PHARMACEUTICAL MICROBIOLOGY - V. A. Galynkin - 2015

PART III. MICROBIOLOGICAL ASPECTS OF PHARMACEUTICAL MANUFACTURING

CHAPTER 23. MICROBIOLOGICAL QUALITY REQUIREMENTS FOR DRUG PRODUCTS

23.1 Microbiota of Non-Sterile Medicinal Products

One of the quality indicators for a medicinal product (MP) is its level of microbial purity [39]. Based on this parameter, all MPs are divided into two categories: sterile and non-sterile. Sterile preparations are defined as those that, in accordance with pharmacopoeial requirements, must not contain viable microorganisms. They account for about 20% of the total volume of medicinal products. Non-sterile products are those that may contain living microorganisms, the quantity and qualitative composition of which depend on the type and intended use of the product and are regulated by relevant documentation. Non-sterile products account for about 80% of all manufactured medicines. Microbiological quality requirements for non-sterile products were established following reported cases of human illness resulting from The Use of microbially contaminated drugs (Table 48).

Class="center">Table 48. Examples of contaminant microbes detected in pharmaceutical products

Contamination of medicinal products can occur both during manufacturing and throughout their use, particularly in clinical settings where products are frequently contaminated with nosocomial strains of microorganisms, such as Pseudomonas aeruginosa.

Due to the action of microbial Enzymes resulting from improper manufacturing and storage conditions, medicinal products may undergo biodegradation. The rate of this process depends on The chemical composition of the drug, the presence of substances that are readily assimilated by microorganisms or possess biocidal activity, the quantity and species composition of contaminants, and environmental conditions (humidity, Temperature). Certain components of medicinal products (starch, gelatin, kaolin, magnesium trisilicate, aluminum hydroxide, Surfactants, Proteins) can protect microbial Cells from preservatives.

The susceptibility to biodegradation is also influenced by the packaging design, which must prevent the ingress of contaminants and control moisture levels.

23.2 The Role of Contaminant Microorganisms in Human Pathology

The main adverse consequences for patients using preparations containing microorganisms may include a reduction or complete loss of therapeutic efficacy, The Development of diseases and adverse drug reactions, as well as the transmission and spread of drug-resistant Bacteria. These pathologies can be of infectious or non-infectious origin. Non-infectious conditions may be caused by substance breakdown products and microbial toxins, which can trigger toxicoinfections and intoxications (poisoning). Toxicoinfections represent a broad group of acute intestinal disorders that develop after the oral ingestion (per os) of medicinal products heavily contaminated with pathogenic and opportunistic bacteria containing endotoxins. Toxicoinfections are polyetiological and can be caused by various microorganisms: enterotoxigenic strains of Escherichia coli, Proteus, enterococci, Bacillus cereus, Staphylococcus aureus, Clostridium perfringens, and less frequently, bacteria of the genera Klebsiella, Enterobacter, and Pseudomonas. Intoxications result from the ingestion of exotoxins produced by certain bacteria (S. aureus, C. botulinum), Fungi, and toxic products of microbial drug degradation.

Infectious diseases resulting from the use of contaminated medicines can present with various localizations and clinical forms:

1) pyogenic and septic infections (local or generalized);

2) fungal infections of the Skin, mucous membranes, eyes, and other Organs;

3) viral infections (e.g., resulting from the administration of contaminated Blood products);

4) intestinal infections (escherichiosis, salmonellosis).

The onset, progression, and outcome of an infection depend on the level of microbial contamination, the biological Properties of the contaminant (its virulence), the patient's immune status, and the route of administration. The mode of application of contaminated products plays a critical role in disease development. The greatest hazard is associated with administration into the bloodstream, eyes, or Body Cavities that are normally sterile. In topical Applications, the likelihood of an infection sharply increases in the presence of extensive tissue Damage caused by trauma, Burns, or surgery. For instance, Staphylococcus aureus introduced onto damaged skin and mucous membranes via a contaminated drug can cause purulent inflammatory processes; inhalation can lead to staphylococcal Pneumonia; oral administration can result in toxicoinfection or intoxication; and entry into the bloodstream can cause a generalized infection (Sepsis).

23.3 Microbiological Quality Requirements for Finished Dosage Forms

Current requirements for the microbiological purity of medicinal products are presented in Table 49 [37].

In addition to the established allowable limits for contaminant microorganisms in various drugs, there is currently an active Structure/133.html">Discussion regarding the Introduction of an additional category to restrict contamination by extraneous bacteria and fungi in immunobiological preparations containing live microorganisms, such as Vaccines, Bacteriophages, and Probiotics. These challenges are associated not only with standard Setting, but also with the development and validation of microbiological Methods for evaluating the quality of such products.

The Selection of microorganisms regulated by the Pharmacopoeia is determined by their hazard to public health and their utility as indicators for assessing GMP-compliant manufacturing hygiene. While economic constraints limit this selection, it is currently considered sufficient to adequately reflect product quality through microbiological indicators, though it may be expanded in the future. Examples include testing for Candida albicans in intravaginal medicinal products, a requirement newly introduced for domestic products of this type, whereas it has long been established in foreign pharmacopoeias. Testing of medicinal products for microbiological purity, in accordance with the currently effective 12th Edition of the Pharmacopoeia, is performed under aseptic conditions using the methods and culture media described below, which are compositionally equivalent to the media of the current edition of the European Pharmacopoeia.

23.4 Medicinal Product Quality Assessment

The quality assessment Procedure for medicinal products and pharmaceutical substances includes:

— Methods for determining antimicrobial activity and ways to neutralize it;

— methods of preparation of various dosage forms;

— sampling for analysis;

— Methods for the Quantitative determination of viable contaminating microorganisms;

— methods for the detection and identification of specific bacterial species whose presence in medicinal products is unacceptable or limited;

— methods for the detection of Candida albicans in intravaginal medicinal products;

— recipes for culture media, solutions, and Reagents used;

— quality control methods for culture media prior to use and their quality requirements.

The standard temperature for incubating cultures on bacterial media is (32.5±2.5) °С, and for fungi, (22.5±2.5) °С, unless otherwise specified in individual pharmacopoeial monographs.

Table 49. Microbiological purity of medicinal products

Notes to Table 49:

1. Regulatory documents may, as an exception, specify other limits depending on the COMPOSITION OF THE medicinal product and the Specific features of its manufacturing process.

2. Regulatory documents for pediatric medicinal products may introduce stricter limits, specifically:

— in 1 g (ml) of medicinal products for infants (aged 0 to 1 year) — not more than 50 aerobic bacteria, Yeasts, and Molds (combined) in the absence of bile-tolerant gram-negative bacteria, Pseudomonas aeruginosa, and Staphylococcus aureus

— in 1 g (ml) of medicinal products for children (over 1 year of age) — not more than 500 aerobic microorganisms and 50 yeasts and molds (combined) in the absence of bile-tolerant gram-negative bacteria, Pseudomonas aeruginosa, and Staphylococcus aureus.

3. If other pathogenic bacteria, in addition to those specified above, are detected during testing, the quality of the medicinal products, substances, and excipients is considered not to comply with the requirements for "Microbiological purity".

