible are the infectious proteins [prions, such as the scrapie agent and PrP(Sc)]. The remaining categories of pathogens are the (oo)cysts of enteric protozoa (e.g., Cryptosporidium parvum and Giardia lamblia), bacterial endospores (e.g., Clostridioides difficile), non-enveloped viruses (e.g., human adenoviruses, noroviruses, rotaviruses, and rhinoviruses), mycobacteria (e.g., Mycobacterium tuberculosis), fungi [e.g., Candida auris, Stachybotrys chartarum (Black mold), and Trichophyton interdigitale], Gram-negative bacteria (e.g., Pseudomonas aeruginosa, Acinetobacter baumannii), and Gram-positive bacteria (e.g., Staphylococcus aureus).

Fig 2.

Hierarchy of susceptibility of pathogens to microbicidal active ingredients. Certain formulated microbicides may include combinations of active ingredients, resulting in synergistic virucidal efficacy greater than that displayed by the individual active ingredients [data are from reference (12)].

It should be noted that the general order of microbicide susceptibility shown in Fig. 2 is not absolute. Even within the broader categories, there may be distinct variations in susceptibility to certain chemical microbicides across and even within families (13–15). Therefore, the hierarchy of pathogen susceptibility should be viewed as a general guideline to be used, with the above limitations in mind.

A microbicide is considered broad-spectrum if it displays lethal efficacy against bacterial, fungal, mycobacterial, spore, and viral targets. In practice, as shown in Fig. 2, many of the microbicides used in IPAC are more limited in spectrum. Selection of the appropriate microbicide for a certain application (i.e., pathogen) should be based on consideration of the hierarchy of pathogen susceptibility.

As already mentioned in Table 1, a variety of factors may play a role in the efficacy or speed of action of any given microbicide against one of these categories of pathogens. These include the specific species or strain of the pathogen, the test method used to assess inactivation, the pathogen culture preparation, the presence and type of soil load comprising the inactivation matrix, the microbicide contact time, the temperature and relative humidity (RH), the type of surface being disinfected (i.e., hard, soft, porous, or non-porous), the delivery method of the microbicide (applicator) and the intended usage of the microbicide (16). Further, the microbicidal agent alone may not exert the desired extent of inactivation, and the specific formulation in which the microbicidal active is applied may optimize the kinetics or spectrum of inactivation across different types of pathogens targeted. Formulation goals for product development may consider factors such as cost of ingredients, speed of microbicidal action (minimization of required contact time), spectrum of microbicidal activity, materials compatibility, method of application (e.g., spraying vs incorporation into wipes), regulatory requirements, and workplace safety (8), including possible synergy between microbicidal actives. To achieve this, additives may include potentiators, preservatives, pH modulators, and synergistic compounds (11). For example, the spectrum of microbicidal activity of ethanol or a chelator (e.g., EDTA) can be potentiated through the addition of certain metallic ions and alteration of pH (12, 17–21). An understanding of formulation science is therefore crucial to providing IPAC products with optimal microbicidal efficacy and rapid kinetics of microbicidal activity, while considering materials compatibility and workplace safety. Unfortunately, there is no rule of thumb to predict the beneficial microbicidal impact of adding a specific formulation excipient to an active. Determination of additional benefits relies on experimental testing.

MECHANISMS OF INACTIVATION BY CHEMICAL MICROBICIDES

A variety of chemical microbicides are used in IPAC, despite our incomplete understanding of the mechanisms by which some of these agents inactivate target pathogens. The study of these mechanisms has been hampered by the generally rapid and indiscriminate types of damage that microbicides cause, and which subsequently lead to microbicidal effect. In the following sections of this review, we discuss what is known or has been hypothesized regarding the mechanisms of inactivation of pathogens by the microbicidal active ingredients commonly used for IPAC. A summary of mechanisms of action is shown in Table 2.

TABLE 2.

An overview of mechanisms of action of microbicidal actives^a

Microbial targets	Microbicidal actives	Mode of microbicidal activity
Cell envelope (cell wall, outer membrane)	Glutaraldehyde	Cross-linking of proteins.
Cytoplasmic (inner) membrane	QAC Chlorhexidine Diamines PHMB, alexidine Phenols	Generalized membrane damage. Low concentrations affect membrane integrity and high concentrations cause congealing of cytoplasm. Induction of leakage of amino acids. Phase separation and domain formation of membrane lipids. Leakage.
Macromolecules (proteins, nucleic acids)	Formaldehyde Glutaraldehyde	Cross-linking of proteins, RNA, and DNA. Cross-linking of proteins in cell envelope and elsewhere in the cell.
DNA	Acridines	Intercalation of an acridine molecule between two layers of base pairs in DNA.
Thiol groups	Copper/silver compounds	Membrane-bound enzymes (interaction with thiol groups).
Nucleic acids	Halogens Hydrogen peroxide, Silver ions	Inhibition of DNA synthesis. DNA strand breakage.
Proteins	Halogens Peroxygens	Oxidation of thiol groups to disulfides, sulfoxides, or disulfoxides. Hydrogen peroxide: activity due to formation of free hydroxyl radicals (·OH), which oxidize thiol groups in enzymes and proteins; Peracetic acid: disruption of thiol groups in proteins and enzymes.

^a Modified from references (22, 23).

