PHARMACEUTICAL MICROBIOLOGY - V. A. Galynkin - 2015

PART II. ANTIMICROBIAL AGENTS

CHAPTER 18. MECHANISM OF ACTION OF DISINFECTANTS AND ANTISEPTICS

18.1 Action of antiseptics on the microbial Cell

The Effect of antiseptics on microbial Cells is non-specific, meaning their molecular targets may also be present in mammalian cells. The sites of antiseptic action are diverse and located within The Cell wall, membranes, and Cytoplasm.

At low concentrations, antiseptics induce microbial cell lysis, likely by interacting with Enzymes involved in cell wall synthesis, thereby altering their function and disintegrating the wall. Cell wall lysis in E. coli, staphylococci, and streptococci has been observed in the presence of the following agents (concentrations given in %): formalin 0.12, phenol 0.32, mercuric chloride 0.0008, sodium hypochlorite 0.005, and merthiolate 0.0004. Glutaraldehyde disrupts The Cell wall Structure of Gram-positive Bacteria, inducing irreversible cross-linking within it.

Effects on the cytoplasmic membrane involve the disruption of Membrane Potential, membrane-bound enzymes, and permeability. Compromising the membrane potential uncouples electron transport from phosphorylation and hinders proton translocation across the membrane, halting energy-generating processes required for ATP synthesis. Similar phenomena are observed in the presence of compounds such as tetrachlorosalicylanilide, tricarbanilide, trichlorocarbanilide, pentachlorophenol, and 2-phenoxyethanol.

The inhibition of membrane-associated enzymes disrupts numerous metabolic pathways. Hexachlorophene suppresses The activity of Electron Transport Chain enzymes, thereby inhibiting the METABOLIC ACTIVITY OF aerobic bacteria. Chlorhexidine inhibits membrane-bound ATPase, thereby interfering with anaerobic processes. Antiseptics containing mercury, as well as bronopol and other agents, inhibit enzymes containing thiol groups (-SH). In the presence of an excess of thiol-bearing compounds (such as Cysteine or thioglycolates), these antiseptics lose their activity.

Many antiseptics (such as quaternary ammonium compounds, phenol, and hexylresorcinol) impair membrane permeability, leading to the leakage of cytoplasmic contents. The cell loses potassium, Purines, Pyrimidines, sugars, and other metabolites. If exposure to the antiseptic is brief, only a bacteriostatic effect is observed.

The cytoplasm represents a complex, multi-component system of molecules and subcellular structures, each of which may be affected by antiseptics to varying degrees. High concentrations of biocides—such as chlorhexidine, phenol, and mercury salts—cause general cytoplasmic coagulation. Hydrogen peroxide and p-chloromercuribenzoate induce the dissociation of Ribosomes into subunits. Acridine Dyes are capable of intercalating into the Introduction/20.html">DNA Structure, thereby disrupting its normal Functions. Many antiseptics interact with protein thiol groups; for instance, halogens can oxidize them. Formaldehyde, glutaraldehyde, and sulfur dioxide react with amino groups. Highly reactive agents affect multiple cellular systems. For example, β-propiolactone alkylates amino, imino, hydroxyl, and carboxyl groups, and interacts with thiol and disulfide groups, disrupting The structure of Proteins and other macromolecules. Ethylene oxide exhibits similar activity. Sulfur dioxide, sulfites, and bisulfites are also characterized by high reactivity.

Table 36 presents data on the cellular targets affected by various antiseptics.

Class="center">Table 36. Action of antiseptics on cellular targets

Note: + Antiseptic is active at low concentrations; +++ Antiseptic is active at high concentrations

18.2 RESISTANCE OF MICROORGANISMS to Antiseptics and disinfectants

Microorganisms vary significantly in their resistance to biocides (Table 37). Similar to Antibiotics, both intrinsic (natural) and acquired resistance to antiseptics and disinfectants are distinguished.

Table 37. Susceptibility of microorganisms to chlorhexidine

Intrinsic resistance is associated with the natural structural and metabolic Features of the microbial cell, such as protective surface layers, biofilm formation, enzymatic degradation, or the active efflux of xenobiotics from the cell. All types of Surfactants and other disinfectants are subject to microbial degradation at sub-inhibitory concentrations, and occasionally even at working concentrations; for example, Pseudomonas aeruginosa utilizes benzalkonium chloride and other surfactants as a carbon source. This microorganism is most frequently isolated from disinfectant solutions alongside representatives of other genera (Table 38).

Table 38. Microorganisms most frequently isolated from disinfectants

18.3 Permeability of cell envelopes and resistance

The cell wall permeability of Gram-negative bacteria is largely determined by the presence of an outer membrane that protects the cell against the penetration of chemical agents. However, surfactants—particularly quaternary ammonium compounds—disrupt the lipopolysaccharide layer of the outer membrane and penetrate the cell interior. Electron Microscopy reveals that cells treated with quaternary ammonium compounds develop surface blebs that increase in size and pinch off as vesicles composed of lipopolysaccharides, proteins, and Phospholipids. Partial compensation for outer membrane damage is possible through phospholipids forming a bilayer. Nevertheless, if the damage is too extensive, the cell loses its outer permeability barrier, and the biocide enters the cell, causing irreversible alterations to structures and metabolites.

Changes in the outer Membrane Structure can affect its permeability and the overall resistance of the cell to biocides. A prime example is Pseudomonas aeruginosa, which exhibits superior resistance compared to other Gram-negative bacteria. A potential reason for this is the elevated content of phosphate groups in lipid A, which is characteristic of this microorganism.

A key feature of Gram-negative bacteria that determines their biocide resistance is their ability to adhere to surfaces and form biofilms—organized communities of cells enmeshed in an exopolysaccharide matrix (glycocalyx). The outer layers of the glycocalyx protect the interior from biocide penetration. Cells residing within a biofilm experience nutrient limitation and grow slowly. These factors contribute to enhanced resistance against unfavorable environmental conditions.

Gram-positive bacteria are generally more susceptible to biocides, although resistant strains also emerge within this group. For instance, the resistance of Staphylococcus aureus to phenols and quaternary ammonium compounds (QACs) depends on the presence of surface Lipids, which protect the microorganism from biocide penetration.

Spores can withstand biocide concentrations several thousand times higher than those effective against vegetative cells. Mercury compounds, QACs, chlorhexidine, phenols, and alcohols exhibit virtually no sporocidal activity, although they may inhibit spore germination. Ethylene oxide, β-propiolactone, formaldehyde, glutaraldehyde, hydrogen peroxide, and halogens kill spores, but their action is relatively slow, requiring a sterilization process lasting from 30 minutes to several hours. Spore resistance is conferred by a unique cell wall that prevents biocides from entering the cell and possibly neutralizes the action of some of them. Spores of different microorganisms vary in their sensitivity to sterilizing agents. In addition to genotypic Variability, there is also a phenotypic dependence of spore resistance on the cultivation conditions of the microorganism.

Mycobacteria are highly resistant to disinfectants (phenols being the most effective); whenever possible, heat Treatment should be used to eradicate them. Their protective properties are attributed to the cell wall, which contains a high concentration of wax-like lipids forming hydrophobic layers. Mycolic acids play a crucial role among these lipids. The cell wall provides acid-fastness, which serves as the basis for their differential staining using carbol fuchsin with heating. At room Temperature, this process requires an 18-hour exposure. Stained cells are resistant to decolorization by alcohol and dilute acids, which is the Water/144.html">Origin of the term "acid-fastness."

Acquired resistance arises from changes in the bacterial genetic apparatus through the Selection of resistant mutants in a biocide-containing environment. Horizontal Gene Transfer between different species and genera of bacteria via transmissible Plasmids and conjugative Transposons plays a major role in the dissemination of resistance genes. Plasmids can confer multiple resistance to biocids. In Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, plasmids conferring resistance to mercury compounds have been described. Narrow-spectrum plasmids control The production of a reductase that converts Hg⁺⁺ into metallic mercury. Broad-spectrum plasmids, in addition to the reductase, encode one or more Hydrolases that release Hg⁺⁺ from organomercury compounds by cleaving the mercury-carbon bond. The resulting metallic mercury evaporates from the medium. The existence of such transformation processes for organomercury compounds makes their use as pharmaceutical preservatives problematic.

The broad host range of resistance genes facilitates their persistence in nature, and such genes can remain stable even in the absence of selective pressure, i.e., in an environment free of biocides. Selective conditions are created in disinfectant solutions with concentrations below the recommended levels and when their storage guidelines are violated. For example, chlorine-containing solutions frequently exhibit a decrease in active chlorine content.

In addition to biocide concentration, population resistance is influenced by medium composition (presence of protective agents, growth factors), developmental phase, and Cell Division rate. For instance, Pseudomonas aeruginosa grown in a magnesium-deficient medium is highly resistant to benzalkonium chloride, whereas cells grown under carbon limitation are highly sensitive. Temperature and cultivation time are also of great importance. Slow-growing cells are less sensitive to biocides than rapidly growing ones. Therefore, standard testing conditions for antimicrobial activity must be strictly observed.

The potential for rapid development of population resistance must be taken into account in practical disinfection Applications. With prolonged use of a particular antimicrobial agent, the resident microbiota of a given facility (hospital, pharmacy, production floor, laboratory, surrounding equipment, walls, and floors, etc.) may acquire high resistance to that agent. To ensure effective aseptic working conditions, biocide rotation is implemented, meaning that several chemical substances are used in a defined sequence.