PHARMACEUTICAL MICROBIOLOGY - V. A. Galynkin - 2015

PART II. ANTIMICROBIAL AGENTS

CHAPTER 16. DISINFECTANTS, ANTISEPTICS, AND PRESERVATIVES

16.1 Classification and Terminology of Antimicrobial Agents

Disinfectants, antiseptics, and preservatives are chemical agents capable of killing microbial Cells or inhibiting their growth [29].

Disinfectants are used to treat inanimate surfaces, equipment, or Materials.

Antiseptics are applied to human Skin and mucous membranes; therefore, they must not be toxic at the concentrations used [30].

Preservatives are incorporated into pharmaceutical formulations to prevent microbial degradation and maintain the microbial burden at a low, safe level. The effective concentration of a preservative in a finished medicinal product must be significantly lower than the toxic dose for humans.

16.2 Factors Determining the Choice of an Antimicrobial Agent

The Selection of an antimicrobial agent is determined by the PHYSICOCHEMICAL PROPERTIES OF the chemical substance, The Nature of the microbiota, and environmental factors.

Properties of the chemical substance. The biocidal efficacy depends on its chemical Structure, concentration, Temperature, pH, and contact time with the contaminated object. When a substance is used as an antiseptic, its toxicity must also be taken into account.

Nature of the microbiota. The microbial profile directly influences the effectiveness of a chemical agent. Key factors include the susceptibility of the microorganism to the given substance and the initial level of microbial contamination. In practice, it is not always possible to identify all microorganisms present on a surface being disinfected; therefore, the efficacy of an antimicrobial agent is evaluated against the most resistant species.

Vegetative bacterial forms. At working concentrations, all disinfectants must eradicate vegetative Bacteria within a specified exposure time. Gram-negative bacteria are generally more resilient than Gram-positive ones. Pseudomonas aeruginosa is particularly resistant due to lipid A, a characteristic component of its outer membrane that acts as a barrier against the penetration of many chemical substances.

Mycobacterium tuberculosis. The tubercle bacillus and other acid-fast microorganisms exhibit high resistance to numerous bactericidal agents. Tuberculosis remains a major public health concern. The risk of transmission from undiagnosed patients is substantial. Because the causative agent may contaminate diagnostic equipment, reliable disinfection is essential.

Bacterial spores are the most resistant to disinfectants. Certain aldehydes, hypochlorites, and hydrogen peroxide are active against them. These substances can be utilized for the chemical sterilization of thermolabile equipment.

The efficacy of disinfectants is assessed by their ability to neutralize the most resistant forms of microorganisms (Table 26).

Table 26. Antibacterial activity of selected chemical substances

Fungi are comparable to vegetative bacteria in their susceptibility to disinfectants. Table 27 presents data on the efficacy of certain substances against pathogenic fungi (Trichophyton mentagrophytes, Candida albicans) and the mold representative Aspergillus niger.

Table 27. Antifungal activity of selected disinfectants and antiseptics

Viruses. The susceptibility of viruses to biocides is difficult to evaluate due to the specific conditions required for their cultivation, as tissue cultures can also be damaged by chemical agents. As a rule, enveloped viruses are more susceptible than non-enveloped viruses (Table 28).

Table 28. Antiviral activity* of selected disinfectants and antiseptics

*Viral inactivation after 10–30 min of contact at 21–25°C, titer reduction of 10^3

**No data

16.4 Environmental Factors.

Organic matter (blood, pus, milk, food residues, etc.) significantly reduces the efficacy of biocidal agents through adsorption and inactivation, or by preventing their penetration into microbial cells. Therefore, whenever possible, equipment, glassware, and instruments should be thoroughly washed prior to disinfection.

Many materials (fabrics, rubber, and other polymers) can adsorb biocides, thereby decreasing their concentration. Biocidal activity [31] requires the Presence of Water to ensure Cell penetration and depends on the ion content of the medium. Some Factors influencing the activity of the most common biocides are summarized in Tables 29 and 30.

Table 29. Characteristics of Certain Disinfectants and Antiseptics

Table 30. Main Properties of Various Groups of Microbiologically Active Substances

Notes: (++) — very pronounced; (+) — moderately pronounced; (+-) — weakly pronounced; (-) — absent.

16.5 Groups of Chemical Compounds of Disinfectants (Fig. 69)

16.5.1 Phenols

Phenols were among the first compounds to be used as disinfectants and preservatives. They rapidly kill bacteria, though not their spores. Their activity decreases markedly upon dilution and in the presence of organic matter, and they are more active at acidic pH values. Their primary drawback is toxicity. Substituted phenols are less toxic but also less active than simple phenols, particularly against gram-negative bacteria.

Phenol (carbolic acid) is currently used to a limited extent. It was first introduced by Lister in 1867 as an antiseptic and is used as a standard for evaluating other disinfectants in the phenol coefficient test.

Synthetic phenols. Modification of the phenol molecule (Fig. 70) yields derivatives with improved properties. For instance, chlorocresol is used at a concentration of 0.1% for preserving injection solutions and at 0.2% during thermal sterilization. Chloroxylenol is used for skin disinfection in formulations containing soap and terpineol to enhance water solubility, exhibiting relatively weak antimicrobial activity; 2-phenylphenol serves as a cosmetic preservative; and resorcinol is used as an antiseptic.

All disinfectants are classified into the following groups: halogen-containing compounds, oxygen-containing compounds, surfactants, alkalis, acids, guanidines, aldehydes, alcohols, and phenols.

Fig. 69. Classification of disinfectants by chemical composition [32, 33]

Fig. 70. Phenol and its derivatives.

Bisphenol derivatives, such as hexachlorophene and triclosan, are used in medical soaps and cleansing pastes. They exhibit bacteriostatic action, though their effect on Pseudomonas spp. is weak.

16.5.2 Alcohols

Aliphatic alcohols (ethanol, isopropanol) kill vegetative forms of bacteria, including mycobacteria, but not spores. Their cleansing ability and volatility make them useful for skin preparation prior to injections or surgical Procedures. Alcohol derivatives (Fig. 71) are predominantly used as preservatives.

Ethanol (СН₃ СН₂ ОН) is widely used as a preservative and antiseptic, exhibiting bactericidal and virucidal activity at concentrations of 60–95%. A 70% solution is typically applied for the disinfection of skin, instruments, and surfaces. Its combination with iodine and chlorhexidine enhances its biocidal activity. It is widely employed in the pharmaceutical and cosmetic industries as a solvent and preservative.

Fig. 71. Alcohols used as disinfectants and preservatives.

Isopropanol (CH₃CHOHCH₃) exhibits greater activity than ethanol while being twice as toxic. Its effect on viruses is weak. It is used at a 70% concentration for skin antisepsis and as a preservative in cosmetic products.

Benzyl alcohol (C₆H₅CH₂OH) possesses antibacterial and mild local anesthetic properties. At a 1% concentration, it is recommended for preserving injectable solutions.

Chlorbutol (trichlorobutanol) is used as a preservative for injectable solutions and eye drops at a concentration of 0.5%. It crystallizes at low temperatures, degrades at low pH during autoclaving, and at alkaline pH under room temperature conditions.

Phenylethanol (C₆H₅CH₂CH₂OH) is active against Gram-negative bacteria and is typically used as a preservative in combination with other biocides.

Phenoxyethanol (C₆H₅OCH₂CH₂OH) is active against Pseudomonas aeruginosa. It is used as a preservative at a 1% concentration, usually in combination with other biocides.