Quaternary ammonium compounds

QACs are cationic (net positively charged) molecules that are widely used as microbicides, due to their microbicidal efficacies, their cleaning and deodorization attributes, and their relatively benign impacts on materials to be disinfected (i.e., their materials compatibility). In general, QACs are effective for inactivating vegetative bacteria, fungi, and enveloped viruses. QACs poorly inactivate non-enveloped viruses, are not sporicidal and do not kill or inactivate prions (7). A review of various QAC structural types has been published by Falk (24). Benzalkonium chloride (BKC) is an example of a QAC with a 12- to 18-carbon alkyl chain hydrophobic tail. The cationic charge and the surfactant properties contributed by the alkyl chain make QAC effective both for disrupting bacterial cell walls and viral lipid envelopes. Greater lipophilicity provides greater efficacy in QAC. These include QAC having chain lengths of 12–16 carbons (25). While the efficacy of QAC against specific microorganisms depends on their n-alkyl chain lengths (n = 10–12 for Gram-positive bacteria and yeast; n = 14–16 for Gram-negative bacteria; n < 4 or n > 18 inactive), their modes of action depend on their concentrations. At low concentrations, QACs bind to anionic sites on the membrane, leading to potassium ion and proton leakage and loss of osmoregulation. At a higher concentration, QACs interfere with respiration, solute transport, and cell wall peptidoglycan biosynthesis. At concentrations usually found in formulated disinfectants, QACs solubilize the membrane, releasing cytoplasmic contents (26).

The mechanisms of action of many QACs have been elucidated (25). All are membrane-active agents and display different interactions with the membrane, leading to membrane disruption depending upon the specific QAC. The surfaces of bacteria are typically negatively charged, allowing the cationic head group of the QAC to be adsorbed onto the surface of the bacteria (27). Once adsorbed, QACs are able to diffuse through the cell wall and bind to the cytoplasmic membrane, causing disruption. Cytoplasmic contents then are released, along with potassium (K⁺) ions; ultimately resulting in death of the cell (27). Gram-positive bacteria appear to be more susceptible to QAC than Gram-negative bacteria. QACs also display efficacy against enveloped viruses, but less universally against non-enveloped viruses (26).

Microbicidal activity of QAC

Microbial inactivation by QAC is initiated by adsorption through electrostatic interaction with the cytoplasmic membrane of bacteria or the plasma membrane of yeast. This adsorption reflects the affinities of QAC for irreversible binding to phospholipids (7, 22, 28). This affinity is mediated through the binding interaction of the positively charged groups of the QAC with membrane phospholipids, which typically are anionic (negatively charged) at pH above 3 or 4. For instance, exposure of a bacterial cell to a QAC (Fig. 3) leads to the following sequence of events: adsorption and penetration of the agent through the cell wall (if present); interaction and binding to the cytoplasmic membrane (lipid or protein), resulting in disorganization (distortion) of the membrane; leakage of intracellular low-molecular-weight constituents such as potassium ion through the damaged membrane; degradation of intracellular proteins and nucleic acids and disruption of proton motive force; and finally, cell lysis caused by autolytic enzymes (22, 27, 29, 30).

Fig 3.

The rapid effect of the QAC/bacterial cell membrane interaction results in disruption of the bacterial membrane and inactivation upon contact. Blue spheres represent positively charged nitrogen atoms [based on reference (31)].

As an example, Ioannou et al. (30) studied the modes of action of alkyldimethylbenzyl-ammonium chloride (ADBAC) (a blend of 12-, 14-, and 16-carbon homologs) and didecyldimethylammonium chloride (DDAC). In that study, S. aureus/QAC interactions leading to lethality occurred rapidly (i.e., within the first 30 s, with inactivation peaking by 5 min). The minimum inhibitory concentrations (MIC_{48 h}) were similar [0.4–1.8 µg/mL over the S. aureus range of 1 × 10⁵ to 1 × 10⁹ CFU (colony-forming units)/mL]. Inactivation of S. aureus by both QACs was concentration-, time-, and temperature-dependent, with kinetics being faster for higher QAC concentrations and at higher temperatures (Fig. 4). Similar results were obtained for measures of leakage of intracellular components (intracellular potassium and 260 nm absorbing material). Autolysis, an intracellular event in which a microbial cell enters a self-destructive state resulting ultimately in death, was observed after exposure both to ADBAC and DDAC. This autolysis was associated with QAC concentrations ≥9 µg/mL and coincided with loss of ~30% of the intracellular potassium pool. Figure 4 shows a microbicidal effect within 30 s following contact with S. aureus produced by both types of QACs used, as indicated by upward direction of the slopes. Quantitative parsing of the contributions of cell leakage and autolysis to reduction in viable cell count was not possible (30).

Fig 4.

Inactivation versus time (log₁₀ reduction equivalents) studies for ADBAC (top panel) and DDAC (bottom panel) versus 2 × 10⁹ CFU/mL S. aureus at 25°C (—) and 35°C (----) in a HEPES buffer medium. Final challenge concentrations studied were 35 µg/mL (▲ and Δ), 45 µg/mL (■ and □), and 55 µg/mL (● and ○), and each symbol indicates the mean ± standard error (SE) for five observations [data are from reference (30)].

In summary, following exposure to QAC, there is a general and rapid loss of ultrastructural organization and integrity of the cytoplasmic membrane in Gram-positive bacteria and of the outer membrane of Gram-negative bacteria, of the plasma membrane of yeast, together with other damaging effects to the pathogens, including initiation of autolysis.

Virucidal activity of QAC

QACs interact with the lipid of enveloped viruses to disrupt the envelope integrity. Enveloped viruses are thought to interact with QAC primarily through hydrophobic and, to a lesser extent, electrostatic interactions involving the lipid envelope (31, 32) as shown in Fig. 5. Tsao et al. (32) reported that the mechanism for inactivation of herpes simplex virus 1 (family Herpesviridae, an enveloped virus) following QAC binding to virus involved disruption of the lipid envelope, with subsequent release of the nucleocapsid. Schrank et al. (31) reviewed the effectiveness of QAC for inactivating, on surfaces or in suspension, mouse hepatitis virus (MHV), human coronavirus (HCoV), canine coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), transmissible gastroenteritis virus (TGEV), and HCoV 229E. Depending on the specific QAC used and its concentration and dwell (contact) time, ≥3 log₁₀ inactivation of each of these viruses (family Coronaviridae, enveloped viruses) was observed. Other enveloped viruses have been shown to be susceptible to QAC inactivation, including murine coronavirus (Coronaviridae), hepatitis C virus (Flaviviridae), influenza virus (Orthomyxoviridae), human immunodeficiency virus (Retroviridae), hepatitis B virus (Hepadnaviridae) (33), and SARS-CoV-2 (31). In addition, when formulated appropriately, QACs have been shown to exhibit efficacy in hard surface testing against a wide range of non-enveloped viruses (34), such as murine norovirus, feline calicivirus (Caliciviridae) (35), rotavirus (Reoviridae), poliovirus (Picornaviridae), and adenovirus (Adenoviridae) (36).

