Mechanisms of Action

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Chapter: Pharmaceutical Microbiology : Non-Antibiotic Antimicrobial Agents: Mode Of Action And Resistance

In any consideration of mechanism of action, due regard should be given to the initial health of the organism, duration of contact with the biocide, and the concentration of biocide employed.


MECHANISMS OF ACTION

 

In any consideration of mechanism of action, due regard should be given to the initial health of the organism, duration of contact with the biocide, and the concentration of biocide employed. Antibacterial effects may progress from early, sublethal events to multiple lesions of bactericidal consequence. Figure 20.2 identifies events in order of severity, but should not be interpreted as defining the normal progression of cell injury. As disclosed in the following sections, the biocide interaction may induce particular lesions over others; this will most certainly be in a concentration-dependant manner


 

a)   Oxidation Reactions

 

Biocides with oxidizing (electron-withdrawing) ability are widely used as disinfectants and chemical sterilants, and include the halogens (chlorine, hypochlorites, bromine, iodophors) and peroxygens (hydrogen peroxide, peracetic acid and chlorine dioxide). They can exert specific effects on essential microbial macromolecules causing, variously: strand breakage and adduct formation on DNA and RNA with disruption of replication, transcription and and translocation processes; degradation of, particularly, unsaturated fatty acids leading to loss of membrane fluidity and subsequent reduced functionality of membrane-bound proteins; and specific modifications to amino acid residues, most notably disulphide bonds, leading to changes in protein primary structure and con-formation with consequent disruption of structural enzymic functions. An accumulation of these effects can be particularly devastating to the microbial cell.

 

b)    Cross-Linking Reactions

 

The aldehydes formaldehyde, glutaraldehyde and ortho-phthalaldehyde, and the sterilant alkylating agents ethylene oxide and propylene oxide, are both highly reactive chemical classes. The alkylating agents exhibit particularly strong reactions with guanine residues causing cross-linking between DNA strands, inhibiting DNA unwinding and RNA translation. The amino, carboxyl, sulphydryl and hydroxyl groups of structural or enzymic proteins are also susceptible to alkylation, causing cross-links between adjacent amino acid chains and also with other amino acid-containing structures such as peptidoglycan. The aldehydes are generally more specific with greatest effect against the amino groups of surface exposed lysine or hydroxylysine residues of proteins, again causing extensive cross-linking.

 

In all instances, progressive cross-linking leads to macromolecule malfunction causing inhibition or arrest of essential cell functions. It is safe to say that there is no single fatal reaction but that death results from the accumulated effect of many reactions in a manner similar to oxidizing agents.

 

c)     Coagulation

 

The cross-linking reactions give rise to macromolecule denaturation which can be recognized under electron microscopy as intracellular coagulation. Coagulative effects are not unique to aldehydes and alkylating agents, however, and high concentrations of disinfectants such as chlorhexidine, phenol, ethanol and mercuric salts will also coagulate the cytoplasm. This most likely arises from the precipitation of protein caused by a variety of interactions including ionic and hydrophobic bonding and the disruption of hydrogen bonds.

 

d)   Disruption Of Functional Structures 

The integrity and functions of the bacterial cell are dependant upon critical macromolecular structural arrangements including within the cell wall and cytoplasmic membrane . A number of biocides can have a profound effect on these organelles.

 

          i)   Cell wall

 

This structure is the traditional target for a group of antibiotics which includes the penicillins, but a little-noticed report which appeared in 1948 showed that low concentrations of disinfectant substances caused cell wall lysis such that a normally turbid suspension of bacteria became clear. It is thought that these low concentrations of disinfectant cause enzymes whose normal role is to synthesize the cell wall to reverse their role in some way and effect its disruption or lysis. In the original report, these low concentrations of disinfectants (formalin, 0.12%; phenol, 0.32%; mercuric chloride, 0.0008%; sodium hypochlorite, 0.005% and merthiolate, 0.0004%) caused lysis of Escherichia coli, streptococci, and staphylococci.

