Aerobic organisms are those organisms that are able to live and develop only in the presence of free oxygen in the environment, which they use as an oxidizing agent. Aerobic organisms include all plants, most protozoa and multicellular animals, almost all fungi, that is, the vast majority of known species of living beings.

In animals, life in the absence of oxygen (anaerobiosis) occurs as a secondary adaptation. Aerobic organisms carry out biological oxidation primarily through cellular respiration. Due to the formation of incomplete oxygen reduction during the oxidation of toxic products, aerobic organisms have a number of enzymes (catalase, superoxide dismutase) that ensure their decomposition and are absent or poorly functioning in obligate anaerobes, for which oxygen is therefore toxic.

The most diverse respiratory chain is found in bacteria that possess not only cytochrome oxidase, but also other terminal oxidases.

A special place among aerobic organisms is occupied by organisms capable of photosynthesis - cyanobacteria, algae, and vascular plants. The oxygen released by these organisms ensures the development of all other aerobic organisms.

Organisms that can develop at low oxygen concentrations (≤ 1 mg/l) are called microaerophiles.

Anaerobic organisms are able to live and develop in the absence of free oxygen in the environment. The term “anaerobes” was introduced by Louis Pasteur, who discovered butyric acid fermentation bacteria in 1861. They are distributed mainly among prokaryotes. Their metabolism is determined by the need to use oxidizing agents other than oxygen.

Many anaerobic organisms that use organic substances (all eukaryotes that obtain energy as a result of glycolysis) carry out various types of fermentation, which produce reduced compounds - alcohols, fatty acids.

Other anaerobic organisms - denitrifying (some of them reduce iron oxide), sulfate-reducing, methane-forming bacteria - use inorganic oxidizing agents: nitrate, sulfur compounds, CO 2.

Anaerobic bacteria are divided into butyric acid groups, etc. in accordance with the main product of exchange. A special group of anaerobes are phototrophic bacteria.

In relation to O2, anaerobic bacteria are divided into obligate, who are unable to use it in exchange, and optional(for example, denitrifying), which can transition from anaerobiosis to growth in an environment with O 2.

Per unit of biomass, anaerobic organisms produce many reduced compounds, of which they are the main producers in the biosphere.

The sequence of formation of reduced products (N 2 , Fe 2+, H 2 S, CH 4), observed during the transition to anaerobiosis, for example in bottom sediments, is determined by the energy output of the corresponding reactions.

Anaerobic organisms develop in conditions where O2 is completely used by aerobic organisms, for example in wastewater and sludge.

The influence of the amount of dissolved oxygen on the species composition and abundance of aquatic organisms.

The degree of oxygen saturation of water is inversely proportional to its temperature. The concentration of dissolved O2 in surface waters varies from 0 to 14 mg/l and is subject to significant seasonal and daily fluctuations, which mainly depend on the ratio of the intensity of the processes of its production and consumption.

In the case of high intensity of photosynthesis, water can be significantly oversaturated with O 2 (20 mg/l and above). In an aquatic environment, oxygen is the limiting factor. O 2 makes up 21% (by volume) in the atmosphere and about 35% of all gases dissolved in water. Its solubility in sea water is 80% of its solubility in fresh water. The distribution of oxygen in a reservoir depends on temperature, the movement of layers of water, as well as the nature and number of organisms living in it.

The tolerance of aquatic animals to low oxygen levels varies among species. Among fish, four groups have been established according to their relationship to the amount of dissolved oxygen:

1) 7 - 11 mg/l - trout, minnow, sculpin;

2) 5 - 7 mg/l - grayling, gudgeon, chub, burbot;

3) 4 mg/l - roach, ruff;

4) 0.5 mg/l - carp, tench.

Some species of organisms have adapted to seasonal rhythms in O2 consumption associated with living conditions.

Thus, in the crustacean Gammarus Linnaeus it was found that the intensity of respiratory processes increases with temperature and changes throughout the year.

Animals living in places poor in oxygen (coastal silt, bottom silt) have respiratory pigments that serve as an oxygen reserve.

These species are able to survive by switching to a slow life, to anaerobiosis, or due to the fact that they have d-hemoglobin, which has a high affinity for oxygen (daphnia, oligochaetes, polychaetes, some elasmobranch molluscs).

Other aquatic invertebrates rise to the surface for air. These are the imago of swimming beetles and water-loving beetles, smoothies, water scorpions and water bugs, pond snails and spool (gastropods). Some beetles surround themselves with an air bubble held by a hair, and insects can use air from the aerial sinuses of aquatic plants.

Anaerobes are bacteria that appeared on planet Earth earlier than other living organisms.

They play an important role in the ecosystem, are responsible for the life of living beings, and participate in the process of fermentation and decomposition.

At the same time, anaerobes cause the development of dangerous diseases and inflammatory processes.

What are anaerobes

Anaerobes are usually understood as micro- and macroorganisms that are able to live in the absence of oxygen. They obtain energy as a result of the process of substrate phosphorylation.

The development and reproduction of anaerobes occurs in purulent-inflammatory foci, affecting people with weak immunity.

Classification of anaerobes

There are two types of these bacteria:

• Facultative, which are able to live, develop and reproduce in both oxygen and oxygen-free environments. Such microorganisms include staphylococci, E. coli, streptococci, shigella;
• Obligate animals live only in environments where there is no oxygen. If this element appears in the environment, then the death of obligate anaerobes occurs.

In turn, obligate anaerobes are divided into two groups:

• Clostridia are bacteria that form spores; stimulate the development of infections - botulism, wounds, tetanus.
• Non-clostridial - bacteria that are not able to form spores. They live in the microflora of people and animals and are not dangerous to living beings. Such bacteria include eubacteria, peillonella, peptococcus, and bacteriodes.

Often, non-clostridial anaerobes cause purulent and inflammatory processes, including peritonitis, pneumonia, sepsis, otitis media, etc. All infections caused by this type of bacteria occur under the influence of internal causes. The main factor in the development of infections is a decrease in immunity and the body’s resistance to pathogens. This usually occurs after operations, injuries, or hypothermia.

Examples of anaerobes

Prokaryotes and protozoan microorganisms. Mushrooms. Seaweed. Plants. Helminths – flukes, tapeworms and roundworms. Infections - intra-abdominal, intracranial, pulmonary, wound, abscesses, in the neck and head, soft tissues, cerebrospinal fluid. Aspiration pneumonia. Periodontitis.

Infections that are provoked by anaerobic bacteria cause the development of necrosis, abscess formation, sepsis and gas formation. A lot of anaerobes create enzymes in tissues that produce paralytic toxins.

Anaerobic bacteria cause the development of the following diseases: Oral infections. Sinusitis. Acne. Inflammation of the middle ear. Gangrene. Botulism. Tetanus. In addition to dangers, anaerobes have benefits for humans. In particular, they convert harmful sugars of toxic origin into beneficial enzymes in the colon.

Differences between anaerobes and aerobes

Anaerobes mainly live in an environment where there is no oxygen, while aerobes are able to live, develop and reproduce only in the presence of oxygen. Anaerobes include birds, mushrooms, several types of mushrooms, and animals. Oxygen in anaerobes takes part in all life processes, which contributes to the formation and production of energy.

Recently, scientists from Holland discovered that anaerobes living at the bottom of reservoirs can oxidize methane. In this case, nitrates and nitrites are reduced, which release molecular nitrogen. Archaeobacteria and eubacteria take part in the formation of this substance.

Microbiologists cultivate anaerobic microorganisms. This process requires specific microflora and a certain degree of concentration of metabolites.

The cultivation of anaerobes occurs on nutrients - glucose, sodium sulfate, casein.

Anaerobes have different metabolisms, which makes it possible to distinguish several subgroups of bacteria based on this characteristic. These are organisms that use anaerobic respiration, solar energy, and catabolism of high molecular weight compounds.

Anaerobic processes are used to decompose and disinfect wastewater sludge to ferment sugars to produce ethyl alcohol.

conclusions

Anaerobes can bring both benefit and harm to humans, animals and plants. If conditions arise for the development of pathogenic processes, then anaerobes will provoke infections and diseases that can be fatal. In industry and microbiology, scientists are trying to use the anaerobic properties of bacteria to obtain useful enzymes and purify water and soil.

Anaerobic infection

Etiology, pathogenesis, antibacterial therapy.

Preface........................................................ ..................................... 1

Introduction........................................................ ............................................... 2

1.1 Definition and characteristics.................................................... .... 2

1.2 Composition of the microflora of the main human biotopes.................................. 5

2. Pathogenicity factors of anaerobic microorganisms......... 6

2.1. The role of anaerobic endogenous microflora in pathology

person........................................................ ........................................………. 8

3. Main forms of anaerobic infection...................................................... 10

3.1. Pleuropulmonary infection................................................................... ......….. 10

3.2. Diabetic foot infection..................................................... . 10

3.3. Bacteremia and sepsis.................................................... ................. eleven

3.4. Tetanus................................................. .................................... eleven

3.5. Diarrhea................................................. ......................................... 12

3.6. Surgical infection of wounds and soft tissues.................................... 12

3.7. Gas-forming soft tissue infection.................................................... 12

3.8. Clostridial myonecrosis................................................................. ... 12

3.9. Slowly developing necrotizing wound infection...13

3.10. Intraperitoneal infection...................................…………….. 13

3.11. Characteristics of experimental anaerobic abscesses.....13

3.12. Pseudomembranous colitis................................................................. ..........14

3.13. Obstetric and gynecological infection....................................................14

3.14. Anaerobic infection in cancer patients……………..15

4. Laboratory diagnostics.................................................... ................15

4.1. Material under study......................................................... .....................15

4.2. Stages of material research in the laboratory...................................16

4.3. Direct study of the material .............................................................. .......16

4.4. Methods and systems for creating anaerobic conditions....................................16

4.5. Nutrient media and cultivation ..............................................17

5. Antibiotic therapy for anaerobic infection .............................................. 21

5.1. Characteristics of the main antimicrobial drugs,

used in the treatment of anaerobic infection ..........................................21

5.2. Combination of beta-lactam drugs and inhibitors

beta-lactamases................................................... ....................................24

5.3. Clinical significance of determining the sensitivity of anaerobic

microorganisms to antimicrobial drugs.......…………...24

6. Correction of intestinal microflora...................................……………….26

1. Conclusion................................................. ........................................27
2. Authors…………………………………………………………….27

Preface

Recent years have been characterized by the accelerated development of many areas of general and clinical microbiology, which is probably due to both our more adequate understanding of the role of microorganisms in the development of diseases and the need for doctors to constantly use information about the etiology of diseases, the properties of pathogens with the goal of successful management of patients and obtaining satisfactory final results of chemotherapy or chemoprophylaxis. One of these rapidly developing areas of microbiology is clinical anaerobic bacteriology. In many countries around the world, significant attention is paid to this section of microbiology. Sections devoted to anaerobes and anaerobic infections are included in the training programs for doctors of various specialties. Unfortunately, in our country, insufficient attention has been paid to this section of microbiology, both in terms of training specialists and in the diagnostic aspect of the work of bacteriological laboratories. The methodological manual “Anaerobic infection” covers the main sections of this problem - definition and classification, characteristics of anaerobic microorganisms, the main biotopes of anaerobes in the body, characteristics of forms of anaerobic infection, directions and methods of laboratory diagnostics, as well as comprehensive antibacterial testing -rapia (antimicrobial drugs, resistance/sensitivity of microorganisms, methods for determining and overcoming it). Naturally, the methodological manual does not aim to provide detailed answers to all aspects of anaerobic infection. It is quite clear that microbiologists who want to work in the field of anaerobic bacteriology need to undergo a special training cycle, to more fully master the issues of microbiology, laboratory technology, methods of indication, cultivation and identification of anaerobes. In addition, good experience is gained by participating in special seminars and symposiums dedicated to anaerobic infection at the national and international levels. These methodological recommendations are addressed to bacteriologists, doctors of various specialties (surgeons, therapists, endocrinologists, obstetricians-gynecologists, pediatricians), students of medical and biological faculties, teachers of medical universities and medical schools.

Introduction

The first ideas about the role of anaerobic microorganisms in human pathology appeared many centuries ago. Back in the 4th century BC, Hippocrates described in detail the clinic of tetanus, and in the 4th century AD, Xenophon described cases of acute necrotizing ulcerative gingivitis in Greek soldiers. The clinical picture of actinomycosis was described by Langenbeck in 1845. However, at that time it was not clear which microorganisms caused these diseases, what their properties were, just as the concept of anaerobiosis was absent until 1861, when Louis Pasteur published a classic work on the study of Vibrio butyrigue and called the organisms living in the absence of air "anaerobes" (17). Subsequently, Louis Pasteur (1877) isolated and cultivated Clostridium septicum , and Israel in 1878 he described actinomycetes. The causative agent of tetanus is Clostridium tetani - identified in 1883 by N. D. Monastyrsky, and in 1884 by A. Nikolayer. The first studies of patients with clinical anaerobic infection were performed by Levy in 1891. The role of anaerobes in the development of various medical pathologies was first described and argued more fully by Veiloon and Zuber in 1893-1898. They described various types of severe infections caused by anaerobic microorganisms (lung gangrene, appendicitis, abscesses of the lung, brain, pelvis, meningitis, mastoiditis, chronic otitis, bacteremia, parametritis, bartholinitis, purulent arthritis). In addition, they developed many methodological approaches to the isolation and cultivation of anaerobes (14). Thus, by the beginning of the 20th century, many anaerobic microorganisms became known, an idea of ​​their clinical significance was formed, and an appropriate technique for cultivating and isolating anaerobic microorganisms was created. From the 60s to the present, the relevance of the problem of anaerobic infections continues to increase. This is due to both the etiological role of anaerobic microorganisms in the pathogenesis of diseases and the development of resistance to widely used antibacterial drugs, as well as the severe course and high mortality of the diseases they cause.

1.1. Definition and characteristics

In clinical microbiology, microorganisms are usually classified based on their relationship to atmospheric oxygen and carbon dioxide. This can be easily verified by incubating microorganisms on blood agar under various conditions: a) in normal air (21% oxygen); b) under CO 2 incubator conditions (15% oxygen); c) under microaerophilic conditions (5% oxygen) d) anaerobic conditions (0% oxygen). Using this approach, bacteria can be divided into 6 groups: obligate aerobes, microaerophilic aerobes, facultative anaerobes, aerotolerant anaerobes, microaerotolerant anaerobes, obligate anaerobes. This information is useful for the initial identification of both aerobes and anaerobes.

Aerobes. For growth and reproduction, obligate aerobes require an atmosphere containing molecular oxygen in a concentration of 15-21% or CO; incubator. Mycobacteria, Vibrio cholerae and some fungi are examples of obligate aerobes. These microorganisms obtain most of their energy through the process of respiration.

Microaerophiles(microaerophilic aerobes). They also need oxygen to reproduce, but in concentrations lower than what is present in the room atmosphere. Gonococci and Campylobacter are examples of microaerophilic bacteria and prefer an atmosphere with an O2 content of about 5%.

Microaerophilic anaerobes. Bacteria that can grow in anaerobic and microaerophilic conditions, but are unable to grow in a CO 2 incubator or air environment.

Anaerobes. Anaerobes are microorganisms that do not need oxygen to live and reproduce. Obligate anaerobes are bacteria that grow only under anaerobic conditions, i.e. in an oxygen-free atmosphere.

Aerotolerant microorganisms. Able to grow in an atmosphere containing molecular oxygen (air, CO2 incubator), but they grow best in anaerobic conditions.

Facultative anaerobes(facultative aerobes). Able to survive in the presence or absence of oxygen. Many bacteria isolated from patients are facultative anaerobes (enterobacteria, streptococci, staphylococci).

capnophiles. A number of bacteria that grow better in the presence of elevated CO 2 concentrations are called capnophiles, or capnophilic organisms. Bacteroides, fusobacteria, hemoglobinophilic bacteria are capnophiles, as they grow better in an atmosphere containing 3-5% CO 2 (2,

19,21,26,27,32,36).

The main groups of anaerobic microorganisms are presented in Table 1 (42, 43,44).

TableI. The most significant anaerobic microorganisms

Genus	Kinds		a brief description of
Bacteroides	IN. fragilis IN. vulgatus IN. distansonis IN. eggerthii		Gram-negative, non-spore-forming rods
Prevotella	P. melaninogenicus P. bivia P. buccalis P. denticola P. intermedia
Porphyromonas	P. asaccharolyticum P. endodontalis P. gingivalis			Gram-negative, non-spore-forming rods
Ctostridium	C. perfringens C. ramosum C. septicum C. novyi C. sporogenes C. sordelii C. tetani C. botulinum C. difficile		Gram-positive, spore-forming rods, or bacilli
Actinomyces	A. israelii A. bovis
Pseudoramibacter *	P. alactolyticum		Gram-positive, non-spore forming rods
		E. lentum E. rectale E. limosum	Gram-positive, non-spore forming rods
Bifidobacterium		B. eriksonii B. adolescentis B. breve	Gram-positive rods
Propionobacterium		P. acnes P. avidum P. granulosum P. propionica**	Gram-positive. non-spore forming rods
Lactobacillus		L. catenaforme L. acidophylus	Gram-positive rods
Peptococcus		P. magnus P. saccharolyticus P. asaccharolyticus
Peptostreptococcus		P. anaerobius P. intermedius P.micros P. productus	Gram-positive, non-spore forming cocci
Veilonella		V. parvula	Gram-negative, non-spore forming cocci
Fusobacterium		F. nucleatum F. necrophorum F. varium F. mortiferum	Fusiform sticks
campylobacter		C. fetus C.jejuni	Gram-negative, thin, spiral-shaped, non-spore-forming rods

* Eubacterium alaclolyticum reclassified as Pseudoramibacter alactolyticum (43,44)

** previously Arachnia propionica (44)

*** synonyms F. pseudonecrophorum, F. necrophorum biovar WITH(42,44)

1.2. Composition of the microflora of the main human biotopes

The etiology of infectious diseases has undergone significant changes in recent decades. As is well known, previously the main danger to human health was highly infectious infections: typhoid fever, dysentery, salmonellosis, tuberculosis and many others, which were transmitted predominantly exogenously. Although these infections still remain socially important and their medical importance is now increasing again, in general their role has decreased significantly. At the same time, there is an increasing role of opportunistic microorganisms, representatives of the normal microflora of the human body. The normal human microflora includes more than 500 species of microorganisms. The normal microflora that lives in the human body is largely represented by anaerobes (Table 2).

Anaerobic bacteria inhabiting the skin and mucous membranes of humans, carrying out microbial transformation of substrates of exo- and endogenous origin, produce a wide range of various enzymes, toxins, hormones and other biologically active compounds that are absorbed and bind to complementary receptors and influence the function of cells and organs. Knowledge of the composition of the specific normal microflora of certain anatomical areas is useful for understanding the etiology of infectious processes. The set of species of microorganisms that inhabit a certain anatomical area is called indigenous microflora. Moreover, the detection of specific microorganisms in significant quantities at a distance or in an unusual place only emphasizes their participation in the development of the infectious process (11, 17,18, 38).

Respiratory tract. The microflora of the upper respiratory tract is very diverse and includes more than 200 species of microorganisms included in 21 genera. 90% of salivary bacteria are anaerobes (10, 23). Most of these microorganisms are unclassified by modern taxonomy methods and are not of significant importance for pathology. The respiratory tract of healthy people is most often colonized by the following microorganisms - Streptococcus pneumonia- 25-70%; H aemophilus influenzae- 25-85%; Streptococcus pyogenes- 5-10%; Neisseria meningitidis- 5-15%. Anaerobic microorganisms such as Fusobacterium, Bacteroides spiralis, Peptostreptococcus, Peptococcus, Veilonella and some types Actinomyces found in almost all healthy people. Coliform bacteria are found in the respiratory tract of 3-10% of healthy people. Increased colonization of the respiratory tract by these microorganisms was detected in alcoholics, people with severe illness, in patients receiving antibacterial therapy that suppresses normal microflora, as well as in people with impaired immune system functions.

Table 2. Quantitative content of microorganisms in biotopes

normal human body

Populations of microorganisms in the respiratory tract adapt to certain ecological niches (nose, pharynx, tongue, gingival crevices). Adaptation of microorganisms to given biotopes is determined by the affinity of bacteria to certain types of cells or surfaces, that is, determined by cellular or tissue tropism. For example, Streptococcus salivarius is well attached to the epithelium of the cheek and dominates the composition of the buccal mucosa. adhesion bacte-

ry can also explain the pathogenesis of some diseases. Streptococcus pyogenes adheres well to the epithelium of the pharynx and often causes pharyngitis; E. coli is affinity for the epithelium of the bladder and therefore causes cystitis.

Leather. The indigenous microflora of the skin is represented by bacteria mainly of the following genera: Staphylococcus, Micrococcus, Corynobacterium, Propionobacterium, Brevibacterium And Acinetobacter. Yeasts of the genus are also often present Pityrosporium. Anaerobes are represented largely by gram-positive bacteria of the genus propi- onobacterium (usually Propionobacterium acnes). Gram-positive cocci (Peptostreptococcus spp.) And gram-positive bacteria of the genus Eubacterium present in some individuals.