Bronopol (2-bromo-2-nitropropane-1,3-diol) exhibits a broad spectrum of antibacterial activity, including against pseudomonads, and is widely used as a preservative in pharmaceutical and cosmetic formulations at concentrations of 0.01–0.02%. It is readily soluble in water and remains active over a wide pH range and in the presence of surfactants. However, at alkaline pH and elevated temperatures, it degrades to form formaldehyde and nitrites; the latter can react with secondary and tertiary amines to yield potentially carcinogenic nitrosamines.

16.5.3 Aldehydes

Many aldehydes possess antimicrobial properties; however, in practice, only glutaraldehyde and formaldehyde are utilized, as their high potency makes them suitable for chemical sterilization.

Glutaraldehyde (CHO(CH₂)₃CHO) destroys vegetative forms of bacteria within a one-minute exposure time. Spore eradication requires 3 hours or more, depending on their thermal resistance. It is not significantly inactivated by Organic compounds. The presence of two highly reactive aldehyde groups in the molecule is of critical importance. The monomer exists in equilibrium with polymeric molecular forms, a balance governed by temperature and medium pH. At pH 8, biocidal activity reaches its peak, but the solution becomes unstable due to polymerization. Conversely, acidic solutions are stable yet less active, although raising the temperature increases the concentration of free dialdehyde and enhances biocidal activity. In practice, glutaraldehyde is supplied as a stable 2% acidic solution, which is activated immediately prior to use by adding an appropriate buffer system. The activated solution has a shelf life of 2 weeks. It is employed for the chemical sterilization of medical devices that cannot be sterilized by other Methods.

Formaldehyde (HCHO) is utilized for disinfection in either liquid or gaseous states. Gaseous formaldehyde is employed for the decontamination of infected facilities, while mixtures with low-temperature steam are used for sterilizing thermolabile materials. Formaldehyde vapors are highly toxic and potentially carcinogenic upon inhalation, necessitating strict SAFETY PRECAUTIONS DURING handling. Formalin (a 34–38% aqueous solution of formaldehyde) is used for preserving anatomical specimens. The toxicity of formalin, its skin-irritating properties, and its tendency to polymerize on surfaces limit its utility as a disinfectant. These drawbacks can be partially mitigated by formulating preparations that contain masked forms of formaldehyde. Among this group of substances, noxitiolin (N-hydroxy-N-methylthiourea) has found the most widespread application. It is supplied as a powder. In aqueous solutions, it slowly decomposes into formaldehyde and N-methylthiourea, with both components exhibiting antimicrobial activity. Noxitiolin is applied topically, within Body Cavities, and in the Treatment of Peritonitis. Polynoxylin (poly[methylenedi(hydroxymethyl)urea]), available in gel and tablet forms, exhibits analogous properties.

Taurorolidine (bis-[1,1-dioxoperhydro-1,2,4-thiadiazinyl-4]methane) contains two taurine molecules and three formaldehyde molecules. It is more stable in solution than noxitiolin and is employed for the same indications, while exhibiting higher potency than formaldehyde.

Hexamine (methenamine) generates formaldehyde in acidic solutions via the transformation of urea. It was formerly used to treat Urinary Tract infections; however, it produces side effects and possesses limited bacteriostatic efficacy.

Rongalite (sodium formaldehyde sulfoxylate) Functions as a reducing agent and is utilized as a preservative and antioxidant.

16.5.4 Biguanides

Chlorhexidine base (Fig. 72) is sparingly soluble in water and is therefore employed in the form of salts—acetate, gluconate, and hydrochloride. It exhibits maximal antimicrobial activity at pH 7–8 in the diconic form. Its efficacy is diminished in the presence of anionic compounds, such as soaps and other anions that form insoluble salts. Consequently, hard water must not be used for preparing its solutions; only deionized or distilled water is suitable. Organic matter also decreases its activity. Solution containers must not be sealed with cork stoppers, as the tannin contained within them inactivates chlorhexidine. It possesses potent antibacterial activity, although it is inactive against viruses, spores, and mycobacteria. It is widely used as an antiseptic and disinfectant and is non-irritating to the skin and mucous membranes.