 

Divalent cations, in addition to their role as enzyme cofactors, also stabilize cell wall, membrane and ribosomal structures. In particular, magnesium serves to link the lipopolysaccharide (LPS) of Gramnegative bacteria to the outer membrane. Chelators, particularly ethylenediamine tetraacetic acid (EDTA), have been used to disrupt this link and cause the release of LPS into the medium. The loss of outer membrane integrity and subsequent permeabilization has been exploited in the potentiation of biocides, including combinations of EDTA with chloroxylenol, cetrimide, phenylethanol and the parahydroxy benzoic acid esters.

 

         ii)  Cytoplasmic membrane

 

The bacterial cytoplasmic membrane consists of an impermeable, negatively-charged, fluid phospholipid bilayer incorporating an organized array of membrane associated proteins. Through the membrane-bound electron transport chain aerobically, or the membrane-bound adenosine triphosphatase (ATPase) anaerobically, the bacterium succeeds in maintaining a transmembrane gradient of electrical potential and pH such that the interior of the cell is negative and alkaline. This proton motive force, as it is called, drives a number of energy-requiring functions which include the synthesis of ATP, the coupling of oxidative processes to phosphorylation, a metabolic sequence called oxidative phosphorylation, and the transport and concentration in the cell of metabolites such as sugars and amino acids. This, put briefly, is the basis of the chemiosmotic theory linking metabolism to energy-requiring processes.

 

Certain chemical substances have been known for many years to uncouple oxidation from phosphorylation and to inhibit active transport, and for this reason they are named uncoupling agents. They are believed to act by partitioning into the membrane and rendering it permeable to protons, hence short-circuiting the potential gradient or protonmotive force. Some examples of antibacterial agents which owe at least a part of their activity to this ability are tetra-chloro-salicylanilide (TCS), tricarbanilide, trichlorocarbanilide (TCC), pentachlorophenol, di-(5-chloro-2-hydroxyphenyl) sulphide (fentichlor), 2-phenoxyethanol, and lipophilic acids and esters.

 

The membrane, as well as providing a dynamic link between metabolim and transport, serves to maintain the pool of metabolites within the cytoplasm. A general increase in membrane permeability brought about by the association and likely insertion of biocide molecules into the lipid bilayer was recognized early as being one effect of many disinfectant substances.

 

Treatment of bacterial cells with appropriate concentrations of such substances as cetrimide and other QACs, chlorhexidine, polyhexamethylene biguanides, phenol and hexylresorcinol causes a leakage of a group of characteristic chemical species. The potassium ion, being a small entity, is the first substance to appear when the cytoplasmic membrane is damaged. Amino acids, purines, pyrimidines and pentoses are examples of other substances which will leak from treated cells. If the action of the drug is not prolonged or exerted only in low concentration, the damage may be reversible and leakage may only induce bacteriostasis. There is however, evidence that a depletion of intracellular potassium caused by membrane damage can lead to the activation of latent ribonucleases and the consequent breakdown of RNA. Several biocides, including cetrimide and some phenols, are known to cause the release of nucleotides and nucleosides following an autolytic process. This is irreversible and has been proposed as an autocidal (suicide) process, committing the injured cell to death (Denyer & Stewart, 1998).

 

Surface-associated proteins within the membrane fulfil a number of important roles including wall biosynthesis, nutrient transport and respiration. Usually enzymes, these macromolecules are often topologically organized and uniquely exposed to disruption by biocidal agents. Thus, hexachlorophane inhibits the electron transport chain in bacteria, chlorhexidine has been shown to inhibit ATPase, and thiol-containing membrane dehydrogenases are highly susceptible to mercury-containing antibacterials, silver,2-bromo-2-nitropropan-1,3-diol (bronopol) and isothiazolinones.

 

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