Alcohols



Alcohols


Günter Kampf

James W. Arbogast



Alcohol is perhaps the oldest of antiseptic agents being used for wound treatment as described by Claudius Galen (131-201 ad) and Guy de Chauliac (1363)1 and as a hand disinfectant for surgeons in 1888 by Fürbringer.2 Today, alcohols are used frequently for pathogen reduction on skin worldwide, and that is growing globally, especially in health care. Skin applications most frequently occur before blood draw, surgical procedures, and vascular catheter insertion as well as for routine hand hygiene with alcohol-based hand rubs (also known as “hand sanitizers” or “hygienic hand rubs”) or prior to surgery (also known as surgical hand rubs). These products typically contain >60% ethanol, isopropanol, n-propanol, or mixtures thereof in a variety of formulation formats (rinses, gels, foams, and wipes).

Alcohols have also been used as hard surface (non-skin) disinfectants, including low-level disinfectants in health care settings as far back as the late 1800s.3,4,5 Despite the lack of appreciable sporicidal effect that limits sterilization applications for alcohols, their general antimicrobial properties can be useful against surface-borne bacteria, including Mycobacteria, and other microorganisms. A variety of surface-disinfectant spray products claim effectiveness against bacteria, yeasts, fungi, and some viruses. Given the volatility and time-exposure requirements of alcohol, some of these claims are questionable. Seventy percent isopropanol is used as a surface spray in some food-handling situations in Europe as well as many other industrial disinfection applications. A recent review of alcohols as surface disinfectants in health care identified an effective solution with 29.4% ethanol5; however, the total formulation is important to the overall observed antimicrobial efficacy. Although alcohols are now nearly universally recognized as effective antimicrobial agents, the history of alcohol use for pathogen reduction and infection prevention involves controversy around the spectrum of antimicrobial activity, concentration levels, formulation impact, and dose/time of action. For all types of application, it is critically important to consider experimental conditions and methods in detail when considering the antimicrobial performance of formulations based on alcohols.

As a group, the alcohols possess many features desirable for a disinfectant or antiseptic. They have excellent bactericidal efficacy as well as bacteriostatic action as a preservative, some virucidal efficacy (especially against enveloped viruses), and fungicidal efficacy. The low-molecular-weight alcohols evaporate readily and are therefore, under regular use conditions, not considered a risk for acquired microbial resistance (as compared to actives claiming persistent effect), are relatively inexpensive, are usually easily obtainable, are colorless but can be easily colored if needed, and are relatively nontoxic with topical (skin) application. Many alcohols can also have a surface cleaning action due to their lipid solvency and low surface tension.


MODE OF ACTION

Alcohols have a nonspecific mode of action with a variety of toxic effects.6,7 The main effect is often considered as the coagulation or denaturation of proteins.6 This can occur in bacterial cells on the cell wall, on the cytoplasmic membrane, as well as on various cytoplasmic proteins or enzymes.6 In 1939, it was already shown that both ethanol (2.5%-10%) and isopropanol (6.6%-20%) significantly reduced or abolished the dehydrogenase activity of Escherichia coli.8 Coagulation of enzymes supports the loss of cellular activity. Enzymes for the production of bioethanol are also inactivated by ethanol in a concentration-dependent manner,9 which supports the hypothesis of the unspecific nature of protein denaturation by alcohols. Specific cellular mechanisms, such as the inhibition or inactivation of cholinesterase activity, can additionally be influenced by the opening of the bacterial cell by ethanol.10


Protein denaturation is so nonspecific that basically all proteins and membrane components are coagulated by exposure to the alcohols. As early as 1921, Kamm11 showed that both ethanol and n-propanol (1%-10%) can almost completely coagulate egg albumen in aqueous solution. Potato protein is also denatured by ethanol.12 In addition, for ubiquitin, protein denaturation is triggered by pressure, temperature, and alcohols.13 This nonspecific effect has been known for decades as a “protein deficit” in the efficacy testing of chemical disinfectants.14 With alcohol-based disinfectants, the “protein deficit” occurs both in the determination of efficacy under protein load as well as during its application, for example, when high levels of protein are present on the hands or noncleaned surfaces in the sense of a pollution.14 As a result, a higher concentration of active ingredient or a longer exposure time via higher doses are means to achieve equivalent antimicrobial efficacy when proteinaceous soil is present.