Fig. 72. Chlorhexidine.

Polyhexamethylene biguanides represent a mixture of polymerized hexamethylene biguanides:

where n averages 5.5. They exhibit high antibacterial activity and low toxicity.

16.5.5 Surface-Active Agents

Surface-active agents (surfactants) are classified into anionic, cationic, and ampholytic (amphoteric) compounds based on The ionization of the hydrophilic group within the molecule, which also contains a hydrophobic group. Anionic and amphoteric surfactants exhibit weak activity against microorganisms or are entirely inactive. Nevertheless, some of them enhance microbial susceptibility to other biocides by altering cell membrane permeability. Cationic surfactants possess antimicrobial activity, with quaternary ammonium compounds (QACs, Fig. 73) being of primary importance. Their activity depends on the carbon chain length of the radical, reaching a maximum in compounds possessing radicals from C₈ to C₁₈. These substances are most active at neutral or mildly alkaline pH and are inactivated at pH levels below 3.5; consequently, they are incompatible with anionic and amphoteric surfactants, likely due to micelle formation. Organic matter also inactivates QACs.

Fig. 73. Quaternary ammonium compounds: A — general formula; B — benzalkonium chloride (n = 8–18); C — cetrimide (n = 12, 14, or 16); D — cetylpyridinium chloride.

Their bactericidal action against Gram-positive bacteria and fungi is manifested at dilutions up to 1:200,000, and against Gram-negative bacteria up to 1:30,000; eradicating Pseudomonas aeruginosa requires higher concentrations. QACs at working concentrations are ineffective against spores and viruses.

QACs do not irritate the skin and mucous membranes, which is why they are used for wound care as well as a preservative for certain preparations. Benzalkonium chloride and cetrimide are widely used in surgery, urology, and gynecology in the form of aqueous and alcoholic solutions and creams, sometimes combined with chlorhexidine. In hospitals, QACs are used for the sanitary treatment of premises and equipment.

16.5.6 Halogens

Chlorine and iodine preparations have been used for disinfection since the early 19th century. Later, their compounds were obtained, which are characterized by high activity, stability, and convenience for Practical Application [4].

Chlorine serves as a source for obtaining many antimicrobial agents. The activity of chlorine-containing disinfectants is expressed in units of free chlorine.

Hypochlorites — one of the longest-known groups of disinfectants, rapidly acting on bacteria, fungi, and viruses, and at high concentrations and with prolonged exposure — on acid-fast bacteria and bacterial spores. They are compatible with cationic and anionic surfactants. Their disadvantages include corrosiveness, The ability to be inactivated by organic matter, and relative instability. Hypochlorites are available in powder or solution form, mainly as potassium or sodium salts of hypochlorous acid (HOCl). In solution, sodium hypochlorite exists in an equilibrium state:

NaOCl + Н₂О ⇄ НОСl + NаОН.

Hypochlorous acid is a strong oxidizing agent; upon ionization in an acidic environment, ions are formed:

НОСl ⇄ Н⁺ + ОСl^-.

The concentration of the OCl^- ion determines the antimicrobial activity of the solution. It is maximal at pH ~5; however, the preparation is unstable in an acidic environment. Therefore, it is stored as an alkaline solution and converted into the active form just before use. The working solution is used within 24 hours.

Organic chlorine compounds. This group includes N-chloro derivatives of sulfonamides, chloramine and dichloramine, halazone (Fig. 74), and N-chloro derivatives of heterocyclic compounds containing a nitrogen atom in the ring, such as dichloroisocyanuric acid. The latter is produced in dry form as sodium or potassium salts. Before use, they are dissolved in an acidic medium.

Fig. 74. Halazone

Chloroform (CHCl₃) is used to a limited extent for the preservation of solutions. Due to its volatility, it can evaporate from solutions, which may be accompanied by Microbial growth.