Fig 5.

The rapid effect of the QAC/viral envelope interaction results in disruption of the lipid envelope (analogous to the microbial membrane) and inactivation upon contact [adapted from reference (37) with permission of American Chemical Society].

Alcohols

A variety of alcohols are used in IPAC. These include ethanol (ethyl alcohol), isopropanol (iso- or 2-propyl alcohol), and 1-propanol (n-propyl alcohol). Each is an effective microbicidal agent. Commercial formulations with high levels of alcohol are typically marketed in the form of aerosol sprays and hand rubs and are formulated in combination with other ingredients (e.g., pH modulators, potentiators, preservatives, or synergistic compounds) to enhance microbicidal efficacy. Alcohols are typically present in commercial formulations at concentrations of 60%–90%. Products having lower (<60%) alcohol concentrations retain effectiveness using formulation science. Efficacy claims for such products include pathogens with lower susceptibility to chemical microbicides, such as mycobacteria and non-enveloped viruses, including poliovirus, rhinovirus, rotavirus, norovirus, and hepatitis A virus (10, 17–22, 35, 38, 39).

Microbicidal activity of alcohols

In general, alcohols are thought to inactivate microbes through membrane damage and rapid denaturation of cellular proteins, leading to changes in permeability, interference with metabolism, and subsequent death of microbial cells. Cheeseman et al. (40) investigated the mechanisms underlying the microbicidal efficacy of commercially available alcohol-based hand rubs (ABHRs) on S. aureus NCIMB 9518 (reference strain), and two methicillin-susceptible S. aureus clinical isolates (S1 and S2). The ABHRs were shown to produce 4 log₁₀ inactivation within 10 s to 5 min for isolate S1, 30 s to 5 min for the reference strain, and 1 to >5 min for isolate S2. Early cytoplasmic damage was assessed through potassium ion leakage. Gross damage to cell structure was assessed using transmission electron microscopy. The earliest indicators of alcohol damage included increased cell wall thickness/fuzzy appearance and disruption of membrane integrity. These occurred within 10 s for the S2 isolate, and by 1 min for the other isolate and for strain NCIMB 9518. Within 5 min of exposure, microbial cell walls thinned and leaked intracellular contents. There was also the presence of ghost cells, and coagulation of cytoplasm (40) (Fig. 6).

Fig 6.

Ultrastructural changes in S. aureus NCIMB 9518 following 5 min exposure to an alcohol hand rub, as observed using transmission electron microscopy. Cell wall damage (WD) leading to leakage of intracellular material (L) is evident, as shown by arrows; UEM, unidentified extracellular material; scale bar, 157 nm [reprinted from reference (40), with permission of the publisher (copyright Elsevier)].

Virucidal activity of alcohols

Enveloped viruses are the most susceptible of viruses to inactivation by alcohol (41). Viral inactivation by alcohols is attributed to envelope damage and denaturation of envelope proteins (42). For instance, RNA integrity and core protein in hepatitis C virus (HCV, family Flaviviridae) were not impacted following exposure to ethanol or isopropanol, while the lipid envelope was partially but not completely disrupted (42). A detailed study of SARS-CoV-2 found that both ethanol and propanol significantly weakened the virus envelope, causing important changes in its structure (43).

Alcohols are not considered to be as effective for inactivating non-enveloped viruses, but such generalizations may be misleading. In the case of the Caliciviridae, inter-family differences in susceptibility to alcohols have been reported (14). Alcohols are effective against certain caliciviruses (14), as well as enterovirus, astroviruses, and rotavirus (44). Recently, an alcohol spray disinfectant was shown to be effective against human norovirus using assays in human intestinal enteroid epithelial cells (45). Studies of susceptibility of human norovirus conducted using RNase-RT-qPCR indicate that alcohols act to damage the capsid, rendering the encapsulated genomic material susceptible to RNase degradation (45)

When formulated with other ingredients, increased efficacy for non-enveloped viruses has been achieved. Formulation is critical in ensuring the efficacy of alcohol. Factors include the type of alcohol (ethanol and isopropyl), concentration, and the addition of potentiators, pH modulators, etc. (46). Examples include the formulation of alcohols with copper ions (17–21) or zinc ions (21), with 2.0% urea and 2.0% citric acid (47), or with phosphoric acid (48). Alcohol formulated with copper displayed activity against poliovirus and rhinovirus (Picornaviridae), and feline calicivirus (Caliciviridae) (17–20). Alcohol formulated with zinc displayed activity against bacteriophage F116 and against adenovirus type 2 (family Adenoviridae) (21). A variety of mechanisms have been proposed for the synergistic effects of inclusion of metal ions, including the generation of free radicals, a variety of possible interactions with viral genomic material, and structural damage to the viral capsid (21).

