Infection Control Today - 09/2001: The Effects of Germicides onMicroorganisms

The Effects of Germicides on Microorganisms

By Kirsten M. Buck

Healthcare workers (HCWs) often take for granted the action of disinfectantswithout fully understanding their mechanism of action. Causing microbial celldeath, in reality, is a complicated process. Not only are there differences inthe action of the antimicrobial ingredients, but there are differences dependingon the concentration of chemical that is used. It is not easy to determine theexact mechanism of action of a chemical agent or physical process. The reason isthat more than one part of the microbial cell may be affected and consequently,the problem is to distinguish which of these effects ultimately contributed tothe cell death. Information as to the cellular target can be obtained in variousways. Biocide treatment of cells under conditions of growth or non-growthindicates whether the test substance inhibits some biosynthetic process, inwhich case non-growing cells are unaffected. Further preliminary experiments canthen be carried out to determine whether the agent inhibits synthesis of thecell wall or nucleic acids. Other useful experiments include studies on thepossible leakage of low-molecular weight materials, monitored by an experimentaltechnique to study proton flux across the cytoplasmic membrane.¹

Chlorine

Chlorine in an aqueous solution, even in very small amounts, exhibits fastbactericidal action. The mechanism of this activity has not been fullydetermined, despite much research. When chlorine is added to water, it formshypochlorous acid. Exactly how hypochlorous acid destroys microorganisms hasnever been demonstrated experimentally, but it has been speculated thathypochlorous acid allows oxygen to emerge, which in turn supposedly combineswith components of cell protoplasm, destroying the organism. Researchers haveassumed that because of the low chlorine level required for bactericidal action,chlorine must inhibit some key enzymatic reactions in the cell. The inhibitionof these essential cytoplasmic metabolic reactions is largely responsible forthe destruction of both bacterial and fungal cells. Very few chemicals areconsidered sporicidal; however, bacterial spores are affected by disinfectantsat different stages in the sporulation process. While not considered sporicidal,chlorine compounds have demonstrated some activity at the outgrowth stage, buthigher concentrations also may prevent germination. Chlorine compounds have beenshown to affect surface antigen in enveloped viruses and DNA as well asstructural alterations in non-enveloped viruses.

Iodine

Iodine, mainly in its molecular form (I²), can penetrate the cellwall of microorganisms rapidly. The actual killing of the microorganism byiodine could be the result of the inability to synthesize proteins due tooxidation in an important amino acid,² the increasing of the bulk ofthe amino acid molecules which leads to the denaturation of DNA,³ orthe addition of iodine to unsaturated fatty acids could to lead to a change inthe physical properties of the lipids.⁴ Electron microscopyobservations support the conclusion that iodine, by interacting with the doublebonds of phospholipids causes damage of the cell wall which lead to a loss ofintracellular material. Halogens such as chlorine and iodine react not only withliving microorganisms but also with dead ones and with dissolved proteins. Incontrast to chlorine, where oxidizing and bactericidal N-chloro compoundsemerge, with iodine the efficacy is diminished because N-iodo compounds are notformed.⁵

Alcohol

Like many disinfectants, alcohols are generally considered to be non-specificantimicrobials because of their many toxic effects. The predominant mode ofaction appears to stem from protein coagulation/denaturation. Disruption of thecytoplasmic membrane, cell lysis, and interference with cellular metabolism hasbeen reported. Protein coagulation occurs within concentration limits around anoptimum alcohol level. In the absence of water, proteins are not denatured asreadily as when water is present. Therefore, mixtures of alcohol with waterexhibit much better efficacy than straight alcohol alone. Alcohol-inducedcoagulation of proteins occurs at the cell wall, the cytoplasmic membrane andamong the various plasma proteins. Alcohols target the bacterial cell wall, withresultant lysis of the cytoplasmic membrane and release of cellular contents.The antifungal action of alcohol is very similar, resulting in the attachment tothe plasma membrane and leakage of cell contents. The inhibition of sporegermination by ethanol and other alcohols may be due to the inhibition ofenzymes necessary for germination. This inhibition is reversible because onlythe removal of alcohol from the environment is necessary for germination to takeplace; therefore, alcohols are not appropriate as chemical sterilants.⁶