Iodine has a wide spectrum of antimicrobial activity. It is used as a tincture containing 2.5% iodine and 2.5% potassium iodide in 90% ethanol, as well as Lugol's solution (5% iodine in a 10% potassium iodide solution). The activity of iodine depends less on temperature, pH, and the presence of organic matter than that of chlorine. Its disadvantage is the irritating effect on the skin and mucous membranes, as well as skin staining.

Iodophors — preparations containing iodine (phor meaning carrier), exhibit a lower irritating effect while retaining the activity of iodine. Polymer compounds (polyethylene oxide, polypropylene, polyvinylpyrrolidone) and certain surfactants are used as carriers. Nonionic or cationic surfactants increase the solubility of iodine; these preparations are stable, and adding phosphoric or citric acid before use to a pH below 5 increases their activity. Iodine in them is present in the form of micellar aggregates, which disperse upon dilution of the solution, releasing iodine.

In a complex with polyvinylpyrrolidone, part of the iodine binds to the polymer, but the bulk of it exists in the form of triiodide. Dilution of the solution leads to a weakening of the bond with the carrier and the release of iodine.

16.5.7 Acids and Esters

In pharmaceutical practice, organic acids are mainly used, which do not dissociate completely in solution. The acid in its non-dissociated form possesses antimicrobial activity; therefore, when choosing application conditions, it is necessary to take into account the dissociation constant $K$ and the pK value (the pH at which the degree of dissociation is 50%).

Benzoic acid (C₆ H₅ COOH) alone or in combination with other preservatives is frequently used in pharmaceutical practice. The pK value for benzoic acid is 4.2; therefore, it should be used for the preservation of solutions whose pH is no more than 5.0, preferably 4.0. For oral preparations, it is used at a concentration of 0.05–0.1%. Resistance may develop. Benzoic acid in combination with, for example, salicylic acid is used for the treatment of superficial mycoses.

Sorbic acid — a widely used preservative both as an acid and its potassium salt, has a pK value of 4.8; its activity, like that of benzoic acid, decreases with increasing pH. It is most effective at pH 4 or lower. It is commonly used for the preservation of syrups and gel-like pharmaceutical products.

Sulfur dioxide, sulfites, and metabisulfites. Sulfur dioxide is widely used as a food preservative and in the brewing industry. In pharmaceutical preparations, sodium sulfite and metabisulfite act as preservatives and antioxidants.

Boric acid is used as an antiseptic in the form of a solution or powder for treating the skin and mucous membranes. Good absorption and slow elimination from the body limit its application.

Acetic acid in the form of a 0.25–2% solution is used for treating the External ear and lower urinary tract. It is active against Pseudomonas spp.

Salicylic acid is used in alcoholic solutions (1–2%), powders, ointments, and pastes as an antiseptic, for instance, in dermatomycoses.

Esters of p-hydroxybenzoic acid (parabens) have a pK value of 8–8.5; therefore, their activity depends less on the pH of the medium than the activity of acids. Parabens (methyl, ethyl, propyl, and butyl esters, Fig. 75) can be used for the preservation of solutions with a pH of 7–8, although optimal activity is manifested in the acidic pH range. They are active against fungi and, to a lesser extent, bacteria; pseudomonads are capable of utilizing parabens as a carbon source. Parabens are used for the preservation of emulsions and creams. For heterophasic preparations, it is convenient to use combinations of esters: the water-soluble methyl ester (0.25%) protects the aqueous phase, while the propyl and butyl esters protect the hydrophobic phase. Parabens are incompatible with nonionic surfactants.

Fig. 75. Parabens (R — methyl, ethyl, propyl, butyl, or benzyl).

16.5.8 Oxidizing Agents

Hydrogen peroxide and peracids are powerful antimicrobial agents effective against spores. Hydrogen peroxide (H₂O₂) is used at 3–6% concentrations for disinfection, and at higher concentrations (up to 25%) for chemical sterilization. Peracetic acid is also a potent biocide; however, it is corrosive to metals, toxic, and inactivated by organic matter.

Potassium permanganate in 0.01–0.5% solutions exhibits strong bactericidal activity.

Heavy metals. Mercury- and silver-containing preparations were among the first to be used as antiseptics; however, they are gradually being phased out and replaced by less toxic alternatives. Mercury compounds are highly efficacious, but resistant strains readily emerge, and they also pose environmental pollution hazards. Medical Applications include merthiolate (thiomersal) and phenylmercuric nitrate or acetate (Fig. 76). Phenylmercuric salts at concentrations of 0.001–0.004% are used as preservatives for eye drops, injection solutions, and contact lens solutions, occasionally in combination with other substances. Organomercury compounds are significantly adsorbed from solutions by rubber and polymeric materials.

Fig. 76. Organic mercury compounds: A — merthiolate, B — phenylmercuric acetate.

Additionally, silver nitrate (lunar caustic), protargol (containing 7.8–8.3% silver), collargol (a colloidal solution containing 70% silver), copper sulfate (0.25% solution), and zinc oxide in the form of powders and ointments are utilized as antiseptics.

Diamidines (propamidine, dibromopropamidine, Fig. 77) are used as preservatives: propamidine (0.1%) in eye drops, and dibromopropamidine (0.15%) in creams and eye ointments. Their activity decreases at an acidic pH and in the presence of organic matter. The Development of resistance is also possible.

Fig. 77. Propamidine.

16.5.9 Dyes

Triphenylmethane derivatives exhibit bacteriostatic activity primarily against Gram-positive microorganisms. Organic matter reduces their efficacy. Crystal violet (gentian violet) and brilliant green are most commonly used as 0.5% aqueous or alcoholic solutions for treating minor wounds, Burns, and superficial bacterial or mycotic skin infections.

Acridine dyes (Fig. 78) act equally against Gram-positive and Gram-negative microorganisms. Their action develops slowly. They are inactive against bacillary spores and fungi. Activity levels depend on the degree of molecular ionization. For the most frequently used 3,6-diaminoacridine (proflavine) hemisulfate and 9-aminoacridine (aminacrine) hydrochloride, the optimal pH is 7.5. These substances are not inactivated by serum and are used for treating wounds and burns.

Fig. 78. Acridine dyes: proflavine (A), aminacrine hydrochloride (B).

Quinoline derivatives are most active against Gram-positive bacteria and fungi. 8-hydroxyquinoline derivatives—such as potassium hydroxyquinoline sulfate, clioquinol, chlorquinaldol, and halquinol—are used topically. These substances act as chelating agents and are active only in the presence of divalent metals, such as copper and iron.

Among 4-aminoquinaldine derivatives, dequalinium chloride (a bis-quaternary ammonium compound) is used for oral and pharyngeal infections, and dequalinium acetate is used in eye drops. Laurolinium acetate (QAC) serves as a skin disinfectant. As cationic surfactants, they are incompatible with anionic agents, phenol, and chlorocresol.

A detailed Overview of disinfectants and antiseptics can be useful when selecting the optimal agent to combat microbial contamination in manufacturing environments or clinical practice.

16.6 Application of Antimicrobial Chemicals as Antiseptics

Antiseptics at working concentrations exert a bacteriostatic or bactericidal effect that manifests quite rapidly; when applied correctly, they cause no adverse effects on The Human Body.

The main antiseptics and their scope of application are listed in Table 31.

Table 31. Application of Antiseptic Substances

16.7 Application of Antimicrobial Chemicals as Preservatives

Table 32. Preservatives in Medicinal Products

Note* — organic mercury compounds may exhibit neurotoxic effects and cause keratopathy, and therefore are not recommended for prolonged use. In vaccine manufacturing, thiomersal is currently being replaced by phenoxyethanol or other alternative compounds.