PHARMACEUTICAL MICROBIOLOGY - V. A. Galynkin - 2015

PART II. ANTIMICROBIAL AGENTS

CHAPTER 17. METHODS OF DISINFECTION AND EVALUATION OF THE EFFICACY OF DISINFECTANTS, ANTISEPTics, AND PRESERVATIVES

17.1 Methods of Disinfection

Our knowledge regarding The properties of pathogens, the routes by which they are shed from the host Organism, and The Role of various factors in the transmission of infection determines the choice of a disinfection method—physical, chemical, or biological (Fig. 77) [28].

Class="center">Fig. 77. Classification of methods and modes of disinfection

The physical method involves The Use of disinfection agents such as mechanical means, thermal energy, radiant energy, and ionizing radiation.

Mechanical methods of disinfection primarily ensure the removal rather than the destruction of microorganisms. These include shaking, beating, brushing, washing, filtration, and ventilation. The best results are achieved using vacuum cleaners. Filtration, in particular, is an integral part of tap Water purification. Another example of filtration is a respirator, which effectively protects individuals from airborne microorganisms. For instance, a two-layer gauze mask traps up to 74% of microorganisms, a four-layer mask traps up to 88%, and a six-layer mask traps up to 97%. Ventilation leads to a sharp decrease in the concentration of airborne microflora. At the same time, airing rooms through transom windows, vents, and casements cannot be considered a reliable disinfection measure, as The rate of air exchange depends on numerous parameters that are difficult to account for and regulate (the Temperature difference between the indoor and outdoor air, atmospheric wind speed, and the size and Location of windows, etc.). In this regard, room ventilation (airing) is considered an auxiliary measure and is used in practice only if maintained for a sufficient duration (at least 30–60 minutes).

Thermal action. The death of microorganisms exposed to high temperatures is associated with protein coagulation. Heat sources that can be utilized as disinfection agents include fire, water, dry or moist hot air, and water vapor.

Fire is used as a thermal agent to destroy contaminated items and to flame them. Items of low value are subjected to incineration. In certain cases (such as particularly dangerous infections), animal carcasses are incinerated. Flaming objects is a standard sterilization Procedure in laboratory practice.

Pasteurization is used to destroy vegetative forms of microorganisms in various food products by heating them to 70–80°C for 30 minutes.

For disinfection purposes, hot water is used as a thermal agent, as it rapidly denatures microbial Proteins. Vegetative forms of pathogens are killed at a water temperature of +60°C. Due to qualitative differences among the proteins of individual microorganisms, the exposure time required to achieve lethality for specific species ranges from 10 to 45 minutes at this temperature. As water temperature increases, this time naturally decreases, and at 100°C, all vegetative forms of microorganisms are killed instantaneously or within 1–2 minutes.

Special attention should be paid to the disinfection of laundry using this method. Water heating cannot occur rapidly due to the hindered Movement of water particles within fabrics. Heating in the interior of the items occurs via the thermal conductivity of the textile. Due to the poor thermal conductivity and high heat capacity characteristic of fabrics, the Heat transfer process is very slow. In practice, to ensure thorough heating of laundry to boiling temperature, it must be boiled for at least 30–90 minutes, depending on the volume, thermal conductivity, and heat capacity of the fabrics. To accelerate heating, the laundry must be agitated, which is successfully accomplished in modern washing machines. Boiling laundry is the simplest, most effective, and gentlest method of disinfection.

Dry air, like water, is heated by convection; however, unlike water, its thermal conductivity is 25 times lower. Each particle of dry air carries four times less heat than water. Therefore, heating an object with dry hot air is a very slow process. When disinfecting clothing, which has low thermal conductivity and high heat capacity, achieving a disinfectant effect with dry hot air requires high temperatures (at least 140°C) and extended exposure times. Under these conditions, clothing may char. Dry hot air is used for the sterilization of medical instruments in microbiological practice. Exposure to hot air above 100°C dehydrates and coagulates the protoplasm of microbial Cells, leading to their destruction. The properties of hot air are utilized in hot-air sterilizers for medical devices, in drying chambers for disinfecting items, and when ironing various fabrics. Depending on the heat Setting, iron temperatures reach 200–300°C. However, hot air is inferior in efficacy to steam, as its action is predominantly superficial.

Moist hot air exhibits manifold greater bactericidal activity compared to dry air. This is due to the action of heat in a humid environment and the fact that moist hot air carries a large reserve of heat owing to water vapor, which releases latent heat of vaporization upon Condensation within Materials. Consequently, moist hot air penetrates and heats items faster and deeper than dry air.

Water vapor is the gaseous state of water. Water vapor at a temperature of 100°C and above is one of the most reliable disinfecting agents because of its ability to penetrate deep into the objects being disinfected. The degree of bactericidal efficacy of water vapor depends on its temperature, pressure, and degree of saturation. Under METABOLISM/18.html">The Influence of water vapor, microbial Cell proteins swell and coagulate, resulting in cell death. The properties of water vapor are utilized in disinfection chambers and steam sterilizers. Evaporation—the transition of water from a liquid to a gaseous state—occurs at any temperature, but exclusively at the water surface. During boiling, the transition of water into steam occurs throughout its entire volume. Steam generated by boiling is used for disinfection. The application of water vapor in disinfection practice is based on the fact that upon condensing into water, it releases a large amount of latent heat of vaporization. Steam is most commonly used for chamber disinfection. Chamber disinfection of items as a mandatory anti-epidemic measure is prescribed for A number of infectious diseases (tuberculosis, plague, anthrax, cholera, epidemic typhus, typhoid fever, etc.). Items subjected to chamber disinfection are those that, for various reasons, cannot be disinfected by boiling, soaking in chemical disinfectant solutions, or other methods (outerwear, bedding such as mattresses, pillows, blankets, and other bulky soft goods).

For the disinfection of items retrieved from a patient's residence for terminal disinfection, and for the sanitization of individuals who have been in contact with patients, the sanitary-epidemiological service is equipped with sanitary inspection stations featuring various disinfection chambers. During the sanitary Processing of individuals, their personal belongings are likewise subjected to chamber disinfection. Disinfection chambers are installed in infectious disease and other healthcare facilities. They are used to disinfect outerwear and bedding as needed, as well as for preventive purposes.

17.1.1 The Steam Method

The steam method of disinfection and steam chambers have become widespread, gradually replacing hot-air chambers, which are now used to a very limited extent. A disadvantage of this method is the practical difficulty of using a steam-air mixture for disinfecting fur and leather goods, as temperatures exceeding 80°C and a relative humidity of 80% or more damage leather and fur. Currently, more favorable combinations of temperature and relative humidity have been developed, enabling reliable disinfection of fur and leather items without the use of formalin. Another negative factor associated with the steam-air mixture is a certain degree of dampening of the items disinfected in the chamber. However, these disadvantages are negligible compared to the advantages offered by this physical disinfection agent (simplicity of chamber operation, cost-effectiveness, straightforward safety protocols, absence of toxicological factors, etc.).

Leather and fur products, industrial raw materials (wool, bristles), any garments, bedding, and other soft inventory can be disinfected in steam-formalin chambers. The active agent in steam-formalin disinfection is a steam-air mixture combined with formaldehyde at temperatures ranging from 40°C to 59°C. The use of formaldehyde combined with steam allows disinfection to be performed at lower temperatures, which makes it possible to sterilize leather, fur, and rubber goods without damaging them. During steam-formalin chamber disinfection, formaldehyde vapors exert a disinfecting effect on the pores of fabrics.

17.1.2 Ultraviolet Radiation

The bactericidal effect of solar energy is associated with the ultraviolet rays of the solar spectrum. Ultraviolet rays with wavelengths ranging from 2500 to 2600 angstroms exhibit the highest bactericidal activity. The bactericidal effect is most likely related to the direct photochemical action of ultraviolet rays on the protoplasm of microbial cells. Bactericidal efficacy depends on wavelength, photon fluence, exposure time, the biological CHARACTERISTICS OF THE microorganism, and the qualitative Properties of the medium containing the microorganisms. Experiments demonstrate that after irradiating air with ultraviolet rays for more than 30 minutes, The amount of microflora decreases sharply.

Ultraviolet radiation consists of electromagnetic waves with lengths ranging from 205 to 315 nm. The highest bactericidal activity is exhibited by radiation at a wavelength of 265 nm, which predominantly causes photochemical damage to the DNA of microbial cells.

The outcome of ultraviolet radiation on a microorganism depends on the species of the microorganism and the radiation energy absorbed by The Cell, i.e., the radiation dose. The ratio of radiation energy to the irradiated surface area is referred to as the surface bactericidal dose (Hs); the ratio of radiation energy to the volume of the irradiated medium is referred to as the volumetric bactericidal dose (Hv).

17.1.3 Ionizing Radiation

Ionizing radiation is used in specific cases.

In global practice, ultrasound is widely used for the pre-sterilization cleaning of medical devices. Under the influence of ultrasound, acoustic flows in a confined volume generate a vast number of micro-flows of the solution, which provide a repeated hydromechanical impact on micro-areas of the treated surface, penetrating hard-to-reach places.

The cavitation effect—the continuous formation and collapse of bubbles combined with thermal Shock—causes the destruction of surface contamination.

A beneficial physical phenomenon during ultrasonic surface Treatment is degassing, i.e., reducing the dissolved gas content in the solution and eliminating gas bubbles. At high concentrations, gas bubbles degrade treatment quality by hindering the solution's access to the targeted surface areas.

17.1.4 Biological Method

The biological method is applied to a small group of objects. Examples of such disinfection include: water filtration at water treatment plants (the biological film formed on the filter surface), treatment of sewage wastewater (biological wastewater treatment plants), and the biothermal method for processing solid organic waste (composting, biothermal chambers).

17.1.5 Chemical Method

The chemical method is applied more frequently than others for disinfection purposes, meaning that chemical substances—disinfectants—are used.

They must possess a broad spectrum of activity; exhibit a microbicidal effect; readily dissolve in water or form stable, active Suspensions, emulsions, aerosols, and fogs with water or air; feature low toxicity and allergenicity; retain their activity in the environment being disinfected; and cause no damage to the treated items. The raw materials used to manufacture disinfectants must be accessible, and the disinfectant itself should be cost-effective.

More than 35% of chemical disinfection products belong to the surfactant group. While possessing a number of valuable qualities (low toxicological and ecological-hygienic hazard, detergent properties, and sufficient bactericidal efficacy against a wide range of Gram-positive and Gram-negative Bacteria, etc.), such disinfectants are characterized by insufficient virucidal activity. This limits their scope of application under modern conditions of epidemiological adversity regarding tuberculosis, hepatitis, etc., as well as the ongoing threat of bioterrorism. The latter circumstance appears particularly important in connection with the use of spore-forming microbial agents (anthrax) for terrorist purposes.

17.2 Disinfection Dynamics

The process of death of microbial cells placed in an environment with an antimicrobial agent can be represented graphically (Fig. 78).

Fig. 78. Dynamics of microbial cell death in a medium containing a disinfectant.

At the same time, a situation is possible where the death process follows first-order kinetics laws (A), and the effectiveness of disinfection can be evaluated using the constant:

where K is the cell death rate constant; No is the initial Number of viable cells; N is the number of viable cells at time t.

However, cases are more frequently observed where the graph represents a sigmoidal curve (B), reflecting the death of the least resistant part of the population in the initial period, the death of the bulk of the population possessing an average level of resistance in the middle period, and the survival of the most resistant cells in The final stage of the experiment.

At high disinfectant concentrations, rapid death of the main part of the population occurs in the initial period of time (C).

The Selection/32.html">Genetic heterogeneity of the bacterial population prevents the use of first-order kinetics laws for evaluating the effectiveness of disinfectants; however, Methods based on determining the number of viable cells or the population death time yield quite adequate results. At the same time, it is necessary to take into account the Influence of Environmental factors (temperature, pH, medium composition), as well as the microbial load (the number of microbial cells in a given volume).

17.3 Methods for Testing the Antimicrobial Activity of Antiseptics and disinfectants

When testing liquid formulations, the microbicidal concentration of the antiseptic at a specified exposure time, THE SPECTRUM OF antimicrobial action, and the antiseptic's effectiveness under conditions approximating practical use are determined [28]. Tests are carried out using standard test cultures of the following species: Staphylococcus aureus, Escherichia coli, Enterococcus faecium, Pseudomonas aeruginosa, Proteus mirabilis, Candida albicans, Trichophyton mentagrophytes, and Mycobacterium terrae.

M. terrae is cultivated on Lowenstein-Jensen medium, while other bacteria and Fungi are grown on media recommended for determining antibiotic susceptibility and studying the microbial contamination of medicinal products. The density of the microbial suspension should be 10⁸ -10¹¹ CFU/mL (CFU — colony-forming unit). The suspension is prepared in a 0.5% sodium chloride solution with a neutral pH. For experiments involving a protein load, 20% Blood serum or 0.2% albumin is added to the suspension.

Exposure is set from 30 s to 5 min. To determine the concentration active within this timeframe, solutions containing 100, 75, 50, 25, and 12% of the antiseptic are prepared.

In parallel, a control experiment is conducted to determine the sensitivity of the test culture to phenol at concentrations of 1, 0.5, 0.25, 0.125, and 0.06%. A suspension of the test culture is added to the antiseptic solution (0.1 ml of suspension per 1 ml of solution). After the exposure time elapses, the antiseptic is neutralized, and the mixture is inoculated onto a standard medium specific for the given strain. A neutralizer is used that inactivates a specific class of compounds or all classes of antiseptic agents. The latter type includes a mixture containing 3% Tween-80 and 0.1% each of saponin, Cysteine, and Histidine.

When approving new preparations for medical practice, the following tests are performed:

a) qualitative suspension test;

b) quantitative suspension test;

c) test simulating the PRACTICAL USE OF the antiseptic.

The qualitative suspension test is performed According to the method described above. Test cultures: St. aureus, E. coli, P. mirabilis, P. aeruginosa, C. albicans. The density of the microbial suspension must correspond to 10⁸-10⁹ CFU/ml. Incubation period for cultures after inoculation: for bacteria, 48 hours at 37°C; for C. albicans, 72 hours at 20-25°C.

For hygienic antisepsis, a preparation is considered suitable if it inhibits the growth of all test strains after an exposure of 0.5-1 min at a tolerable concentration. A preparation intended for surgical hand antisepsis must inhibit the growth of all test cultures after an exposure of 3-5 min.

Quantitative suspension test.

Test cultures — St. aureus, E. coli; suspension density 1-1.5 x 10»’ CFU/ml. For each culture, two sets of antiseptic dilutions are prepared: without protein protection and with The addition of 0.2% albumin. The exposure times of the preparation and culture are 0.5, 1, and 5 min. Next, the mixture is neutralized and inoculated onto nutrient Agar.

The activity of the preparation is evaluated based on the degree of reduction in CFU/ml compared to the control (microbial suspension without antiseptic). Preparations for hygienic antisepsis must reduce the CFU/ml by at least 10⁵-fold at the recommended application concentration with an exposure of 0.5-1 min, while preparations for surgical antisepsis must provide the same degree of CFU/ml reduction within 3-5 min.

The simulated-use test is performed on human volunteers. A suspension of E. coli (1 x 10¹¹ CFU/ml) is applied to the Skin of the hands, air-dried for 3 min, and the hands are treated with the test antiseptic solution: for hygienic antisepsis, for 1 min using 3 ml of the preparation; for surgical antisepsis, for 5 min, 2-3 times, using 5 ml of the preparation each time. Subsequently, a hand wash is collected using a liquid nutrient medium. In the control group,

a) a hand wash is collected from contaminated hands without antiseptic treatment,

б) the hands are treated with 3 ml of a standard antiseptic (isopropanol) twice for 30 s, after which a wash is collected.

Dilutions of the wash samples (10¹, 10^-2, 10^-3) are inoculated onto nutrient agar and incubated at 37°C for 48 hours. The efficacy of the test preparation is evaluated by comparing the degree of reduction in the count of the test microorganism after hand treatment with the test and standard antiseptics.

Determination of the antimicrobial activity of antiseptics in semisolid and solid forms is carried out on a solid nutrient medium inoculated with the test culture. The samples are placed On the surface of the medium or in wells, similar to the agar diffusion method used for antibiotic testing. The activity of the preparation is judged by the diameter of the growth inhibition zone around the sample compared to a standard preparation.

The test preparation can be dissolved or emulsified in a liquid medium (e.g., in a buffer solution with an appropriate emulsifier) and its activity determined by the methods described above for liquid forms.

17.4 Determination of the Efficacy of Preservatives in Medicinal Products

Preservatives are incorporated into both sterile and non-sterile medicinal products (Table 32) to prevent the growth of microorganisms introduced during the manufacturing process or upon repeated use [28]. They must not be used to mask poor manufacturing practices. Given the potential for adverse effects, preservatives should be used only when strictly necessary. Requirements for preservatives:

✵ broad spectrum of antimicrobial activity;

✵ rapid biocidal action;

✵ absence of interaction with the Components of the medicinal product;

✵ stability;

✵ absence of irritating or toxic effects from the biocide or its degradation products.

However, few substances meet these ideal requirements. When using a preservative, a number of factors affecting its efficacy must be taken into account: microbial load, temperature, pH, and the COMPOSITION OF THE medicinal product.

In a multiphase system, the preservative is distributed unevenly according to its hydrophilic or hydrophobic nature. The preservative may adsorb onto the primary packaging material, thereby reducing its activity. The concentration of volatile substances (e.g., chloroform) may decrease upon repeated opening of the container.

All medicinal products containing preservatives are divided into the categories presented in Table 33.

Table 33. Categories of medicinal products containing preservatives

Table 34. Cultivation conditions of test microorganisms for inoculum preparation

* — alternative liquid and agar culture media of domestic and foreign production are permitted to be used.

The effectiveness of antimicrobial preservatives is currently determined in accordance with FS 42-0069-07 "Determination of the effectiveness of antimicrobial preservatives in medicines", State Pharmacopoeia XII ed., part 1, pp. 216-219.

The effectiveness of preservatives is determined against specific types of bacteria and fungi. Strains of bacteria and fungi that are the most frequent contaminants of medicinal products are used as test microorganisms:

Escherichia coli ATCC 8739 (or ATCC 25922),

Pseudomonas aeruginosa ATCC 9027,

Staphylococcus aureus ATCC 6538,

Candida albicans ATCC 10231 (or NCTC 885653),

Aspergillus brasiliensis ATCC 16404 (or ATCC 9642).

In addition to the listed test strains, other microorganisms may be used, provided they are typical in their cultural, morphological, staining, and biochemical properties. The set of test microbial strains may be reduced or expanded depending on the route of administration or the composition of the test medicinal product. All test microbial strains obtained from State Collections with the manufacturer's certificate in ampoules, on disks, or in any other form should be revived using the methods described in the instructions supplied with the test strains or in accordance with the State Pharmacopoeia XII ed. General Pharmacopoeia Article "Microbiological Purity". The cultivation conditions of the test strains for inoculum preparation are presented in Table 34.

The growth-promoting properties of the culture media used are tested in accordance with the State Pharmacopoeia XII ed. General Pharmacopoeia Article "Microbiological Purity".

When preparing the inoculum, 24-hour cultures of bacterial test strains and C. albicans are washed off The surface of the agar slant with a sterile 0.9% sodium chloride solution. The concentration of bacterial cells is adjusted to 10⁹ CFU/ml, and that of C. albicans to 10 ⁷ CFU/ml, using a turbidity standard or instrumental methods, including turbidimetry.

To wash off A. brasiliensis conidia, a sterile 0.9% sodium chloride solution containing 0.05% Tween-80 is used. The number of A. brasiliensis conidia in 1 ml of the wash suspension is determined using a Goryaev chamber or the plate agar method. The resulting suspension is diluted to a concentration of 10⁷ conidia per 1 ml.

Standardized suspensions of all test microbial strains are diluted to a concentration of 10-10 ⁸ CFU/ml, and for category 4 products — to 10⁵-10 ⁶ CFU/ml.

Ready-to-use medicinal products in intact packaging are used to determine the effectiveness of preservatives.

A sufficient amount of the test product is placed into 5 sterile vials. 0.1 ml (but not less than 0.5% of the volume of the test product) of one of the prepared test strain inocula (E. coli, P. aeruginosa, S. aureus, C. albicans, A. brasiliensis) is added to each vial to obtain a concentration of 10⁵-10 ⁶ CFU per 1 ml or 1 g of the test sample, and mixed thoroughly.

Medicinal product samples with a hard ointment base are heated to a temperature of (47.5±2.5)°C. The inoculum of each standardized microbial suspension is mixed with the product sample for at least 1 minute until a homogeneous emulsion is formed. To improve mixing, a specific (validated) amount of a sterile surfactant, such as Tween-80, may be added, provided it does not affect the viability of microorganisms or the preservative's effectiveness.

The actual initial concentration of bacteria and fungi in the contaminated samples is determined immediately after contamination. For this purpose, inoculation onto appropriate culture media (Table 34) is performed using the plate agar method, employing suitable dilutions to obtain 30 to 300 bacterial colonies and 10 to 100 fungal colonies per plate. The Membrane filtration method can also be used for this purpose, provided the medicinal product is soluble in aqueous Solvents or isopropyl myristate.

The contaminated product samples are kept at a temperature of (22.5±2.5)°C in a dark place for a specified period of time. After 7, 14, and 28 days following the inoculation of category 1 product samples, and after 14 and 28 days for category 2 and 3 products, the number of viable microorganisms per 1 ml of the sample is determined by plating on Petri dishes.

If necessary, an inactivator (neutralizer) of the antimicrobial action of a specific substance, as specified in the General Pharmacopoeia Article "Microbiological Purity", is introduced into the agar medium plates or into the appropriate dilution of the medicinal product prior to plating.

Accounting for test results is carried out after plating by the agar plate method, determining the number of CFU/ml for each test strain after the aforementioned incubation periods of the contaminated sample. The change in the number of microbial cells compared to the initial concentration in 1 ml is expressed in decimal Logarithms (lg). When evaluating the effectiveness of the antimicrobial action of preservatives, an increase in CFU/ml is not recorded if the subsequent measurement does not differ from the previous one by more than 50%. The antimicrobial preservatives of medicines are considered effective if a decrease in the number of bacterial cells is observed in accordance with the criteria described in Table 35.

Between 7 and 14 days, there should be no increase in the bacterial count. The count of Yeast and mold cells must not increase throughout the entire test period for all categories of medicinal products.

Table 35. Evaluation Criteria for the antimicrobial preservation efficacy of pharmaceutical products against bacteria

The test is performed using the following culture media:

✵ Casein Soya Bean Digest agar

Alternative domestic medium for cultivating aerobic bacteria: Medium No. 1 for microbial contamination control

✵ Casein Soya Bean Digest Broth

Alternative domestic medium for cultivating bacteria: Medium No. 8 for microbial contamination control

✵ Sabouraud 2% Dextrose Broth

Alternative domestic medium for cultivating Yeasts and Molds: Medium No. 2 (Sabouraud glucose agar) for microbial contamination control

✵ Sabouraud 4% Glucose Agar

To prevent bacterial growth prior to sterilization, add 50 mg of chloramphenicol per 1 liter of medium, or add 0.1 g of benzylpenicillin sodium salt and 0.1 g of tetracycline per 1 liter of medium as sterile solutions immediately before use.