Alcohol formulated with urea and citric acid inactivated non-enveloped viruses in a soil load within one minute. The challenge viruses included poliovirus (Picornaviridae), adenovirus type 5 (Adenoviridae), murine norovirus (Caliciviridae), and SV40 (Polyomaviridae) (47). While the mechanism involved in the enhancement by urea and citric acid of viral inactivation by alcohol was not identified by the authors, it may relate to acidification. Alcohols formulated with phosphoric acid also inactivate poliovirus and hepatitis A virus (Picornaviridae), rotavirus (Reoviridae), and feline calicivirus (Caliciviridae) (48).

Oxidizing agents

Oxidizing agents represent another broad category of microbicidal agents that are widely used in IPAC. Oxidizing agents are substances that remove electrons from a reactant in a redox reaction. The oxidizing agent is “reduced” by accepting electrons, while the reactant is “oxidized” in the course of donating electrons to the oxidizer. The broader term “oxidizing agents” includes such commonly employed actives as chlorine-releasing agents (CRAs) and peroxygens. Iodine and iodophors are less commonly used, due to their decreased solubility and for the higher concentrations required for efficacy. As a result, iodine and iodophors are used primarily as topical microbicidal agents. Generally, oxidizing agents are powerful broad-spectrum microbicides (Fig. 2).

Chlorine-releasing agents

The most commonly used CRAs are sodium hypochlorite, chlorine dioxide, and the N-chloro compounds such as sodium dichloroisocyanurate. Stabilized hypochlorous acid formulations also recently have become available (49, 50).

Microbicidal activity of CRA

Despite their common use in both domestic and institutional settings, the mechanisms of action of CRA are not fully understood. Being oxidizers, they denature cellular proteins with acidic pH, potentiating oxidation. Further, CRAs interact with bacterial DNA, forming chlorinated derivatives of nucleotide bases. Hypochlorous acid disrupts oxidative phosphorylation and other membrane-associated enzyme activities (22, 23, 28). The sporicidal activity of CRA is observed at relatively high concentrations (≥3,000 ppm) and appears to involve separation of the spore coat from the cortex, resulting in spore lysis (28).

Virucidal activity of CRA

The virucidal activity of CRA against the non-enveloped poliovirus type 1 (Picornaviridae) was observed using transmission electron microscopy and stain penetration to visualize the rapid concentration-dependent degradation of the capsid (51). The mode of action of chlorine dioxide appears to involve primarily or initially the denaturation of specific regions of viral capsids for some viruses (e.g., MS2 bacteriophage and poliovirus) and for other viruses, the nucleic acid (52–55). These studies have indicated that chlorine dioxide denatures proteins through the oxidation of tyrosine, tryptophan, and cysteine residues in proteins. Time- and chlorine dioxide concentration-dependent degradation of murine norovirus capsid protein was observed by Yeap et al. (55). Degradation of viral genomic RNA was also proposed as a mechanism of action in that study. The impacts on viral structure occurred within 5 min of exposure to 2 mg/L chlorine dioxide gas (55). These results confirmed a conclusion made previously by Alvarez and O’Brien (56) that chlorine dioxide inactivates poliovirus through interactions with the genomic RNA that impair the ability of the RNA to act as a template for RNA synthesis. Hypochlorous acid also appears to result in morphological changes resulting from interaction with the protein coat of human papillomaviruses (50).

In contrast, chlorine appears to act both on the viral nucleic acid and capsid (51). Initial damage of the exterior proteins in the capsid may lead to damage of interior proteins and action against the nucleic acid (52). Certain regions of the 5′-non-translated region (5′-NTR) of hepatitis A virus have been found to be more sensitive to damage by chlorine and chlorine dioxide, while the antigenicity of the capsid was not lost, indicating resistance of the protein (57). The site of action on the capsid and nucleic acid may be different for different types of viruses, which could be related to the relative resistance of different viruses to oxidizing agents (13, 52). For example, impacts of chlorine on the capsid allow the chlorine to penetrate and damage the nucleic acid of rotavirus more rapidly than Tulane virus (a surrogate for human norovirus), resulting in greater resistance of the Tulane virus to chlorine (58).

Prion inactivation by CRA

A 5-min exposure to a 40% dilution of household bleach was found to prevent chronic wasting syndrome prion (CWD) seeding activity associated with both CWD-positive brain homogenates as well as stainless steel wires (surrogates for surgical instruments contaminated with CWD) (59). It has been proposed that the inactivation of prions by sodium hypochlorite is due to its ability to block the self-propagating activity of prions and other amyloids (60).

Peroxygens

Commonly used peroxygens include solutions of hydrogen peroxide (H₂O₂) and peracetic acid (PAA). Depending upon the concentration, peroxygens display broad-spectrum microbicidal activity. Peroxides are considered a “Green” (environmentally friendly) microbicide, in that their breakdown products are water and oxygen (22). PAA also decomposes to the safe by-products of acetic acid and oxygen. Unlike H₂O₂, PAA is not affected by peroxidases and remains active in the presence of organic (soil) loads, which impact the microbicidal activity of H₂O₂. Hydrogen peroxide and PAA solutions are commonly used as microbicides for IPAC and are available commercially in concentrations ranging from 1% to up to 90%. Formulations have been created that “activate” the hydrogen peroxide. These activated or accelerated hydrogen peroxides (AHPs) are formulated with additional ingredients that transfer energy to the hydrogen peroxide, increasing or accelerating its decomposition into the ultimate reactive species (free radicals). Room disinfection can be achieved through fogging with vaporous hydrogen peroxide or vaporous peracetic acid (61).