The penetration of the alcohols into the bacterial cell is explained by various mechanisms.15 On the one hand, the alcohols can lower the surface tension of bacterial cells, which can lead to disruption of linkages in the cell membrane or membrane structure.15 The alcohols ultimately penetrate into the hydrocarbon components of the bacterial membrane lipid bilayer, resulting in loss of function of the cell membrane and ultimately lysis of the cell.16 Two factors of the cell membrane have proved to be relevant in yeast cells for tolerance to ethanol or isopropanol: strengthening the opposing potassium and proton electrochemical membrane gradients.17 Whether these factors are of comparable relevance to bacterial cells is, according to current knowledge, unclear. In individual hospital isolates of Enterococcus faecium, a mutation (H486N/Y substitution in the rpoB gene) for increased tolerance to isopropanol at 23% was detected, possibly caused by exposure to rifampicin16; however, this mutation alone does not explain the increased tolerance of individual isolates to isopropanol, suggesting that isopropanol tolerance is a polygenic phenotype with multiple genetic changes across different loci.16 This finding also suggests that there is no specific mode of action to explain the antimicrobial activity of the alcohols. Alcohols can also dissolve hydrogen bonds in the cell membrane.15,18 In addition, it has been demonstrated in E coli that cell lysis can be explained by the weakening of hydrophobic interactions rather than from the intercalation of ethanol into membranes.19

Other nonspecific modes of action include the destruction of cytoplasmic integrity, lysis of the cell, and interference with cellular metabolism.6 Lysis of the cells by alcohols has also been described in yeasts such as Candida albicans, Torulopsis glabrata, and Saccharomyces cerevisiae.20 Depending on their concentration, ethanol and n-propanol can prolong the lag phase for bacterial multiplication, as demonstrated by Aerobacter aerogenes.21 This effect is explained by a slowing of cell metabolism required for rapid cell division.7 The halophilic bacterium Halobacterium cutirubrum has also been shown to increase envelope dissolution by 10% ethanol.22 Similar effects can predict the effects on other types of microorganisms, such as the efficacy of alcohols against enveloped viruses due to their exposed lipid/protein surface in comparison to the greater resistance observed with non-enveloped viruses due to their unique protein exposed surfaces.7


PHYSICAL CHEMICAL PROPERTIES

As the chain length and molecular weight of alcohol increases their specific gravity, boiling point, melting point, and lipophilicity generally increases, whereas solubility in water decreases. A nonlinear, branched carbon chain structure can improve the water solubility. These values are summarized for various alcohols in Table 19.1. Alcohols at higher chain lengths are solids at room temperature, which limits their applications as antimicrobial agents. The short-chain alcohols are inflammable liquids. The flash points for methanol, ethanol, propanols, and tert butanol are lower than 15°C. Therefore, caution should be exercised in handling these alcohols. Safety data for a range of alcohols are presented in Table 19.2.

The concentrations of alcohols are generally given in percentage by weight (gram per gram [g/g] or weight per weight [wt/wt]) or percentage in volume (milliliter per milliliter [mL/mL] or volume per volume [vol/vol]). Table 19.3 illustrates the percentages by volume and weight for ethanol, isopropanol, and n-propanol. Weight per weight has advantages of being easier to compare between products for end users and in manufacturing due to volume contraction on mixing. However, some parts of the world have regulations that specify vol/vol percentages to be specified on product labeling.23


REGULATIONS AND GUIDELINES


European Regulations and Guidelines

In almost all countries within the European Union, products for hygienic and surgical hand disinfection are considered biocidal products (product type 1, intended for human hygiene). Products for surface disinfection belong to product type 2 (disinfectants and algaecides not intended for direct application to humans or animals) or product type 4 (food and feed area). Isopropanol has been approved in 2015 as an active biocidal agent for product types 1, 2, and 4.24 n-Propanol has also been approved (November 2017) as an active biocidal agent for product types 1, 2, and 4.25 Ethanol remains under review (June 2018) as an active biocidal agent for product types 1, 2, 4, and 6 (preservatives for products during storage).26









TABLE 19.1 Physical properties of alcohols



























































































































































































Alcohol


Molecular Weight


Specific Gravity at 20°C


Boiling Point (°C)


Melting Point (°C)


Solubility in Water at 20°C (g/100 g Water)


Partition Coefficient (log P)a


Viscosity


(mPa · s)b


(°C)b


Methanol


32.04


0.791


64.7


-98


Unlimited


NA


0.52


20


Ethanol


46.07


0.789


78.4


-117


Unlimited


-0.58


1.22


20


Propan-1-olc


60.10


0.805


97.2


-127


Unlimited


0.28


2.75


20


Propan-2-old


60.10


0.785


82.0


-89


Unlimited


-0.19


2.27


20


Butan-1-ole


74.12


0.810


117.0


-89


7.7


0.89


2.95


20


Butan-2-olf


74.12


0.808


99.8


-114


12.5


0.84


4.21


20


2-Methylpropan-1-olg


74.12


0.802


108


-108


9.5


0.65


6.68


20


2-Methylpropan-2-olh


74.12


0.779


82.9


24


Unlimited


0.34


3.30


30


Pentan-1-oli


88.15


0.815


138


-79


2.7


NA


3.68


20


Pentan-2-olj


88.15


0.808


119


-50


13.5


NA


NA


NA


3-Methylbutan-1-olk


88.15


0.816


131


-117


2.5


NA


NA


NA


2-Methylbutan-1-oll


88.15


0.816


128


70


3.6


NA


5.10


20


2-Methylbutan-2-olm


88.15


0.800


102


-12


17.7 (30°C)


NA


3.70


25


Hexan-1-ol


102.20


0.814


157


-45


0.6


NA


0.45


24


Heptan-1-ol


116.21


0.824


175


-34


Slight


NA


NA


NA


Octan-1-ol


130.23


0.827


195


-16


Slight


NA


8.40


20


Benzyl alcohol


108.13


1.042


204.7


-15.3


3.5


NA


NA


NA


Abbreviation: NA, not available.


a System: diethyl ether/water.

b Centipoise.

c n-Propanol.

d Isopropanol.

e n-Butanol.

f sec-Butanol.

g lsobutanol.

h tert-Butanol.

i n-Amyl alcohol.

j sec-Amyl alcohol.

k Isoamyl alcohol.

l act-Amyl alcohol.

m tert-Amyl alcohol.


Skin antiseptics are commonly regulated as medicinal products. The efficacy requirements for a disinfectant depend on the intended use. In human medicine, a product for disinfection has to show at least bactericidal and yeasticidal activity. Additional claims such as fungicidal, tuberculocidal, mycobactericidal, virucidal (three levels of efficacy), and sporicidal activity are possible but not mandatory. The application of European efficacy standards is summarized in EN 14885.27


United States Regulations

In the United States the regulation of alcohols depends mostly on the product application. Skin antiseptics fall under the purview of the US Food and Drug Administration and are regulated as drugs, either as an over-the-counter under a drug monograph guideline or as a new drug approval, whereas alcohol-based antimicrobials for inanimate surface applications are regulated by the US Environmental Protection Agency.


World Health Organization Guidelines

Alcohol-based hand rubs are recommended as the first choice treatment for the decontamination of clean hands in health care since 2009.28 In 2015, the World Health Organization (WHO) described alcohol-based hand rubs as essential medicines,29 referencing the simple formulations using 80% ethanol or 75% isopropanol,28 although they do not meet European efficacy requirements when applied
with 3 mL for 30 seconds.30 In 2016, WHO recommended the use of alcohol-chlorhexidine-skin-antiseptics for surgical site preparation in patients undergoing surgical procedures.31 In the same guideline, “suitable” alcohol-based hand rubs are recommended next to surgical hand scrubs for the preoperative treatment of hands.31








TABLE 19.2 Safety data of alcohols used for antiseptic purposes













































































































































Alcohol


Vapor Pressure at 20°C hPa


Vapor Density at 20°C


Flash Pointa (°C)


Explosion Limits in Air (vol %)


Autoignition Temperature (°C)


Odor Threshold (ppm)


Methanol


128


1.1


11


6.0-36.0


385


5


Ethanol


59


1.6


13


3.3-19.0


363


10


Propan-1-ol


19


2.1


15


2.2-13.7


410


30


Propan-2-ol


43


2.1


12


2.0-12.7


399


40


Butan-1-ol


7


2.5


30


1.4-11.3


343


10


Butan-2-ol


17


2.6


23


1.7-9.8


405


0.6


Isobutanol


12


2.6


29


1.7-10.9


430


40


tert-Butanol


40


2.6


11


2.3-8.0


478


73


Pentan-1-ol


3


3.0


48


1.3-10.5


300


1


Pentan-2-ol


3


3.0


41


1.2-9.0


347


70


akt-Amyl alcohol


51b


NA


50


1.9-9.3


340


NA


tert-Amyl alcohol


13


3.0


20


1.3-9.6


425


2.3


Hexan-1-ol


1


3.5


63


2.1-7.7


292


5.2


Heptan-1-ol


0.5


4.0


70


NA


350


0.5


Octan-1-ol


0.3


4.5


81


0.8


270


NA


Benzyl alcohol


0.1


3.7


101


NA


436


NA


Abbreviation: NA, not available.


a For concentrated solutions.

b At 50°C.


Data from Hommel G. Handbuch der Gefährlichen Güter. 4th ed. Berlin, Germany: Springer; 1980, and Threon JF. Alcohols, Industrial Hygiene and Toxicology. 2nd ed. New York, NY: John Wiley & Sons; 1963. Toxicology; Vol 2.



Other National Guidelines

Other regions of the world may regulate alcohol use for antimicrobial purposes differently. Other national specific guidelines are not considered further in this review.


ANTIMICROBIAL PROPERTIES OF ALCOHOLS


Measurement Methods

The spectrum of antimicrobial activity is usually determined in suspension tests or time kill tests. The test principle is the same. The disinfectant or antiseptic must demonstrate a minimum log10 reduction of culturable microorganisms or reduction of viral infectivity within a specific exposure time compared to a control (eg, pre-exposure or exposure to negative control).

In Europe, the spectrum of antimicrobial activity must include bactericidal activity according to EN 13727 and yeasticidal activity according to EN 13624. The same spectrum of antimicrobial activity is required in the United States, although the number of bacterial species to be investigated to determine bactericidal activity is much higher with at least 20 species compared to 4 species in EN 13727.23 A mycobactericidal or virucidal activity is optional.27 In veterinary medicine, the products must be at least bactericidal according to EN 13727.27 Hygienic hand rub products used in food, industrial, domestic, and institutional areas have in Europe no specific minimum spectrum of antimicrobial activity. Tests are available to demonstrate bactericidal (EN 1276) and yeasticidal (EN 1650) activity.27


Spectrum of Bactericidal Activity

In the following sections, the alcohol concentrations are given as wt/wt unless otherwise indicated.









TABLE 19.3 Concentrations as percentages by weight and volume for aqueous ethanol, propan-2-ol, and propan-1-ol






























































































Weight (% g/g)


Concentrations by Volume at 20°C (% mL/mL)


Alcohol


Ethanol


Propan-2-ol


Propan-1-ol


40


47.4


47.0


46.5


50


57.8


57.0


56.5


60


67.7


67.2


66.2


70


76.9


76.5


76.1


80


85.4


85.0


84.5


90


93.2


93.8


93.5


95


96.7


97.5


97.2


100


100.0


100.0


100.0


Volume at 20°C (% mL/mL)


Weight (% g/g)


40


33.4


33.5


34.0


50


42.6


43.0


43.4


60


52.1


53.7


53.9


70


62.6


62.8


63.5


80


73.4


73.5


74.5


90


85.8


86.0


86.0


95


92.5


92.8


93.0


100


100.0


100.0


100.0


Ethanol at 78% or more has comprehensive bactericidal activity within 30 seconds against Acinetobacter baumannii,32 Acinetobacter lwoffii,32 Bacteroides fragilis,32 Burkholderia cenocepacia,33 Burkholderia cepacia,32 Campylobacter jejuni,34 the vegetative cell form of Clostridium difficile,32 Corynebacterium jeikeium,35 Enterobacter aerogenes,32 Enterobacter cloacae,32 Enterococcus faecalis,32,36 E faecium,32,36 Enterococcus hirae,37,38 E coli,32,37,38 Haemophilus influenzae,32 Haemophilus parasuis,39 Helicobacter pylori,40 Klebsiella pneumoniae,32 Klebsiella oxytoca,32 Listeria monocytogenes,32 Micrococcus luteus,32 Proteus mirabilis,32 Pseudomonas aeruginosa,32,37,38,41 Serratia marcescens,32 Staphylococcus aureus,32,37,38,41 Staphylococcus epidermidis,32,35 Staphylococcus haemolyticus,32 Staphylococcus hominis,32 Staphylococcus saprophyticus,32 Streptococcus pneumoniae,32 Streptococcus pyogenes,32 Salmonella enteritidis,32 Salmonella Typhimurium,32,42 and Shigella sonnei.32 Lower concentration of ethanol may require longer exposure times to reach an equivalent efficacy, depending on the formulation.

Published data with isopropanol indicates a strong bactericidal activity of a 70% solution beginning after 15 seconds against C jejuni,34 Enterococcus species,43 H parasuis,39 S aureus,44 and S epidermidis.45 Additional data with mixed propanols (eg, 45% isopropanol plus 30% n-propanol) indicate comprehensive bactericidal activity within 30 seconds.37,46

n-Propanol at 60% has a strong bactericidal activity beginning after 15 seconds against A baumannii,47 Enterococcus species,43 and S aureus.48 Additional data with mixed propanols (eg, 30% n-propanol plus 45% isopropanol) indicate comprehensive bactericidal activity within 30 seconds.37,46 The bactericidal activity of 60% n-propanol is considered to be equal to isopropanol at 70%, whereas lower concentrations such as 50% or 40% have a lower bactericidal activity.49


Spectrum of Fungicidal Activity

Ethanol is usually effective at 70% to 90% within 30 seconds to 5 minutes against fungi typically found in health care setting such as C albicans,38,50 Aspergillus niger,38 Cryptococcus neoformans,50 or Cryptococcus uniguttulatus.50 Food-related fungi such as ascospores,51 conidia,51 or yeasts51,52 were often more resistant to ethanol with a variable efficacy of 70% ethanol in 10 minutes. In mixed suspensions of environmental isolates (Rhodotorula rubra, C albicans, C uniguttulatus) and clinical isolates (R rubra, C albicans, C neoformans), 70% ethanol is still fungicidal (log10 reduction ≥6) in 5 minutes, although the effect was somewhat less against the environmental mix.50

Isopropanol at 60% or 70% was mostly effective with 5 to 10 minutes against various types of yeasts including
food-associated yeasts.51,52 The efficacy of 70% isopropanol against food-associated fungal spores such as ascospores or conidia is low even at 10-minute exposure.51

Fourteen percent n-propanol has been described to inhibit multiplication of C albicans suggesting a levurostatic activity at that concentration.53 At 89.5% (vol/vol), n-propanol is effective against C albicans.54 A commercial product based on n-propanol (>30%) and isopropanol (15%-30%) has been investigated for yeasticidal activity against 25 strains isolated from food or food processing. Within 5 minutes, the product reduced the number of yeast cells by at least 4 log10 steps of 19 species; some species were less susceptible.52


Spectrum of Mycobactericidal Activity

Ethanol of 70% or more has sufficient activity against selected mycobacterial species such as Mycobacterium chelonae,35 Mycobacterium nonchromogenicum,35 Mycobacterium smegmatis,35,55 Mycobacterium terrae,38,56 and Mycobacterium tuberculosis56,57 within 1 to 5 minutes. Only few data were found against aquatic nontuberculous mycobacteria. For example, Mycobacterium marinum was effectively reduced by ethanol at 50% and 70% in 1 minute.58

Early data from 1953 indicate a tuberculocidal activity of isopropanol at 50% to 70%.59 In suspension tests, little has been published on the mycobactericidal activity of isopropanol. At 60%, it was mostly effective against both M tuberculosis and M terrae within 5 minutes.56 Whether the tuberculocidal efficacy covers other mycobacterial species is not clear.

n-Propanol at 20% has been described to reduce Mycobacterium avium on frosted glass strips by >6 log in 20 minutes.60 No other data were found to describe the efficacy of n-propanol against mycobacteria.


Spectrum of Virucidal Activity

Ethanol has been shown to be effective against various enveloped viruses.61 Beginning at a concentration of 42.6%, ethanol was effective within 30 seconds against severe acute respiratory syndrome coronavirus62; Middle East respiratory syndrome coronavirus63; Ebola virus63; influenza A virus,61,64,65 including the human type H3N2,66 the avian type H3N8,66 and human type H1N163; influenza B virus67; human immunodeficiency virus (HIV)38,68,69,70,71; hepatitis B virus (HBV)72,73; vaccinia virus61,64,74,75,76,77,78; duck HBV77; togavirus79; pseudorabies virus71; Newcastle disease virus80; bovine viral diarrhea virus71,78,81; Zika virus63; herpes simplex viruses (HSVs)66 types 1 and 2; and respiratory syncytial virus (RSV).67 Ethanol is effective at 73.6% against hepatitis C virus (HCV) in 15 seconds81 and 30 seconds63 but not in 40%.82

Ethanol as a solution has mostly sufficient activity in 30 seconds against adenovirus type 5 (test virus of EN 14476) at concentrations between 45% (contains additional active ingredients) and 95%,78,81,83,84 although one study indicates sufficient activity only in 1 minute with 77% ethanol.85 Ethanol at 70% as a gel was sufficiently active in 2 minutes.84 Adenovirus type 2 was less susceptible, requiring 2 minutes at 55%67 and 85%.38 In 30 seconds, 62.4% ethanol was not sufficiently effective86 and at 50% required a 10-minute exposure time to be effective.61 The combination of ethanol 73.6% and peracetic acid (0.2%) was effective in 30 seconds,87 but this would be expected based on the potency of peracetic acid alone (see chapter 18). Data with other adenoviruses suggests that ethanol at 72.5% to 77% was effective in 60 seconds against adenovirus type 7 but not against adenovirus type 8.88

The effectiveness of ethanol on nonenveloped viruses has also been found to vary depending on the virus type. Gels based on 85% ethanol or the combination of 62.4% ethanol with additional citric acid were effective against rotavirus in 30 seconds38,86 as well as a hand rub based on 55% ethanol, 0.7% phosphoric acid, and three other alcohols.67 Today, murine norovirus (MNV), an enteric (gastrointestinal virus), is commonly used as a surrogate to assess activity of disinfectants and antiseptics against human noroviruses. Ethanol at concentrations between 62.4% and 85.8% is usually effective in 30 seconds78,81,83,89,90,91 or 1 minute91; lower concentrations were less effective. A gel based on 53.2% ethanol was only effective in 1 hour.92 Ethanol at 42.6% reduces MNV according to one study in 1 minute by 0.3 log10 and in 5 minutes by 0.4 log10.91 In another study, 42.6% ethanol demonstrated approximately 3 log10 reduction in 30 seconds, whereas ethanol at 24.7% or 8% was ineffective within 3 minutes.89 Feline calicivirus (FCV) has often been used in the past as a surrogate for human noroviruses, although it is not preferred because it is a respiratory tract virus and is highly sensitive to acidity.93 The FCV is difficult to inactive by ethanol. Ethanol solutions at 42.5% and 62.4% were effective at 3 minutes, whereas 73.6% required 5 minutes.94 Formulations with 72.5% ethanol did not reach a 4 log10 reduction in 60 seconds.88 A solution based on 77% ethanol showed little activity against FCV in 60 seconds (<1 log10).88 Even after 5 minutes, the efficacy was still insufficient (<2 log10).85 A hand rub based on >85.8% ethanol was practically ineffective in 30 seconds (<1 log10).78 Finally, ethanol at 42.6%, 62.4%, and 85.8% had insufficient efficacy in 5 minutes with the highest log10 reduction of 2.6.91 Whenever different types of acid are added, ethanol in hand rubs has been described to be effective in 30 seconds at 45%,78 50.2%,67,78 55%,95 62.4%,86 and 67.9%.95 Malic acid at 0.35% has also been described to improve the efficacy of ethanol against FCV to some extent.96 Data on the efficacy of ethanol against coxsackievirus are conflicting. Ethanol at 72.5% to 92.4% has been described to be effective against coxsackievirus B5 within 60 seconds88,97 and also against coxsackievirus B1 within 10 minutes but not against coxsackievirus A7.88 Higher
ethanol concentrations (85%-90%) are effective against coxsackievirus B3 in 15 to 60 seconds.98 The efficacy of ethanol against echoviruses is rather good. One study shows that ethanol at 92.4% is effective against echovirus 11 in 20 seconds.99 Another study found a log10 reduction ≥3 within 1 minute for ethanol at 92.4% against the same virus, whereas ethanol at 67.9% was almost ineffective (<1 log10) at the same exposure time.100 Against echovirus 6, ethanol was effective at 50% within 10 minutes.61 With human enterovirus 71, ethanol had only little activity at 62.4% and 67.9% within 10 minutes (<1 log10). At 79.6%, a 3.2 log10 reduction was achieved in the same exposure time, and at 92.4%, this increased to 5.8 log10.101

Formulations with ethanol up to 80% and without acid were not sufficiently effective in up to 5 minutes against poliovirus type 1 when tested under standard conditions with 80% product proportion in the suspension. When the product proportion is increased in the suspension test to 97%, some formulations with 73.5% or 80% ethanol were effective within 1 minute. One formulation with 95% ethanol was effective in 30 seconds. Gels were mostly less effective compared to solutions. The addition of various types of acids can substantially improve the activity of ethanol against poliovirus type 1 so that the formulations are often sufficiently effective in 30 seconds.102 Poliovirus type 2 is somewhat less susceptible to ethanol compared to poliovirus type 1.103 A hand rub based on 55% ethanol, 0.7% phosphoric acid, and three other alcohols was effective in 30 seconds against rhinoviruses.67

A gel based on 54.2% ethanol showed no effect at all (0 log10) against hepatitis A virus (HAV) after 30 seconds.86 Ethanol at 80% or 95% was not sufficiently effective within 2 minutes.104 A hand rub based on 80% ethanol showed only little reduction of viral infectivity within 30 seconds (0.47) but increased efficacy on exposure at 2 minutes (≥2.2 log10).71 When 0.7% phosphoric acid and three other alcohols are added to ethanol at 55%, the formulation was effective against HAV in 30 seconds.67 Ethanol at 62.4% with additional 0.25% citric acid, however, was not sufficiently effective in 30 seconds with 1.75 log10.86 The infectivity of the foot-and-mouth disease virus was insufficiently reduced by ethanol between 55.2% and 72.5% within 5 minutes.105 Hand rubs with 70% to 75.2% ethanol and additional phosphoric acid (0.6%) or 50% ethanol and additional citric acid (0.5%) were effective within 30 seconds.105

Ethanol has little activity against polyomavirus SV40, an additional test virus that was chosen due to its resistance to alcohols by experts for the German test method to determine virucidal activity.106 A gel based on 85% ethanol revealed sufficient activity in 15 minutes,38 but a hand rub based on 78.2% ethanol had insufficient activity within 10 minutes (approximately 2 log10).103 When 0.7% phosphoric acid and three other alcohols are added to ethanol at 55%, the formulation was able to reduce infectivity sufficiently in 60 seconds.67 Ethanol at 73.6% in combination with 0.2% peracetic acid was effective in 30 seconds.87 Ethanol has almost no virucidal activity against parvoviruses. At 80% ethanol, the infectivity of the canine parvovirus, as an example was reduced in 5 minutes only by 0.1 log10.71

Isopropanol at 40% is effective against the enveloped viruses duck HBV, vaccinia virus,76 and the modified vaccinia virus Ankara.77 At 75%, isopropanol is effective within 15 seconds against bovine viral diarrhea virus (a surrogate for HCV).81 Seventy percent isopropanol was also shown to be effective against influenza A virus,65 whereas 30% isopropanol was effective at a 10-minute exposure time.61 The HIV can easily be inactivated by 70% isopropanol.107 For inactivation of RSV, 35% isopropanol was effective within 1 minute.108 The HSVs seem somewhat more resistant. The HSV types 1 and 2 were inactivated by 70% isopropanol in 5 minutes,109 whereas another study indicated sufficient efficacy against HSV type 1 within 1 minute for isopropanol at 60% and 70%.110 A concentration of 20% was still effective against HSV type 1 but required a 10-minute exposure.61

As for other alcohols, the results with other viruses vary depending on their structure. Isopropanol is considered to be ineffective against enteroviruses.111 Against poliovirus type 1, human enterovirus 71, and coxsackieviruses B2 and B3, no efficacy was found at concentrations between 70% and 100% within 10 minutes.61,98,101,112 The efficacy against echovirus61,100,113 and astrovirus113 is poor. Fifty percent isopropanol was, however, described as effective against adenovirus within 10 minutes.61 The data with FCV, used as a surrogate for human noroviruses before the advent of MNV culture in vitro, are overall conflicting. The FCV was inactivated by isopropanol between 50% and 70% in 3 minutes, but at 80%, no sufficient efficacy was found after 5 minutes.94 Another study indicated that isopropanol at 50%, 70%, and 90% for 5 minutes gave a maximum reduction of 0.8 log10.91 The data with MNV are also not consistent. One study shows that isopropanol at 80% is effective against MNV in 30 seconds, whereas 50%, 60%, 70%, and 90% were not,89 indicating that 80% may be the optimum concentration. Another study describes isopropanol at 70% in 5 minutes as effective (log10 reduction ≥2.6) and greater at 90% after 1 minute.91 A third study shows that 60% isopropanol is effective against MNV in 60 seconds, but concentrations of 30% and 10% are not.92 In contrast, rotaviruses have been found to be inactivated quite easily as shown with a mixture of 45% isopropanol and 30% n-propanol.92

Thirty percent and 60% n-propanol are effective within 1 minute against enveloped viruses such as HCV.114 n-Propanol at 70% was described as effective against influenza A virus H1N1 within 1 minute.65 A combination of 30% n-propanol and 45% isopropanol was effective within 15 seconds against various enveloped virus species.66


The FCV is inactivated within 30 seconds by n-propanol at 50% to 70%, but at 80%, an exposure time of 1 minute was necessary.94 The MNV is also inactivated within 30 seconds by n-propanol at 50% to 90%.89 The type 1 poliovirus is inactivated by n-propanol at 33% in 4 minutes by 3 log10.115 The addition of 0.2% peracetic acid improved the efficacy resulting in a 4 log10 reduction within 1.5 minute.115


Spectrum of Sporicidal Activity

Bacterial spores are regarded to be resistant to ethanol,116 isopropanol,117 and n-propanol.


Activity Against Protozoans

Sixty-three percent and 80% isopropanol and ethanol at 5-minute contact have been shown to have some effects against cysts of Giardia duodenalis and Entamoeba invadens (a nonpathogenic model for Entamoeba histolytica), resulting in an almost complete collapse of the cyst wall of Giardia. Treatment with 80% isopropanol also prevented an oral infection of gerbils by 1000 G duodenalis cysts.118


RESISTANCE


Acquired Bacterial or Fungal Resistance

In typical disinfectant applications, alcohols have an advantage of acting and evaporating rapidly and not leaving residue that allows time for bacteria to develop resistance. Apart from bacterial spores, no other bacteria with a notable resistance to n-propanol or propanol have been reported so far.28,119,120 Conidia of Penicillium chrysogenum, Penicillium digitatum, and Penicillium italicum were shown to resist ethanol vapors under nonoptimal natural conditions probably caused by a reduced intracellular water activity of dry-harvested conidia,121 but differences in the intrinsic resistance profiles of bacteria/fungi to alcohols may be demonstrated at low-level exposures.


Effect of Low-Level Exposure to Alcohols

Ethanol at 1.25% to 2.5% has been shown to significantly enhance S aureus biofilm formation by upregulation of some proteins with adhesive functions and others with cell maintenance functions and virulence factor EsxA.122 Similar findings were reported with L monocytogenes. When cells were exposed to 5% (vol/vol) ethanol for 60 minutes, they were significantly more difficult to kill by ethanol at 17.5%.123 The adapted cells were also more difficult to kill by 0.1% hydrogen peroxide.123 Attachment of cells can be significantly increased in some L monocytogenes strains when exposed to 2.5% ethanol, mostly at 10°C.124 When the Pseudomonas species strain DJ-12 was exposed to 5% ethanol for 10 minutes, the cells were significantly more difficult to kill by 20% ethanol.125 Cells treated with ethanol displayed irregular rod shapes with wrinkled surfaces.125 Exposure of a Pseudomonas putida strain to toluene increased the tolerance to ethanol, which was explained by an inhibitory effect of ethanol on the biosynthesis of saturated fatty acids.126 In Bacillus subtilis cells, the transfer of the mobile genetic element Tn916, a conjugative transposon, and the prototype of a large family of related elements was increased 5-fold by exposure to 4% ethanol for up to 2 hours. This may also result in a transfer of Tn916-like elements and any resistance genes they contain.127

The maximum ethanol tolerance of S cerevisiae has been described to be at 25% (vol/vol).128 The presence of 2.5% ethanol was found to increase S cerevisiae colony growth by 20%, whereas 5% ethanol or more inhibited yeast colony growth.129 When S cerevisiae cells were exposed for 30 minutes to sublethal concentrations of ethanol (8%, vol/vol), they became less susceptible to ethanol at a previously lethal concentration (14%, vol/vol).130 Specific activities of glycolytic and alcohologenic enzymes within intact living cells remained high by the presence of sublethal ethanol.128 Trehalose plays a role in ethanol tolerance at lethal ethanol concentrations but not at sublethal ethanol concentrations.131 Using atomic force microscopy, it was shown that challenge of S cerevisiae with 9% ethanol (vol/vol) for 5 hours reduced the observed stiffness of glucose-grown yeast cells, suggesting that changes in the cell membrane due to the presence of ethanol could modify the biophysical properties of yeast cells.132

Less has been published on the effects of low-level exposure of isopropanol on microorganisms. One study indicated that attachment of cells can be significantly increased in some L monocytogenes strains when exposed to 2.5% isopropanol, mostly at 10°C.124 Biofilm formation with 37 clinical icaADBC-positive S epidermidis isolates was investigated after exposure to isopropanol at 1%, 2%, 4%, or 6%. In 14 of the 37 strains, biofilm formation was inducible by isopropanol exposure.133 With C albicans, it was described that 2% isopropanol inhibited to some extent biofilm development.134 In E coli, it was shown that low-level exposure to isopropanol concentrations up to 2.7% for up to 24 days reduced the susceptibility of the six tested strains.135

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May 9, 2021 | Posted by in MICROBIOLOGY | Comments Off on Alcohols
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