Colony morphology

• Differentiating colonies:

Important classes of characteristics include:

• size;

• type of margin;

• colony elevation;

• colony texture;

• light transmission;

• colony pigmentation.

Colony size

• Colony size is dependent not just on the type of organism but also on the growth medium and the number of colonies present on a plate (that is, colonies tend to be smaller when greater than as certain amount are present) and on culture medium characteristics.

• Usually stabilizes after few days'.

Colony size usually stabilizes after a day or two of incubation. Exceptions include:

• slow growing microorganisms;

• during growth under conditions that promote slow growth. With slow growth colonies may continue to experience growth past this time, especially if an effort is made to prevent solid medium from drying out.

Type of margin

• Colonies can vary in the shape of their margins (Fig. 8).

Figure 8. Shape of margins of bacteria

(Source: www. mansfield. ohio-state. edu/~sabedon/ bio. 14035. htm#pure_culture_technique)

Illustration, variation in colony margins Colony elevation

• Colonies can vary in their elevations both between microorganisms and growth conditions, and within individual colonies themselves

(Fig. 9).

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Figure 9. Variations in colony elevation of bacteria

(Source: www. mansfield. ohio-state. edu/~sabedon/ bio14035. htm#pure_culture_technique)

Colony texture

• Surface appearance:

Colonies can vary in their texture.

Possible textures include:

• Shiny to dull;

• Smooth to wrinkled;

• Rough;

• Granular;

• Mucoid.

• A shiny, smooth, and/or mucoid appearance tends to be associated with the presence of capsular material.

Colony light transmission

• The light transmission through colonies can range from:

Complete (transparent);

Through intermediate (transparent);

Through completely lacking (opaque).

Colony pigmentation

• Colonies can come in a rainbow of colors.

Purity of colonies must also be controlled by microscopy. For this purpose, fixed smear is prepared, followed by staining procedure with Gram stain. Cells of pure cultures of bacteria are usually homogenous by size and Gram stain. However, it should be remembered that some of the bacteria could be Gram- variable and smear might contain spores, etc.

Purity of cultures can also be confirmed by inoculation on selective media,

thus providing selective growth of microorganisms of particular interest. In this case, homogeneity of grown colonies is used as a criterion of purity.

Specific features of physiology of anaerobes

Oxygen is a universal component of cells and is always provided in large amounts by H₂O. However, prokaryotes display a wide range of responses to molecular oxygen O₂ (Table 9).

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Table 9. TERMS USED TO DESCRIBE O₂ RELATIONS OF MICROORGANISMS

Group	Aerobic	Anaerobic	O2 effect
Obligate aerobe	Growth	No growth	Required (utilized for aerobic
			respiration)
Microaerophile	Growth if level	No growth	Required but at levels below
	not too high		0. 2 atm
Obligate anaerobe	No growth	Growth	Toxic
Facultative anaerobe	Growth	Growth	Not required for growth but
(facultative aerobe)			utilized when available
Aerotolerant anaerobe	Growth	Growth	Not required and not utilized

Obligate aerobes require O₂ for growth; they use O₂ as a final electron acceptor in aerobic respiration.

Obligate anaerobes (occasionally called aerophobes) do not need or use O₂ as a nutrient. In fact, O₂ is a toxic substance, which either kills or inhibits their growth. Obligate anaerobic prokaryotes may live by fermentation, anaerobic respiration, bacterial photosynthesis, or the novel process of methanogenesis.

Facultative anaerobes (or facultative aerobes) are organisms that can switch between aerobic and anaerobic types of metabolism. Under anaerobic conditions (no O₂) they grow by fermentation or anaerobic respiration, but in the presence of O₂ they switch to aerobic respiration.

Aerotolerant anaerobes are bacteria with an exclusively anaerobic (fermentative) type of metabolism but they are insensitive to the presence of O₂. They live by fermentation alone whether or not O₂ is present in their environment.

The response of an organism to O₂ in its environment depends upon the occurrence and distribution of various enzymes which react with O₂ and various oxygen radicals that are invariably generated by cells in the presence of O₂. All cells contain enzymes capable of reacting with O₂. For example, oxidations of flavoproteins by O₂ invariably result in the formation of H₂O₂ (peroxide) as one major product and small quantities of an even more toxic free radical, superoxide or O₂ -. Also, chlorophyll and other pigments in cells can react with O₂ in the presence of light and generate singlet oxygen, another radical form of oxygen which is a potent oxidizing agent in biological systems.

In aerobes and aerotolerant anaerobes the potential for lethal accumulation of superoxide is prevented by the enzyme superoxide dismutase (Fig. 10). All organisms which can live in the presence of O₂ (whether or not they utilize it in their metabolism) contain superoxide dismutase. Nearly all organisms contain the enzyme catalase, which decomposes H₂O₂. Even though certain aerotolerant bacteria such as the lactic acid bacteria lack catalase, they decompose H₂O₂ by means of peroxidase enzymes which derive electrons from NADH₂ to reduce peroxide to H₂0. Obligate anaerobes lack superoxide dismutase and catalase

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Figure 10. The action of superoxide dismutase, catalase and peroxidase

and/or peroxidase, and therefore undergo lethal oxidations by various oxygen radicals when they are exposed to O₂.

The distribution of these enzymes in cells determines their ability to exist in the presence of O₂ and presented in Table 10.

Table 10. DISTRIBUTION OF SUPEROXIDE DISMUTASE, CATALASE AND PEROXIDASE IN PROKARYOTES WITH DIFFERENT O₂ TOLERANCES.

Group	Superoxide dismutase Catalase		Peroxidase
Obligate aerobes and most	+	+	—
facultative anaerobes
(e.g. Enterobacteriaceae)
Most aerotolerant anaerobes	+	-	+
(e.g. Streptococcus spp. )
Obligate anaerobes (e.g.	-	-	-
Clostridium spp., Bacteroides spp. )

Methods of creation of anaerobic conditions

Physical methods are based on creation of oxygen-free conditions of growth by the following means:

• Inoculation of media containing reducers and easily oxidative substances;

• Inoculation of microorganisms into depth of solid media;

• Mechanical removal of air from incubating cameras/flasks;

• Replacement of air by special gas mixture.

Usually peaces of animal or plants are used as reducers (e.g. liver, brain, blood, potato, etc. ). Those are binding diluted oxygen in nutrient media and also absorbing bacteria. In order to decrease of concentration of oxygen in nutrient medium, before inoculation it should be boiled for 10-15 min., then quickly cooled down and sealed with sterile liquid paraffin. Glucose, lactose and aminoformic sodium are used as easily oxidative substances.

One of the best examples of broth with reducers is Kitt-Tarozzi medium

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which is primarily used for accumulation of anaerobes under primary inoculation and support of growth of pure cultures.

Inoculation of microorganisms into depth of solid media is done by Weinberg and Venyal-Venyon methods.

Weinberg method is based on cultivation of anaerobes into tube glucose agar. Glucose agar is poured into tube in column fashion up to 2/3 of tube volume. Then it allowed to cool down to +42-45°C. Studied specimen is added to agar and thoroughly mixed. Tube is placed in tube stand. After solidification of media, good anaerobic conditions are created (especially in bottom part of the tube).

Venyal-Venyon method is based on mechanical protection of anaerobic cultures from atmospheric oxygen. For this purpose, a long (30 cm in length and 3-6 mm is diameter) glass tube. One side of tube is stretched out as a Pasteur pipette and on second side constriction is made. The wide side of tube is sealed with cotton plug. Studied material is inoculating into tubes with melted and cooled up to +50°C agar. Inoculated agar then is sucked into sterile Venyal- Venyon tubes. Capillary side of the tube is sealed by flaming and tubes are placed into incubator, thus creating suitable conditions for inoculating even strict anaerobes. For isolation of separate colony tube is cut with file in aseptic conditions, on the level of colony, cut, then colony is taken by sterile bacteriological loop and taken to tube with nutrient medium.

Removal or replacement of air is performed mechanically from anaerobic jars.

Chemical methods are based on absorption of oxygen in hermetically sealed flask (anaerobic or candle jar) by pyrogallol or sodium bisulfite.

Biological methods (Fortner' method) are based on concomitant incubation of anaerobes and obligate aerobes. For this purpose approximately 1 cm wide strip is cut by sterile scalpel from solidified agar (by diameter of Petri dish). By this approach, 2 agar semi-disks are obtained per 1 plate. On one side of agar strip, aerobe is inoculated (e.g. Staphylococcus aureus or Serratia marcescens), on the other one - anaerobe. Edges of Petri dishes are sealed with liquid paraffin, and Petri dishes are placed in the incubator. In case of suitable conditions, aerobes are starting to multiply thus consuming oxygen. After 3-4 days, when all oxygen is consumed, anaerobes are starting to grow.

Combined methods are based on various combinations of physical, chemical and biological methods.

Influence of physical factors on microorganisms

(adapted from http://textbookofbacteriologv.net/control.html)

Microorganisms have been found growing in virtually all environments where there is liquid water, regardless of its temperature. Subsequently, procaryotes have been detected growing around black smokers and hydrothermal vents in the deep sea at temperatures at least as high as +115°C. Microorganisms have been found growing at very low temperatures as well. In supercooled solutions of H₂O as low as -20°C, certain organisms can extract water for growth,

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and many forms of life flourish in the icy waters of the Antarctic, as well as household refrigerators, near 0°C.

Considering the total span of temperature where liquid water exists, the procaryotes may be subdivided into several subclasses on the basis of one or another of their cardinal points for growth. For example, organisms with an optimum temperature (T) near +37°C (the body temperature of warm-blooded animals) are called mesophiles. Organisms with an optimum T between about +45°C and +70°C are thermophiles. Some Archaea with an optimum T of +80°C or higher and a maximum T as high as +115°C, are now referred to as extreme thermophiles or hyperthermophiles. The cold-loving organisms are psychrophiles defined by their ability to grow at 0°C, A variant of a psychrophile (which usually has an optimum T of +10-15°C) is a psychrotroph, which grows at 0°C but displays an optimum T in the mesophile range, nearer room temperature. Psychrotrophs are the scourge of food storage in refrigerators since they are invariably brought in from their mesophilic habitats and continue to grow in the refrigerated environment where they spoil the food. Of course, they grow slower at +2°C than at +25°C.

The control of microbial growth is necessary in many practical situations, and significant advances in agriculture, medicine, and food science have been made through study of this area of microbiology.

«Control of growth», as used here, means to prevent growth of microorganisms. This control is effected in two basic ways:

• By killing microorganisms or

• By inhibiting the growth of microorganisms.

Control of growth usually involves the use of physical or chemical agents which either kill or prevent the growth of microorganisms. Agents which kill cells are called cidal agents; agents which inhibit the growth of cells (without killing them) are referred to as static agents. Thus the term bactericidal refers to killing bacteria and bacteriostatic refers to inhibiting the growth of bacterial cells. A bactericide kills bacteria, a fungicide kills fungi, and so on.

Sterilization is the complete destruction or elimination of all viable organisms (in or on an object being sterilized). There are no degrees of sterilization: an object is either sterile or not. Sterilization procedures involve the use of heat, radiation or chemicals, or physical removal of cells.

Methods of sterilization

Heat: most important and widely used. For sterilization always consider type of heat, time of application and temperature to ensure destruction of all microorganisms. Endospores of bacteria are considered the most thermoduric of all cells so their destruction guarantees sterility.

1. Incineration: bums organisms and physically destroys them. Used for needles, inoculating wires, glassware, etc. and objects not destroyed in the incineration process.

2. Boiling: + 100°C for 30 minutes. Kills everything except some endospores

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(Actually, for the purposes of purifying drinking water +100°C for five minutes is probably adequate though there have been some reports that Giardia cysts can survive this process). To kill endospores, and therefore sterilize the solution, very long or intermittent boiling is required.

3. Autoclaving (steam under pressure or pressure cooker): +121°C for 15 minutes (15#/in² pressure). Good for sterilizing almost anything, but heat-labile substances will be denatured or destroyed.

4. Dry heat (hot air oven): +160°C/2 hours or +170°C/1 hour. Used for glassware, metal, and objects that won’t melt.

The protocol and recommendations for the use of heat to control microbial growth are given in Table 11.

Irradiation: usually destroys or distorts nucleic acids. Ultraviolet light is usually used (commonly used to sterilize the surfaces of objects), although X-rays and microwaves are possibly useful.

Table 11. RECOMMENDED USE OF HEAT TO CONTROL BACTERIAL GROWTH

Treatment	Temperature	Effectiveness
Incineration	>500°C	Vaporizes organic material on nonflammable surfaces but may destroy many substances in the process
Boiling	1OO°C	30 minutes of boiling kills microbial pathogens and vegetative forms of bacteria but may not kill bacterial endospores
Intermittent boiling	100°C	Three 30-minute intervals of boiling, followed by periods of cooling kills bacterial endospores
Autoclave and pressure	121°C/15 minutes	Kills all forms of life including bacteria]
cooker (steam under pressure)	at 15# pressure	endospores. The substance being sterilized must be maintained at the effective T for the full time
Dry heat (hot air oven)	160°C/2 hours	For materials that must remain dry and which are not destroyed at T between 121°C and 170°C Good for glassware, metal, not plastic or rubber items
Dry heat (hot air oven)	170°C/1 hour	Same as above, Note increasing T by 10 degrees shortens the sterilizing time by 50 percent
Pasteurization (batch method)	63°C/30 minutes	Kills most vegetative bacterial cells including pathogens such as streptococci, staphylococci and Mycobacterium tuberculosis
Pasteurization (flash method)	72°C/15 seconds	Effect on bacterial cells similar to batch method; for milk, this method is more conducive to industry and has fewer undesirable effects on quality or taste

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Filtration: involves the physical removal (exclusion) of all cells in a liquid or gas, especially important to sterilize solutions which would be denatured by heat (e.g. antibiotics, injectable drugs, amino acids, vitamins, etc. )

Chemical and gas: (formaldehyde, glutaraldehyde, ethylene oxide) toxic chemicals kill all forms of life in a specialized gas chamber.

The lethal temperature varies in microorganisms. The time required to kill depends on the number of organisms, species, nature of the product being heated, pH, and temperature. Whenever heat is used to control microbial growth inevitably both time and temperature are considered.

1. Sterilization (boiling, autoclaving, hot air oven) kills all microorganisms with heat; commonly employed in canning, bottling, and other sterile packaging procedures.

2. Pasteurization is the use of mild heat to reduce the number of microorganisms in a product or food. In the case of pasteurization of milk the time and temperature depend on killing potential pathogens that are transmitted in milk, i.e., staphylococci, streptococci, Brucella abortus and Mycobacterium tuberculosis. For pasteurization of milk: batch method: +63°C for 30 minutes; flash method: +72°C for 15 seconds.

Low temperature (refrigeration and freezing): Most organisms grow very little or not at all at 0°C. Store perishable foods at low temperatures to slow rate of growth and consequent spoilage (e.g. milk). Low temperatures are not bactericidal. Psychrotrophs, rather than true psychrophiles, are the usual cause of food spoilage in refrigerated foods. Listeria monocytogenes is of great concern in refrigerated foods and has been the topic of recent news articles and FDA action.

Drying (removal of H₂O): Most microorganisms cannot grow at reduced water activity (Aw < 0.90). Often used to preserve foods (e.g. fruits, grains, etc. ). Methods involve removal of water from product by heat, evaporation, freeze-drying, and addition of salt or sugar.

Irradiation (microwave, UV, X-ray): destroys microorganisms as described under sterilization. Many spoilage organisms are easily killed by irradiation. In some parts of Europe, fruits and vegetables are irradiated to increase their shelf life up to 500 percent.

Main objectives of the session

1. To study respiration of bacteria and classify microorganisms based on respiration type.

2. To learn principles of inoculation and isolation of pure cultures of aerobes and anaerobes.

3. To study the character of influence of physical and chemical factors.

4. To learn sterilization methods and get acquainted with principles of work of autoclave and hot air oven.

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Educational tasks

To know: 1. Principles of respiration of bacteria. Classification of

microorganisms based on type of respiration. Aerobic and anaerobic respiration.

2. Principles of cultivation of anaerobic bacteria.

3. Stages of isolation of pure cultures of microorganisms and their identification.

4. Action of physical and chemical factors. Sterilization and disinfection. Asepsis and antiseptics.

To be 1. To be able to prepare laboratory ware for sterilization in hot air

capable: oven and autoclave.

2. To describe cultural properties of bacteria.

3. To learn methods of creation of anaerobic conditions.

Methodical guidelines

To take out from the incubator and examine Petri dishes with solid agar, inoculated on the first day and look for presence of colonies of different types of shape, color, size, consistence. Each microorganism is characterized by specific type of colony, which helps to select colony of microorganism of interest and establish diagnosis. Take down isolated colony by bacteriological loop and inoculate agar slant. Remaining part of the colony is used for preparation of the smear and Gram stain.

Demonstrations

1. To describe the colonies of solid agar in Petri dishes plate. Inoculate one colony onto agar slant

2. To get acquainted with different methods of cultivation of anaerobes: anaerobic jar, Forner’ method, inoculation Kitt-Tarozzi medium, column of Wilson-Blair medium, blood agar (Venyal-Venyon method), usage of pyrogallol solution.

3. Demonstration of apparatus for sterilization: autoclaves, hot air oven, electric sterilizer, porcelain candles, Zeits’ filters. Incubator and thermoregulators. Koch roller. Tests for control of sterilization.

Students’ activities

1. Students in groups are examining plates with bacterial cultures.

2. Each student examines and describes in detail different type of colonies on Petri dishes. Re-inoculation of one colony into agar slant, preparation of smear, Gram stain and microscopy. Draw the observation into the workbook.

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Control questions

1. What is sterilization?

2. What is disinfection?

3. What is pasteurization

4. What are sterilization conditions in autoclave?

5. What are sterilization conditions in hot air ovens (Pasteur’ ovens)?

6. What tests for control of sterilization do you know?

7. What is fractional sterilization and what is it used for?

8. Why in some cases inoculation of one colony leads to growth of mixed culture?

9. What characteristics are necessary to consider during the examination of colonies?

10. How the growth of bacteria can be characterized in broth and semi- solid media?

11. What is the basis for aerobic type of oxidation?

12. What is the basis for anaerobic type of oxidation?

13. What media are used for cultivation of anaerobes?

14. What methods are used for cultivation of anaerobes?

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PRACTICAL SESSION No. 7

Methods of isolation and identification of pure cultures of aerobic bacteria (continuation): biochemical activity of bacteria. Antibiotics.

Plan of the session

1. Identification of isolated pure cultures of bacteria based on biochemical properties.

2. Antagonism of microorganisms.

3. Classification of microorganisms.

4. Mechanisms of action of main groups of antimicrobial agents.

5. Methods of detection of antibiotics in body fluids.

6. Quantitative and qualitative determination of susceptibility of bacteria to antibiotics.

7. Mechanisms of development of antibiotic resistance in bacteria and approaches to its overcoming.

8. Side effects of antimicrobial therapy.

Foreword notes

1. Identification of bacteria grown on agar slant is performed only after establishment of purity (homogeneity) of culture based on morphological, tinctorial and cultural properties. In addition, fermentative (biochemical) activities, susceptibility to phages, toxigenicity and other characteristic properties of bacteria are determined. In some cases, epidemiological markers (serotype, phage type, biotype, etc. ) are determined in order to establish source of infection and ways of its spread.

2. In natural conditions, microorganisms exist in complex associations within which here are different types of relationship are established. There are determined, primarily, by physiological and biochemical properties of members of association and also by different ecological factors. Relationships between microorganisms can be symbiotic (symbiosis, metabiosis, satellitism, synergism) and competitive (antagonism, parasitism, etc. ).

Symbiosis is a relationship between microorganisms when two or more species in concomitant existence are creating beneficial conditions for each other. Typical example of such relationship is concomitant growth of aerobic and anaerobic bacteria.

During metabiosis products of vital functions of one microorganism which contain a substantial amount of energy are consumed by other microorganisms

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as nutrient substances. Between such microorganisms so called syntrophic connections are created.

A special sort of metabiosis is satellitism, which is characterized by the fact that one type of microorganisms is secreting factors (amino acids, vitamins, etc. ) which are stimulating growth of others. A typical example is stimulation of growth of Haemophilus influenzae on blood agar by hemolytic Staphylococcus aureus.

Apart from symbiotic relationships between microorganisms there are also types in which one type of bacteria is partially or completely inhibiting growth of others — so called antagonism. There are the following types of antagonism:

• Antagonism during the concomitant development of different species which need in the same nutrient substances. In such type of antagonism advantages will have bacteria growth rate of which will be faster on comparison with others. For example, during the concomitant inoculation of eubacteria and actinomyces, eubacteria will grow faster.

• Antagonisms connected with the production of organic acids, alcohols and other products of vital functions which change environments creating unsuitable conditions for growth of other microorganisms. When fresh milk is left out of fridge, propagation of microorganisms, including lactobacteria and others, is started generally at the same rate. Later, because of propagation of lactobacteria. Lactic acid concentration is increasing, thus inhibition of growth of all bacteria, except of lactobacteria, is observed.

• Antagonisms associated with production and excretion into the environment of inhibitory substances (antibiotics, bacteriocins, etc. ).

Process of predatoriness include destruction by some microorganisms cell of others, followed by their use as a nutrient substrate (e.g. Myxobacteria spp. )

Parasitism is characterized when one type of bacteria (parasite) is invading other (host) and feed on it. Obligate parasites cannot exist in the absence of host. The typical examples of parasites are bacteriophages.

The practical use of antagonism is use of products of vital functions of some microorganisms, which partially or completely inhibit growth of others.

Examples of those are antibiotics which are used either unchanged and undergo by chemical modification to increase spectrum of their activities, etc.

Antibiotics (adapted from http: //textbookofbacteriology. net/control. html with additions and amendments) are antimicrobial agents produced by microorganisms that kill or inhibit other microorganisms. This is the microbiologist’s definition. A more broadened definition of an antibiotic includes any chemical of natural origin (from any type of cell), which has the effect to kill or inhibit the growth of other types cells. Since most clinically- useful antibiotics are produced by microorganisms and are used to kill or inhibit infectious Bacteria, we will follow the classic definition.

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Antibiotics are low molecular-weiglit (non-protein) molecules produced as secondary metabolites, mainly by microorganisms that live in the soil. Most of these microorganisms form some type of a spore or other dormant cell, and there is thought to be some relationship (besides temporal) between antibiotic production and the processes of sporulation. Among the molds, the notable antibiotic producers are Penicillium and Cephalosporium, which are the main source of the beta-lactam antibiotics (penicillin and its relatives). In the bacteria, the Actinomycetes, notably Streptomyces species, produce a variety of types of antibiotics including the aminoglycosides (e.g. streptomycin), macrolides (e.g. erythromycin), and the tetracyclines. Endospores-forming Bacillus spp. produce polypeptide antibiotics such as polymyxin and bacitracin. The Table 12 is a summary of the classes of antibiotics and their properties including their biological sources.

Antimicrobial agents used in the treatment

of infectious diseases

The modern era of antimicrobial chemotherapy began in 1929 with Fleming’s discovery of the powerful bactericidal substance penicillin, and Domagk’s discovery in 1935 of synthetic chemicals (sulfonamides) with broad antimicrobial activity. In the early 1940”s, spurred partially by the need for antibacterial agents in World War II, penicillin was isolated, purified and injected into experimental animals, where it was found to not only cure infections but also to possess incredibly low toxicity for the animals. This fact ushered into being the age of antibiotic chemotherapy and an intense search for similar antimicrobial agents of low toxicity to animals that might prove useful in the treatment of infectious disease. The rapid isolation of streptomycin, chloramphenicol and tetracycline soon followed, and by the 1950’s, these and several other antibiotics were in clinical usage.

The most important property of a clinically-useful antimicrobial agent, especially from the patient’s point of view, is its selective toxicity, i.e., that the agent acts in some way that inhibits or kills bacterial pathogens but has little or no toxic effect on the animal taking the drug This implies that the biochemical processes in the bacteria are in some way different from those in the animal cells, and that the advantage of this difference can be taken in chemotherapy. Antibiotics may have a cidal (killing) effect or a static (inhibitory) effect on a range of microbes. The range of bacteria or other microorganisms that are affected by a certain antibiotic are is expressed as its spectrum of activity. Antibiotics effective against procaryotes which kill or inhibit a wide range of Gram-positive and Gram-negative bacteria are said to be broad spectrum. If effective mainly against Gram-positive or Gram-negative bacteria, they are narrow spectrum. If effective against a single organism or disease, they are referred to as limited spectrum.

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Kinds of antimicrobial agents and their

primary modes of action

1. Cell wall synthesis inhibitors Cell wall synthesis inhibitors generally inhibit some step in the synthesis of bacterial peptidoglycan. Generally they exert their selective toxicity against eubacteria because human cells lack cell walls.

Beta-lactam antibiotics Chemically, these antibiotics contain a 4-membered beta lactam ring. They are the products of two groups of fungi, Penicillium and Cephalosporium molds, and are correspondingly represented by the penicillins and cephalosporins. The beta-lactam antibiotics inhibit the last step in peptidoglycan synthesis, the final cross-linking between peptide side chains, mediated by bacterial carboxypeptidase and transpeptidase enzymes. Beta lactam antibiotics are normally bactericidal and require that cells be actively growing in order to exert their toxicity.

Natural penicillins, such as Penicillin G or Penicillin V, are produced by fermentation of Penicillium chrysogenum. They are effective against streptococcus, gonococcus and staphylococcus, except where resistance has developed. They are considered narrow spectrum since they are not effective against Gram-negative rods.

Semisynthetic penicillins first appeared in 1959. A mold produces the main part of the molecule (6-aminopenicillahic acid) which can be modified chemically by the addition of side chains. Many of these compounds have been developed to have distinct benefits or advantages over penicillin G, such as increased spectrum of activity (effectiveness against Gram-negative rods), resistance to penicillinase, effectiveness when administered orally, etc. Amoxicillin and ampicillin have broadened spectra against Gram- negatives and are effective orally; oxacillin is penicillinase-resistant. Clavulanic acid is a chemical sometimes added to a semisynthetic penicillin preparation to inhibit beta-lactamase enzymes and has given extended life to penicillinase-sensitive beta-lactams.

Although nontoxic, penicillins occasionally cause death when administered to persons who are allergic to them. In the U.S. there are 300-500 deaths annually due to penicillin allergy. In allergic individuals the beta lactam molecule attaches to a serum protein which initiates an IgE-mediated inflammatory response.

Cephalolsporins are beta lactam antibiotics with a similar mode of action to penicillins that are produced by species of Cephalosporium. The have a low toxicity and a somewhat broader spectrum than natural penicillins. They are often used as penicillin substitutes, against Gram-negative bacteria, and in surgical prophylaxis. They are subject to degradation by some bacterial beta-lactamases, but they tend to be resistant to beta- lactamases from S. aureus.

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Bacitracin is a polypeptide antibiotic produced by Bacillus species. It prevents cell wall growth by inhibiting the release of the muropeptide subunits of peptidoglycan from the lipid carrier molecule that carries the subunit to the outside of the membrane Teichoic acid synthesis, which requires the same carrier, is also inhibited. Bacitracin has a high toxicity which precludes its systemic use. It is present in many topical antibiotic preparations, and since it is not absorbed by the gut, it is given to «sterilize» the bowel prior to surgery.

2. Ceil membrane inhibitors disorganize the structure or inhibit the function of bacterial membranes. The integrity of the cytoplasmic and outer membranes is vital to bacteria, and compounds that disorganize the membranes rapidly kill the ceils. However, due to the similarities in phospholipids in eubacterial and eukaryotic membranes, this action is rarely specific enough to permit these compounds to be used systemically. The only antibacterial antibiotic of clinical importance that acts by this mechanism is polymyxin, produced by Bacillus polymyxis. Polymyxin is effective mainly against Gram-negative bacteria and is usually limited to topical usage. Polymyxins bind to membrane phospholipids and thereby interfere with membrane function. Polymyxin is occasionally given for urinary tract infections caused by Pseudomonas aeruginosa that are gentamicin, carbenicillin and tobramycin resistant. The balance between effectiveness and damage to the kidney and other organs is dangerously close, and the drug should only be given under close supervision in the hospital.

3. Protein synthesis inhibitors Many therapeutically useful antibiotics owe their action to inhibition of some step in the complex process of translation. Their attack is always at one of the events occurring on the ribosome and rather than the stage of amino acid activation or attachment to a particular tRNA. Most have an affinity or specificity for 70S (as opposed to 80S) ribosomes, and they achieve their selective toxicity in this manner. The most important antibiotics. with this mode of action are the tetracyclines, chloramphenicol, the macrolides (e.g. erythromycin) and the aminoglycosides (e.g. streptomycin).

The aminoglycosides are products of Streptomyces species and are represented by streptomycin, kanamycin, amikacin and gentamicin. These antibiotics exert their activity by binding to bacterial ribosomes and preventing the initiation of protein synthesis. Aminoglycosides have been used against a wide variety of bacterial infections caused by Gram- positive and Gram-negative bacteria. Streptomycin has been used extensively as a primary drug in the treatment of tuberculosis. Gentamicin is active against many strains of Gram-positive and Gram- negative bacteria, including some strains of Pseudomonas aeruginosa. Kanamycin (a complex of three antibiotics, A, B and C) is active at low

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concentrations against many Gram-positive bacteria, including penicillin-resistant staphylococci. Gentamicin and amikacin are mainstays for treatment of pseudomonas infections. An unfortunate side effect of aminoglycosides has tended to restrict their usage: prolonged use is known to impair kidney function and cause damage to the auditory nerves leading to deafness.

The tetracyclines consist of eight related antibiotics which are all natural products of Streptomyces, although some can now be produced semisynthetically. Tetracycline, chlortetracyciine and doxycycline are the best known. The tetracyclines are broad-spectrum antibiotics with a wide range of activity against both Gram-positive and Gram-negative bacteria. The tetracyclines act by blocking the binding of aminoacyl tRNA to the A site on the ribosome. Tetracyclines inhibit protein synthesis on isolated 70S or 80S (eukaryotic) ribosomes, and in both cases, their effect is on the small ribosomal subunit. However, most bacteria possess an active transport system for tetracycline that will allow intracellular accumulation of the antibiotic at concentrations 50 times as great as that in the medium. This greatly enhances its antibacterial effectiveness and accounts for its specificity of action, since an effective concentration cannot be accumulated in animal cells. Thus a blood level of tetracycline which is harmless to animal tissues can halt protein synthesis in invading bacteria. The tetracyclines have a remarkably low toxicity and minimal side effects when taken by animals. The combination of their broad spectrum and low toxicity has led to their overuse and misuse by the medical community and the widespread development of resistance has reduced their effectiveness. Nonetheless, tetracyclines still have some important uses, such as in the treatment of Lyme disease.

Chloramphenicol has a broad spectrum of activity but it exerts a bacteriostatic effect. It is effective against intracellular parasites such as the rickettsia. Unfortunately, aplastic anemia, which is dose related develops in a small proportion (1/50,000) of patients. Chloramphenicol was originally discovered and purified from the fermentation of a Streptomyces, but currently it is produced entirely by chemical synthesis. Chloramphenicol inhibits the bacterial enzyme peptidyl transferase thereby preventing the growth of the polypeptide chain during protein synthesis. Chloramphenicol is entirely selective for 70S ribosomes and does not affect 80S ribosomes. Its unfortunate toxicity towards the small proportion of patients who receive it is in no way related to its effect on bacterial protein synthesis. However, since mitochondria probably originated from prokaiyotic cells and have 70S ribosomes, they are subject to inhibition by some of the protein synthesis inhibitors including chloramphenicol. This likely explains the toxicity of chloramphenicol. The eukaryotic cells most likely to be inhibited by

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chloramphenicol are those undergoing rapid multiplication, thereby rapidly synthesizing mitochondria. Such cells include the blood forming cells of the bone marrow, the inhibition of which could present as aplastic anemia. Chloramphenicol was once a highly prescribed antibiotic and a number of deaths from anemia occurred before its use was curtailed. Now it is seldom used in human medicine except in life- threatening situations (e.g. typhoid fever).

The macrolides are a family of antibiotics whose structures contain large lactone rings linked through glycoside bonds with amino sugars. The most important members of the group are erythromycin, azithromycin and clarithromycin. Erythromycin is active against most Gram-positive bacteria, Neisseria and Legionella, but not against the Enterobacieriaceae and Haemophilus. Macrolides inhibit bacterial protein synthesis by binding to the 50S ribosomal subunit. Binding inhibits elongation of the protein by peptidyl transferase or prevents translocation of the ribosome or both. Macrolides are bacteriostatic for most bacteria but are cidal for a few Gram-positive bacteria.

2. Effects on Nucleic Acids Some chemotherapeutic agents affect the synthesis of DNA or RNA, or can bind to DNA or RNA so that their messages cannot be read. Either case, of course, can block the growth of cells. The majorities of these drugs are unselective, however, and affect animal cells and bacterial cells alike and therefore have no therapeutic application. Two nucleic acid synthesis inhibitors which have selective activity against procaryotes and some medical utility are nalidixic acid and rifamycins.

Nalidixic acid is a synthetic chemotherapeutic agent which has activity mainly against Gram-negative bacteria. Nalidixic acid belongs to a group of compounds called quinolones. Nalidixic acid is a bactericidal agent that binds to the DNA gyrase enzyme (topoisomerase) which is essential for DNA replication and allows supercoils to be relaxed and reformed. Binding of the drug inhibits DNA gyrase activity.

Some quinolones penetrate macrophages and neutrophils better than most antibiotics and are thus useful in treatment of infections caused by intracellular parasites. However, the main use of nalidixic acid is in treatment of lower urinary tract infections (UTI). The compound is unusual in that it is effective against several types of Gram-negative bacteria such as Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae and Proteus spp. which are common causes of UTIs. It is not usually effective against P. aeruginosa, and Gram-positive bacteria are resistant. Newer compounds are characterized by wider spectrum of activity. For example, ciprofloxacin has an excellent primary activity against P. aeruginosa, levofloxacin and moxifloxacin both have very good activity against gram-positives (especially Streptococcus pneumoniae).

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The rifamycins are also the products of Streptomyces. Rifampicin is a semisynthetic derivative of rifamycin that is active against Gram-positive bacteria (including Mycobacterium tuberculosis) and some Gram-negative bacteria. Rifampicin acts quite specifically on eubacterial RNA polymerase and is inactive towards RNA polymerase from animal cells or towards DNA polymerase. The antibiotic binds to the beta subunit of the polymerase and apparently blocks the entry of the first nucleotide which is necessary to activate the polymerase, thereby blocking mRNA synthesis. It has been found to have greater bactericidal effect against M. tuberculosis than other anti-tuberculosis drags, and it has largely replaced isoniazid as one of the front-line drugs used to treat the disease, especially when isoniazid resistance is indicated. It is effective orally and penetrates well into the cerebrospinal fluid and is therefore useful for treatment of tuberculosis meningitis and meningitis caused by Neisseria meningitidis.

2. Competitive Inhibitors The competitive inhibitors are mostly all synthetic chemotherapeutic agents. Most are «growth factor analogs» which are structurally similar to a bacterial growth factor but which do not fulfill its metabolic function in the cell. Some are bacteriostatic and some are bactericidal.

Sulfonamides were introduced as chemotherapeutic agents by Domagk in 1935, who showed that one of these compounds (prontosil) had the effect of curing mice with infections caused by beta-hemolytic streptococci. Chemical modifications of the compound sulfanilamide gave compounds with even higher and broader antibacterial activity. The resulting sulfonamides have broadly similar antibacterial activity, but differ widely in their pharmacological actions. However, due to severe side effects, their clinical significance currently is very limited. The sulfonamides and trimethoprim are inhibitors of the bacterial enzymes required for the synthesis of tetrahydrofolic acid (THF), the vitamin form of folic acid essential for 1 -carbon transfer reactions. Sulfonamides are structurally similar to para aminobenzoic acid (PABA), the substrate for the first enzyme in the THF pathway, and they competitively inhibit that step. Trimethoprim is structurally similar to dihydrofolate (DHF) and competitively inhibits the second step in THF synthesis mediated by the DHF reductase. Animal cells do not synthesize their own folic acid but obtain it in a preformed fashion as a vitamin. Since animals do not make folic acid, they are not affected by these drugs, which achieve their selective toxicity for bacteria on this basis.

Three additional synthetic chemotherapeutic agents have been used in the treatment of tuberculosis: isoniazid (INH), paraaminosalicylic acid (PAS), and ethambutol. The usual strategy in the treatment of tuberculosis has been to administer a single antibiotic (historically streptomycin, but now, most commonly, rifampicin is given) in conjunction with INH and ethambutol. Since the tubercle bacillus rapidly

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develops resistance to the antibiotic, ethambutol and INH are given to prevent outgrowth of a resistant strain. It must also be pointed out that the tubercle bacillus rapidly develops resistance to ethambutol and INH if either drug is used alone. Ethambutol inhibits incorporation of mycolic acids into the mycobacterial cell wall. Isoniazid has been reported to inhibit mycolic acid synthesis in mycobacteria and since it is an analog of pyridoxine (vitamin B₆) it may inhibit pyridoxine catalyzed reactions as well. Isoniazid is activated by a mycobacterial peroxidase enzyme and destroys several targets in the cell. PAS is an anti-folate. PAS was once a primary anti-tuberculosis drug, but now it is a secondary agent,

Main objectives of the session

1. To study essence and mechanism of action of different fermentative systems in bacteria: to get acquainted with methods of their studies and use for identification of pure cultures of bacteria.

2. To learn peculiarities of relationships between bacteria as a main basis for usage of antibiotics.

3. To get acquainted with methods of determination of susceptibility of bacteria.

Educational tasks

To know: 1. Classification of enzymes in bacteria. Mechanisms of their

action and methods of study.

2. Stages of isolation of pure cultures of microorganisms and their identification based on biochemical properties.

3. Chemotherapy. Understanding of chemotherapeutic index. Principles of antimicrobial chemotherapy

4. Symbiotic and competitive relationships between microorganisms.

5. