PHARMACEUTICAL MICROBIOLOGY - V. A. Galynkin - 2015

PART III. MICROBIOLOGICAL ASPECTS OF PHARMACEUTICAL MANUFACTURING

CHAPTER 21. MICROBIAL ECOLOGY AND ITS RELATION TO THE PHARMACEUTICAL INDUSTRY

21.1 Human Normal Microbiota

Under physiological norms, The Human Body harbours hundreds of species of microorganisms, among which Bacteria dominate, while Viruses and Protozoa are represented by a significantly smaller number of species [37, 39]. The overwhelming majority of such microorganisms are commensal saprotrophs and cause no visible harm to the host. Just as in the environment, microbes in the human body exist in the form of biocenoses. In total, the human biocenosis is inhabited by 10 ¹⁴-10 ¹⁵ microbial Cells represented by more than 500 species, some of which remain unstudied to this day. Each individual is characterised by specific microbial communities formed in the course of their life activity.

The term "normal microbiota" encompasses species frequently isolated from the body of a healthy human (Table 43). Information on the Qualitative and quantitative COMPOSITION OF THE healthy human microbiota is extremely important for pharmacists and biotechnologists, since humans are permanent participants in production processes and a potential source of microbial Introduction into them. The composition of the microbiota in different biotopes (topos — place) is influenced by the state of the hormonal, immune, nervous, and other systems of the macroorganism. A significant role in altering the composition of microbial associations is played by dietary habits, irrational drug therapy, the presence of somatic (non-infectious) diseases, and working conditions [40].

Class="center">Table 43. Representatives of the human normal microbiota and their isolation frequency

++++ — isolated almost always.

+++ — usually isolated.

++ — frequently isolated.

+ — isolated occasionally.

Organs and Tissues that do not come into contact with the external environment are free of microorganisms. Normally, The Heart, Blood, Lymph, Brain, cerebrospinal and synovial fluids, Urinary Bladder, Uterus, and deep tissues are sterile. The main PARTS OF THE body colonised by bacteria include the skin, Upper Respiratory Tract, gastrointestinal tract, and Urogenital System.

21.2 Skin Microbiota

Transient microbiota can be numerous and diverse, including opportunistic and pathogenic microorganisms originating from other biotopes of the body or the external environment, which rapidly perish due to the bactericidal Properties of the skin, sebaceous secretions, and antagonistic interactions with the resident microbiota.

Permanent residents include Staphylococcus epidermidis, St. saprophyticus, micrococci (sarcinae), and diphtheroids. Their permanent habitat is the stratum corneum of the skin, sebaceous gland ducts, and Hair follicles. The microbiota of the hair cover has an identical composition. Typically, 10³-10⁴ microorganisms are detected per 1 cm², but in areas with high humidity, this figure can reach 10⁶. In some individuals, streptococci and Gram-positive spore-forming rods are found on the skin.

Streptococci are fastidious regarding nutrient media and require The addition of special supplements (growth factors); upon cultivation, they are detected on media supplemented with animal erythrocyte mass (sheep). Based on their reaction with haemoglobin, streptococci are divided into 3 groups:

α-streptococci (viridans) grow on the medium as colonies surrounded by greenish zones, with a greyish-green colony colour;

β-streptococci form colonies surrounded by transparent zones due to erythrocyte lysis;

γ-streptococci do not cause erythrocyte haemolysis.

Streptococci can be considered transiently circulating (inconstant) species.

The highest number of microorganisms inhabits skin folds, where Fungi of the genus Candida are found. In areas where Sebaceous Glands accumulate (External ear, genitalia), non-pathogenic acid-fast mycobacteria, corynebacteria, and lipophilic Yeasts are discovered.

On the scalp, Pityrosporum ovale are frequently present (P. orbiculare on hairless skin), while dermatophytes of the genera Epidermophyton, Microsporum, and Trichophyton are also found. Among the permanent inhabitants of the skin, St. aureus is found in 5-10% of healthy individuals.

21.3 Oral Cavity Microbiota

In this biotope, the microbiota is particularly diverse, which is facilitated by the presence of moisture and nutrients, as well as mildly alkaline pH values. Many opportunistic microorganisms belonging to the normal microbiota play a significant role in The Development of dental caries, periodontal diseases, and Diseases of the oral mucosa. The presence of saliva in the oral cavity, with its bactericidal components, Lysozyme, IMMUNOGLOBULINS, and certain lytic Enzymes, limits the ability of oral microbes to act as disease agents. Among the microorganisms of the oral cavity, autochthonous (native to the biotope) and allochthonous species — immigrants from other biotopes (nasopharynx, intestine) — are encountered.