Microbicidal activity of peroxygens

The oxidizing activity of H₂O₂ is attributed to the Fenton reaction-mediated generation of hydroxyl free radicals (·OH), which target cellular lipids, proteins, and DNA. Sulfhydryl (thiol) groups and double bonds are especially vulnerable to attack (22, 62, 63). Linley et al. (62) proposed that the oxidation of DNA by H₂O₂ is also mediated by the generation of the ferryl radical formed from iron associated with DNA. The inactivating effects of H₂O₂ occur very rapidly. For instance, >6 log₁₀ reduction of healthcare-associated pathogens, including community-acquired methicillin-resistant S. aureus (MRSA), vancomycin-resistant Enterococcus (VRE), and multidrug-resistant A. baumannii, by improved H₂O₂ microbicides in the presence of a soil load consisting of 5% fetal bovine serum occurred within 30 s (64). Similar efficacy within a short contact time (30 s) has been reported against a wide range of Gram-positive and Gram-negative bacteria, including MRSA and VRE (65). The primary effect of peracetic acid is on the bacterial cell membrane, resulting in cell death (66). PAA also can be effective against bacterial spores, causing damage to the inner spore membrane and germination receptor proteins, but not to the DNA (67). Combining PAA with hydrogen peroxide has been found to lead to synergism, in which PAA damage to the spore coat is believed to result in better penetration by the hydrogen peroxide (68). PAA effectiveness also is enhanced in the presence of metals such as copper ion, which act as catalysts in the extracellular solution that have an impact on membrane permeability, resulting in the generation of reactive oxygen species (ROS), and impacting membrane permeability (68). This, subsequently, enhances entry of PAA and ROS into the cell. The resulting increasing levels of intracellular ROS then damage intracellular structures, including proteins and nucleic acid (68). The sporicidal activity of peracetic acid is associated with interaction with spore membrane and germination receptors (69).

Virucidal activity of peroxygens

Virucidal activity against non-enveloped viruses, including poliovirus type 1 and human rhinovirus type 37 (family Picornaviridae), feline calicivirus (Caliciviridae), and human rotavirus (Reoviridae) of AHP in the presence of a 5% soil load was achieved within 1 min (64). Investigators have reported that the inactivation of viruses by PAA is mediated primarily through interactions with the capsid proteins (70, 71). Fogging with PAA gas (2.0 mL/m³) for 60 min resulted in 9 log₁₀ inactivation of reovirus (family Reoviridae), 6.4 log₁₀ inactivation of mouse minute virus (family Parvoviridae), and 7.7 log₁₀ inactivation of avian polyomavirus (family Polyomaviridae) (61). These are non-enveloped viruses that are less susceptible to many chemical-based microbicides. Studies with coliphage MS2 and murine norovirus (both being small, non-enveloped viruses) indicate that the primary reaction is with the capsid proteins, and these agents do not readily damage viral nucleic acids (72). The efficacy of hydrogen peroxide can be enhanced in the presence of metal ions, organic acids, or amino acids (73). Hydrogen peroxide was found to allow for greater penetration of copper ions, resulting in the loss of SARS-CoV-2 envelope integrity and oxidative damage to lipids, proteins, and nucleic acid (74).

Acids and alkalis

Pathogen inactivation by low pH (acids) and high pH (caustics/bases) treatment has been utilized as a disinfection approach for facilities, equipment, and liquids. In general, high pH conditions are effective against a broader range of pathogens than are low pH conditions (75). As with other classes of microbicides, the efficacies of low and high pH for inactivating pathogens depend upon contact time, concentration (pH), and temperature, including potentiators and preservatives. Greater efficacy is obtained at longer contact times, greater extremes in pH, and higher temperatures (75).

Low pH inactivation

Acids are substances that dissociate in water to provide hydrogen ions (H⁺), measured on the pH scale as <7. In the case of low pH treatment, it is generally assumed that this inactivation approach will be effective primarily for vegetative bacteria and for enveloped viruses. Certain non-enveloped viruses (e.g., rhinoviruses) may also be susceptible to low pH (14), as are certain bacterial spores (76). Several mechanisms have been proposed (77) for the bactericidal activity of organic acids: (i) impacts on the cell wall; (ii) decreases in cytoplasmic pH; (iii) chelation of metal ions from the cell wall; and (iv) disruption of membrane function.

Individual acids may exert their microbicidal activities through a single mechanism or multiple mechanisms, based on their chemistries and, therefore, combinations of organic acids are sometimes formulated together (78). Lactic acid disrupts bacterial cell functions by creating osmotic imbalance across the cell wall. Stress to the bacterial cell occurs as energy is consumed in order to rebalance pH. During this process, free radicals are generated which can damage cellular structures (78). In the case of citric acid, chelation of ions from the cell wall has been proposed to cause damage to cellular structures (79). The uncoating of viral capsids through interactions of acid with capsid proteins is thought to lead to exposure of viral protein and nucleic acid to the damaging effects of proteases and nucleases (80). For instance, certain rhinoviral proteins appear to be disordered following exposure to salicylic acid. The magnitude of this disordering increases with longer exposures to the acid. These effects have been observed on a time scale of ≥6 min post-exposure (80).

The inactivating effects of low pH vary according to the type of virus. Non-enveloped viruses are less susceptible to acidic pH than enveloped viruses, implying that lipid envelope disruption is important mechanistically (75). Even among a series of enveloped or non-enveloped viruses, those that are enteric (i.e., surviving in and infecting the gastrointestinal tract) are much less susceptible to acidic pH, compared with non-enteric (e.g., respiratory) viruses (15, 75). Examples of this include the picornaviruses, enterovirus versus rhinovirus. Both are non-enveloped viruses, though the former is much less susceptible to low pH than the latter (15, 75, 80). Where susceptibility is expected, the inactivation is typically observed at pH ≤4 (75). Acid washes have been investigated for inactivation activity against rhinovirus 2–3 h post-application and found to reduce infection (80).

Acids may be added to formulations of other microbicidal actives. For example, acids act synergistically with anionic surfactants at pH levels of 3 and below (81). Under these conditions, bacteria become positively charged and interact electrostatically with anionic surfactants. In a manner analogous to QAC, anionic surfactants can alter the permeability of cell membranes in acidic environments. The sporicidal action of low pH requires strong acids and relatively high temperatures and is thought to involve changes in the permeability of the inner spore membrane, allowing access of genomic material to low pH and nucleases (76).