Peroxygen Compounds

Hydrogen peroxide is effective against a wide variety of organisms: bacteria,yeast, fungi, viruses, and spores. Anaerobes are even more sensitive becausethey do not produce catalase to break down the peroxide. In general, hydrogenperoxide has a greater activity against gram-negative than gram-positivebacteria. Unlike like most disinfectants, hydrogen peroxide is unaffected by theaddition of organic matter and salts.⁷

Hydrogen peroxide, the superoxide ion radical, and the hydroxyl radical areintermediate products in the reduction of oxygen to water. The hydroxyl radicalis said to be the strongest oxidant known, and it is by this mechanism thathydrogen peroxide is believed to do the actual killing of bacteria. The hydroxylradical, being highly reactive, can attack membrane lipids, DNA, and otheressential cell components. Transition metals are believed to catalyze theformation of the hydroxyl radical, therefore the addition of iron, copper,cobalt, chromium, or manganese increases the efficacy of hydrogen peroxide. Itis important to note that many metal ions are inherent in the microbial cell aswell as in water, therefore this increased activity is essentially predictable.⁸However, because of the increased activity, metal contamination of concentratedperoxygen chemistries will cause degradation and instability of the formula.

Peracetic acid is another peroxygen compound of great importance in infectioncontrol. It is typically formulated with hydrogen peroxide, and likewise hassimilar stability issues. As with hydrogen peroxide, the formation of thehydroxyl radical is the lethal species. Both peracetic acid and hydrogenperoxide may react with small, acid-soluble proteins to leave the bacterial DNAunprotected and susceptible to other disinfectants, such as chlorine or iodine.The destruction of spores is greatly increased with both a rise in temperatureand an increase in concentration. One of the most striking characteristics ofperacetic acid in comparison to other disinfectants is the low concentrationneeded to achieve the desired antimicrobial efficacy.⁹ Virucidaleffects include the alteration of DNA as well as structural alterations.

Phenol

The free hydroxyl group has been determined to be the reactive components ofthe phenol molecule. Introduction of the different chemical groups into thenucleus of the phenol molecule modifies this reactivity in different respects.It is these small changes that give the different phenol derivatives theirmembrane-active properties and also contributes to their varying degrees ofactivity.¹⁰

Phenol and its derivatives exhibit several types of bactericidal action. Athigher concentrations, the compounds penetrate and disrupt the cell wall andprecipitate cell proteins. Generally, gram-positive bacteria are more sensitivethan gram-negative bacteria, which in turn are more sensitive than Mycobacteria.The initial reaction between a phenolic derivative and bacteria involves bindingof the active phenol species to the cell surface.¹¹ Once the activehas bound to the exterior of the cell, it needs to penetrate to its targetsites--either by passive diffusion (gram-positive) or by the hydrophobic lipidbilayer pathway (gram-negative).¹² One of the initial events to occurat the cytoplasmic membrane is the inhibition of membrane bound enzymes. Thenext level in the damage to the cytoplasmic membrane is the loss in themembrane's ability to act as a permeability barrier. There is limitedinformation regarding the action of phenolics against viruses. The molecularmechanisms probably do not differ from those that occur in bacteria. Phenols actat the germination stage of bacterial spore development; however, this effect isreversible--therefore the sporicidal activity of phenolic compounds is low. Aswith many disinfectants, the activity of phenols is highly formulation dependantand affected by factors such as temperature, concentration, pH and the presenceof organic matter.

Quaternary Ammonium Compounds

Although the mode of action of quaternary ammonium compounds has not yet beencompletely described in detail, there are definitive explanations of theantimicrobial mode of action of cationic disinfectants in general.