Streptococci dominate among bacteria, accounting for 30–60% of the total microorganism count. Most frequently, these are viridans group streptococci of low virulence, exhibiting specific tissue tropism: Str. mitior colonizes the buccal epithelium, Str. salivarius adheres to the lingual papillae, and Str. mutans and Str. sanguis target the tooth surfaces. They ferment CARBOHYDRATES and produce peroxides. A pH shift toward the acidic side leads to the decalcification of tooth enamel. Another important characteristic is their ability to synthesize Polysaccharides from sucrose. The glucose moiety of the molecule is converted into dextran (α-1,6-glucan), whereas fructose is transformed into fructan (levan). Insoluble dextran promotes The formation of dental plaques, which facilitate the attachment of microbial cells to dental tissue. Fructan can serve as a substrate for subsequent acid-production processes even in the absence of exogenous dietary carbohydrates.

Other Gram-positive cocci include peptococci, which exhibit weak saccharolytic activity but break down peptone and Amino Acids. Peptococci are most commonly found in mixed infections associated with fusobacteria and spirochetes in cases of dental caries, pulpitis, and periodontitis.

Gram-negative anaerobic cocci are represented by the genus Veillonella; they do not utilize Disaccharides, yet they break down Pyruvate, lactate, and acetate into carbon dioxide and Water, thereby helping to elevate the pH and inhibit the proliferation of lactic acid-producing cariogenic bacteria. The highest amounts of lactic acid are produced by Gram-positive bacilli of the genus Lactobacillus. Gram-negative Microorganisms in the oral cavity are represented by the genera Bacteroides, Fusobacterium, and Leptotrichia. They ferment sugars into CO₂, and peptones into foul-smelling metabolic products.

Bacteroides produce the enzymes collagenase and hyaluronidase, which destroy tissue and contribute to the progression of periodontal diseases. Fusobacteria are spindle-shaped bacilli that inhabit gingival pockets in association with spirochetes.

Leptotrichia appear as paired "granular" bacilli, often taking a filamentous form.

Actinomycetes form the matrix of dental calculus; they are integral components of dental plaque, participating in plaque formation and the development of dental caries.

Corynebacteria are capable of lowering the oxidation-reduction potential, thereby fostering an environment favorable for the growth of anaerobes.

Oral microorganisms are capable of entering the bloodstream, for instance, following tooth extraction or even during routine toothbrushing.

Oral spirochetes are represented by the genera Treponema (T. denticola, T. orale) and Borrelia (B. buccalis), as well as Leptospira; protozoa are represented by the genera Entamoeba (E. buccalis, E. dentalis) and Trichomonas (T. buccalis); among Mycoplasmas, Mycoplasma orale and M. salivarium are frequently encountered.

21.4 Microbiota of the Upper Respiratory Tract (URT)

The upper respiratory tract carries a particularly heavy microbial load, as it is anatomically adapted for the deposition of bacteria from inhaled air.

The nasal cavity harbors non-hemolytic and viridans streptococci, non-pathogenic Gram-negative cocci such as neisseriae, staphylococci, and corynebacteria. In some individuals, Staphylococcus aureus is constantly present, representing resident carriage.

Non-hemolytic and α-hemolytic streptococci, non-hemolytic variants of Str. pyogenes (found in 100% of individuals), non-hemolytic staphylococci, and diphtheroids are found in the Larynx. Mycoplasmas and Adenoviruses inhabit the Tissues of the Tonsils.

The small Bronchi, alveoli, and lung parenchyma are free of microorganisms.

21.5 Microbiota of the Gastrointestinal Tract (GIT)

The qualitative and quantitative composition of the gut microbiota varies across different Regions of the gastrointestinal tract. Due to low pH levels, The Stomach of a healthy individual contains 10³-10⁴ cells/mL of contents, primarily acid-tolerant microorganisms such as lactobacilli, Helicobacter pylori, and yeasts; their numbers increase in pathological conditions due to an elevated pH.

The upper sections of the Small Intestine harbor 10⁴-10⁵ cells/mL. Qualitatively, these consist of lactic acid bacteria—differing in adhesive properties from those found in the oral cavity and stomach—as well as bifidobacteria and fecal enterococci. The surface area of the small intestinal mucosa spans 180-200 m². A variety of substances, including those of microbial origin, are continuously absorbed through it into the bloodstream, and the translocation of microbial cells itself is possible even in healthy individuals, not to mention pathological states.

The microbiota of the large intestine is the most abundant and diverse. Microbial counts reach 10⁹-10¹¹ cells/mL (occasionally 10¹² cells/mL) belonging to roughly 260 species. Anaerobes are numerically predominant: bifidobacteria, bacteroides, lactobacilli, veillonellae, peptococci, and clostridia. Facultative anaerobes are represented by coliform bacteria (coliforms) and fecal enterococci.