High pH inactivation

High pH is used more often as a disinfection approach for facility and equipment surfaces than low pH. Bases, such as sodium hydroxide (NaOH), readily dissolve in water to give rise to hydroxyl ions (OH⁻) measured on the pH scale as >7. At very high pH (≥11), even pathogens displaying the least susceptibility to microbicides (e.g., prions and bacterial spores or parasitic ova/(oo)cysts) (Fig. 2) can be inactivated. Prions are normally resistant to most microbicides; however, 1–2 N NaOH is used to denature prions (82, 83). High pH can cause breakage of peptide bonds and denaturation of proteins. For instance, inactivation of Bacillus subtilis spores by high pH is thought to involve the inactivation of lysosomal enzymes which degrade the cortex upon spore germination (76).

High pH is effective for inactivation of a broad range of enveloped and non-enveloped viruses (35, 75, 84–87). For instance, the complete inactivation (>4 log₁₀) of a non-enveloped virus, feline calicivirus, was observed following a 1-min contact time at pH 12 (35). The mechanism of inactivation of viruses by high pH is not well understood. High pH may exhibit a mechanism of inactivation similar to heat and low pH, involving capsid opening and subsequent genomic damage resulting from exposure to nucleases ubiquitously present in the environment (75). Alternatively, high pH may damage viral genomic material directly (87).

Detergents/soaps

The effects of surfactants, detergents, and soaps (all hydrophobic and designed to reduce surface tension) on bacteria or viruses include both facilitating the removal and killing/inactivation of targeted pathogens (11, 88, 89). Physical removal is mediated through the suspension and removal of the bacteria or viruses, often associated with bodily fluids or other types of soil loads, from a contaminated surface (88, 89). This action is mediated by the hydrophobic tails of the surfactant/detergent molecules, which are associated with the lipophilic bacterial cell wall and viral lipid envelope (88–91). These lipid structures of bacteria and viruses are solubilized by surfactants (89, 91), resulting in structural disruption. The disruption may lead to leakage of intracellular material from the bacteria or protein or nucleic acid from the viruses. Furthermore, the disruption may facilitate the entry of nucleases and proteases, or additional microbicidal active(s) present in the inactivation matrix to enter the bacterial cell or virus (89).

Gram-positive bacteria have no outer membrane. Instead, there is a thick layer composed of peptidoglycan and teichoic acid, which does not present a barrier for surfactant to pass into the bacterial cell (92). Disruption of this structure does, however, lead to leakage of cellular constituents and, ultimately, to microbicidal activity.

Disruption of the capsid protein of non-enveloped viruses (e.g., norovirus, hepatitis A) presents a greater challenge to microbicides. Anionic and cationic surfactants have been shown to influence the aggregation of norovirus virus-like particles as a consequence of altering the surface charge of the capsids (93). The ultimate disassembly of capsid proteins occurs at surfactant concentrations above the critical micelle concentration. This activity has not been observed with non-ionic surfactants (93).

The nucleocapsid of enveloped viruses is enclosed within a lipid envelope derived from the infected host cell (virally modified phospholipid bilayer containing glycoproteins) during the virion assembly and release from the host cell during the budding process (exocytosis). The continuity of the viral envelope is disrupted by surfactants during solvation of the lipid within and among the surfactant micelles (11, 89). This ultimately impacts the host-cell receptor-binding ability of the infectious virus, preventing the initiation of infection of a host cell. Certain surfactants, such as sodium lauryl sulfate, possess strongly hydrophilic heads that readily target the capsid proteins, unfolding and extracting them before solubilizing the lipid envelope (94).

Aldehydes and ortho-phthalaldehyde

Glutaraldehyde exhibits broad-spectrum microbicidal activity (against bacteria, bacterial spores, fungi, and viruses). It has been used as a disinfectant and sterilant, particularly for low-temperature disinfection and sterilization of endoscopes and surgical equipment, and as a fixative in electron microscopy. Glutaraldehyde forms a strong association with the outer layers of bacterial cells (Gram-positive and Gram-negative bacteria), cross-linking amino groups in proteins, and with nucleotides (95) and inhibiting transport into the cell (22).

Ortho-phthalaldehyde (OPA) is an aromatic compound with two aldehyde groups and bactericidal and sporicidal activity. It has been suggested as a replacement for glutaraldehyde. The mechanism of action for OPA appears similar to that of glutaraldehyde (22), although others have suggested that OPA is less of a cross-linker than glutaraldehyde, enabling better penetration into the cell structure (96).

Phenols

Chloroxylenol (p-chloro-m-xylenol, PCMX) is often formulated into soaps, hand hygiene agents, and surface disinfectants (38, 97, 98). This phenolic agent has been used as an active in skin hygiene agents, surgical scrubs, and agents for pre-operative skin preparation. PCMX is a phenolic with activity against both Gram-positive (S. aureus) and Gram-negative (P. aeruginosa, Escherichia coli) bacteria and enveloped viruses (99–102). At high concentrations, PCMX is a cellular poison that penetrates the cell wall and precipitates proteins in the cytoplasm. At lower concentrations, PCMX disrupts critical bacterial enzymes and induces leakage of the cell wall (97). A commercially available hygiene agent containing PCMX has been reported to display microbicidal activity against a wide range of Gram-positive and Gram-negative bacteria, and enveloped viruses within 5 min when applied at dilutions of 1:48 to 1:600, far less concentrated than the recommended dilution of 1:20, on skin (97). PCMX exhibited complete virucidal activity against enveloped viruses (e.g., influenza virus, Ebola virus, and murine hepatitis virus [MHV]), even in the presence of a soil load (97, 101). Surprisingly, the activity of PCMX against human coronavirus was found to be low, similar to tested non-enveloped viruses (102). In contrast, PCMX at 0.12% completely inactivated the coronavirus MHV within 30 s (99). Wijesinghe and Weerasinghe (100) reported that a commercially available PCMX-containing microbicidal product exhibited efficacy against S. aureus and P. aeruginosa at a dilution (1:40) that is greater than that recommended (1:20) within a 1-min contact time. More recently, a PCMX-containing microbicidal product has been shown to be effective for inactivating the Ebola virus (family Filoviridae, an enveloped virus) (101). Despite its widespread use, little is known about the mechanism of action of PCMX against viruses (103). This is a knowledge gap that needs further investigation.

FUTURE TRENDS

To keep environmental surfaces (fomites) hygienically clean is challenging, as current disinfection methodologies must be re-applied periodically, since each application achieves only a temporary state of sanitization which is short-lived (104). Following such decontamination, re-contamination can occur. To address this, current trends include:

1. The formulation of products that result in continuously active surfaces retaining microbicidal activity over time (105–109). An example of such a formulation includes surface-anchored polymeric QAC microbicides, used to create a sustained virucidal effect with mechanisms of action including virion destruction and viral RNA degradation (108). It has recently been shown (109) that such a coating provides broad-spectrum microbicidal and virucidal activity for months, with mechanisms of inactivation including cell membrane disintegration and binding to proteins. A QAC spray has been described which, when applied to equipment surfaces, provides 24 h of residual microbicidal activity against a wide range of antibiotic-resistant bacteria, spores, and fungi (105). Little has been published on the mechanism of action of these microbicides that leave a residual and display long-term effects on microbial sites of action. The possibility that the use of continuously active (residual) microbicides that prolonged exposure of microorganisms to the active might create conditions conducive to the development of cross-resistance (106) or increased tolerance needs further investigation.

2. The use of microbicidal formulations containing natural products as active ingredients or as excipients increases the efficacy of microbicidal active agents (110). A better understanding of the mechanism could lead to enhancement of chemical microbicides currently in use and targeting of sites of action for specific pathogens.

3. Concern has been voiced about the overuse of microbicides in infection control and the development of microbicide resistance. However, despite the widespread use of microbicides for more than 100 years, these agents are still effective in controlling environmental transmission of pathogens. While increased tolerance has been noticed to some microbicides in the laboratory, these still are effective in controlling the spread of pathogens in practice (9, 111). Application of knowledge of the mechanism of action of commonly used microbicides can aid in the development of formulations that can be modified over time to present continued challenges to pathogens of concern in built environments (hospitals, homes, schools, etc.).

CONCLUSIONS

It is clear from this review that a wide variety of formulated microbicidal actives can be used for IPAC of pathogens. Understanding how they work is paramount in developing more effective microbicides, increasing their spectrum of action against different pathogens, and reducing the concentrations and contact times required for achieving the desired efficacy. Because viruses depend on attachment to a specific host cell, any microbicide that affects the receptor-binding domain on the virus capsid or envelope will prevent infection. These are the primary targets for most virucidal microbicides and, as such, enveloped viruses are susceptible to a broader range of microbicides than are non-enveloped viruses. In some cases, both the capsid/envelope and the nucleic acid may be targeted during inactivation.

Bacteria and fungi are more complex in structure than viruses and require nutrients and other substances to grow and survive. This increased complexity creates more targets for microbicides, allowing the microbes to survive the effects of certain microbicides. Induction of overexpression of efflux pumps and alternative metabolic pathways can reduce the effects of certain microbicides (112). The primary site of attack for the most commonly used microbicides on bacteria is the cell wall or membrane of bacteria, with the ultimate outcome (i.e., microbistasis vs microbicidal action) depending on a number of factors, including the number of structural targets affected, the importance of these targets, and the severity of the damage imparted. These are critical structures that actively transport nutrients into the cell and protect the bacteria against harmful substances. Damage to the enzymes or integrity of lipo-proteinaceous components can result in leakage of intracellular components, resulting in cell death. Once these barriers have been penetrated, damage to essential metabolic enzymes and the nucleic acids can accrue.

Formulation of microbicides (i.e., addition of potentiators, pH modulators, preservatives, and synergism) is useful for obtaining products that attack multiple sites on targeted pathogens or increase the spectrum of microbicidal action, while reducing both the concentrations and times required for inducing microbicidal activity. Thus, an understanding of the relevant mechanisms of action can result in the formulation of smart microbicides that are both more effective and require lower applied concentrations for IPAC and the reduction of the spread of pathogens through the environment.

Biographies

Charles P. Gerba is a professor in the Department of Environmental Science at the University of Arizona. He obtained his PhD. In Microbiology from the University of Miami (Florida). He was a postdoctoral fellow and Assistant Professor of Virology at Baylor College of Medicine from 1973-1981. He is a fellow of the American Academy of Microbiology and the American Association for the Advancement of Science. He has served on the science advisory committees of the U.S. Environmental Protection Agency and the Food and Drug Administration. He conducts research on control and transmission of pathogens transmitted by water, food, fomites, and air. He is co-author on serval textbooks, including Environmental Microbiology, Quantitative Microbial Risk, and Environmental and Pollution Science.

Stephanie Boone has a PhD in Environmental Microbiology, and an MS degree in both Molecular Cell Sciences and Psychology. She has published over 30 publications in molecular engineering, agricultural systems, and microbiology. She was employed as a molecular biologist for the United States Agricultural Research Service (USDA-ARS) from 2007-2021 where she coordinated and designed experimental studies, wrote grants and publications while collaborating with industry, farms, and academia. Dr. Boone currently works at the University of Arizona Water and Energy Sustainable Technology Center (WEST) where she also collaborates and writes grants with industrial partners conducting research in various areas of Environmental Microbiology including Built Environment disease transmission, Viral resuspension, surface contamination and disinfection, and natural product disinfection of fomites.

