©2009 Mark Tuttle
Immunity and infection, 2009
Independent Project weeks 4-5
Disinfectants and Antisepsis
See also accompanying PowerPoint
Isabel S. Novella
Objectives
Describe the physical processes being used to control microbial growth and compare the efficiency of both wet vs. dry heating and UV vs. g-irradiation for sterilization.
Explain why filtration of air or liquids does not remove virus particles
Define “antiseptic” and “disinfectant.”
Describe the mode of action of phenol, alcohols, halogens, surfactants, alkylating agents (e.g. aldehydes such as glutaraldehyde), and heavy metals when used for antisepsis or as disinfectants
Disinfection is the antimicrobial reduction of the number of microorganism (the bioburden) on or in a material. Successful disinfection refers to a reduction of bacteria to a level appropriate for use (i.e. not necessarily complete sterilization). In general, disinfection refers to a process, and a disinfectant refers to a product, that acts against most pathogens. It may not be very effective against bacterial spores.
High-level disinfectants kill viruses, bacteria, including mycobacteria, and fungi. They may not be effective against large numbers of bacterial spores.
Intermediate-level disinfectants kill most viruses, bacteria and fungi, but some bacterial spores and some fungal spores survive.
Low-level disinfectants are effective against most bacteria, some viruses (especially enveloped), some fungi, but do not kill mycobacteria or bacterial spores
Resistance differs with different types of organisms:
Physical Disinfection:
Heat:
Wet heat
60-65º C kills most bacteria (not spores) but bacteria are killed faster at higher temperatures. Pasteurization often uses a low temp to avoid food flavor/texture deterioration due to heat.
65º C 30 min
72º C 15 sec (flash pasteurization)
recommended surgical/medical device disinfection
100 min 70º C
10 min 80º C
1 min 90º C
0.1 min 100º C
Note bacterial spores need to be killed by higher temperature (e.g. 121ºC)
Dry heat
Not as effective as wet heat; wet heat penetrates the organism more effectively. Longer periods needed if using the same temperatures.
Radiation:
Ionizing (X rays and g rays) High energy, penetrating radiation that can be used for sterilization. Cause release of electrons from target atoms.
Non-ionizing (mainly ultraviolet; UV) U.V. tends not to penetrate well, but causes DNA and RNA damage. Short wavelength UV (200-280 nm or UV-C radiation) is germicidal. 265 nm most effective wavelength.
Filtration
Gases and liquids. Filters remove, but don’t inactivate microbes.
Filtration is widely used for removing microbes from air using HEPA (high efficiency particulate air) filters, usually where the particle size that is retained is >0.3 mm diam.
Used in preparing heat sensitive liquids, (e.g. pharmaceuticals). Some membrane filters are available that can remove particles that are below the size of a virus.
Chemical disinfectants
Antimicrobial weak acids (often used as preservatives in foods, soaps, lotions, eye drops, and cosmetics):
Benzoic
Citric RCOOH (in acid soln) → RCOO- + H+ (within cell)
Sorbic
p-hydroxybenzoic acid esters
Action:
These are most germicidal at low pH when they are not dissociated into ions: this allows them to pass through lipid membranes surrounding cells. Once inside cell, the higher pH leads to ionization and acidification of the cell. Some used in ointments to treat infections
Aldehydes
Action:
Crosslink and denature cell molecules reactive with aldehydes, especially proteins.
Glutaraldehyde, orthophthaldehyde (OPA), formaldehyde. Cross link and denature proteins. Kill most things, but do not inactivate prions, and also may not inactivate bacterial endospores and some mycobacteria. Tend to be less effective with clumps (clumped polio virus vaccine – live virus protected within clumps of viral particles).
Note that one outbreak of Creutzfeldt Jakob disease was believed to occur after prions were fixed to neurological electrodes by formaldehyde, then spread to several others.
Typical use: Glutaraldehyde, orthophthaldehyde (OPA),
Used as low temperature liquid disinfectants for temperature sensitive devices (e.g. flexible endoscope.
Some strains of bacteria resist low concentrations of formaldehyde via production of formaldehyde dehydrogenase (E. coli) or changing outer membrane structure (Pseudomonas).
Note: contaminants (e.g. blood/dirt) in area or on device reduce activity by competing for aldehyde groups.
Alcohols (especially ethanol and isopropanol)
Used for cleaning, drying, disinfection and antisepsis.
Kills bacteria including mycobacteria and enveloped viruses. Fairly effective against fungi and some naked viruses. Does not kill bacterial spores.
Action:
Probably mainly act via disruption of lipids in membranes and denaturation of proteins.
Biguanides: Especially Chlorhexidine
Action:
Bind and insert into cell membranes leading to disruption of cell activity.
Widely used for antisepsis and in soaps (Hibiclens and Hibiscrub for surgical scrubs).
Act against both Gram+ and Gram- bacteria, and most mycobacteria, but do not kill bacterial spores. Active against enveloped viruses, not naked virions.
Halogens and Halogen-Releasing Agents
Numerous products used in hospitals (e.g. betadine scrub contains iodine). Note that competition from dirt or proteins reduces effectiveness, e.g. protein in a blood spill. Bleach is used for controlling bacteria during purification of drinking water and in swimming pools. Most organisms and bacterial spores are susceptible, provided that a high enough concentration is used.
Chlorine, Bromine and Iodine (Oxidizers)
Cl2 + H20 → HOCl + H+ + Cl-
Br2 + H20 → HOBr + H+ + Br-
I2 + H20 → HOI + H+ + I-
Items in bold arte antimicrobial
pH dependence: High pH with increased ionization is less active.
pH 6.5: 90% of chlorine will be hypochlorous acid
pH 7.5: 50% of chlorine will be hypochlorous acid
pH 8.0: 20% of chlorine will be hypochlorous acid (not ionized and most active)
Phenolics
Early introduction of phenol by Lister in 19th century as an antiseptic was a revolution for surgery. Several phenolics are now only used as disinfectants, as they are excessively toxic for routine antisepsis
Antisepsis:
Bisphenols (hexachlorophene, triclosan)
Extremely common component of antibacterial soaps. Triclosan, at low concentrations (in soaps) affects fatty acid biosynthesis. Some bacteria becoming triclosan-tolerant have altered membrane fatty acids.
Action:
Phenolics: Affect cell membranes and poison many cellular activities.
Soaps and Surfactants
Molecules that have hydrophobic (non-polar or lipophilic) portion and a hydrophilic (polar) portion. Reduce surface tension by forming micelles when they interact with water, allowing water to penetrate better. Chemical modifications to the hydrophilic region give rise to a wide range of different types.
Mainly cleaning action, but quaternary ammonium compounds which are strong cations (e.g. benzalkonium chloride) have some antimicrobial activity.
Metals (Silver and copper ions: Mercury is little used nowadays)
Bind sulfhydryl groups on cell surfaces; e.g. Silver nitrate is used to prevent eye infection of newborn.
Peroxides
Hydrogen peroxide, peracetic acid, benzyl peroxide, ozone, chlorine dioxide
Ozone used for wastewater disinfection.
Action: Strong oxidizing agents affecting cell surface.
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