The coliform group comprises bacteria from 4 genera of the family Enterobacteriaceae: Escherichia, Enterobacter, Citrobacter, and Klebsiella. These microorganisms are grouped together based on shared characteristics: they are Gram-negative, non-spore-forming, oxidase-negative rods that ferment glucose and lactose into acid and gas at 37°C within 24 hours.

Coliforms (predominantly Escherichia coli) rank second in Abundance after bifidobacteria, while enterococci—Enterococcus faecalis and E. faecium—rank third. Clostridia, such as Clostridium perfringens and C. sporogenes, are present in significantly smaller numbers.

The intestine also hosts the intestinal amoeba (Entamoeba coli), enteric viruses, and, in some individuals, Yeast-like fungi of the genus Candida.

21.6 Microbiota of the Urogenital System

The composition of the microbiota in these biotopes varies depending on sex, age, and organ. The Kidneys, Ureters, and urine in the urinary bladder are sterile. The lower Urethra contains non-spore-forming anaerobes: peptococci, peptostreptococci, bacteroides, mycobacteria, and Gram-negative bacteria of intestinal origin. The vaginal microbiota establishes itself with the onset of Puberty. It consists of lactic acid bacteria (Döderlein's bacilli), corynebacteria, non-hemolytic streptococci, yeast-like fungi, and protozoa, all of which thrive in the acidic environment of this organ (pH 4.0-4.2).

21.7 Significance of the Normal Microbiota

The normal microbiota of a healthy human plays a vital role in maintaining health and ensuring the proper functioning of the entire Organism. Most microorganisms inhabiting various biotopes exhibit antagonistic properties against other bacteria and viruses, particularly pathogens. For instance, bifidobacteria and lactobacilli produce organic acids (lactic and acetic acids), alcohols, lysozyme, and bacteriocins; they can actively suppress the proliferation of putrefactive bacteria in the gut and inhibit the secretion of heat-labile enterotoxin by enteropathogenic Escherichia strains.

An important mechanism of pathogen suppression is the selective binding of epithelial Cell surface receptors by members of the normal microbiota. The normal intestinal microbiota AIDS in Digestion: it breaks down complex organic substances that are difficult to digest, and influences mucosal Morphology and absorptive capacity. Lipid METABOLISM, Bile acid degradation, protein breakdown into end products, nutrient absorption processes, peristalsis, and many other Functions are closely linked to microbial activity. Microorganisms participate in the detoxification of xenobiotics entering from the environment as well as toxic metabolic byproducts. Butyric acid, produced during the anaerobic Fermentation of dietary fiber by intestinal microorganisms, promotes the formation of a specific enzyme that inactivates the Gene responsible for malignant cell transformation.

The normal microbiota enhances the intestinal absorption of Fe⁺² and Ca⁺² ions, as well as vitamin D, and participates in the synthesis of Vitamins K and B-complex vitamins, particularly B₁, B₂, Folic acid, nicotinic acid, and pantothenic acid. A high concentration of bifidobacteria and lactobacilli prevents the development of numerous pathological processes and even carcinogenesis.

The normal microbiota contributes to the maturation and Maintenance of the immune system. In germ-free animals (gnotobiotics), the mass of Lymph Nodes is reduced several-fold compared to conventional animals. Gnotobiotics cannot survive in ordinary environments exposed to microbial flora and perish from bacteria and viruses that pose no threat to animals raised under standard conditions. Even in a sterile environment, gnotobiotics rapidly die from intestinal dysfunction associated with impaired digestion, absorption, and detoxification caused by the absence of normal microbiota.

During normal colonization of mucous membranes, bacteria act as Antigens, inducing The production of Antibodies (IgA), which form The basis of local Immunity, prevent pathogens from penetrating tissues, and help maintain mucosal Homeostasis.

Heat generated during the metabolism of intestinal microbes helps maintain a constant body Temperature in warm-blooded animals.

Metabolic products of the normal microbiota constantly enter the bloodstream, influencing the metabolism of the macroorganism. A disruption in the Symbiosis between the micro- and macroorganism leads to severe consequences: Metabolic Disorders, allergic, skin, and oncological diseases, and even psychiatric disorders.

The normal microbiota can trigger the development of infectious diseases, most of which are opportunistic in nature. For instance, intestinal anaerobes (bacteroides) entering the intestinal wall through trauma can cause abscesses, while the primary causative agents of post-Influenza Pneumonia are considered to be microorganisms residing in the nasopharynx of any healthy individual. The leading role in the development of such lesions is played not by the virulence of the pathogen itself, but by the weakening of the macroorganism's defense systems (immunodeficiency).

21.8 Dysbacteriosis (Dysbiosis)

Dysbacteriosis manifests as a disruption in the qualitative and quantitative composition of the microbiota and its translocation into other biotopes. The development of dysbiosis is facilitated by the prolonged use of Antibiotics and antiseptics, which suppress certain microbial species while leaving others unaffected. Antimicrobial therapy is accompanied by dysbiosis in 90% of cases. An important contributing factor is the suppression of local and systemic immunity resulting from hormone therapy, immunosuppressants, Radiation therapy, infectious and allergic diseases, and inflammatory processes. Stress is another major cause of dysbiosis. High-risk groups include individuals under constant pressure—pilots, sailors, businesspeople, athletes, doctors, journalists—as well as residents of ecologically unfavorable areas and the elderly. General environmental degradation, poor water quality, and inadequate, unbalanced Nutrition can also trigger dysbiosis. Intestinal dysbiosis is the most common form, manifesting as digestive dysfunction, general malaise, abdominal pain, and flatulence. Prolonged imbalance of intestinal microorganisms can provoke allergic conditions such as Bronchial Asthma, Chronic Bronchitis, and rheumatoid Arthritis, among others.

Intestinal dysbiosis is diagnosed using microbiological Methods. Based on the culture results of the intestinal microbiota, the following are determined:

1) the total count of Escherichia coli strains with typical enzymatic activity;

2) the presence of hemolytic E. coli strains;

3) the presence of other opportunistic microorganisms;

4) the presence of bacteria of the genus Proteus;

5) the presence of fungi of the genus Candida;

6) the quantitative content of bifidobacteria, lactobacilli, and bacteroides.

Table 44 shows the composition of the normal intestinal microbiota. Eubiotic (probiotic) preparations containing live, freeze-dried bacterial cells are used to correct dysbiosis (Table 45).

Table 44. Content of various bacteria in the feces of healthy adults

Table 45. Eubiotics (Probiotics) used for the Treatment and Prevention of dysbiosis

21.9 Environmental Microbiota. Sanitary Indicator Microorganisms

Microorganisms are ubiquitous in the Earth's biosphere, and their metabolic activity plays a crucial role in the biogeochemical cycles of carbon, nitrogen, sulfur, phosphorus, and other elements, while maintaining dynamic equilibrium in the biosphere. The natural habitats of microorganisms include water, soil, and the bodies of plants, animals, and humans. Sanitary microbiology studies environmental microbiota—including free-living and parasitic microorganisms—as well as The impact of microbiota on ecological conditions and human health. The primary objective of sanitary microbiology is the early detection of pathogenic microbiota in the environment. Humans and warm-blooded animals serve as the Main sources of infectious disease agents. The largest quantities of these pathogens enter the environment via airborne droplets and fecal matter.

Despite the development of rapid and direct quantitative detection methods, the direct identification of pathogenic microorganisms presents several challenges:

— pathogenic microorganisms are not constantly present in the environment; they are easily detected during epidemic outbreaks but difficult to find during inter-epidemic periods;

— the number of pathogenic microorganisms entering the environment is significantly lower than that of the resident microbiota, and pathogens are distributed unevenly across environmental objects;

— when inoculated onto nutrient media, pathogens suffer from competition with saprotrophs; being poorly adapted to environmental survival, they require The Use of "rich" and therefore costly culture media.

A negative result when testing for pathogens in environmental objects does not definitively rule out their presence. In sanitary microbiology, the assessment of various objects is carried out indirectly by establishing contamination via Human and Animal excreta; the heavier the contamination, the higher the probability of pathogens being present in the object.

The composition of the resident microbiota in various biotopes of the human body is relatively stable and changes very little during infectious diseases. For many species, the oral cavity, intestine, and upper respiratory tract serve as their sole habitat. The detection of such microorganisms in any given object indicates contamination with the corresponding excreta. For example, detecting normal intestinal inhabitants allows one to conclude that fecal contamination is present, along with the potential risk of typhoid fever bacilli, dysentery bacilli, and other causative agents of intestinal infections.

Microbes isolated in such cases serve as indicators of sanitary unreliability and the potential hazard of the tested objects, and are therefore referred to as sanitary-indicator microorganisms (SIMs). However, not all microorganisms comprising the resident microbiota of the human body qualify as SIMs.

21.9.1 Basic Requirements for Sanitary-Indicator Microorganisms [28, 37]

1. Such microorganisms must be constantly present in the excreta of humans and warm-blooded animals and enter the environment in large quantities.

2. They must not have any natural reservoir other than the human and animal organism.

3. Following release into the environment, they must remain viable for periods comparable to the survival times of pathogens entering the environment via the same route.

4. They must not multiply in the environment.

5. Microbes must not have environmental "doppelgängers" or analogs with which they can be confused.

6. They must not alter their biological properties in the external environment.

7. They must be sufficiently typical so that their identification can be performed without undue difficulty.

8. Identification methods must be simple, accessible, and cost-effective.

21.9.2 Principles and Methods of Sanitary-Microbiological Research

When conducting sanitary-microbiological studies, the following requirements must be met.

1. Proper sampling.

Sampling is carried out in compliance with all necessary aseptic rules; storage and transport must prevent both the death and additional proliferation of microorganisms. If immediate analysis is not possible, the material should be stored for no longer than 6–8 hours.

2. Serial analyses.

Microorganisms in environmental objects are distributed extremely unevenly. To obtain adequate results, a series of samples is taken from different parts of the object. During analysis, all samples are mixed to obtain a representative composite sample.

3. Repeat sampling.

To obtain comparable results, repeat sampling is performed because the composition of the microbiota in the tested samples changes quite rapidly.

4. Use of standard Research Methods.

The use of standardized and approved methodologies ensures that comparable results can be obtained across different laboratories.

5. Use of a test battery is necessary to obtain adequate information by combining Direct and Indirect METHODS OF MICROORGANISM detection, taking into account the Influence of Environmental factors and the object's own indigenous microbiota.

Modern sanitary microbiology strives to employ simple, precise, and reliable methods. These are aimed at determining total microbial contamination and identifying indicator microorganisms (IMs), and include:

— direct microscopic counting of microorganisms in the sample;

— methods for isolating and identifying microorganisms;

— biological methods using laboratory animals.

Direct counting is used in emergency cases when There is a need

for a rapid assessment of bacterial concentrations (e.g., during water supply system failures or when evaluating the efficiency of wastewater treatment plants, etc.). Its main drawback is the inability to obtain an exact count due to bacteria forming clumps or attaching to environmental particles. Furthermore, this method does not distinguish between live and dead bacteria.

Inoculation onto nutrient media is performed for quantitative enumeration. On solid media, the number of grown colonies is counted, assuming that each colony originates from a single viable cell. This method is imprecise because it only detects groups of microorganisms capable of growing on specific media at a given temperature. It is impossible to create a universal medium suitable for all microorganisms. In addition, not all microorganisms present in the sample form colonies on a nutrient medium due to competition and antagonism.

The concentration of viable cells in a sample is expressed by the total bacterial count (TBC) per g (mL) of the analyzed material. The concentration of IMs is expressed in titers and indices:

IM titer — the smallest volume of the test material in g (mL) in which a single viable IM cell is detected.

IM index — the number of IM cells detected in a specified volume (mass): per 1 L for water, and per 1 g for soil.

The index is the reciprocal of the titer.

21.10 CHARACTERISTICS OF THE Main Groups of IMs

IMs are conventionally divided into 3 groups.

The first group includes inhabitants of the human intestine, which serve as indicators of fecal contamination. This group comprises coliform bacteria, enterococci, sulfite-reducing clostridia (including Clostridium perfringens), and coliphages.

The second group includes inhabitants of the upper respiratory tract and nasopharynx. They serve as indicators of airborne environmental contamination. This group comprises α- and β-streptococci and staphylococci.

The third group includes saprotrophic microorganisms inhabiting the external environment. These are indicators of self-purification processes. This group comprises ammonifying and nitrifying bacteria, certain spore-forming bacteria, actinomycetes, cyanobacteria, and fungi.

21.10.1 Coliform bacteria

The advantage of these bacteria as IMs is that they are permanent inhabitants of the intestine and are constantly excreted with feces into the environment in large quantities, with their numbers far exceeding those of other intestinal microorganisms.

Currently, in accordance with regulatory and technical documentation, the IMs are:

a) coliform bacteria (total), which ferment only lactose with the production of acid and gas at 37°C within 24 hours;

б) fecal coliforms (thermotolerant), which ferment only lactose with the production of acid and gas at +43–+44.5°C.

Escherichia coli itself, as a sanitary-indicator microorganism, has several drawbacks.

1. Its environmental counterparts can be found in nature, creating The Need for additional biochemical tests for identification.

2. It is less resistant to adverse environmental factors (pH fluctuations, elevated chemical concentrations) than certain pathogens.

3. Escherichia coli is capable of multiplying in water when organic matter concentrations are at least 0.28 µg/mL.

4. It is not always possible to accurately assess the epidemic risk based solely on the E. coli count; for instance, waterborne salmonellosis outbreaks have been recorded at bacterial concentrations of 17 cells/L, whereas the E. coli count did not exceed 4 cells/L.

21.10.2 Enterococci

All species and variants of enterococci are of sanitary indicator value and meet A number of criteria required for sanitary indicator microorganisms (SIM).

1. They are permanent inhabitants of the intestine, although their numbers are lower than those of E. coli.

2. They are incapable of multiplying in the external environment (more precisely, they can multiply when organic matter is present at 375 µg/L and the temperature is 20°C or higher).

3. They do not exhibit marked Variability in the external environment, which facilitates their identification.

4. They have no analogues in the external environment.

5. They die off in the external environment much earlier than E. coli and therefore consistently indicate recent fecal contamination.

Their primary advantage is resistance to adverse environmental factors. They withstand heating up to 65°C for 30 min, making them an indicator of pasteurization efficiency. Enterococci tolerate high NaCl concentrations (6.5-17%), allowing their use in seawater analysis. Enterococci are stable across a pH range of 3-12, which can be utilized when analyzing acidic and alkaline wastewater.

Currently, quantitative enterococcimetry has been adopted by international water standards as an additional indicator of fecal contamination, and as the primary method for detecting fecal contamination when atypical E. coli are identified.

The difficulties in detecting enterococci lie in the need to use complex media and the fact that their identification requires more time than that of coliform bacteria.

21.10.3 Clostridia

Clostridia are released into the environment with feces, but their numbers are lower than those of coliforms and enterococci, ranging from 10⁵-10 ⁶ cells/g. Clostridium perfringens and Cl. sporogenes are classified as SIM. The main biochemical feature used to identify clostridia is their ability to form black colonies on iron-sulfite medium due to FeS production. This medium allows the differentiation of fecal clostridia from those inhabiting the external environment. Intestinal clostridia reduce sulfites and cause the medium to blacken, whereas free-living ones lack sulfite reductase and do not change the color of the medium. It should be noted that other bacteria, such as E. coli, can also cause the medium to blacken. To suppress the growth of accompanying microbiota, cultures are incubated at 43-44.5°C or heated at 80°C for 15-20 min. The simplicity of detection on Wilson-Blair and certain other media is a significant advantage of intestinal clostridia as SIM. However, they also have certain limitations.

1. Cl. perfringens can persist in the external environment for a long time due to spore formation. The detection of this microbe indicates prior fecal contamination. Given that a residual chlorine concentration of 1.2-1.7 mg/L is lethal to Cl. perfringens spores, as it is to enteroviruses, the detection of this microbe suggests the possible presence of enteroviruses in water.

2. Cl. perfringens can multiply in the external environment at temperatures not lower than 18-20°C and with a sufficient supply of nutrients, for example, in humus-rich soils of southern latitudes. Spore germination requires a thermal Shock, i.e., heating at 70°C for 15-30 min. Without heating, only 0.1-3% of spores germinate.

It has been proposed to assess the age of fecal contamination by comparing the levels of spore and vegetative forms. For this purpose, the number of clostridia is determined in unheated and heated samples. Heated samples contain only Spore Forms, which indicates long-standing fecal contamination. Unheated samples reveal both vegetative and spore forms. A high number of vegetative forms indicates fresh fecal contamination. In practice, however, the count of Cl. perfringens in unheated samples is sometimes lower than in heated ones. This is due to the antagonistic effect of the accompanying microbiota or the lack of thermal shock in unheated samples.

In domestic practice, the age of fecal contamination is judged by comparing the coliform and Cl. perfringens indices. If both indicators are high, it is concluded that fresh fecal contamination is present. A high coliform index combined with a low clostridial index points to old contamination.

21.10.4 Streptococci and Staphylococci

Hemolytic and viridans streptococci inhabit the upper respiratory tract and enter the air via saliva and sputum.

The main difficulty in using these microorganisms as SIM is that streptococci comprise a large group of disease-causing microorganisms (scarlet fever, tonsillitis, Erysipelas, etc.).

Blood Agar is used for cultivation; α-hemolytic streptococci are found in 100% of the population, and β-streptococci in 25-76%, which is why both α- and β-streptococci are considered SIM. In terms of survival time, streptococci coincide with the diphtheria pathogen and other airborne pathogenic microbes entering the human body.

The detection of α-streptococci (the least resistant) indicates recent airborne droplet contamination. Streptococci are not detected in the air of unoccupied indoor spaces.

Staphylococci enter the air from the skin surface, as well as from mucous membrane secretions of the upper respiratory tract during speech and coughing. For swimming pool discharge areas, the staphylococcal count is an important indicator of water sanitary conditions. Staphylococci survive in water longer than coliform bacteria and enterococci. However, staphylococci are primarily used as SIM for indoor air.

The superiority of these bacteria over streptococci as SIM lies in their simpler and faster detection, as well as their lack of fastidious nutritional requirements. Staphylococci exhibit resistance to various Physical and Chemical factors, making them suitable for use as water SIM in recreational areas and swimming pools.

21.10.5 Bacteriophages

It has been proposed to use enteric bacteriophages (infecting Escherichia, Salmonella, and Shigella species) as sanitary indicator microorganisms. Enteric phages are consistently found wherever their host bacteria are present. However, as indicators of the potential presence of pathogenic bacteria, they have certain limitations: 1) bacteriophages survive in the environment much longer (8–9 months) than their bacterial hosts (4–5 months); 2) they are capable of adapting to other bacterial species.

Nevertheless, they are of significant value as indicators of fecal contamination, as they are shed in wastewater with a frequency comparable to that of many enteric viruses (such as Coxsackie, hepatitis A, and poliovirus). Their resistance to disinfectants is also comparable to that of enteropathogenic viruses. Furthermore, phages can be detected using straightforward methods.

21.11 Sanitary Microbiology of Water

Water serves as a natural habitat for a diverse array of microorganisms. Representatives of all bacterial taxonomic groups, along with numerous protozoa and fungi, are found in both fresh and marine waters. The qualitative composition of the microbiota is heavily influenced by the Origin of the water body. Aquatic environments are generally classified into surface waters (rivers, lakes, reservoirs, ponds, etc.), groundwaters (soil water, subterranean water, artesian wells), and saline waters (seas, salt lakes).

The microbial communities of water bodies comprise autochthonous (indigenous aquatic) and allochthonous (introduced via contamination) microorganisms. Water bodies can become polluted through the discharge of various wastewaters, including industrial effluents (especially from food Processing plants), domestic sewage, as well as snowmelt and stormwater runoff.

Autochthonous microbiota refers to the community of microorganisms that permanently reside and multiply in water. Typically, the microbial composition of water mirrors that of the surrounding soil it comes into contact with. Common Components of the aquatic microbiota include Micrococcus candicans, M. roseus, Sarcina lutea, Pseudomonas fluorescens, and various species of Proteus and Leptospira.

Unpolluted water bodies often contain Bacillus cereus, Bac. mycoides, Chromobacterium, and Clostridium. Cyanobacteria, aquatic fungi, and protozoa are also frequently detected. Aquatic microorganisms play a vital role in biogeochemical nutrient cycles by breaking down organic matter and providing nourishment for other aquatic life forms.

When untreated municipal waste and sewage enter water bodies, microbial pollution can introduce various human pathogens responsible for infectious diseases, such as cholera, typhoid fever, paratyphoid fevers A and B, leptospirosis, yersiniosis, campylobacteriosis, tularemia, poliomyelitis, and Viral Hepatitis, among others.

Because water is not an optimal habitat for pathogens adapted to human or animal hosts, they gradually die off, allowing the water to purge itself of contaminating microbes. The primary driving force behind this self-purification is competition from the saprophytic microbiota. As Saprophytes rapidly decompose organic matter, the total microbial count—particularly of fecal origin—drops sharply. This self-cleaning capacity relies on the presence of resident species characteristic of a given aquatic biocenosis.

Based on microbial abundance, aquatic zones are categorized into polysaprobic (from sapros meaning putrid), mesosaprobic, and oligosaprobic zones.

Polysaprobic zones (heavily polluted zones) contain high concentrations of decaying organic matter and are almost entirely depleted of dissolved oxygen. Bacterial counts reach up to 10⁶ cells/ml. The species composition is largely restricted to anaerobic bacteria, fungi, and actinomycetes.

Mesosaprobic zones (moderately polluted zones) feature a diverse qualitative microbial composition, predominantly comprising nitrifying bacteria, obligate anaerobes, and representatives of Clostridium, Pseudomonas, Mycobacterium, Flavobacterium, and Streptomyces. Total microbial counts are around 10⁵ cells/ml.

Oligosaprobic zones (clean water zones) are characterized by low organic content and the final stages of mineralization. Bacterial counts typically range from 10 to 10³ cells/ml.

Sanitary and microbiological water analysis involves determining the total bacterial count (TBC)—the Number of viable microorganisms per 1 liter of water—along with the enumeration of sanitary indicator microorganisms.

Sanitary indicator microorganisms for water include total coliforms, thermotolerant coliforms, Clostridium perfringens, Cl. sporogenes, and bacteriophages. When necessary, fecal enterococci (Enterococcus faecalis) are also tested.

According to the Sanitary Rules and Norms SanPiN 2.1.4.1074-01, centralized drinking water supplies must meet the following criteria: a total bacterial count (TBC) not exceeding 50 CFU/ml; total coliforms, thermotolerant coliform bacteria, and coliphages must be entirely absent in 100 ml; sulfite-reducing clostridia must not be detected in 20 ml; and Giardia cysts must be absent in 50 ml.

21.12 Sanitary Microbiology of Soil

Soil serves as the primary reservoir and natural habitat for microorganisms involved in soil formation, self-purification, and global biogeochemical cycles [41]. The qualitative composition of soil microbiota is exceptionally diverse, encompassing predominantly spore-forming bacteria, actinomycetes, spirochetes, archaea, protozoa, cyanobacteria, fungi, viruses, and mycoplasmas. This microbial makeup varies significantly depending on soil type, tillage and management practices, organic matter content, moisture levels, climatic conditions, and other environmental factors.

Sandy soils are generally dominated by aerobic microorganisms, whereas clay soils favor anaerobes. Microbial populations peak in the ROOT zone (rhizosphere), where plant-specific root exudates create a microenvironment that stimulates intense microbial proliferation and heightened metabolic activity.

Microbial abundance in soil can reach several billion cells per gram, with the highest densities found in heavily manured soils (up to 4.8–5.2 billion cells/g) and lower counts in forest soils and sandy soils (0.9–1.2 billion cells/g).

The living biomass of microorganisms in soil can reach up to 1,000 kg per hectare. However, their distribution is uneven. Microbial diversity and density are highest at a depth of 10–20 cm, where active microbial-mediated biochemical transformations of organic matter take place. Microbial numbers decline progressively in deeper soil layers.

Just like water, soil can receive normal flora from humans and animals, as well as pathogenic microorganisms, via wastewater discharge. Typically, these foreign microbes do not survive for long in the external environment. Nevertheless, many bacteria that are part of the normal human microbiota can integrate into the soil biocenosis, and certain species even become permanent residents. Consequently, distinguishing between resident and transient soil microbiota can be challenging. Assessing The Role of soil in the transmission of infectious diseases requires an understanding of how long pathogenic microbes can survive and multiply in the soil matrix.

Depending on their survival time, microorganisms originating from human sources can be divided into three main groups.

1. Pathogenic microorganisms that naturally reside in the soil, such as Clostridium botulinum. When introduced via fecal matter, they can persist in the soil indefinitely.

2. Spore-forming pathogens for which soil acts as a secondary reservoir. These enter the soil via human and animal excreta, as well as animal carcasses. Under favorable environmental conditions, they can multiply and survive in spore form for extended periods.

3. Pathogenic microorganisms introduced with human and animal excreta that persist for several weeks or months. This group comprises non-spore-forming bacteria, their persistence time being influenced by the antagonistic activity of the soil microbiota.

Sanitary microbiological monitoring is based on two parameters: 1) the total microbial count (TMC), indicating the number of viable cells per 1 g of soil; and 2) the presence of sanitary-indicator microorganisms (coliforms, Clostridium perfringens, Cl. sporogenes, Enterococcus faecalis). A high abundance of saprophytic microbiota indicates organic pollution, whereas microbial contamination from human and animal excreta is dominated by sanitary-indicator microorganisms.

21.13 Air Sanitary Microbiology

Air is not a natural habitat for microorganisms; microbes neither feed nor are capable of multiplying in it. The viability of microorganisms in the air is sustained by suspended particles of water, mucus, dust, and soil.

Ambient air and indoor air differ significantly in the qualitative and quantitative composition of their microbiota. Bacterial contamination in residential and certain Types of Industrial premises always exceeds that of atmospheric air. Outdoor air microbiota is conventionally divided into resident (most frequently detected) and transient, which is less resistant to detrimental factors and detected sporadically.

The resident microbiota of atmospheric air is formed by soil organisms and includes Micrococcus roseus, M. flavus, M. candicans, Sarcina flava, S. rosea, Bacillus subtilis, B. mycoides, B. mesentericus, species of Streptomyces, Penicillium, Aspergillus, Mucor, and others.

The transient microbiota is formed by microorganisms originating from soil and water body surfaces.

Contamination of indoor air occurs via droplets as part of aerosols generated during talking, coughing, and sneezing. In addition, microbes are introduced with desquamated skin epithelium, clothing dust particles, and soil particles.

An aerosol is a colloidal system composed of moisture droplets and solid particles with adsorbed microorganisms. Aerosol particle sizes range from 10–100 to 2000 nm. Depending on droplet size, electrical charge, and air motion velocity, the following phases are distinguished:

The droplet phase, represented by fine droplets that persist in the air for long periods and evaporate before settling.

The dust phase, represented by large droplets that rapidly settle and evaporate. This results in the formation of dust capable of becoming airborne.

Droplet nuclei. Fine aerosol droplets (up to 100 nm) that dry out, remain suspended in the air, and form a stable aerodisperse system. They partially retain moisture, which maintains the viability of the microorganisms.

Indoor air is subject to sanitary and microbiological monitoring. It involves determining the total viable count (the number of viable microorganisms per 1 m³ of air). In healthcare facilities, sanitary-indicator microorganisms (staphylococci, α- and β-hemolytic streptococci) are additionally evaluated.

In industrial premises, the total viable count is determined, while sanitary-indicator testing is not performed. The permissible level of microorganisms in the air is determined by the cleanroom Classification required for specific technological operations.