Urethra. Bacteria that colonize the distal urethra are staphylococci, non-hemolytic streptococcus, diphtheroids and, in a small number of cases, various representatives of the Enterobacteriaceae family. Anaerobes are represented to a greater extent by gram-negative bacteria - BacteroidesAndFusobacterium spp..

Vagina. About 50% of bacteria from the secretions of the cervix and vagina are anaerobes. Most of the anaerobes are represented by lactobacilli and peptostreptococci. Prevo-tells are often found - P. bivia And P. disiens. In addition, there are gram-positive bacteria of the genus Mobiluncus And Clostridium.

Intestines. Of the 500 species that inhabit the human body, approximately 300 - 400 species live in the intestines. The following anaerobic bacteria are found in the largest numbers in the intestines - Bacteroides, Bifidobacterium, Clostridium, Eubacterium, LactobacillusAndPeptostrepto- coccus. Bacteroides are the dominant microorganisms. It has been established that for one E. coli cell there are a thousand bacteroid cells.

2. Pathogenicity factors of anaerobic microorganisms

Pathogenicity of microorganisms means their potential ability to cause disease. The emergence of pathogenicity in microbes is associated with their acquisition of a number of properties that provide the ability to attach, penetrate and spread in the host’s body, resist its defense mechanisms, and cause damage to vital organs and systems. At the same time, it is known that the virulence of microorganisms is a polydeterminate property, which is fully realized only in the body of a host sensitive to the pathogen.

Currently, several groups of pathogenicity factors are distinguished:

a) adhesins, or attachment factors;

b) adaptation factors;

c) invasins, or penetration factors

d) capsule;

e) cytotoxins;

f) endotoxins;

g) exotoxins;

h) enzymes, toxins;

i) factors modulating the immune system;

j) superantigens;

l) heat shock proteins (2, 8, 15, 26, 30).

The stages and mechanisms, the spectrum of reactions, interactions and relationships at the molecular, cellular and organismal levels between microorganisms and the host organism are very complex and diverse. Knowledge about the pathogenicity factors of anaerobic microorganisms and their practical use to prevent diseases is not yet sufficient. Table 3 shows the main groups of pathogenicity factors of anaerobic bacteria.

Table 3. Pathogenicity factors of anaerobic microorganisms

Interaction stage		Factor		Kinds
Adhesion		Fimbria capsular polysaccharides Hemagglutinins
Invasion		Phospholipase C Proteases
Damage fabrics	Exotoxins Hemolysins Proteases Collagenase Fibrinolysin Neuraminidase Heparinase Chondriitin sulfate glucoronidase N-acetyl-glucosaminidase Cytotoxins Enterotoxins Neurotoxins		P. melaninogenica P. melaninogenica
Factors that suppress the immune system	Metabolic products Lipopolysaccharides (O-antigen) Immunoglobulin proteases (G, A, M) C 3 and C 5 convertases Protease a 2 -microglobulin Metabolic products Fatty acids of anaerobes Sulfur compounds Oxidoreductases Beta-lactamases		Most anaerobes
Activators of damage factors	Lipopolysaccharides (O-antigen) Surface structures

It has now been established that the pathogenicity factors of anaerobic microorganisms are determined genetically. Chromosomal and plasmid genes, as well as transposons encoding various pathogenicity factors, have been identified. The study of the functions of these genes, mechanisms and patterns of expression, transmission and circulation in a population of microorganisms is a very important problem.

2.1. The role of anaerobic endogenous microflora in human pathology

Anaerobic microorganisms of the normal microflora very often become the causative agents of infectious processes localized in various anatomical parts of the body. Table 4 shows the frequency of anaerobic microflora in the development of pathology. (2, 7, 11, 12, 18, 24, 27).

A number of important generalizations can be formulated regarding the etiology and pathogenesis of most types of anaerobic infection: 1) the source of anaerobic microorganisms is the normal microflora of patients from their own gastrointestinal, respiratory or urogenital tract; 2) changes in tissue properties caused by trauma and/or hypoxia provide appropriate conditions for the development of a secondary or opportunistic anaerobic infection; 3) anaerobic infections, as a rule, are polymicrobial and are often caused by a mixture of several types of anaerobic and aerobic microorganisms that synergistically have a damaging effect; 4) the infection is accompanied by the formation and release of a strong odor in approximately 50% of cases (non-spore-forming anaerobes synthesize volatile fatty acids that cause this odor); 5) the infection is characterized by the formation of gases, tissue necrosis, the development of abscesses and gangrene; 6) the infection develops during treatment with aminoglycoside antibiotics (bacteroides are resistant to them); 7) the exudate is stained black (porphyromonas and prevotella produce dark brown or black pigment); 8) the infection has a protracted, sluggish, often subclinical course; 9) there are extensive necrotic changes in tissue, a discrepancy between the severity of clinical symptoms and the amount of destructive changes, and little bleeding on the incision.

Although anaerobic bacteria can cause serious and fatal infections, the initiation of infection generally depends on the state of the body's defense factors, i.e. functions of the immune system (2, 5, 11). The principles of treatment of such infections include removal of dead tissue, drainage, restoration of adequate blood circulation, removal of foreign substances, and the use of active antimicrobial therapy appropriate to the pathogen, in an adequate dose and duration.

Table 4. Etiological role of anaerobic microflora

in development diseases

Diseases	Number of people examined	Frequency of anaerobes excretion
Head and neck Non-traumatic head abscesses Chronic sinusitis Perimandibular space infections
Rib cage Aspiration pneumonia Lung abscess
Abdomen Abscesses or peritonitis Appendicitis Liver abscess
Female genital tract Mixed types Pelvic abscesses Inflammatory processes			33 (100%) 22 (88%)
Soft fabrics Wound infection Skin abscesses Diabetic limb ulcers Non-clostridial cellulitis
Bacteremia All cultures Intra-abdominal sepsis Septic abortion

3. Main forms of anaerobic infection

3.1. Pleuropulmonary infection

Etiologically significant anaerobic microorganisms in this pathology are representatives of the normal microflora of the oral cavity and upper respiratory tract. They are the causative agents of various infections, including aspiration pneumonia, necrotizing pneumonia, actinomycosis and pulmonary abscess. The main causative agents of pleuropulmonary diseases are presented in Table 5.

Table 5. Anaerobic bacteria that cause

pleuropulmonary infection

Factors that contribute to the development of anaerobic pleuropulmonary infection in a patient include aspiration of normal microflora (as a result of loss of consciousness, dysphagia, the presence of mechanical objects, obstruction, poor oral hygiene, necrotization of lung tissue) and hematogenous spread of microorganisms. As can be seen from Table 5, aspiration pneumonia is most often caused by organisms previously designated as “oral bacteroides” species (currently Prevotella and Porphyromonas species), Fusobacterium and Peptostreptococcus. The spectrum of bacteria isolated from anaerobic empyema and pulmonary abscess is almost the same.

3.2. Diabetic foot infection

Among the more than 14 million diabetics in the United States, bad foot is the most common infectious cause of hospitalization. This type of infection is often ignored by the patient at the initial stage, and sometimes inadequately treated by doctors. In general, patients do not strive to carefully and regularly examine their lower extremities and do not follow doctors’ recommendations for care and walking regimen. The role of anaerobes in the development of foot infections in diabetics was established many years ago. The main types of microorganisms that cause this type of infection are presented in Table 6.

Table 6. Aerobic and anaerobic microorganisms that cause

foot infection in diabetics

Aerobes	Anaerobes
Proteus mirabili	Bacteroides fragilis
Pseudomonas aeruginosa	other species of the B. fragilis group
Enterobacter aerogenes	Prevotella melaninogenica
Escherichia coli	other species of Prevotella\ Porphyromonas
Klebsiella pneumonia	Fusobacterium nucleatum
	other fusobacteria
	Peptostreptococcus
Staphylococcus aureus	other types of clostridia

It has been established that 18-20% of diabetic patients have a mixed aerobic/anaerobic infection. On average, 3.2 aerobic and 2.6 anaerobic species of microorganisms were detected per patient. Of the anaerobic bacteria, peptostreptococci were dominant. Bacteroides, Prevotella and Clostridia were also often detected. An association of bacteria was isolated from deep wounds in 78% of cases. In 25% of patients, gram-positive aerobic microflora (staphylococci and streptococci) were detected, and in approximately 25% - gram-negative rod-shaped aerobic microflora. About 50% of cases of anaerobic infection are mixed. These infections are more severe and most often require amputation of the affected limb.

3.3. Bacteremia and sepsis

The share of anaerobic microorganisms in the development of bacteremia ranges from 10 to 25%. Most studies indicate that IN.fragilis and other species of this group, as well as Bacteroides thetaiotaomicron are a more common cause of bacteremia. The next most frequently isolated species are clostridia (especially Clostridium perfringens) and peptostreptococci. They are often isolated in pure culture or in associations. In recent decades, in many countries of the world there has been an increase in the frequency of anaerobic sepsis (from 0.67 to 1.25 cases per 1000 hospital admissions). The mortality rate of patients with sepsis caused by anaerobic microorganisms is 38-50%.

3.4. Tetanus

Tetanus has been a well-known serious and often fatal infection since the time of Hippocrates. For centuries, this disease has been a pressing problem associated with gunshot, burn and traumatic wounds. Controversy Clostridium tetani are detected in human and animal feces and are widespread in the environment. Ramon and his colleagues in 1927 successfully proposed immunization with toxoid to prevent tetanus. The risk of developing tetanus is higher in people over 60 years of age due to a decrease in the effectiveness/loss of protective post-vaccination antitoxic immunity. Therapy includes the administration of immunoglobulins, wound treatment, antimicrobial and antitoxic therapy, constant nursing care, the use of sedatives and analgesics. Particular attention is currently paid to neonatal tetanus.

3.5. Diarrhea

There are a number of anaerobic bacteria that cause diarrhea. Anaerobiospirillum succiniciproducens- mobile spiral-shaped bacteria with bipolar flagella. The causative agent is excreted in the feces of dogs and cats with asymptomatic infections, as well as from people with diarrhea. Enterotoxigenic strains IN.fragilis. In 1984, Mayer showed the role of toxin-producing strains IN.fragilis in the pathogenesis of diarrhea. Toxigenic strains of this pathogen are isolated from diarrhea in humans and animals. They cannot be differentiated from common strains by biochemical and serological methods. In the experiment, they cause diarrhea and characteristic lesions of the large intestine and distal small intestine with crypt hyperplasia. Enterotoxin has a molecular weight of 19.5 kD and is thermolabile. The pathogenesis, spectrum and frequency of incidence, as well as optimal therapy, have not yet been sufficiently developed.