When conducting tests to determine the antimicrobial activity of medicinal products, the quality of culture media, and the biochemical identification of isolated microorganisms, it is necessary to use reference strains of microorganisms obtained from official culture collections (50):

— American Type Culture Collection, USA (ATCC);

— National Collection of Type Cultures, UK (NCTC);

— Collection de l'Institut Pasteur, France (CIP);

— All-Russian Collection of Microorganisms of the RAS, Russia (VKM);

— State Collection of Pathogenic Microorganisms, Russia (GKPМ);

— Collection of the All-Russian Myrological Center, Russia.

Table 50. List of microbial reference strains used in testing

In addition to the test strains of microorganisms listed in the table, other cultures with typical morphological, tinctorial, and biochemical properties may be used, provided that the method is properly validated. The set of test microorganisms can be reduced or expanded if necessary.

Lyophilized test microorganisms in ampoules, as well as in tubes on semi-solid Agar, should be stored at temperatures ranging from 2 to 8°C. Microbial cultures on discs must be stored at a temperature not exceeding minus 20°C.

No more than five passages from the original culture are permitted. All work with microorganisms must be carried out in strict compliance with the State Pharmacopoeia. At the same time, the specific Characteristics of Individual test strains described in the certificates (strain passports) and manufacturer's instructions must be taken into account.

23.5 Determination of Antimicrobial Activity

Prior to testing for microbiological purity, it is necessary to determine whether the medicinal product exhibits antimicrobial activity against specific species of microorganisms [28].

The METHOD FOR DETERMINING antimicrobial activity is based on comparing the growth intensity of test microbial strains in the presence or absence of the test product.

Inocula are prepared as follows: 24-hour broth cultures of bacteria grown in soybean-casein digest broth or medium No. 8, and a 48-hour culture of C. albicans grown in liquid Sabouraud medium, are diluted with a sterile 0.9% sodium chloride solution to a concentration of approximately 10⁴ CFU/mL:

— 1:1000 (B. cereus, C. albicans);

— 1:100000 (E. coli, S. abony, P. aeruginosa, S. aureus).

B. subtilis spore suspension is likewise diluted to a concentration of 10⁴ CFU per 1 mL.

The A. brasiliensis culture grown on Sabouraud dextrose agar slants or medium No. 2 is washed off using phosphate buffer solution containing 0.05% Tween-80. The number of conidia per 1 mL of the wash is determined using a Goryaev chamber or the pour plate agar method, and diluted to a concentration of 10 ⁴ conidia per 1 mL.

The medicinal product sample is prepared as a solution, suspension, or emulsion, depending on the Physical Properties of the dosage form, by adding an appropriate diluent to obtain a 1:10 dilution. As a rule, the diluent used is a phosphate buffer solution containing sodium chloride and peptone (pH 7.0) or the same buffer solution containing no more than 5% Tween-80.

A neutralizing fluid is used to dilute products with known antimicrobial activity. Starting from the 1:10 dilution, serial dilutions of 1:50, 1:100, 1:500, 1:1000, etc., are prepared.

Testing for antimicrobial activity is performed using one of the methods described below.

23.6 Determination of Antimicrobial Activity Under Conditions of Microbiological Purity Testing

A 1 mL portion of each dilution of the product is added to six Petri dishes (90 mm in diameter). To two of these dishes, 0.2 mL of the B. cereus suspension (or B. subtilis spore suspension) is added; to another two, 0.2 mL of the working suspension of the C. albicans culture is added; and to the final two, 0.2 mL of the A. brasiliensis conidial suspension is added. The dishes with bacteria are poured over with 10–15 mL of soybean-casein digest agar or medium No. 1, melted and cooled to (42.5±2.5) °C; the dishes with fungal cultures are poured over with the same volume of Sabouraud agar or medium No. 2.

A 1.0 mL portion of each product dilution is added to tubes containing 10 mL of liquid media—Mossel broth and soybean-casein digest broth (or equivalent media, such as medium No. 3 and medium No. 8). Then, 1 mL of the test strain suspension (E. coli, S. abony, P. aeruginosa, S. aureus, tested separately) is added to each tube containing the medium appropriate for the Nutritional Requirements of the microorganism being tested.

Control dishes and tubes are prepared by substituting an equal volume of the diluent for the product dilutions.

The inoculated media are incubated under standard conditions for 48 hours for bacteria and 72 hours for fungi.

The replica plate method is recommended for determining the antimicrobial activity of water-insoluble (Suspensions, emulsions, etc.) or colored medicinal products.

A 1 mL portion of each dilution of the test product is placed into sterile Petri dishes. An equal volume (1 mL) of the diluent used for preparing the dilutions is added to the control dishes. To both the test and control Petri dishes, 10–15 mL of soybean-casein digest agar or medium No. 1 (melted and cooled to (42.5±2.5) °C) or an equal amount of Sabouraud agar or medium No. 2 is added, and the contents are thoroughly mixed. After the agar solidifies, the dishes are dried in an incubator or laminar flow cabinet to remove Condensation from The surface of the medium. The working suspension of each bacterial and fungal test strain is then applied to the surface as droplets using a bacteriological loop, pipette, or replicator. The inoculated media are incubated under standard conditions for 48 hours for bacteria and 72 hours for fungi.

Following the incubation period, visual inspection is performed to check for typical growth of the test Microorganisms in the control dishes and tubes (without the product) as well as in the test samples (containing various dilutions of the product). In cases where result evaluation is hindered (e.g., turbidity or color changes of the liquid medium resulting from an interaction between the medicinal product and the nutrient medium), subculturing onto solid agar media must be performed.

If growth of E. coli, S. abony, P. aeruginosa, or S. aureus is observed on the nutrient media, it indicates the absence of antimicrobial activity in the test product.

The presence of test microorganism growth in the test dishes and tubes comparable to the controls is designated by a "+" sign, while the absence of growth is designated by a "-" sign. If media containing the product show a reduced colony count on the dishes or a complete lack of test microorganism growth, it is concluded that antimicrobial activity is present. The first of the serial product dilutions that exhibits no antimicrobial activity is used for inoculation onto the corresponding nutrient medium.

The following methods may be used to eliminate the antimicrobial activity of medicinal products:

✵ increasing the dilution of the product by using a larger volume of diluent or culture medium within the limits of acceptable microbial contamination (using a neutralizing fluid of laboratory or industrial manufacture (cl. 9) instead of the standard phosphate buffer solution as a diluent);

✵ using specific inactivators (e.g., β-lactamase for certain Antibiotics and p-aminobenzoic acid (PABA) for sulfonamides) that neutralize the antimicrobial activity of the product without inhibiting the growth of microorganisms contaminating the medicinal product;

✵ for products containing preservatives, the use of nonspecific inactivators is recommended. Following validation, Tween-80, soy or egg lecithin, etc., may be added to the buffer solution and/or culture media.

✵ for products soluble in water or isopropyl myristate (IPM), the Membrane filtration method is applied, followed by washing of the filters.

23.7 Sampling of Medicinal Products for Quality Analysis by the "Microbiological Purity" Indicator

The required number of samples is taken from each test batch of the medicinal product in accordance with the product category from a sufficient number of different packages (at least 3–10).

For aerosols based on liquid or solid substances, 10 containers are sampled; for transdermal patches, 10 patches.

In certain cases (high cost of the product and/or small batch size), the Sample size may be reduced to 2 g (ml), unless otherwise specified in the individual pharmacopoeial monograph. Any reduction in sample size, along with the testing method, must be validated and approved in regulatory documentation in the established manner.

For solid medicinal forms, unless otherwise specified in individual monographs, the Analysis of the product requires:

— 10.0 g of the sample to determine the total count of bacteria and fungi per 1 g of the product, and to test for the absence of P. aeruginosa, S. aureus, and E. coli;

— 25.0 g of the sample to determine bacteria of the genus Salmonella;

— 10.0 g for the enumeration of bile-tolerant gram-negative bacteria.

For tablets, coated tablets, granules, powders, etc., 10.0 g of the sample (unless another amount is specified in individual monographs) is pulverized (if necessary) and transferred into 100 ml of buffer solution. Subsequently, quantitative and qualitative determination of microorganisms is carried out.

For capsules, 10.0 g of the sample is transferred into 100 ml of buffer solution containing not more than 5% Tween-80 and preheated to a temperature not exceeding 40°C. After suspending the capsules in the buffer solution, quantitative and qualitative determination of microorganisms is performed.

For semisolid medicinal forms, unless otherwise specified in individual monographs, the analysis of the product requires:

— 10.0 g of the product to determine the total count of bacteria and fungi, and to test for the absence of P. aeruginosa, S. aureus, and E. coli per 1 g of the product;

— 10.0 g of the sample to test for the absence or for the enumeration of bile-tolerant gram-negative bacteria per 1 g of the product.

For ointments, liniments, creams, and suppositories that are easily miscible with water, 10.0 g of the sample is placed in a sterile flask containing 100 ml of buffer solution and Glass beads 5–6 mm in diameter. The mixture is heated on a water bath to a temperature not exceeding 40°C and shaken vigorously until a homogeneous emulsion is obtained, which is used for the quantitative and qualitative determination of microorganisms.

For ointments, liniments, creams, and suppositories that are difficult to mix with water, 10.0 g of the sample is mixed with sterile Tween-80 in an amount not exceeding 1/2 of the sample volume (in this case, 5 g). The mixture is heated on a water bath or in an incubator to a temperature not exceeding 40°C (in exceptional cases up to 45°C) and mixed gently. The heating time must not exceed 30 minutes. The required amount of sterile phosphate buffer solution, preheated to the appropriate temperature and containing glass beads, is added. The mixture is stirred gently to obtain a homogeneous emulsion at a 1:10 dilution, which is used for the quantitative and qualitative determination of microorganisms. Other technical means and homogenization methods may be used, provided that aseptic rules and incubation conditions are strictly observed.

For liquid medicinal forms, unless otherwise specified in individual monographs, the analysis of the product requires:

— 10.0 ml of the sample to determine the total count of microorganisms and fungi per 1 ml of the product, and to test for the absence of E. coli, P. aeruginosa, and S. aureus;

— 25.0 ml for testing for the absence of bacteria of the genus Salmonella;

— 10.0 ml for the enumeration of bile-tolerant gram-negative bacteria.

For solutions, suspensions, syrups, and mixtures, 10.0 ml of the sample is transferred into 90 ml of buffer solution, mixed, and subjected to quantitative and qualitative determination of microorganisms.

For oily solutions and emulsions, 10.0 ml of the sample is placed in a sterile flask containing 90 ml of buffer solution with not more than 5% Tween-80 and glass beads. The mixture is heated on a water bath to a temperature not exceeding 40°C and shaken vigorously until a homogeneous emulsion is obtained, which is used for the quantitative and qualitative determination of microorganisms.

23.7.1 For medicinal products in the form of aerosols

For aerosols based on alcohols and solid substances, 3.0 g of the sample (after evaporation of the propellant) is transferred to 30 ml of buffer solution, mixed, and used for the quantitative and qualitative determination of microorganisms. Not less than 1.0 g of the sample is used for the quantitative determination of bile-tolerant gram-negative bacteria.

For oil-based aerosols, 3.0 g of the sample (after evaporation of the propellant) is transferred to 30 ml of buffer solution containing not more than 5% of Tween-80 and glass beads. The mixture is heated on a water bath to a temperature not exceeding 40 °C and shaken vigorously until a homogeneous emulsion is obtained, which is then used for the quantitative and qualitative determination of microorganisms. Not less than 1.0 g of the sample is used for the quantitative determination of bile-tolerant gram-negative bacteria.

23.7.2 For transdermal patches

When sampling transdermal patches, a sample consisting of 10 units is used. The release liner is removed from each of the 10 patches using sterile instruments. If necessary, the patches are cut into smaller pieces with sterile scissors and transferred into a 1000 ml flask containing 500 ml of sterile buffer solution and glass beads. The flask is heated on a water bath to a temperature not exceeding 40 °C and shaken vigorously for 30 min. A 50 ml portion of the resulting washings is used for the quantitative determination of microorganisms by membrane filtration and for testing for the absence of P. aeruginosa and S. aureus.

If the patch is known to have antimicrobial activity, a suitable inactivator (Tween-80 and/or lecithin) is added to the diluent.

If the washings from transdermal patches cannot be used for membrane filtration testing, the direct inoculation method on nutrient media is applied.

23.7.3 For herbal medicinal products (HMPs)

Herbal medicinal products include medicinal preparations, including herbal teas, packaged in boxes, bags, briquettes, etc.

From each tested batch of the medicinal product, regardless of its size, at least 5 unopened boxes, bags, or briquettes are sampled. Before testing, the packages are opened using sterile instruments, samples are taken from them in equal amounts, mixed, and transferred into a sterile container. The mass of the sample must be at least 50.0 g.

For the enumeration of aerobic microorganisms and fungi, a sample of 10.0 g (fruits, bark, roots and rhizomes, buds, etc.) or

2.0 g (herbs, leaves, flowers, and others with a high water absorption coefficient) is transferred to a sterile flask. When using a 10.0 g sample, 100 ml of sterile 0.9% sodium chloride solution is added to the flask. The flask with the test sample is shaken on a reciprocal shaker or shaking apparatus for at least 15 min. The resulting washing is considered a 1:10 dilution. When using a 2.0 g sample, 200 ml of sterile 0.9% sodium chloride solution is added to the flask. The resulting washing is considered a 1:100 dilution.

If the sample is difficult to wet, a surfactant—sterile Tween-80 in an amount of 0.1% of the solution volume—is added to the flask.

From the resulting HMP washings corresponding to 1:10 or 1:100 dilutions, subsequent tenfold dilutions are prepared in the same diluent and used for the enumeration of aerobic bacteria and fungi, as well as for tests for the absence of E. coli, Salmonella, and bile-tolerant gram-negative bacteria.

23.8 Methods for enumeration of aerobic microorganisms

Depending on The Nature of the medicinal product and its physicochemical properties, one of the agar plate methods (pour-plate, double-layer, surface-spread, or modified pour-plate), membrane filtration, or the Most Probable Number (MPN) tube method is used.

23.8.1 Agar plate methods

For the CULTIVATION OF MICROORGANISMS, agar nutrient media are used in accordance with the formulations of the State Pharmacopoeia, 12th ed.: soybean-casein digest agar or medium No. 1, dry, for microbial contamination control—for the cultivation of bacteria; Sabouraud dextrose agar or medium No. 2, dry, for microbial contamination control—for the cultivation of yeasts and molds.

At least two Petri dishes with the specified medium are used for each sample dilution.

23.8.2 Pour-plate method

Transfer 1 ml of the test sample prepared for analysis into a sterile Petri dish 90 mm in diameter. Add 15–20 ml of melted agar nutrient medium cooled to 42.5±2.5 °C and mix thoroughly by swirling. For Petri dishes of larger diameter, the volume of the medium is increased proportionally to 20–25 ml. After the agar has solidified, invert the dishes and incubate the cultures.

23.8.3 Double-layer method

Pour 15–20 ml of melted agar nutrient media into each sterile Petri dish 90 mm in diameter and leave until solidified. For larger Petri dishes, increase the volume of the medium accordingly. Dry the surface of the agar in the dishes.

Transfer 1 ml of the sample prepared for analysis into a test tube containing 4 ml of the appropriate nutrient medium melted and cooled to 42.5±2.5 °C, and mix the Contents of the tube thoroughly. Then, pour the contents of the tube over the surface of the solidified and dried agar in a Petri dish, distributing the top layer of the medium evenly with rotary movements. After solidification, invert the dishes and place them in an incubator.

23.8.4 Surface-spread method

Molten nutrient media cooled to 42.5±2.5°C are poured in amounts of 15-20 mL into each sterile 90 mm Petri dish and left to solidify. The agar surface in the dishes is then dried.

A 0.1 mL volume of the sample prepared for analysis is applied to the agar and evenly distributed over the medium surface using a spreader.

The Petri dishes are inverted and placed into an incubator.

23.8.5 Modified Pour-Plate Method

A 1.0 mL volume of the sample prepared for analysis is placed into a sterile 90 mm Petri dish. Then, 7-10 mL of molten nutrient medium cooled to 42.5±2.5°C is added and quickly mixed with circular motions. After the agar solidifies, the dishes are inverted and incubated. Results are read after 48 h and finally after 5 days.

23.9 Counting and Interpretation of Results for Agar Plate Methods

The cultures are examined daily. Colony counting is performed after 48-72 h (preliminary result) and after 5 days (final result).

To obtain reliable results, select plates where the number of bacterial colonies does not exceed 250, and fungal colonies do not exceed 50. If the number of colonies on the plates in two consecutive dilutions falls within the specified limits, calculate the results from the lower dilution.

If an average of more than 250 bacterial colonies or more than 50 fungal colonies grow on the plates, a series of further consecutive dilutions of the sample is prepared, selecting the one appropriate for plating.

If an average of less than 15 and more than 250 bacterial colonies, or less than 15 and more than 50 fungal colonies grow on the plates, a series of further consecutive dilutions of the sample is prepared, selecting the one appropriate for plating.

If fungal colonies are additionally detected on soybean-casein digest agar (or Medium No. 1), they are added to the bacterial count to determine the total aerobic microbial count, which is limited for each category of medicinal products.

If no Microbial growth is observed on the nutrient medium, the results are recorded as follows: for a 1:10 dilution of the medicinal product — "1 g (or 1 mL) of the medicinal product contains less than 10 bacteria (or fungi)"; for a 1:100 dilution of the medicinal product — "1 g (or 1 mL) of the medicinal product contains less than 100 bacteria (or fungi)", and so forth.

The number of microorganisms per 1 g or 1 mL is calculated using the formula:

Where N is the number of microorganisms in 1.0 g or 1.0 mL, c is the sum of colonies on all Petri dishes, n is the number of Petri dishes, d is the sample dilution factor, and 10 is the conversion factor for plating a 0.1 mL volume.

Example. When plating from a 10^-2 dilution, 168 and 215 colonies grew on two plates

The obtained result is recorded as 1.9×10⁴ colony-forming units (CFU).

If it is necessary to calculate the total number of microorganisms (total bacteria and fungi) per 1 g or 1 mL of the medicinal product, the number of aerobic bacteria should be added to the number of fungi.

Due to the fact that medicinal plant materials (MPM), representing medicinal plants or parts thereof (leaves, flowers, herbs, fruits, seeds, bark, roots, rhizomes, etc.), are heterogeneous regarding the content of aerobic bacteria and fungi, the acceptable limits of microbial contamination for MPM are interpreted as follows:

• If the microbial count per 1 g does not exceed 10⁵ CFU, a maximum of 5 x 10⁵ CFU is permitted

• If the microbial count per 1 g does not exceed 10⁷ CFU, a maximum of 5 x 10⁷ CFU is permitted, and so forth

For other categories of medicinal products (excluding MPM), the acceptable limits of microbial contamination are interpreted as follows:

• If the microbial count per 1 g or 1 mL does not exceed 10² CFU, a maximum of 2 x 10² CFU is permitted

• If the microbial count per 1 g or 1 mL does not exceed 10³ CFU, a maximum of 2 x 10³ CFU is permitted, and so forth

Variations of the pour plate method (deep, double-layer, and modified) can be used for testing various dosage forms, regardless of the level of microbial contamination. The surface spread plate method is preferred when testing medicinal products with a high level of microbial contamination. To shorten the time required to obtain quantitative results for bacteria and fungi prone to swarming, a modified plating method is used.

23.10 Membrane Filtration Method

is used for the quantitative and qualitative determination of microorganisms in medicinal products with or without antimicrobial activity, in particular for solutions and water-soluble medicinal products, as well as for fat-containing preparations soluble in isopropyl myristate.

23.10.1 Test Conditions

The membrane filtration unit must be designed to allow easy removal of the filter and its subsequent transfer to nutrient media. Membrane filters with a pore diameter of not more than 0.45 µm, capable of effectively retaining microorganisms, must be used, which must be verified by validation. The membrane material should be selected so that the Components of the test preparation do not affect its efficacy. Cellulose nitrate filters are used for aqueous, oily, and dilute alcoholic solutions (less than 30%), while cellulose acetate filters are used for alcoholic solutions (greater than 30%), acids, and alkalis. Membrane filtration is carried out under aseptic conditions using a vacuum.

During the test, the sample is generally dissolved in a buffer solution at a 1:10 ratio. First, a rinsing fluid (approximately 5 mL) is introduced into the filter funnel to wet the filter. An amount of the sample solution corresponding to 1 g of the test sample is added and immediately filtered. If the medicinal product exhibits antimicrobial activity, 0.9% sodium chloride solution or the fluids described below (No. 1, No. 2, No. 3) are used to wash the membrane, by passing at least three 100 mL portions of a suitable sterile rinsing fluid through the filter. If necessary, surfactants (e.g., Tween-80) or antimicrobial inactivators may be added to the rinsing fluid. No more than 500 mL of liquid should be passed through a single membrane. Fewer than three portions of rinsing fluid may be used for membrane washing provided the method has been validated.

Washings from transdermal patches are passed through membrane filters in 50 mL portions (corresponding to 1 patch) through each membrane.

Upon completion of the filtration process, the membranes are transferred to appropriate nutrient media poured into Petri dishes or flasks containing liquid nutrient media. The plates with filters are inverted. Inoculations in plates and flasks are incubated under standard conditions.

Colony counting is performed after 48-72 h (preliminary results) and after 5 days (final results). Plates where the number of bacterial colonies on the filters does not exceed 100 and fungal colonies does not exceed 50 are selected, and the number of microorganisms per 1.0 g or 1.0 mL of the sample or per 1 patch is calculated. If the filter contains a higher number of microorganisms, a series of consecutive dilutions of the sample is prepared and a suitable one is selected.

Evaluation of results on liquid nutrient media is carried out in accordance with Section 6.

To determine whether the membranes have been completely washed free of the filtered preparation possessing antimicrobial activity, 1 mL of a test microorganism suspension corresponding to the category of the test sample is added to the last portion of rinsing fluid after solution filtration. The number of each individual microorganism added must not exceed 100 CFU per 1 mL.

Growth of the test strains on the filters confirms the absence of antimicrobial activity of the medicinal product. If antimicrobial activity persists, specific or nonspecific inactivators are used, or the volume of the rinsing fluid is increased.

23.10.2 Filter Rinsing Fluids

✵ Sterile 0.9% sodium chloride solution, pH 7.0

✵ Fluid No. 1: Dissolve 1 g of meat peptone in 1000 mL of water, filter or centrifuge for clarification, dispense into flasks, and sterilize. pH after sterilization 7.0±0.2

✵ Fluid No. 2: Add 1 mL of Tween-80 to 1000 mL of Fluid No. 1, dispense into flasks, and sterilize. pH after sterilization 6.9±0.2. Fluid No. 2 is used if the preparation contains oil.

✵ Fluid No. 3: Dissolve 5 g of meat peptone, 3 g of meat extract, and 10 g of Tween-80 in 1000 mL of water. Dispense into flasks and sterilize. pH after sterilization 6.9±0.2.

23.11 Most Probable Number (MPN) Method

The MPN method is used when testing medicinal products with a low level of microbial contamination, as well as in cases where other methods cannot be applied. The MPN method is less sensitive and precise compared to the pour plate method or membrane filtration method. The method is used only for determining the total bacterial count, since results obtained for determining the total fungal count, especially molds, are considered unreliable.

When performing the test, the test sample is prepared as a solution, suspension, or emulsion in dilutions of 1:10, 1:100, 1:1000 using a suitable solvent. Liquid nutrient medium is dispensed into 12 sterile tubes, 9 mL in each. The tubes are placed in a rack in 4 rows of 3 tubes each.

1 mL of the test sample at a 1:10 dilution is added to the first row of tubes, 1 mL at a 1:100 dilution to the second row, and 1 mL at a 1:1000 dilution to the third row. 1 mL of the diluent used for dissolving, suspending, or emulsifying the sample is added to the tubes of the fourth row. Inoculated tubes are incubated under standard conditions for not more than 3 days.

Upon completion of the test, the number of tubes in the first, second, and third rows showing visual evidence of microbial growth is recorded. The medium in the fourth row tubes (diluent control) must remain sterile. The resulting three-digit number corresponds to the most probable Number of viable microorganisms in 1.0 g or 1.0 mL of the medicinal product (Table 51).

Table 51. Most Probable Number (MPN) of Microorganisms

Example. Microbial growth is observed in three tubes in the first row, in two tubes In the second row, and in one tube in the third row. The resulting number "321" corresponds to the value "150" according to Table 5.

Therefore, the most probable number of bacteria in 1 g or 1 ml of the test sample is 150. If the results cannot be determined precisely due to the Nature of the test preparation (e.g., medium turbidity, color change, etc.), subculturing is performed onto an appropriate liquid or agar medium to confirm the presence of microbial growth.

23.12 Enumeration and Detection of Specific Microorganisms in Medicinal Products: Specified and Quantified Microorganisms

The test involves the use of selective and diagnostic culture media described in the State Pharmacopoeia of the USSR, XII ed. [43, 44].

23.12.1 Test for the absence of bile-tolerant gram-negative bacteria

To restore the viability of potential contaminant microorganisms, preliminary incubation of the medicinal product sample in a liquid culture medium is used.

10.0 g or 10.0 ml of the test sample is transferred to 100 ml of soybean-casein digest broth (or medium no. 8), mixed, and incubated at a temperature of (22.5±2.5) °C for generally two hours, but not more than five hours. After incubation, the contents of the container are mixed (homogenizate A), and 10 ml (an amount corresponding to 1 g or 1 ml of the sample) is transferred to 100 ml of enrichment medium (Mossel broth). The cultures are incubated for 24-48 h under standard conditions. If growth appears, subculturing is performed using an inoculating loop onto Mossel agar or medium no. 4, which is incubated for 18-24 h.

If typical bacterial colonies are detected on Mossel agar, which by their staining properties are gram-negative non-spore-forming bacilli possessing cytochrome c oxidase, the sample is considered to be contaminated with bile-tolerant gram-negative bacteria.

23.12.2 Enumeration of bile-tolerant gram-negative bacteria

Three tubes containing 9 ml of Mossel broth each are used for inoculation. 1 ml of homogenizate A (corresponding to 0.1 g or 0.1 ml of the sample) is added to the first tube, thoroughly mixed, and 1 ml (corresponding to 0.01 g or 0.01 ml of the sample) is transferred to the second tube, mixed again, and 1 ml (corresponding to 0.001 g or 0.001 ml of the sample) is transferred to the third tube, changing the pipette after each step. The cultures are incubated for 24-48 h. To confirm the absence of bile-tolerant enterobacteria, subculturing is performed with an inoculating loop from each tube showing visible growth onto Mossel agar (medium no. 4), and the Petri dishes are incubated for 18-24 h. Microscopic examination of the colonies found on the solid medium is carried out. The detection of gram-negative non-spore-forming rod-shaped bacteria indicates the presence of bile-tolerant enterobacteria in the medicinal product. The most probable number of bile-tolerant enterobacteria in 1 g or 1 ml of the sample is determined from Table 52.

Table 52. Interpretation of quantitative determination results for bile-tolerant enterobacteria

Designations: + — growth present; - — growth absent

23.12.3 Test for the absence of E. coli

10 g of the test sample, dissolved or diluted 1:10 with sterile phosphate-buffered saline, is transferred in an amount of 10 ml, corresponding to 1 g or 1 ml, into 100 ml of soybean-casein digest broth (or medium no. 8). Mix and incubate for 18-24 h. If growth is present, 1 ml of the container contents is transferred into 100 ml of MacConkey broth (or medium no. 3) and incubated for 24-48 h at a temperature of (43±1) °C.

If growth is present, subculture using an inoculating loop onto MacConkey agar or medium no. 4. Incubate the cultures for 18-72 h (MacConkey agar) or 18-24 h (medium no. 4). If colonies typical of E. coli are detected on the solid nutrient media after incubation, they are examined microscopically. If gram-negative rods are found in the smears, individual typical colonies are subcultured onto sloped soybean-casein agar or medium no. 1 in tubes and incubated for 18-24 h to obtain a pure culture of the microorganism.

To identify the isolated bacteria, biochemical tests are used to determine the presence of cytochrome c oxidase, indole, and The ability to utilize sodium citrate. From the tubes with the pure culture, subcultures are made onto Simmons citrate agar (or medium no. 14) and soybean-casein digest broth (or medium no. 15). After 18-24 h of incubation, bacterial growth or its absence on Simmons agar (or medium no. 14) is recorded. Citrate utilization is indicated by a shift in the pH

of the medium toward the alkaline side (change in medium color from green to blue). The presence of indole is determined by the appearance of a red ring On the surface of the soybean-casein digest broth (or medium no. 15) upon The addition of Kovac's reagent.

If typical bacteria are found during the study, which by their staining properties are gram-negative rods, possess the enzyme cytochrome c oxidase, do not utilize sodium citrate, and produce indole, the medicinal product is considered to be contaminated with E. coli.

23.12.4 Quantitative determination of E. coli

The quantitative determination of E. coli is carried out in the same manner as the quantitative determination of bile-tolerant enterobacteria, by subculturing from homogenizate A into tubes containing MacConkey broth (or medium no. 3). If growth is detected in the tubes, subculturing is performed from each tube using an inoculating loop onto MacConkey agar or medium no. 4. The cultures are incubated under standard conditions for 18-48 h (MacConkey agar) or 18-24 h (medium no. 4).

The appearance of typical bacterial colonies on the media, which by their staining properties are gram-negative rods, indicates the presence of E. coli in the medicinal product. The most probable number of E. coli cells in 1 g or 1 ml of the sample is determined according to Table 47.

23.12.5 Test for the absence of Salmonella species

25.0 g or 25.0 ml of the test sample is transferred into 225 ml of soybean-casein digest broth (or medium no. 8), mixed, and incubated for 18-24 h. After mixing, 0.1 ml is transferred into 10 ml of Rappaport-Vassiliadis Salmonella enrichment broth and incubated under standard conditions for 18-24 h. Subculture using an inoculating loop onto one of two solid diagnostic media: xylose Lysine deoxycholate agar or bismuth sulfite agar (medium no. 5), which are incubated for 48 h.

If colonies typical of Salmonella species are detected on the specified media, microscopic examination is performed. When gram-negative rods are found in the smears, characteristic colonies are subcultured onto triple sugar iron agar containing iron salts (or medium no. 13) by applying a large amount of culture with an inoculating loop first to the slanted surface of the agar and then by stabbing the butt without touching the bottom of the tube. After 24 h of incubation under standard conditions, a color change of the medium from red to yellow at the Base of the nutrient medium butt is noted (glucose Fermentation). In the slanted part of the agar, the color of the medium remains unchanged (absence of sucrose and lactose fermentation). Blackening of the medium indicates The production of hydrogen sulfide—a typical characteristic of most species of the genus Salmonella. In parallel, the determination of cytochrome c oxidase activity, as well as other biochemical and serological tests, is carried out if additional confirmation is necessary.

If bacteria typical in their morphological and staining properties, possessing cytochrome c oxidase, failing to ferment sucrose and lactose, and producing hydrogen sulfide are found in the sample, the medicinal product is considered to be contaminated with bacteria of the genus Salmonella.

23.12.6 Test for the absence of Pseudomonas aeruginosa

A 10 ml quantity of the test sample (corresponding to 1 g or 1 ml), dissolved or diluted 1:10 with sterile buffer solution, is transferred to 100 ml of liquid nutrient medium (soybean-casein digest broth or medium No. 8). Mix and incubate under standard conditions for 24-48 h. Following incubation, if growth is observed, subculture using an inoculating loop onto a selective nutrient medium for the isolation of Pseudomonas aeruginosa (cetrimide agar or cetylpyridinium chloride (CPC) agar — medium No. 16). Incubate the cultures under standard conditions for 24-48 h. The isolated colonies of microorganisms exhibiting Gram-negative rod Morphology are subcultured onto an agar medium for the detection of the blue-green pyocyanin pigment (or medium No. 9). Incubate the cultures for 24-48 h.

To confirm the species identity of the isolated bacteria as P. aeruginosa, the presence of the cytochrome oxidase enzyme is determined, along with the ability of the isolated microorganisms to grow in soybean-casein digest broth (or medium No. 8) at (42±1) °C for 18-24 h.

When testing the quality of transdermal patches, 10 patches are placed in 500 ml of phosphate buffer solution and gently shaken for at least 15 min.

A 50 ml quantity of the resulting liquid is filtered through a sterile cellulose nitrate membrane filter with a pore diameter of 0.45 µm, which is then transferred to 100 ml of soybean-casein digest broth (or medium No. 8). Incubate the cultures for 24-48 h. Following incubation, if growth is observed, subculture using an inoculating loop onto selective media, such as cetrimide agar or CPC agar. Further identification is carried out as described above.

If the sample is found to contain bacteria with morphological and staining characteristics typical of P. aeruginosa (Table 48) that produce the blue-green pigment pyocyanin, possess the cytochrome oxidase enzyme, and grow at (42±1) °C, the medicinal product is considered contaminated with P. aeruginosa.

23.12.7 Test for the absence of Staphylococcus aureus

A 10 ml quantity of the test sample (corresponding to 1 g or 1 ml of the sample), dissolved or diluted 1:10 with sterile buffer solution, is transferred to 100 ml of soybean-casein digest broth or medium No. 8. Mix and incubate for 24-48 h. If growth is present, subculture using a loop onto mannitol-salt agar (or medium No. 10) and incubate under standard conditions for 24-48 h.

The appearance of typical golden-yellow colonies surrounded by yellow zones after incubation indicates mannitol fermentation by S. aureus. Perform a microscopic examination of the typical colonies. If Gram-positive cocci are observed in the smears, subculture onto soybean-casein digest agar (or medium No. 1). Incubate under standard conditions for 18-24 h. Perform a coagulase test for identification.

When testing the microbiological purity of transdermal patches, 10 patches are placed in 500 ml of phosphate buffer solution with gentle shaking for at least 15 min.

A 50 ml quantity of the resulting liquid is filtered through a sterile cellulose nitrate membrane filter with a pore diameter of 0.45 µm, which is transferred to 100 ml of soybean-casein digest broth (or medium No. 8) and incubated for 24-48 h. Following incubation, if growth is present, subculture using a loop onto mannitol-salt agar (or medium No. 10) to isolate S. aureus. Incubate the cultures for 48 h.

If bacteria having morphological and staining characteristics typical of S. aureus and possessing the coagulase enzyme are detected in the sample, the medicinal product is considered contaminated with S. aureus.

23.12.8 Test for the absence of Candida albicans

A 10 ml quantity of the test sample (corresponding to 1 g or 1 ml of the sample), dissolved or diluted 1:10 with sterile buffer solution, is transferred to 100 ml of Sabouraud dextrose broth, mixed, and incubated for 3-5 days at a temperature of (32.5±2.5) °C. If growth is present, subculture using an inoculating loop onto Sabouraud dextrose agar (or medium No. 2) and incubate for 24-48 h at the same temperature.

The growth of white, round, convex, shiny colonies may indicate the presence of Candida albicans, which is confirmed during further identification. One of the steps includes microscopic examination (Gram staining) revealing Gram-positive yeast-like budding oval or round cells measuring 4-8 µm. A chromogenic medium designed for the differentiation of C. albicans and other Candida species may be used for identification.

If yeast-like fungi with morphological and staining characteristics typical of C. albicans are not detected in the sample, the medicinal product is considered free from contamination by this fungal species.

23.13 Morphological and Staining CHARACTERISTICS OF MICROORGANISMS

The characteristic morphological and staining properties of certain contaminant microorganisms found in medicinal products are presented in Table 53.

23.13.1 Retesting

If necessary, when contamination of a medicinal product is detected, the section of the test whose results do not meet the regulatory requirements should be repeated. The analysis is performed using twice the number of product samples.

To identify the isolated contaminants, the biochemical tests and nutrient media described in the State Pharmacopoeia of the USSR, XII edition, are used.

23.14 Pathogens of Human Bacterial Diseases

A brief Description of the main prokaryotic representatives causing infectious diseases is given below. The order of presentation corresponds to their description in Bergey's Manual of Determinative Bacteriology.

Spirochetes — thin (0.3-1.5 µm), flexible, helically coiled bacteria. The Cell consists of a protoplasmic cylinder wound with one or more axial fibrils originating from subterminal discs. The cell is covered by an outer elastic sheath containing a thin layer of murein. Motile due to The flexibility of their body. Aerobes, facultative anaerobes, and anaerobes. Saprotrophs and parasites, pathogens causing diseases such as: Treponema pallidum — Syphilis, Leptospira interrogans — leptospirosis, Borrelia recurrentis — relapsing fever, etc.

Aerobic (microaerophilic) motile spiral bacteria. Gram-negative. Possess polar flagella. Aquatic inhabitants,

soils, predators in relation to other bacterial species, and some are pathogenic. Species of the genera Campylobacter and Helicobacter cause Stomach ulcers and gastritis. Spirillum minor causes sodoku, a zoonotic disease.

Gram-negative aerobic (microaerophilic rods and cocci).

This group includes heterotrophic microorganisms that are diverse in morphology and physiology, including the pathogenic species described below.

Bordetella pertussis — small, non-motile coccobacilli measuring 0.2–0.3 × 0.5–1.0 µm. Produces an exotoxin. The CAUSATIVE AGENT OF whooping cough.

Brucella spp. — small coccobacilli measuring 0.5–0.7 × 0.6–1.5 µm. They produce endotoxins with specific allergenic properties and pathogenicity enzymes. They cause brucellosis, a zoonotic infection.

Francisella tularensis — small coccoid, rod-shaped, and pleomorphic cells with a diameter of 0.2–0.7 µm. Causes tularemia, a zoonotic infection.

Legionella pneumophila — a slender, pleomorphic rod measuring 0.5–0.7 × 2–3 µm, motile, with polar and lateral flagella. Produces exo- and endotoxins. Causes legionellosis.

Gram-negative cocci.

Neisseria gonorrhoeae (gonococcus) — Kidney-shaped diplococci measuring 0.7–0.8 × 1.25 µm, occurring in pairs both intracellularly and extracellularly. A facultative anaerobe. Produces an endotoxin. Causes Gonorrhea as well as extragenital infections: endocarditis, meningitis, Arthritis, stomatitis, Conjunctivitis, and septicemia.

Neisseria meningitidis (meningococcus) — kidney-shaped diplococci 0.6–1.0 µm in diameter, possessing a capsule. A facultative anaerobe. Produces exo- and endotoxins. Causes nasopharyngitis, meningitis, and septicemia. It primarily affects children aged 1–5 years.

The genus Pseudomonas includes over 70 species, among which are pathogens, opportunistic infection agents, saprotrophs, and phytopathogenic species. They can contaminate pharmaceutical preparations and produce toxins. Due to their high enzymatic activity and adaptability, they hold promise for industrial waste Treatment systems. In addition, they can be used as producers of Amino acids and antibiotics, and as recombinant DNA recipients in Genetic Engineering.

Pseudomonas aeruginosa (pyocyanic bacterium) — cells measuring 0.5–0.8 × 1.5–3 µm, monotrichous. Aerobic. Can grow at temperatures of 4–41°C. Produces a blue-green pigment. Causes local and generalized purulent processes: otitis media, pyelitis, cystitis, keratitis, meningoencephalitis, and infects wound and burn surfaces. Resistant to antiseptics.

Table 53. Morphological and staining Properties of Individual microorganisms

Pseudomonas mallei — cells measuring 0.5–1.5 × 4.0 µm, non-motile. A facultative anaerobe. The causative agent of glanders, which affects horses, donkeys, and mules. Humans become infected through contact with sick animals.

Pseudomonas pseudomallei — cells measuring 0.8 × 1.5 µm, lophotrichous. A facultative anaerobe. The causative agent of melioidosis, a zoonotic infection.

Facultatively anaerobic Gram-negative rods

The family Enterobacteriaceae includes over 115 species belonging to 30 genera. These are straight rods measuring 0.3–1.8 µm. They are either motile (peritrichous) or non-motile. They are ubiquitous: found in soil, water, on plants, and in animals. Some of them are pathogenic and cause Diseases of the gastrointestinal, respiratory, and urinary tracts, as well as meningitis and wound infections. About 50% of nosocomial infections are caused by species of this family. The most frequently encountered are Escherichia coli, Serratia marcescens, and species of the genera Klebsiella, Enterobacter, Proteus, and Providencia.

The genus Escherichia includes members of the normal microbiota of warm-blooded animals. E. coli is an inhabitant of the human Large Intestine and is subdivided into several groups that differ in their biological properties. Pathogenic strains produce enterotoxins, invasion factors, and colonization factors that ensure their penetration and multiplication in organs. They cause acute intestinal diseases and other escherichioses (Peritonitis, meningitis, enteritis, cystitis, pyelitis, Pyelonephritis, otitis media, foodborne toxicoinfections, etc.). Non-pathogenic strains are used to produce colibacterin for the treatment and Prevention of dysbiosis, serve as producers of certain enzymes, and are widely utilized in genetic engineering. Escherichia coli is a sanitary-indicator microorganism used to assess the state of water and soil.

The genus Salmonella includes species (about 2,000 serovars) that are pathogenic for humans and many animals. In humans, they cause typhoid fever, paratyphoid fever, salmonellosis, and toxicoinfections. They produce endotoxins with pronounced toxic properties.

The genus Shigella. Bacteria of this genus differ from other enterobacteria by the absence of flagella. S. dysenteriae produces an exotoxin with a pronounced tropism for The Nervous system and intestinal mucosa. Other species of shigellae produce endotoxins. They cause dysentery.

The genus Yersinia includes species pathogenic to humans. The plague pathogen Y. pestis is extremely virulent to humans and produces exotoxins. Y. pseudotuberculosis causes pseudotuberculosis, and Y. enterocolitica causes yersiniosis. These microorganisms are capable of surviving for long periods in the external environment. Y. enterocolitica multiplies at -4°C, on food products in refrigerators.

The genus Klebsiella. Members of this genus are distinguished by their ability to form a capsule.

K. pneumoniae produces an exotoxin. It is the causative agent of bronchopulmonary diseases, and occasionally meningitis; in children, it can cause septicemia, cystitis, and other conditions.

K. ozaenae is the causative agent of a chronic respiratory tract disease.

K. rhinoscleromatis causes chronic granulomatous or atrophic processes in the mucous membrane of the Upper Respiratory Tract.

Genus Proteus. Species of this genus inhabit the human intestine and are found in water, soil, and food products. They cause food-borne toxicoinfections, dyspepsia in children, purulent processes (otitis, cystitis, conjunctivitis), and wound infections.

The family Vibrionaceae comprises straight or Curved Rods, 0.3-1.3x1.4-5.0 µm in size, with polar flagella (non-motile species also exist).

Genus Vibrio includes about 10 species pathogenic to humans. They cause wound, intestinal, and extraintestinal infections. V. cholerae is the causative agent of cholera, V. parahaemolyticus causes food poisoning, and V. vulnificus causes septicemia with a high mortality rate. V. cholerae produces an exotoxin (choleragen) and pathogenicity enzymes, while all pathogenic vibrios contain endotoxins.

Family Pasteurellaceae — rods ranging from coccoid to straight, 0.2-0.4x0.4-2.0 µm. Parasites of vertebrates. Haemophilus influenzae causes meningitis in children, septicemia, Bronchitis, and otitis media. H. ducreyi is the causative agent of Chancroid.

Rickettsiae and Chlamydiae

Rickettsiae — polymorphic coccoid cells about 0.5 µm in diameter, rod-shaped cells 1-1.5x3-4 µm, or filamentous cells; they are Gram-negative and reproduce by binary fission. Obligate parasites. Causative agents of rickettsioses: epidemic typhus, endemic (murine) typhus, Mediterranean spotted fever, Q fever, etc.

Chlamydiae — intracellular parasites with a diameter of 0.3-0.45 µm. Their developmental cycle involves The formation of a large (0.8-1.5 µm) cell that multiplies by division. Daughter cells reorganize into small (0.2-0.4 µm) elementary bodies possessing infectious properties. In humans, chlamydiae cause trachoma, conjunctivitis, lymphogranuloma venereum, and psittacosis.

Gram-positive cocci

This group includes saprotrophic microorganisms, including the dextran producer Leuconostoc spp., as well as opportunistic and pathogenic cocci described below.

Staphylococcus aureus — spherical cells typically 0.8-1 µm in diameter, arranged in irregular clusters. Facultative anaerobe. Forms golden, lemon-yellow, and white pigments. Synthesizes over 25 proteins, toxins, and pathogenicity enzymes. Causes inflammatory processes of various localization and severity: abscesses, furunculosis, Osteomyelitis, dermatitis, Pyoderma, peritonitis, enterocolitis, conjunctivitis; consumption of contaminated food leads to toxicoinfections.

Streptococcus pyogenes — spherical cells 0.6-1 µm in diameter, arranged in pairs and chains, forming a microcapsule. Facultative anaerobe. Synthesizes toxins (hemolysins, leukocidin, nephrotoxin, etc.) and pathogenicity enzymes. Causes purulent (pneumonia, erysipelas, impetigo, tonsillitis, sepsis) and non-purulent diseases (scarlet fever, rheumatism, etc.).

Endospore-forming Gram-positive bacilli

This is a diverse group of microorganisms comprising both saprophytic and pathogenic species. It includes producers of enzymes and antibiotics (Bacillus subtilis, B. polymyxa, B. brevis) as well as other BIOLOGICALLY ACTIVE SUBSTANCES. Pathogenic species are described below.

Bacillus anthracis — rods measuring 1.0–1.2 × 3–5 µm, arranged in chains, with truncated or slightly concave ends. Spores are oval and centrally located. Form a capsule. Non-motile. Facultative anaerobe. Produces an exotoxin. The causative agent of the zoonotic infection anthrax.

Clostridium tetani — rods measuring 0.5–1.1 × 2.4–5 µm. Spores are spherical and terminal, giving the microbe a drumstick appearance. Peritrichous. Strict anaerobe. Produces an extremely potent exotoxin. The causative agent of tetanus.

Clostridium perfringens — polymorphic rods with rounded ends, 0.9–1.3 × 3–9 µm. Spores are oval, subterminally located, and their diameter exceeds that of the clostridium itself. Forms a capsule. Non-motile. Anaerobe. Produces a group of toxins and pathogenicity enzymes. The causative agent of anaerobic infection.

Clostridium novyi — polymorphic rods with rounded ends, 1.4–2.5 × 4.7–22.5 µm, arranged in short chains. Spores are oval and subterminally located. Peritrichous. Strict anaerobe. Produces a group of toxins. The causative agent of anaerobic infection.

Clostridium septicum — polymorphic rods, 1.1–1.6 × 3.1–14.1 µm. Spores are rounded, centrally or subterminally located. Peritrichous. Strict anaerobe. Produces a group of toxins and pathogenicity enzymes. The causative agent of anaerobic infection.

Clostridium botulinum — polymorphic rod with rounded ends, 0.3–1.3 × 4.4–8.6 µm. Spores are oval, subterminally located, giving the cell the appearance of a tennis racket. Peritrichous. Strict anaerobe. Produces an extremely potent exotoxin (neurotoxin). Toxin production can occur in food products (sausages, meat, fish, vegetable, and mushroom canned goods, etc.). Botulinum toxin is thermostable and resistant to gastric juice. The causative agent of botulism.

Irregular non-spore-forming Gram-positive bacilli

This group includes microorganisms important in biotechnology as producers of biologically active substances (genera Acetobacterium, Arthrobacter, Brevibacterium), a key member of the normal human microbiota, Bifidobacterium bifidum, as well as pathogenic species.

Corynebacterium diphtheriae — polymorphic straight or slightly curved rods, 0.3–0.8 × 1–8 µm, with club-shaped thickenings at the ends. Facultative anaerobe. Produces potent exotoxins. The causative agent of diphtheria.

Actinomyces bovis — polymorphic rod-shaped or coccoid cells; branching filaments with a diameter of 0.5–1.2 µm may occasionally form. Facultative anaerobe. The causative agent of actinomycosis.

Actinomyces israelii has a structure similar to A. bovis, differing from it in antigenic structure. The causative agent of actinomycosis.

Mycobacteria. This group includes Gram-positive microorganisms capable of forming branched structures. They are acid-fast Saprophytes inhabiting the soil and found in The Human Body, alongside pathogenic species.

Mycobacterium tuberculosis — polymorphic straight or slightly curved rods, 0.3–0.6 × 1–4 µm, occasionally featuring slight swellings at the ends. Aerobic. Produces endotoxin. The causative agent of tuberculosis.

Mycobacterium leprae — cell morphology is similar to M. tuberculosis. An intracellular parasite. Within cells, they are arranged in groups resembling bundles of cigars. Produces endotoxin. The causative agent of leprosy.

Mycoplasmas or Mollicutes: bacteria lacking a Cell wall. The absence of murein and a rigid cell wall determines pronounced cellular polymorphism, allowing for spherical, filamentous, branched, and filterable forms. Anaerobes and facultative anaerobes. Require specialized media for cultivation. Sensitive to biocides. Saprophytes (found in soil and wastewater) as well as parasites of plants, animals, and humans. Pathogenic species (Mycoplasma pneumoniae, M. hominis) affect the respiratory, cardiovascular, urogenital, and central nervous systems.

Conclusion

Microbial contamination of medicinal products (MPs) can lead to a loss of therapeutic activity due to the enzymatic degradation of active ingredients. Microorganisms contaminating MPs may cause infectious and non-functional diseases (such as poisonings caused by microbial toxins and drug degradation products). The microbiological purity of medicinal products is regulated by the XII Pharmacopoeia.