One of the main considerations in examining the mode of action is thecharacterization of quaternary ammonium compounds as cationic surfactants. Thisclass of chemical reduces the surface tension at interfaces, and is attracted tonegatively charged surfaces, including microorganisms. Quaternary ammoniumcompounds denature the proteins of the bacterial or fungal cell, affect themetabolic reactions of the cell and allow vital substances to leak out of thecell, finally causing death.¹³

Classification of the "generation" of quaternary ammonium compoundscan be confusing. The most current definitions of the different generations ofquaternary 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 bethe case that a disinfectant with a fifth-generation quat is better than onewith a third-generation quat. The non-germicide components of a disinfectantalso have an impact on overall performance, and are discussed at the end of thisarticle. Quats are extremely sensitive to hard water, and usually require achelant in the formula to obtain efficacy in these conditions. Although regardedas standard by one authority, the quat generation definitions given above maydiffer from those found elsewhere. Regardless, the examples given should giveone a relative understanding of the evolution of quaternary germicides.¹³

Aldehydes

Glutaraldehyde-protein interactions indicate an effect of the dialdehyde onthe surface of bacterial cells. Many of the studies indicate a powerful bindingof the aldehyde to the outer cell layers. Because of this reaction in the outerstructures of the cell, there is an inhibitory effect on RNA, DNA, and proteinsynthesis as a result.

In reacting with bacterial spores, studies have shown that acidglutaraldehyde could interact at the spores surface and remain there, whereasalkaline glutaraldehyde could penetrate the spore. Thus, the role of theactivator: an alkalinizing agent in facilitating penetration and interaction ofglutaraldehyde with components of the spore cortex or core.¹⁴Inhibition of germination, spore swelling, mycelial growth, and sporulation infungal species at varying concentrations has been demonstrated. The principalstructural wall component of many molds and yeast is chitin, which resembles thepeptidoglycan of bacteria and is thus a potentially reactive site forglutaraldehyde action. In viruses, the main targets for glutaraldehyde arenucleic acid, proteins, and envelope constituents. The established reactivity ofglutaraldehyde with proteins suggests that the viral capsid or viral-specificenzymes are vulnerable to glutaraldehyde treatment.

Ortho-phthalaldehyde is a claimed alternative aldehyde that is currentlyunder investigation.¹⁵ Unlike glutaraldehyde, ortho-phthalaldehyde isodorless, stable, and effective over a wide pH range. It has been proposed that,because of the lack of alpha-hydrogens, ortho-phthalaldehyde remains in itsactive form at alkaline pH.

Synergy with Other Formula Components

Surfactants are often important constituents of disinfectants. They are usedto achieve both uniform wetting of the surface to be treated and frequently forthe additional cleaning effect they provide. Particular attention should begiven to this group of substances when formulating a disinfectant because thereare many ways in which the two groups of compounds can interact. It is generallyknown that anionic surfactants promote the inactivation of cationicantimicrobials.¹⁷ Nonionics can also impair the effectiveness ofantimicrobial substances by binding with the antimicrobial, thereforeinactivating it.

In contrast, low surfactant concentrations may improve the microbiocidaleffect. The reason for the improved action is thought to be an accumulation ofthe agent within micelles of the surfactant, which absorb to the microorganism'scell wall. The active substance thus becomes enriched at the cell wall, whichmeans that a lower dose is required for the desired effect.

EDTA and other chelating agents are often added to the germicide formula toaid in activity in hard water conditions. These ingredients also add to theantimicrobial activity by chelating magnesium and calcium in the organism. EDTAhas been shown to boost the effect of antimicrobial activity againstgram-negative organisms such as Pseudomonas aeruginosa.¹⁸

Kirsten M. Buck is a principal technical specialist and field testcoordinator for Ecolab's Professional Products Division in Mendota Heights,Minn.