3.6. Surgical anaerobic infection of wounds and soft tissues

Infectious agents isolated from surgical wounds largely depend on the type of surgical intervention. The cause of suppuration in clean surgical interventions that are not accompanied by an opening of the gastrointestinal, urogenital or respiratory tracts, as a rule, is St. aureus. In other types of wound suppuration (cleanly contaminated, contaminated and dirty), a mixed polymicrobial microflora of surgically resected organs is most often isolated. In recent years, there has been an increase in the role of opportunistic microflora in the development of such complications. Most superficial wounds are diagnosed later in life between the eighth and ninth days after surgery. If the infection develops earlier - within the first 48 hours after surgery, then this is typical for a gangrenous infection caused by certain species of either clostridia or beta-hemolytic streptococcus. In these cases There is a dramatic increase in the severity of the disease, pronounced toxicosis, rapid local development of infection involving all layers of body tissue in the process.

3.7. Gas-forming soft tissue infection

The presence of gas in infected tissue is an ominous clinical sign, and in the past, this infection was most often associated by physicians with the presence of clostridial gas gangrene. It is now known that gas-forming infection in surgical patients is caused by a mixture of anaerobic microorganisms such as Clostridium, Peptostreptococcus or Bacteroides, or one of the types of aerobic coliform bacteria. Predisposing factors for the development of this form of infection are vascular diseases of the lower extremities, diabetes, and trauma.

3.8. Clostridial myonecrosis

Gas gangrene is a destructive process of muscle tissue associated with local crepitus, severe systemic intoxication caused by anaerobic gas-forming clostridia. Clostridia are gram-positive obligate anaerobes that are widespread in soil contaminated with animal excrements. In humans, they are normally inhabitants of the gastrointestinal and female genital tract. Sometimes they can be found on the skin and in the oral cavity. The most significant species of the 60 known is Clostridium perfringens. This microorganism is more tolerant of air oxygen and is fast-growing. It is an alpha toxin, phospholipase C (lecithinase), which breaks down lecithin into phosphorylcholine and diglycerides, as well as collagenase and proteases, which cause tissue destruction. Alpha-toxin production is associated with high mortality in gas gangrene. It has hemolytic properties, destroys platelets, causes intense capillary damage and secondary tissue destruction. In 80% of cases, myonecrosis is caused by WITH.perfringens. In addition, the etiology of this disease involves WITH.novyi, WITH. septicum, WITH.bifer- mentas. Other types of clostridia C. histoliticum, WITH.sporogenes, WITH.fallax, WITH.tertium have low etiological significance.

3.9. Slowly developing necrotizing wound infection

Aggressive life-threatening wound infection May appear 2 weeks after infection, especially in diabetics

sick. Usually these are either mixed or monomicrobial fascial infections. Monomicrobial infections are relatively rare. in approximately 10% of cases and are usually observed in children. The causative agents are group A streptococci, Staphylococcus aureus and anaerobic streptococci (peptostreptococci). Staphylococci and hemolytic streptococcus are isolated with the same frequency in approximately 30% of patients. Most of them become infected outside the hospital. Most adults have necrotizing fascillitis of the extremities (in 2/3 of cases the extremities are affected). In children, the trunk and groin area are more often involved. Polymicrobial infection includes a number of processes caused by anaerobic microflora. On average, about 5 main types are isolated from wounds. The mortality rate for such diseases remains high (about 50% among patients with severe forms). Older people tend to have a poor prognosis. Mortality rate in people over 50 is more than 50%, and in patients with diabetes - more than 80%.

3.10. Intraperitoneal infection

Intra-abdominal infections are the most difficult for early diagnosis and effective treatment. A successful outcome primarily depends on early diagnosis, rapid and adequate surgical intervention and the use of an effective antimicrobial regimen. The polymicrobial nature of the bacterial microflora involved in the development of peritonitis as a result of perforation in acute appendicitis was first shown in 1938 Altemeier. The number of aerobic and anaerobic microorganisms isolated from areas of intra-abdominal sepsis depends on the nature of the microflora or the injured organ. Generalized data indicate that the average number of bacterial species isolated from the source of infection ranges from 2.5 to 5. For aerobic microorganisms, these data are 1.4-2.0 species and 2.4-3.0 species of anaerobic microorganisms. At least 1 type of anaerobes is detected in 65-94% of patients. The most frequently identified aerobic microorganisms are Escherichia coli, Klebsiella, Streptococcus, Proteus, and Enterobacter, and anaerobic microorganisms are Bacteroides, Peptostreptococcus, and Clostridia. Bacteroides account for 30% to 60% of all isolated strains of anaerobic microorganisms. According to the results of numerous studies, 15% of infection cases are caused by anaerobic and 10% aerobic microflora, and, accordingly, 75% are caused by associations. The most significant of them are E.coli And IN.fragilis. According to Bogomolova N. S. and Bolshakov L. V. (1996), anaerobic infection

was the cause of the development of odontogenic diseases in 72.2% of cases, appendiceal peritonitis - in 62.92% of cases, peritonitis due to gynecological diseases - in 45.45% of patients, cholangitis - in 70.2%. Anaerobic microflora was isolated most often in severe peritonitis in the toxic and terminal stages of the disease.

3.11. Characteristics of experimental anaerobic abscesses

In the experiment IN.fragilis initiates the development of a subcutaneous abscess. The initial events are the migration of polymorphonuclear leukocytes and the development of tissue edema. After 6 days, 3 zones are clearly identified: internal - consists of necrotic masses and degeneratively changed inflammatory cells and bacteria; the middle one is formed from the leukocyte shaft and the outer zone is represented by a layer of collagen and fibrous tissue. The concentration of bacteria ranges from 10 8 to 10 9 in 1 ml of pus. An abscess is characterized by a low redox potential. It is very difficult to treat, since the destruction of antimicrobial drugs by bacteria is observed, as well as evasion of the host’s defense factors.

3.12. Pseudomembranous colitis

Pseudomembranous colitis (PMC) is a serious gastrointestinal disease characterized by exudative plaques on the colon mucosa. This disease was first described in 1893, long before the advent of antimicrobial drugs and their use for medicinal purposes. It has now been established that the etiological factor of this disease is Clostridium difficile. Disruption of intestinal microecology due to the use of antibiotics is the cause of the development of MVP and the widespread spread of infections caused by WITH.difficile, the clinical spectrum of manifestations of which varies widely - from carriage and short-term, self-limiting diarrhea to the development of MVP. The number of patients with colitis caused by S. difficile, among outpatients 1-3 per 100,000, and among hospitalized patients 1 per 100-1000.

Pathogenesis. Colonization of the human intestine with toxigenic strains WITH,difficile is an important factor in the development of MVP. However, asymptomatic carriage occurs in approximately 3-6% of adults and 14-15% of children. Normal intestinal microflora serves as a reliable barrier to colonization by pathogenic microorganisms. It is easily disturbed by antibiotics and very difficult to recover. The most pronounced effect on the anaerobic microflora is 3rd generation cephalosporins, clindamycin (lincomycin group) and ampicillin. As a rule, all patients with MVP suffer from diarrhea. In this case, the stool is liquid with impurities of blood and mucus. There is hyperemia and swelling of the intestinal mucosa. Ulcerative colitis or proctitis, characterized by granulations and hemorrhagic mucosa, is often observed. Most patients with this disease have fever, leukocytosis, and abdominal tension. Subsequently, serious complications may develop, including general and local intoxication, hypoalbuminemia. Symptoms of antibiotic-associated diarrhea begin on days 4-5 of antibiotic therapy. S. is detected in the stool of such patients. difficile in 94% of cases, while in healthy adults this microorganism is isolated only in 0.3% of cases.

WITH.difficile produces two types of highly active exotoxins - A and B. Toxin A is an enterotoxin that causes hypersecretion and accumulation of fluid in the intestines, as well as an inflammatory reaction with hemorrhagic syndrome. Toxin B is a cytotoxin. It is neutralized by polyvalent anti-gangrenous serum. This cytotoxin was found in approximately 50% of patients with antibiotic-associated colitis without pseudomembrane formation and in 15% of patients with antibiotic-associated diarrhea with normal sigmoidoscopic findings. Its cytotoxic effect is based on depolymerization of actin microfilaments and damage to the cytoskeleton of enterocytes. Recently, more and more data have appeared about WITH.difficile as a nosocomial infectious agent. In this regard, it is advisable to isolate surgical patients who are carriers of this microorganism in order to avoid the spread of infection in the hospital. WITH.difficile most sensitive to vancomycin, metronidazole and bacitracin. Thus, these observations confirm that toxin-producing strains WITH.difficile cause a wide range of diseases, including diarrhea, colitis and MVP.

3.13. Obstetric and gynecological infections

Understanding the patterns of development of infections of the female genital organs is possible on the basis of an in-depth study of the microbiocenosis of the vagina. The normal vaginal microflora must be considered in terms of a protective barrier against the most common pathogens.

Dysbiotic processes contribute to the formation of bacterial vaginosis (BV). BV is associated with the development of complications such as anaerobic postoperative soft tissue infections, postpartum and post-abortion endometritis, premature termination of pregnancy, intra-amniotic infection (10). Obstetric and gynecological infection is polymicrobial in nature. First of all, I would like to note the increasing role of anaerobes in the development of acute inflammatory processes of the pelvic organs - acute inflammation of the uterine appendages, postpartum endometritis, especially after surgical delivery, postoperative complications in gynecology (pericultitis, abscesses, wound infection) (5 ). The microorganisms most often isolated during infections of the female genital tract include Bactemides fragilis, as well as types Peptococcus And Peptostreptococcus. Group A streptococci are not found very often in pelvic infections. Group B streptococci more often cause sepsis in obstetric patients whose entry point is the genital tract. In recent years, during obstetric and gynecological infections, WITH.trachomatis. The most common infectious processes of the urogenital tract include pelvioperitonitis, endometritis after cesarean section, vaginal cuff infections after hysterectomy, and pelvic infections after septic abortion. The effectiveness of clindamycin in these infections ranges from 87% to 100% (10).

3.14. Anaerobic infection in cancer patients

The risk of developing infection in cancer patients is incomparably higher than in other surgical patients. This feature is explained by a number of factors - the severity of the underlying disease, immunodeficiency state, a large number of invasive diagnostic and therapeutic procedures, the large volume and traumatic nature of surgical interventions, and the use of very aggressive treatment methods - radiotherapy and chemotherapy. In patients operated on for gastrointestinal tumors, subphrenic, subhepatic and intraperitoneal abscesses of anaerobic etiology develop in the postoperative period. The dominant pathogens are Bacteroides fragi- lis, Prevotella spp.. Fusobacterium spp., gram-positive cocci. In recent years, more and more reports have appeared on the important role of non-sporogenous anaerobes in the development of septic conditions and their release from the blood during bacteremia (3).