Raymond W. Nims is an employee of Syner-G Biopharma, providing subject matter expertise in viral safety and viral inactivation (physical and chemical) for the biologics manufacturing industry. Raymond obtained a PhD in chemistry (chemical toxicology) at The American University, Washington, DC, in 1993. He currently serves on the editorial board for the BioProcessing Journal, served on the ad hoc advisory boards for USP chapters 1237, 1050, and 1050.1, and is a founding member of the International Cell Line Authentication Committee. He is a generalist in the biomedical sciences, with a publication list spanning a wide range of areas in chemistry, carcinogenesis, biochemistry, pharmacology, toxicology, and virology.

Jean-Yves Maillard is a Professor of Pharmaceutical Microbiology at the School of Pharmacy & Pharmaceutical Sciences, Cardiff University, Wales. His field of expertise concerns antimicrobials, particularly biocides, with a particular interest in sporicides, complex biofilms and environmental dry surface biofilms. He has published over 200 peer-reviewed articles, 20 book chapters and 3 books and over 150 refereed conference communications. He has contributed to many national and international committees working on antimicrobial resistance prevention and disinfection protocol development. Over the years, he has been the recipient of multiple innovation and engagement awards.

Syed A. Sattar, PhD, as Professor Emeritus of Microbiology, University of Ottawa, studies the environmental fate of human pathogens and infection prevention. He is a Registered Microbiologist of the Canadian College of Microbiologists, a Fellow of the American Academy of Microbiology and the Royal Society for Public Health. He has been an adviser to the World Health Organization and the Organization for Economic Co-operation and Development on biosafety and infection prevention. He is also an advisor to the Healthcare Surfaces Institute, The Infection Prevention Strategy, International Nosocomial Infection Control Consortium and Virox Tech. His awards include: The Lifetime Achievement Award of the Ottawa Centre for Research and Innovation, Hygeia Gold Medal of the Schülke Foundation (Hamburg, Germany), Queen Elizabeth Diamond Jubilee Medal, Distinguished Microbiologist’s Award of the Canadian College of Microbiology, and an Award of Merit of ASTM International. In addition to being an academic, he is Chief Scientific Officer at CREM Co Labs.

Joseph R. Rubino began working in the industrial microbiology field in 1978, starting with Gibraltar Biological Laboratories, a contract microbiology laboratory in New Jersey where he worked as a microbiologist. In 1987 Joe moved on to Lehn & Fink, now Reckitt, as Group Leader for the Microbiology and Virology Laboratories, raising to the level R&D Director for Equity, Claims and Compliance supporting the Lysol and Dettol brands. Joe retired from Reckitt in July 2022 and is now an independent consultant focusing on home and community hygiene issues. He also works with the International Forum on Home Hygiene (IFH), a US and UK-based scientific authority on home and community hygiene, as a member of their Scientific Advisory Panel. Joe holds a B.A. in Biology from Upsala College, as well as an M.A. in Biology from Montclair State University. He also attended New York Medical College where he studied public health. Joe has both authored and co-authored more than 60 peer-reviewed publications, review articles and papers on antimicrobial agents, transmission of infectious agents and infection control strategies, including several recent pieces on SARS-CoV-2.

Julie McKinney (Global Director of Microbiology & Virology) holds a PhD and MS from Virginia Tech. After completing a Post-Doctoral Fellowship program funded by Pepsi Co and the Sara Lee Corporation, Julie moved to Pfizer Consumer Healthcare. During her time in the Consumer Healthcare space, she quickly moved from bench scientist working on ways to enhance probiotic stability to leadership roles, including Global Director of Formulation for Emulsion and Semi-Solids and finally Global Director of Microbiology. She is currently the Global R & D Director for Equity, Claims, and Compliance at Reckitt Benckiser. She considers her role at Reckitt a dream job, leading a skilled team of Microbiologists, Chemists and Consumer Scientists conducting global research to support the life changing brands Lysol, Dettol, and Harpic.

M. Khalid Ijaz is Senior Research Fellow and Director of Scientific Affairs at Global Research and Development for Lysol and Dettol, Reckitt Benckiser, Montvale, N.J., U.S.A. He obtained his DVM and MSc (Honors) from the University of Agriculture, Faisalabad, Pakistan. Having received a WHO Trainee Award, he obtained his PhD in Microbiology and Immunology (Environmental Virology) from the University of Ottawa. As a recipient of an MRC Fellowship award, he did post-doctoral work at Vaccine and Infectious Disease Organization (VIDO), Saskatoon, Canada. During his 30+ year career, he has worked in academia, research, and industry, focusing on pathogens and the mitigational role of hygiene agents and vaccines. He made contributions to aerobiology, environmental microbiology (air, water, surface, and personal care), immunobiology, vaccinology, virology (including several papers on virucidal activity of microbicides against emerging viruses including SARS-CoV-2) and parasitology, including air pollution R&D and public health, publishing over 100 peer-reviewed papers including book chapters and presentations at scientific meetings, and co-authoring a book on Disinfection of Viruses while receiving patent awards on microbicides, and enteric vaccines. Recently, he has played a key role in reviving research on the aerobiology of indoor air. He developed several microbicidal and aerobiology protocols, received EPA approval of an aerobiology protocol enabling the first EPA registration of an air sanitizer. He also successfully advised the EPA on the topics of disinfection hierarchy for emerging viral pathogens and the testing of Clostridium difficile, and 810 Guidelines. He is a Registered Microbiologist of the Canadian College of Microbiologists, and a Fellow of the Royal Society for Public Health, member of the Global Virus Network, and has recently been nominated for Full Membership in Sigma Xi, The Scientific Research Honor Society.