The Effects of Germicides on Microorganisms
By Kirsten M. Buck
Bacteria after 15 seconds of treatment with 10 ppm chlorine. The dead cells are red, the green are the live cells.
Healthcare workers (HCWs) often take for granted the action of disinfectants without fully understanding their mechanism of action. Causing microbial cell death, in reality, is a complicated process. Not only are there differences in the action of the antimicrobial ingredients, but there are differences depending on the concentration of chemical that is used. It is not easy to determine the exact mechanism of action of a chemical agent or physical process. The reason is that more than one part of the microbial cell may be affected and consequently, the problem is to distinguish which of these effects ultimately contributed to the cell death. Information as to the cellular target can be obtained in various ways. Biocide treatment of cells under conditions of growth or non-growth indicates whether the test substance inhibits some biosynthetic process, in which case non-growing cells are unaffected. Further preliminary experiments can then be carried out to determine whether the agent inhibits synthesis of the cell wall or nucleic acids. Other useful experiments include studies on the possible leakage of low-molecular weight materials, monitored by an experimental technique to study proton flux across the cytoplasmic membrane.1
Chlorine in an aqueous solution, even in very small amounts, exhibits fast bactericidal action. The mechanism of this activity has not been fully determined, despite much research. When chlorine is added to water, it forms hypochlorous acid. Exactly how hypochlorous acid destroys microorganisms has never been demonstrated experimentally, but it has been speculated that hypochlorous acid allows oxygen to emerge, which in turn supposedly combines with components of cell protoplasm, destroying the organism. Researchers have assumed that because of the low chlorine level required for bactericidal action, chlorine must inhibit some key enzymatic reactions in the cell. The inhibition of these essential cytoplasmic metabolic reactions is largely responsible for the destruction of both bacterial and fungal cells. Very few chemicals are considered sporicidal; however, bacterial spores are affected by disinfectants at different stages in the sporulation process. While not considered sporicidal, chlorine compounds have demonstrated some activity at the outgrowth stage, but higher concentrations also may prevent germination. Chlorine compounds have been shown to affect surface antigen in enveloped viruses and DNA as well as structural alterations in non-enveloped viruses.
Iodine, mainly in its molecular form (I2), can penetrate the cell wall of microorganisms rapidly. The actual killing of the microorganism by iodine could be the result of the inability to synthesize proteins due to oxidation in an important amino acid,2 the increasing of the bulk of the amino acid molecules which leads to the denaturation of DNA,3 or the addition of iodine to unsaturated fatty acids could to lead to a change in the physical properties of the lipids.4 Electron microscopy observations support the conclusion that iodine, by interacting with the double bonds of phospholipids causes damage of the cell wall which lead to a loss of intracellular material. Halogens such as chlorine and iodine react not only with living microorganisms but also with dead ones and with dissolved proteins. In contrast to chlorine, where oxidizing and bactericidal N-chloro compounds emerge, with iodine the efficacy is diminished because N-iodo compounds are not formed.5
|Gram-negative bacteria before treatment with a disinfectant|
Like many disinfectants, alcohols are generally considered to be non-specific antimicrobials because of their many toxic effects. The predominant mode of action appears to stem from protein coagulation/denaturation. Disruption of the cytoplasmic membrane, cell lysis, and interference with cellular metabolism has been reported. Protein coagulation occurs within concentration limits around an optimum alcohol level. In the absence of water, proteins are not denatured as readily as when water is present. Therefore, mixtures of alcohol with water exhibit much better efficacy than straight alcohol alone. Alcohol-induced coagulation of proteins occurs at the cell wall, the cytoplasmic membrane and among the various plasma proteins. Alcohols target the bacterial cell wall, with resultant lysis of the cytoplasmic membrane and release of cellular contents. The antifungal action of alcohol is very similar, resulting in the attachment to the plasma membrane and leakage of cell contents. The inhibition of spore germination by ethanol and other alcohols may be due to the inhibition of enzymes necessary for germination. This inhibition is reversible because only the removal of alcohol from the environment is necessary for germination to take place; therefore, alcohols are not appropriate as chemical sterilants.6
Hydrogen peroxide is effective against a wide variety of organisms: bacteria, yeast, fungi, viruses, and spores. Anaerobes are even more sensitive because they do not produce catalase to break down the peroxide. In general, hydrogen peroxide has a greater activity against gram-negative than gram-positive bacteria. Unlike like most disinfectants, hydrogen peroxide is unaffected by the addition of organic matter and salts.7
Hydrogen peroxide, the superoxide ion radical, and the hydroxyl radical are intermediate products in the reduction of oxygen to water. The hydroxyl radical is said to be the strongest oxidant known, and it is by this mechanism that hydrogen peroxide is believed to do the actual killing of bacteria. The hydroxyl radical, being highly reactive, can attack membrane lipids, DNA, and other essential cell components. Transition metals are believed to catalyze the formation of the hydroxyl radical, therefore the addition of iron, copper, cobalt, chromium, or manganese increases the efficacy of hydrogen peroxide. It is important to note that many metal ions are inherent in the microbial cell as well as in water, therefore this increased activity is essentially predictable.8 However, because of the increased activity, metal contamination of concentrated peroxygen chemistries will cause degradation and instability of the formula.
Peracetic acid is another peroxygen compound of great importance in infection control. It is typically formulated with hydrogen peroxide, and likewise has similar stability issues. As with hydrogen peroxide, the formation of the hydroxyl radical is the lethal species. Both peracetic acid and hydrogen peroxide may react with small, acid-soluble proteins to leave the bacterial DNA unprotected and susceptible to other disinfectants, such as chlorine or iodine. The destruction of spores is greatly increased with both a rise in temperature and an increase in concentration. One of the most striking characteristics of peracetic acid in comparison to other disinfectants is the low concentration needed to achieve the desired antimicrobial efficacy.9 Virucidal effects include the alteration of DNA as well as structural alterations.
|Gram-negative bacteria after treatment with a disinfectant. Note the "melted look" after disruption of the cell membrane.|
The free hydroxyl group has been determined to be the reactive components of the phenol molecule. Introduction of the different chemical groups into the nucleus of the phenol molecule modifies this reactivity in different respects. It is these small changes that give the different phenol derivatives their membrane-active properties and also contributes to their varying degrees of activity.10
Phenol and its derivatives exhibit several types of bactericidal action. At higher concentrations, the compounds penetrate and disrupt the cell wall and precipitate cell proteins. Generally, gram-positive bacteria are more sensitive than gram-negative bacteria, which in turn are more sensitive than Mycobacteria. The initial reaction between a phenolic derivative and bacteria involves binding of the active phenol species to the cell surface.11 Once the active has bound to the exterior of the cell, it needs to penetrate to its target sites--either by passive diffusion (gram-positive) or by the hydrophobic lipid bilayer pathway (gram-negative).12 One of the initial events to occur at the cytoplasmic membrane is the inhibition of membrane bound enzymes. The next level in the damage to the cytoplasmic membrane is the loss in the membrane's ability to act as a permeability barrier. There is limited information regarding the action of phenolics against viruses. The molecular mechanisms probably do not differ from those that occur in bacteria. Phenols act at the germination stage of bacterial spore development; however, this effect is reversible--therefore the sporicidal activity of phenolic compounds is low. As with many disinfectants, the activity of phenols is highly formulation dependant and affected by factors such as temperature, concentration, pH and the presence of organic matter.
Quaternary Ammonium Compounds
Although the mode of action of quaternary ammonium compounds has not yet been completely described in detail, there are definitive explanations of the antimicrobial mode of action of cationic disinfectants in general.
One of the main considerations in examining the mode of action is the characterization of quaternary ammonium compounds as cationic surfactants. This class of chemical reduces the surface tension at interfaces, and is attracted to negatively charged surfaces, including microorganisms. Quaternary ammonium compounds denature the proteins of the bacterial or fungal cell, affect the metabolic reactions of the cell and allow vital substances to leak out of the cell, finally causing death.13
Classification of the "generation" of quaternary ammonium compounds can be confusing. The most current definitions of the different generations of quaternary ammonium compounds are as follows:
- First Generation: Benzalkonium chlorides (example: Benzalkonium chloride). First generation quats have the lowest relative biocidal activity are commonly used as preservatives.
- Second Generation: Substituted benzalkonium chlorides (example: alkyl dimethyl benzyl ammonium chloride). The substitution of the aromatic ring hydrogen with chlorine, methyl and ethyl groups resulted in this second generation quat with high biocidal activity.
- Third Generation: "Dual Quats" (example: contain an equal mixture of alkyl dimethyl benzyl ammonium chloride + alkyl dimethyl ethylbenzyl ammonium chloride). This mixture of two specific quats resulted in a dual quat offering increased biocidal activity, stronger detergency, and increased safety to the user (relative lower toxicity).
- Fourth Generation: "Twin or Dual Chain Quats" - dialkylmethyl amines (example: didecyl dimethyl ammonium chloride or dioctyl dimethyl ammonium chloride) Fourth generation quats are superior in germicidal performance, lower foaming, and have an increased tolerance to protein loads and hard water.
- Fifth Generation: Mixtures of fourth generation quats with second-generation quats (example: didecyl dimethyl ammonium chloride + alkyl dimethyl benzyl ammonium chloride) Fifth generation quats have an outstanding germicidal performance, they are active under more hostile conditions and are safer to use.
This information is general in principle. For example, it may not always be the case that a disinfectant with a fifth-generation quat is better than one with a third-generation quat. The non-germicide components of a disinfectant also have an impact on overall performance, and are discussed at the end of this article. Quats are extremely sensitive to hard water, and usually require a chelant in the formula to obtain efficacy in these conditions. Although regarded as standard by one authority, the quat generation definitions given above may differ from those found elsewhere. Regardless, the examples given should give one a relative understanding of the evolution of quaternary germicides.13
Glutaraldehyde-protein interactions indicate an effect of the dialdehyde on the surface of bacterial cells. Many of the studies indicate a powerful binding of the aldehyde to the outer cell layers. Because of this reaction in the outer structures of the cell, there is an inhibitory effect on RNA, DNA, and protein synthesis as a result.
In reacting with bacterial spores, studies have shown that acid glutaraldehyde could interact at the spores surface and remain there, whereas alkaline glutaraldehyde could penetrate the spore. Thus, the role of the activator: an alkalinizing agent in facilitating penetration and interaction of glutaraldehyde with components of the spore cortex or core.14 Inhibition of germination, spore swelling, mycelial growth, and sporulation in fungal species at varying concentrations has been demonstrated. The principal structural wall component of many molds and yeast is chitin, which resembles the peptidoglycan of bacteria and is thus a potentially reactive site for glutaraldehyde action. In viruses, the main targets for glutaraldehyde are nucleic acid, proteins, and envelope constituents. The established reactivity of glutaraldehyde with proteins suggests that the viral capsid or viral-specific enzymes are vulnerable to glutaraldehyde treatment.
Ortho-phthalaldehyde is a claimed alternative aldehyde that is currently under investigation.15 Unlike glutaraldehyde, ortho-phthalaldehyde is odorless, stable, and effective over a wide pH range. It has been proposed that, because of the lack of alpha-hydrogens, ortho-phthalaldehyde remains in its active form at alkaline pH.
Synergy with Other Formula Components
Surfactants are often important constituents of disinfectants. They are used to achieve both uniform wetting of the surface to be treated and frequently for the additional cleaning effect they provide. Particular attention should be given to this group of substances when formulating a disinfectant because there are many ways in which the two groups of compounds can interact. It is generally known that anionic surfactants promote the inactivation of cationic antimicrobials.17 Nonionics can also impair the effectiveness of antimicrobial substances by binding with the antimicrobial, therefore inactivating it.
In contrast, low surfactant concentrations may improve the microbiocidal effect. The reason for the improved action is thought to be an accumulation of the agent within micelles of the surfactant, which absorb to the microorganism's cell wall. The active substance thus becomes enriched at the cell wall, which means that a lower dose is required for the desired effect.
EDTA and other chelating agents are often added to the germicide formula to aid in activity in hard water conditions. These ingredients also add to the antimicrobial activity by chelating magnesium and calcium in the organism. EDTA has been shown to boost the effect of antimicrobial activity against gram-negative organisms such as Pseudomonas aeruginosa.18
Kirsten M. Buck is a principal technical specialist and field test coordinator for Ecolab's Professional Products Division in Mendota Heights, Minn.