4. Laboratory diagnostics

4.1. Material under study

Laboratory diagnosis of anaerobic infection is a rather difficult task. The research time from the moment of delivery of pathological material from the clinic to the microbiological laboratory and until receiving a complete detailed answer ranges from 7 to 10 days, which cannot satisfy clinicians. Often the result of bacteriological analysis becomes known by the time the patient is discharged. Initially, the question should be answered: are anaerobes present in the material? It is important to remember that anaerobes are the main component of the local microflora of the skin and mucous membranes and, moreover, that their isolation and identification must be carried out under appropriate conditions. The successful initiation of research in clinical microbiology of anaerobic infection depends on the correct collection of appropriate clinical material.

In routine laboratory practice, the most commonly used materials are: 1) infected lesions from the gastrointestinal tract or female genital tract; 2) material from the abdominal cavity with peritonitis and abscesses; 3) blood from septic patients; 4) discharge from chronic inflammatory diseases of the respiratory tract (sinusitis, otitis, mastoiditis); 5) material from the lower parts of the respiratory tract during aspiration pneumonia; 6) cerebrospinal fluid for meningitis; 7) contents of a brain abscess; 8) local material for dental diseases; 9) contents of superficial abscesses: 10) contents of superficial wounds; 11) material from infected wounds (surgical and traumatic); 12) biopsy samples (19, 21, 29, 31, 32, 36, 38).

4.2. Stages of material research in the laboratory

Successful diagnosis and treatment of anaerobic infection is possible only with the interested cooperation of microbiologists and clinicians of the appropriate profile. Obtaining adequate sample samples for microbiological testing is critical. Methods for collecting material depend on the location and type of pathological process. Laboratory research is based on the indication and subsequent species identification of anaerobic and aerobic microorganisms contained in the test material using traditional and express methods, as well as on determining the sensitivity of isolated microorganisms to antimicrobial chemotherapeutic drugs (2).

4.3. Direct material examination

There are many quick direct tests that convincingly indicate the presence of anaerobes in large quantities in the material being tested. Some of them are very simple and cheap and therefore have advantages over many expensive laboratory tests.

1. 3 a p a x. Foul-smelling materials always contain anaerobes, only a few of them are odorless.

2. Gas-liquid chromatography (GLC). It is one of the express diagnostic methods. GLC makes it possible to determine short-chain fatty acids (acetic, propionic, isovaleric, isocaproic, caproic) in pus, which cause odor. Using GLC, the spectrum of volatile fatty acids can be used to identify the species of microorganisms present in it.

3. Fluorescence. Examination of materials (pus, tissues) in ultraviolet light at a wavelength of 365 nm reveals intense red fluorescence, which is explained by the presence of black-pigmented bacteria belonging to the Basteroides and Porphyromonas groups, and which indicates the presence of anaerobes.

4. Bacterioscopy. When examining many preparations stained using the Gram method, the smear reveals the presence of cells of the inflammatory focus, microorganisms, especially polymorphic gram-negative rods, small gram-positive cocci or gram-positive bacilli.

5. Immunofluorescence. Direct and indirect immunofluorescence are express methods and allow the identification of anaerobic microorganisms in the material under study.

6. Immunoenzyme method. Enzyme immunoassay allows you to determine the presence of structural antigens or exotoxins of anaerobic microorganisms.

7. Molecular biological methods. The greatest distribution, sensitivity and specificity in recent years has been shown by the polymerase chain reaction (CPR). It is used both to detect bacteria directly in the material, and for identification.

4.4. Methods and systems for creating anaerobic conditions

Material taken from appropriate sources and in suitable containers or transport medium for this purpose should be delivered immediately to the laboratory. However, there is evidence that clinically significant anaerobes in large volumes of pus or in an anaerobic transport medium survive for 24 hours. It is important that the inoculated medium be incubated under anaerobic conditions or placed in a CO2-filled vessel and stored until transferred to a special incubation system. There are three types of anaerobic systems commonly used in clinical laboratories. More widely used are microanaerostat systems (GasPark, BBL, Cockeysville), which have been used in laboratories for many years, especially in small laboratories, and provide satisfactory results. Petri dishes inoculated with anaerobic bacteria are placed inside the vessel simultaneously with a special gas-generating package and an indicator. Water is added to the bag, the vessel is hermetically sealed, and CO2 and H2 are released from the bag in the presence of a catalyst (usually palladium). In the presence of a catalyst, H2 reacts with O2 to form water. CO2 is necessary for the growth of anaerobes, as they are capnophiles. Methylene blue is added as an indicator of anaerobic conditions. If the gas-generating system and catalyst are operating efficiently, then discoloration of the indicator is observed. Most anaerobes require cultivation for at least 48 hours. After this, the chamber is opened and the dishes are examined initially, which does not seem entirely convenient, since anaerobes are sensitive to oxygen and quickly lose their viability.

Recently, simpler anaerobic systems - anaerobic bags - have come into practice. One or two inoculated cups with a gas-generating bag are placed in a transparent, hermetically sealed plastic bag and incubated under thermostatic conditions. The transparency of plastic bags makes it easy to periodically monitor the growth of microorganisms.

The third system for cultivating anaerobic microorganisms is an automatically sealed chamber with a glass front wall (anaerobic station) with rubber gloves and automatic supply of an oxygen-free mixture of gases (N2, H2, CO2). Materials, cups, test tubes, plates for biochemical identification and determination of sensitivity to antibiotics will be placed in this office through a special hatch. All manipulations are performed by a bacteriologist wearing rubber gloves. The material and plates in this system can be viewed daily, and cultures can be incubated for 7-10 days.

These three systems have their advantages and disadvantages, but they are effective for isolating anaerobes and should be in every bacteriological laboratory. Often they are used simultaneously, although the greatest reliability belongs to the method of cultivation in an anaerobic station.

4.5. Culture media and cultivation

The study of anaerobic microorganisms is carried out in several stages. The general scheme for the isolation and identification of anaerobes is presented in Figure 1.

An important factor in the development of anaerobic bacteriology is the presence of a collection of typical bacterial strains, including reference strains from the ATCC, CDC, and VPI collections. This is especially important for monitoring culture media, for biochemical identification of pure cultures and assessing the activity of antibacterial drugs. There is a wide range of basic media available that are used to prepare special culture media for anaerobes.

Nutrient media for anaerobes must meet the following basic requirements: 1) satisfy nutritional needs; 2) ensure rapid growth of microorganisms; 3) be adequately reduced. Primary inoculation of the material is carried out on blood agar plates or election media given in Table 7.

Increasingly, the isolation of obligate anaerobes from clinical material is carried out on media that include selective agents in a certain concentration, allowing the isolation of certain groups of anaerobes (20, 23) (Table 8).

The duration of incubation and the frequency of examination of inoculated dishes depends on the material being studied and the composition of the microflora (Table 9).

Material under study Wound discharge Contents of abscesses, Tracheobronchonal aspirate, etc. Transportation to the laboratory: in Cyprus, in a special transport medium (immediate placement of the material in the medium) Microscopy of material Gram stain

Cultivation and isolation pure culture Aerobic cups for 35±2°С compared with 18-28 hours anaerobes 5-10% C0 2 1. Blood agar Microaerostat Gas-Pak (H 2 + C0 2) 35±2°С from 48 hours to 7 days 2. Schedler's blood agar 35±2°С from 48 hours to 7 days 3. Selective identification environment anaerobes from 48 hours to 2 weeks 4. Liquid medium (thioglycolate)

Identification. Pure cultures from isolated colonies 1. Gram and Ozheshko staining to identify spores 2.Morphology of colonies 3.Colony type relationship with oxygen 4.Preliminary differentiation based on sensitivity to antimicrobial drugs 5.Biochemical tests Determination of sensitivity to antibiotics 1.Method of dilution in agar or broth 2.Paper disc method (diffusion)

Rice. 1. Isolation and identification of anaerobic microorganisms

anaerobic microorganisms

Wednesday	Purpose
Blood agar for Brucella (CDC anaerobic blood agar, Schadler's blood agar) (BRU agar)	Non-selective, for isolating anaerobes present in the material
Bile Esculin Agar for Bacteroides(BBE agar)	Selective and differential; for the isolation of bacteria of the Bacteroides fragilis group
Kanamycin-vancomycin blood agar(KVLB)	Selective for most non-sporeformers gram-negative bacteria
Phenyl Ethyl Agar(PEA)	Inhibits the growth of Proteus and other enterobacteria; stimulates the growth of gram-positive and gram-negative anaerobes
Thioglycol broth(THIO)	For special situations
Yolk agar(EYA)	To isolate clostridia
Cycloserine-cefoxitin-fructose agar(CCFA) or cycloserine mannitol agar (CMA) or cycloserine mannitol blood agar (CMBA)	Selective for C. difficile
Crystal-violet-erythromycin-new agar(CVEB)	For isolation of Fusobacterium nucleatum and Leptotrichia buccalis
Bacteroid gingivalis agar(BGA)	For isolation of Porphyromonas gingivalis

Table 8. Selective agents for obligate anaerobes

Organisms	Selective agents
Obligate anaerobes from clinical material	neomycin (70mg/l) nalidixic acid (10 mg/l)
Actinomyces spp.	metronidazole (5 mg/l)
Bacteroides spp. Fusobacterium spp.	nalidixic acid (10 mg/l) + vancomycin (2.5 mg/l)
Bacteroides urealytica	nalidixic acid (10 mg/l) teicoplanin (20 mg/l)
Clostridium difficile	cycloserine (250 mg/l) cefoxitin (8 mg/l)
Fusobacterium	rifampicin (50 mg/l) neomycin (100 mg/l) vancomycin (5 mg/l)

The results are recorded by describing the cultural properties of grown microorganisms, colony pigmentation, fluorescence, and hemolysis. Then a smear is prepared from the colonies, stained with Gram and thus gram-negative and gram-positive bacteria are identified, microscopically examined and morphological properties are described. Subsequently, the microorganisms of each type of colonies are subcultured and cultivated in thioglycol broth with the addition of hemin and vitamin K. The morphology of colonies, the presence of pigment, hemolytic properties, and the characteristics of bacteria in Gram stains make it possible to preliminarily identify and differentiate anaerobes. As a result, all anaerobic microorganisms can be divided into 4 groups: 1) Gr + cocci; 2) Gr+ bacilli or coccobacilli: 3) Gr- cocci; 4) Gr- bacilli or coccobacilli (20, 22, 32).

Table 9. Duration of incubation and frequency of testing

cultures of anaerobic bacteria

Type of crops	Incubation time*	Study frequency
Blood		Every day before 7 and after 14
Liquids		Daily
Abscesses, wounds		Daily
Airways Sputum Transtracheal aspirate Bronchial discharge		Daily One time Daily Daily
Urogenital tract Vagina, uterus Prostate		Daily Daily Daily One time
Feces		Daily
Anaerobes Brucella Actinomycetes		Daily 3 times a week 1 time per week

*until a negative result is obtained

At the third stage of research, longer identification is carried out. The final identification is based on the determination of biochemical properties, physiological and genetic characteristics, pathogenicity factors in the toxin neutralization test. Although the completeness of identification of anaerobes can vary greatly, some simple tests with a high probability allow the identification of pure cultures of anaerobic bacteria - Gram stain, motility, sensitivity to certain antibiotics using paper discs and biochemical properties.

5. Antibacterial therapy for anaerobic infection

Antibiotic-resistant strains of microorganisms arose and began to spread immediately after the widespread introduction of antibiotics into clinical practice. The mechanisms of formation of resistance of microorganisms to antibiotics are complex and diverse. They are classified into primary and acquired. Acquired resistance is formed under the influence of drugs. The main ways of its formation are the following: a) inactivation and modification of the drug by enzyme systems of bacteria and its transfer to an inactive form; b) decreased permeability of the surface structures of the bacterial cell; c) disruption of transport mechanisms into the cell; d) change in the functional significance of the target for the drug. The mechanisms of acquired resistance of microorganisms are associated with changes at the genetic level: 1) mutations; 2) genetic recombinations. The mechanisms of intra- and interspecific transmission of extrachromosomal factors of heredity - plasmids and transposons, which control the resistance of microorganisms to antibiotics and other chemotherapeutic drugs - play an extremely important role (13, 20, 23, 33, 39). Information on antibiotic resistance in anaerobic microorganisms comes from both epidemiological and genetic/molecular studies. Epidemiological data indicate that since about 1977, there has been an increase in the resistance of anaerobic bacteria to several antibiotics: tetracycline, erythromycin, penicillin, ampicillin, amoxicillin, ticarcillin, imipenem, metronidazole, chloramphenicol, etc. Approximately 50% of bacteroides are resistant to penicillin G and tetracycline.

When prescribing antibacterial therapy for a mixed aerobic-anaerobic infection, it is necessary to answer a number of questions: a) where is the infection localized?; b) what microorganisms most often cause infections in this area?; c) what is the severity of the disease?; d) what are the clinical indications for the use of antibiotics?; e) what is the safety of using this antibiotic?; f) what is its cost?; g) what is its antibacterial characteristic?; h) what is the average duration of use of the drug to achieve cure?; i) does it penetrate the blood-brain barrier?; j) how does it affect normal microflora?; k) are additional antimicrobial drugs needed to treat this process?

5.1. Characteristics of the main antimicrobial drugs used in the treatment of anaerobic infection

PENICILLIONS. Historically, penicillin G has been widely used to treat mixed infections. However, anaerobes, especially bacteria of the Bacteroides fragilis group, have the ability to produce beta-lactamase and destroy penicillin, which reduces its therapeutic efficacy. It has low or moderate toxicity, a negligible effect on normal microflora, but has weak activity against beta-lactamase-producing anaerobes, in addition, it has limitations against aerobic microorganisms. Semi-synthetic penicillins (naflacin, oxacillin, cloxacillin and dicloxacillin) are less active and are inadequate for the treatment of anaerobic infections. A comparative randomized study of the clinical effectiveness of penicillin and clindamycin for the treatment of pulmonary abscesses showed that when using clindamycin in patients, the period of fever and sputum production was reduced to 4.4 versus 7.6 days and to 4.2 versus 8 days, respectively. On average, 8 (53%) of 15 patients treated with penicillin were cured, while all 13 patients (100%) were cured when treated with clindamycin. Clindamycin is more effective than penicillin in the treatment of patients with anaerobic pulmonary abscess. On average, the effectiveness of penicillin was about 50-55%, and clindamycin - 94-95%. At the same time, the presence of microorganisms resistant to penicillin in the material was noted, which became a common cause of the ineffectiveness of penicillin and at the same time showed that clindamycin is the drug of choice for therapy at the beginning of treatment.

T etra c l i n s. Tetracyclines are also characterized by low

no toxicity and minimal effect on normal microflora. Tetracyclines were also previously the drugs of choice, since almost all anaerobes were sensitive to them, but since 1955 there has been an increase in resistance to them. Doxycycline and monocycline are the more active of these, but a significant number of anaerobes are also resistant to them.

C h l o r a m p h e n i c o l. Chloramphenicol has a significant effect on normal microflora. This drug is extremely effective against bacteria of the B. fragilis group, penetrates well into body fluids and tissues, and has average activity against other anaerobes. In this regard, it has been used as a drug of choice for the treatment of life-threatening diseases, especially those involving the central nervous system, as it easily penetrates the blood-brain barrier. Unfortunately, chloramphenicol has a number of disadvantages (dose-dependent inhibition of hematopoiesis). In addition, it can cause idiosencratic, dose-independent aplastic anemia. Some strains of C. perfringens and B. fragilis are capable of reducing the p-nitro group of chloramphenicol and selectively inactivating it. Some strains of B. fragilis are highly resistant to chloramphenicol because they produce acetyltransferase. Currently, the use of chloramphenicol for the treatment of anaerobic infection has decreased significantly due to both the fear of developing side hematological effects and the emergence of many new, effective drugs.

K l i n d a m i tsin. Clindamycin is a 7(S)-chloro-7-deoxy derivative of lincomycin. Chemical modification of the lincomycin molecule led to several advantages: better absorption from the gastrointestinal tract, an eightfold increase in activity against aerobic gram-positive cocci, an expansion of the spectrum of activity against many gram-positive and gram-negative anaerobic bacteria, as well as protozoa (toxoplasma and plasmodium). Therapeutic indications for the use of clindamycin are quite wide (Table 10).

Gram-positive bacteria. The growth of more than 90% of S. aureus strains is inhibited in the presence of clindamycin at a concentration of 0.1 μg/ml. At concentrations that can easily be achieved in serum, clindamycin is active against Str. pyogenes, Str. pneumonia, Str. viridans. Most strains of diphtheria bacillus are also sensitive to clindamycin. This antibiotic is inactive against gram-negative aerobic bacteria Klebsiella, Escherichia coli, Proteus, Enterobacter, Shigella, Serration, and Pseudomonas. Gram-positive anaerobic cocci, including all types of peptococci, peptostreptococci, as well as propionobacteria, bifidumbacteria and lactobacilli, are generally highly sensitive to clindamycin. Clinically significant clostridia are also sensitive to it - C. perfringens, C. tetani, as well as other clostridia, often found in intraperitoneal and pelvic infections.

Table 10. Indications for the use of clindamycin

Biotope	Disease
Upper respiratory tract	Tonsillitis, pharyngitis, sinusitis, otitis media, scarlet fever
Lower respiratory tract	Bronchitis, pneumonia, empyema, lung abscess
Leather and soft tissues	Pyoderma, boils, cellulitis, impetigo, abscesses, wounds
Bones and joints	Osteomyelitis, septic arthritis
Pelvic organs	Endometritis, cellulitis, vaginal cuff infections, tubo-ovarian abscesses
Oral cavity	Periodontal abscess, periodonitis
Septicemia, endocarditis

Gram-negative anaerobes - Bacteroides, Fusobacteria and Veillonella - are highly sensitive to clindamycin. It is well distributed in many tissues and biological fluids, so that in most of them significant therapeutic concentrations are achieved, but does not penetrate the blood-brain barrier. Of particular interest are the concentrations of the drug in the tonsils, lung tissue, appendix, fallopian tubes, muscles, skin, bones, and synovial fluid. Clindamycin is concentrated in neutrophils and macrophages. Alveolar macrophages concentrate clindamycin intracellularly (30 minutes after administration, the concentration exceeds the extracellular one by 50 times). It increases the phagocytic activity of neutrophils and macrophages, stimulates chemotaxis, and suppresses the production of certain bacterial toxins.

M e tr o n i d a z o l. This chemotherapy drug is characterized by very low toxicity, is bactericidal against anaerobes, and is not inactivated by bacteroid beta-lactamases. Bacteroides are highly sensitive to it, but certain anaerobic cocci and anaerobic gram-positive bacilli may be resistant. Metronidazole is inactive against aerobic microflora and in the treatment of intra-abdominal sepsis it must be combined with gentamicin or some aminoglycosides. May cause transient neutropenia. Metronidazole-gentamicin and clindamycin-gentamicin combinations do not differ in effectiveness in the treatment of serious intra-abdominal infections.

Ts e f o k s i t i n. This antibiotic belongs to the cephalosporins, has low and moderate toxicity and, as a rule, is not inactivated by bacteroid beta-lactamase. Although there is information about cases of isolation of resistant strains of anaerobic bacteria due to the presence of antibiotic-binding proteins that reduce the transport of the drug into the bacterial cell. Resistance of B. fragilis bacteria to cefoxitin ranges from 2 to 13%. It is recommended for the treatment of moderate abdominal infections.

C ephot e t a n. This drug is more active against gram-negative anaerobic microorganisms compared to cefoxitin. However, it has been established that approximately 8% to 25% of B. fragilis strains are resistant to it. It is effective in the treatment of gynecological and abdominal infections (abscesses, appendicitis).

C e p h e m e t a z o l. It is similar in spectrum of action to cefoxitin and cefotetan (more active than cefoxitin, but less active than cefotetan). Can be used to treat mild to moderate infections.

C epha r e z o n. It is characterized by low toxicity, higher activity in comparison with the three above drugs, but from 15 to 28% of resistant strains of anaerobic bacteria have been identified. It is clear that it is not the drug of choice for the treatment of anaerobic infection.

C eft i z o k s i m. It is a safe and effective drug in the treatment of leg infections in diabetic patients, traumatic peritonitis, and appendicitis.

M e r o p e n e m. Meropenem is a new carbapenem, which is methylated at position 1, characterized by resistance to the action of renal dehydrogenase 1, which destroys it. It is approximately 2-4 times more active than imipenem against aerobic gram-negative organisms, including representatives of enterobacteria, hemophilus, pseudomonas, neisseria, but has slightly less activity against staphylococci, some streptococci and enterococci. Its activity against gram-positive anaerobic bacteria is similar to that of imipenem.

5.2. Combinations of beta-lactam drugs and beta-lactamase inhibitors

The development of beta-lactamase inhibitors (clavulanate, sulbactam, tazobactam) is a promising direction and allows the use of new beta-lactam agents protected from hydrolysis when administered simultaneously: a) amoxicillin - clavulanic acid - has a wider spectrum of antimicrobial activity than amoxicillin alone and its effectiveness is close to the combination of antibiotics - penicillin-cloxacillin; b) ticarcillin-clavulanic acid - expands the spectrum of antimicrobial activity of the antibiotic against beta-lacgamase-producing bacteria, such as staphylococci, hemophilus, klebsiella and anaerobes, including bacteroides. The minimum inhibitory concentration of this mixture was 16 times lower than that of ticarcillin; c) ampicillin-sulbactam - when combined in a 1:2 ratio, their spectrum expands significantly and includes staphylococci, hemophilus, klebsiella and most anaerobic bacteria. Only 1% of bacteroides are resistant to this combination; d) cefaperazone-sulbactam - in a 1:2 ratio also significantly expands the spectrum of antibacterial activity; e) piperacillin-tazobactam. Tazobactam is a new beta-lactam inhibitor that acts on many beta-lactamases. It is more stable than clavulanic acid. This combination can be considered as a drug for empirical monotherapy of severe polymicrobial infections, such as pneumonia, intra-abdominal sepsis, necrotizing soft tissue infection, gynecological infections; f) Imipenem-cilastatin - Imipenem is a member of a new class of antibiotics known as carbapenems. Used in combination with cilastatin in a 1:1 ratio. Their effectiveness is similar to clindamycin-aminoglycosides in the treatment of mixed anaerobic surgical infection.

5.3. Clinical significance of determining the sensitivity of anaerobic microorganisms to antimicrobial drugs

The increasing resistance of many anaerobic bacteria to antimicrobial agents raises the question of how and when the determination of sensitivity to antibiotics is justified. The cost of this testing and the time required to obtain a final result further increase the importance of this issue. It is clear that initial therapy for anaerobic and mixed infections should be empirical. It is based on the specific nature of infections and a certain spectrum of bacterial microflora during a given infection. The pathophysiological condition and previous use of antimicrobial drugs, which could modify the normal microflora and the microflora of the lesion, must be taken into account, as well as the results of Gram staining. The next step should be early identification of the dominant microflora. Information on the spectrum of species antibacterial sensitivity of the dominant microflora. Information about the spectrum of species-specific antibacterial sensitivity of the dominant microflora will allow us to assess the adequacy of the initially chosen treatment regimen. In treatment, if the course of the infection is unfavorable, it is necessary to use determination of the sensitivity of a pure culture to antibiotics. In 1988, the Anaerobe Task Force reviewed recommendations and indications for antibiotic susceptibility testing of anaerobes.

Determination of the sensitivity of anaerobes is recommended in the following cases: a) it is necessary to establish changes in the sensitivity of anaerobes to certain drugs; b) the need to determine the spectrum of activity of new drugs; c) in cases of providing bacteriological monitoring of an individual patient. In addition, certain clinical situations may also dictate the need for its implementation: 1) in the case of an unsuccessfully chosen initial antimicrobial regimen and persistent infection; 2) when the choice of an effective antimicrobial drug plays a key role in the outcome of the disease; .3) when the choice of drug in a given particular case is difficult.

It should be taken into account that, from a clinical point of view, there are other points: a) increasing the resistance of anaerobic bacteria to antimicrobial drugs is a big clinical problem; b) clinicians have disagreements about the clinical effectiveness of some drugs against anaerobic infection; c) there are discrepancies between the results of the sensitivity of microorganisms to drugs in vitro and their effectiveness in vivo; r) interpretation of results that is acceptable for aerobes may not always be applicable to anaerobes. Observation of the sensitivity/resistance of 1200 strains of bacteria isolated from different biotopes showed that a significant part of them are highly resistant to the most widely used drugs (Table 11).

Table 11. Resistance of anaerobic bacteria to

widely used antibiotics

Bacteria	Antibiotics		Percentage of resistant forms
Peptostreptococcus	Penicillin Erythromycin Clindamycin
Clostridium perfringens	Penicillin Cefoxitin Metronidazole Erythromycin Clindamycin
Bacteroides fragilis	Cefoxitin Metronidazole Erythromycin Clindamycin
Veilonella	Penicillin Metronidazole Erythromycin

At the same time, numerous studies have established minimum inhibitory concentrations of the most common drugs that are adequate for the treatment of anaerobic infections (Table 12).

Table 12. Minimum inhibitory concentrations

antibiotics for anaerobic microorganisms

The minimum inhibitory concentration (MIC) is the lowest concentration of an antibiotic that completely inhibits the growth of microorganisms. A very important problem is the standardization and quality control of determining the sensitivity of microorganisms to antibiotics (tests used, their standardization, preparation of media, reagents, training of personnel performing this test, use of reference cultures: B. fragilis-ATCC 25285; B. thetaiotaomicron - ATCC 29741; C. perfringens-ATCC 13124; E. lentum-ATCC 43055).

In obstetrics and gynecology, penicillin, some 3-4 generation cephalosporins, lincomycin, and chloramphenicol are used to treat anaerobic infections. However, the most effective antianaerobic drugs are representatives of the 5-nitroimidazole group - metronidazole, tinidazole, ornidazole, and clindamycin. The effectiveness of treatment with metronidazole alone is 76-87%, depending on the disease, and 78-91% with tinidazole. The combination of imidazoles with aminoglycosides and 1st-2nd generation cephalosporins increases the rate of successful treatment to 90-95%. Clindamycin plays a significant role in the treatment of anaerobic infection. The combination of clindamycin with gentamicin is the standard method of treating purulent-inflammatory diseases of the female genital organs, especially in cases of mixed infections.

6. Correction of intestinal microflora

Over the last century, the normal microflora of the human intestine has been the subject of active research. Numerous studies have established that the indigenous microflora of the gastrointestinal tract plays a significant role in ensuring the health of the host, playing an important role in the maturation and maintenance of the function of the immune system, as well as in ensuring a number of metabolic processes. The starting point for the development of dysbiotic manifestations in the intestine is the suppression of indigenous anaerobic microflora - bifidobacteria and lactobacilli, as well as stimulation of the proliferation of opportunistic microflora - enterobacteria, staphylococci, streptococci, clostridia, candida. I. I. Mechnikov formulated the basic scientific principles regarding the role of indigenous intestinal microflora, its ecology and put forward the idea of ​​​​replacing harmful microflora with useful ones in order to reduce intoxication of the body and prolong human life. I. I. Mechnikov’s idea was further developed in the development of a number of bacterial preparations used to correct or “normalize” human microflora. They are called "eubiotics" or "probiotics" and contain live or

dried bacteria of the genera Bifidobacterium and Lactobacillus. The immunomodulatory activity of a number of eubiotics has been shown (stimulation of antibody formation and the activity of peritoneal macrophages is noted). It is also important that strains of eubiotic bacteria have chromosomal resistance to antibiotics, and their joint administration increases the survival of animals. The most widespread are fermented milk forms of lactobacterin and bifidumbacterin (4).

7. Conclusion

Anaerobic infection is one of the unsolved problems of modern medicine (especially surgery, gynecology, therapy, dentistry). Diagnostic difficulties, incorrect assessment of clinical data, errors in treatment, implementation of antibacterial therapy, etc. lead to high mortality in patients with anaerobic and mixed infection. All this points to the need to quickly eliminate both the existing lack of knowledge in this area of ​​bacteriology and significant shortcomings in diagnosis and therapy.

Anaerobic bacteria are able to develop in the absence of free oxygen in the environment. Together with other microorganisms that have a similar unique property, they constitute the class of anaerobes. There are two types of anaerobes. Both facultative and obligate anaerobic bacteria can be found in almost all samples of pathological material; they accompany various purulent-inflammatory diseases, can be opportunistic and even sometimes pathogenic.

Anaerobic microorganisms, classified as facultative, exist and multiply in both oxygen and oxygen-free environments. The most pronounced representatives of this class are Escherichia coli, Shigella, staphylococci, Yersinia, streptococci and other bacteria.

Obligate microorganisms cannot exist in the presence of free oxygen and die from its exposure. The first group of anaerobes of this class is represented by spore-forming bacteria, or clostridia, and the second by bacteria that do not form spores (non-clostridial anaerobes). Clostridia are often causative agents of anaerobic infections of the same name. An example would be clostridial botulism and tetanus. Non-clostridial anaerobes are gram-positive and They have a rod-shaped or spherical shape; you have probably seen the names of their prominent representatives in the literature: bacteroides, veillonella, fusobacteria, peptococci, propionibacteria, peptostreptococci, eubacteria, etc.

Non-clostridial bacteria for the most part are representatives of the normal microflora in both humans and animals. They can also participate in the development of purulent-inflammatory processes. These include: peritonitis, pneumonia, abscess of the lungs and brain, sepsis, phlegmon of the maxillofacial area, otitis media, etc. The majority of infections that are caused by anaerobic bacteria of the non-clostridial type tend to exhibit endogenous properties. They develop mainly against the background of a decrease in the body's resistance, which can occur as a result of injury, cooling, surgery, or impaired immunity.

To explain the method of maintaining the vital functions of anaerobes, it is worth understanding the basic mechanisms by which aerobic and anaerobic respiration occurs.

It is an oxidative process based on Respiration leads to the breakdown of the substrate without a residue, the result being broken down into energy-poor representatives of inorganics. The result is a powerful release of energy. Carbohydrates are the most important substrates for respiration, but both proteins and fats can be consumed in the process of aerobic respiration.

It corresponds to two stages of occurrence. At the first stage, an oxygen-free process of gradual breakdown of the substrate occurs to release hydrogen atoms and bind with coenzymes. The second, oxygen stage, is accompanied by further detachment from the substrate for respiration and its gradual oxidation.

Anaerobic respiration is used by anaerobic bacteria. They use not molecular oxygen, but a whole list of oxidized compounds to oxidize the respiratory substrate. They can be salts of sulfuric, nitric, and carbonic acids. During anaerobic respiration they are converted into reduced compounds.

Anaerobic bacteria that carry out such respiration as the final electron acceptor do not use oxygen, but inorganic substances. Based on their belonging to a certain class, several types of anaerobic respiration are distinguished: nitrate respiration and nitrification, sulfate and sulfur respiration, “iron” respiration, carbonate respiration, fumarate respiration.

1. Characteristics of anaerobes

2. Diagnostics of EMKAR

1. Distribution of anaerobic microorganisms in nature.

Anaerobic microorganisms are found everywhere where organic matter decomposes without access to O2: in different layers of soil, in coastal silt, in piles of manure, in ripening cheese, etc.

Anaerobes can also be found in well-aerated soil, if there are aerobes that absorb O2.

Both beneficial and harmful anaerobes are found in nature. For example, in the intestines of animals and humans there are anaerobes that benefit the host (B. bifidus), which plays the role of an antagonist to harmful microflora. This microbe ferments glucose and lactose and produces lactic acid.

But there are putrefactive and pathogenic anaerobes in the intestines. They break down proteins, cause rotting and various types of fermentation, and release toxins (B. Putrificus, B. Perfringens, B. tetani).

The breakdown of fiber in the animal body is carried out by anaerobes and actinomycetes. This process mainly takes place in the digestive tract. Anaerobes are mainly found in the forestomach and large intestine.

A large number of anaerobes are found in the soil. Moreover, some of them can be found in the soil in vegetative form and reproduce there. For example, B. perfringens. As a rule, anaerobes are spore-forming microorganisms. Spore forms have significant resistance to external factors (chemicals).

2. Anaerobiosis of microorganisms.

Despite the diversity of physiological characteristics of microorganisms, their chemical composition is, in principle, the same: proteins, fats, carbohydrates, inorganic substances.

Regulation of metabolic processes is carried out by the enzymatic apparatus.

The term anaerobiosis (an - negation, aer - air, bios - life) was introduced by Pasteur, who first discovered the anaerobic spore-bearing microbe B. Buturis, capable of developing in the absence of free O2 and facultative ones, developing in an environment containing 0.5% O2 and can bind it (for example, B. chauvoei).

Anaerobic processes - during oxidation, a series of dehydrogenations occur, in which “2H” are sequentially transferred from one molecule to another (ultimately O2 is involved).

At each stage, energy is released, which the cell uses for synthesis.

Peroxidase and catalase are enzymes that promote the use or removal of H2O2 formed during this reaction.

Strict anaerobes do not have mechanisms for binding to oxygen molecules, so they do not destroy H2O2. The anaerobic action of catalase and H2O2 is reduced to the anaerobic reduction of catalase iron by hydrogen peroxide and to aerobic oxidation by the O2 molecule.

3. The role of anaerobes in animal pathology.

Currently, the following diseases caused by anaerobes are considered established:

EMKAR – B. Chauvoei

Necrobacillosis – B. necrophorum

The causative agent of tetanus is B. Tetani.

It is difficult to differentiate these diseases based on their course and clinical signs, and only bacteriological studies make it possible to isolate the corresponding pathogen and establish the cause of the disease.

Some anaerobes have several serotypes and each of them causes different diseases. For example, B. perfringens - 6 serogroups: A, B, C, D, E, F - which differ in biological properties and toxin formation and cause different diseases. So

B. perfringens type A – gas gangrene in humans.

B. perfringens type B – B. lamb – dysentery – anaerobic dysentery in lambs.

B. perfringens type C – (B. paludis) and type D (B. ovitoxicus) – infectious enteroxemia of sheep.

B. perfringens type E – intestinal intoxication in calves.

Anaerobes play a certain role in the occurrence of complications in other diseases. For example, with swine fever, paratyphoid fever, foot-and-mouth disease, etc., as a result of which the process becomes more complicated.

4. Methods for creating anaerobic conditions for growing anaerobes.

There are: chemical, physical, biological and combined.

Nutrient media and cultivation of anaerobes on them.

1.Liquid nutrient media.

A) Meat peptone liver broth - Kitt-Torozza medium - is the main liquid nutrient medium

To prepare it, use 1000 g of bovine liver, which is poured with 1.l tap water and sterilized for 40 minutes. At t=110 C

Dilute with 3 times the amount of MPB

I set pH = 7.8-8.2

For 1 l. broth 1.25 g. Nacle

Add small pieces of liver

Vaseline oil is layered onto the surface of the medium.

Autoclave t=10-112 C – 30-45 min.

B) Brain environment

Ingredients: fresh cattle brain (no later than 18 hours), peeled and minced in a meat grinder

Mix with water 2:1 and pass through a sieve

The mixture is poured into test tubes and sterilized for 2 hours at t=110

Solid culture media

A) Zeismer blood sugar agar is used to isolate a pure culture and determine the growth pattern.

Zeissler agar formula

3% MPA is bottled in 100 ml. and sterilize

Add sterile to molten agar! 10 ml. 20% glucose (t.s. 2%) and 15-20 ml. sterile blood of sheep, cattle, horse

Dried

B) gelatin - in a column

To determine the type of anaerobes, it is necessary to study the following characteristics:

Morphological, cultural, pathological and serological, taking into account their potential for variability.

Morphological and biochemical properties of anaerobes

Morphological features - characterized by pronounced diversity. The forms of microbes in smears prepared from organs differ sharply from the forms of microbes obtained in artificial nutrient media. More often they have the form of rods or threads and less often cocci. The same pathogen can be in the form of rods or in grouped threads. In old cultures it can be found in the form of cocci (eg B. necrophorum).

The largest are B. gigas and B. perfringens with a length of up to 10 microns. And the width is 1-1.5 microns.

Somewhat less than B. Oedematiens 5-8 x 0.8 –1.1. At the same time, the length of Vibrion Septicum filaments reaches 50-100 microns.

Among anaerobes, most are spore-forming microorganisms. The spores are located differently in these microorganisms. But more often it is Clostridium type (closter - spindle). The spores can have a round oval shape. The location of the spores is characteristic of certain types of bacteria: in the center - rods B. Perfringens, B. Oedematiens, etc., or subterminally (somewhat closer to the end) - Vibrion Septicum, B. Histolyticus, etc. and also terminally B. Tetani

Spores are produced one at a time per cell. Spores usually form after the death of the animal. This feature is related to the functional purpose of spores as the preservation of the species in unfavorable conditions.

Some anaerobes are motile and the flagella are arranged in a peritric pattern.

The capsule has a protective function and contains reserve nutrients.

Basic biochemical properties of anaerobic microorganisms

Based on their ability to decompose carbohydrates and proteins, anaerobes are divided into saccharolytic and proteolytic.

Description of the most important anaerobes.

Feser - 1865 in the subcutaneous tissue of a cow.

B. Schauvoei is the causative agent of an acute non-contact infectious disease that mainly affects cattle and sheep. The pathogen was discovered in 1879-1884. Arluenk, Korneven, Thomas.

Morphology and coloring: in smears prepared from pathological material (edematous fluid, blood, affected muscles, serous membranes) B. Schauvoei has the appearance of rods with rounded ends 2-6 microns. x 0.5-0.7 microns. Usually the sticks are found singly, but sometimes short chains (2-4) can be found. Does not form threads. It is polymorphic in shape and often has the shape of swollen bacilli, lemons, spheres, and disks. Polymorphism is especially clearly observed in smears prepared from animal tissue and media rich in proteins and fresh blood.

B. Schauvoei is a movable rod with 4-6 flagella on each side. Does not form capsules.

The spores are large, round to oblong in shape. The spore is located centrally or subterminally. Spores are formed both in tissues and outside the body. On artificial nutrient media, the spore appears within 24-48 hours.

B. Schauvoei is stained with almost all dyes. In young cultures G+, in old ones -G-. Rods perceive color granularly.

EMCAR diseases are septic in nature and therefore Cl. Schauvoei are found not only in organs with pathological abnormalities, but also in pericardial exudate, pleura, kidneys, liver, spleen, lymph nodes, bone marrow, skin and epithelial layer, and blood.

In an unopened corpse, bacilli and other microorganisms multiply rapidly, and therefore a mixed culture is isolated.

Cultural properties. On IPPB Cl. Chauvoei produces abundant growth in 16-20 hours. In the first hours there is uniform turbidity, by 24 hours there is a gradual clearing, and by 36–48 hours the broth column is completely transparent, and at the bottom of the test tube there is a sediment of microbial bodies. With vigorous shaking, the sediment breaks up into a uniform turbidity.

On Martin's broth - after 20-24 hours of growth, turbidity and abundant gas evolution are observed. After 2-3 days there are flakes at the bottom, the medium clears.

Cl. Chauvoei grows well on brain medium, producing small amounts of gases. Blackening of the medium does not occur.

On Zeismer agar (blood) it forms colonies similar to a mother-of-pearl button or grape leaf, flat, with a raised nutrient medium in the center, the color of the colonies is pale purple.

B. Schauvoei coagulates milk within 3-6 days. Coagulated milk has the appearance of a soft, spongy mass. Peptonization of milk does not occur. Does not liquefy gelatin. It does not liquefy curdled whey. Indole does not form. Nitrites are not reduced to nitrates.

Virulence on artificial nutrient media is quickly lost. To maintain it, it is necessary to carry out a passage through the body of guinea pigs. In pieces of dried muscle it retains its virulence for many years.

B. Schauvoei decomposes carbohydrates:

Glucose

Galactose

Levulez

Sucrose

Lactose

Maltose

Does not decompose - mannitol, dulcite, glycerin, inulin, salicin. However, it must be recognized that the ratio of Cl. Chauvoei to carbohydrates is fickle.

On Veillon agar + 2% glucose or serum agar, round or lentil-like colonies with shoots form.

Antigenic structure and toxin formation

Cl. Chauvoei has an O - somatic-thermostable antigen, several H-antigens - thermolabile, as well as a spore S-antigen.

Cl. Chauvoei - causes the formation of agglutinins and complement binding antibodies. Forms a number of strong hemolytic, necrotizing and lethal protein toxins that determine the pathogenicity of the pathogen.

Resistance is due to the presence of spores. It can be stored in rotting corpses for up to 3 months, in heaps of manure with remains of animal tissue - 6 months. Spores persist in the soil for up to 20-25 years.

Boiling depending on the nutrient medium 2-12 minutes (brain), broth cultures 30 minutes. – t=100-1050С, in muscles – 6 hours, in corned beef – 2 years, direct sunlight – 24 hours, 3% formalin solution – 15 minutes, 3% carbolic acid solution has a weak effect on spores, 25% NaOH – 14 hours, 6% NaOH – 6-7 days. Low temperature has no effect on spores.

Sensitivity of animals.

Under natural conditions, cattle are sick at the age of 3 months. up to 4 years. Animals up to 3 months do not get sick (colostral immunity), over 4 years old – animals have suffered from the disease in a latent form. Disease up to 3 months cannot be ruled out. and over 4 years old.

Sheep, buffaloes, goats, and deer also get sick, but rarely.

Camels, horses, pigs are immune (cases have been reported).

Humans, dogs, cats, chickens are immune.

Laboratory animals - guinea pigs.

The incubation period is 1-5 days. The progression of the disease is acute. The disease begins unexpectedly, the temperature rises to 41-43 C. Severe depression stops chewing gum. Often the symptoms are causeless lameness, which indicates damage to the deep layers of the muscles.

Inflammatory tumors appear in the torso, lower back, shoulder, less often the sternum, neck, submandibular space - hard, hot, painful, and soon become cold and painless.

Percussion - tempo sound

Palpation - crupitation.

The skin takes on a dark blue color. Sheep - wool sticks out at the site of the tumor.

The duration of the illness is 12-48 hours, less often 4-6 days.

Pat. anatomy: the corpse is very swollen. Bloody foam with a sour smell (rancid oil) is released from the nose. The subcutaneous tissue at the site of muscle damage contains infiltrates, hemorrhage, and gas. The muscles are black-red in color, covered with hemorrhages, dry, porous, and crunch when pressed. Shells with hemorrhages. The spleen and liver are enlarged.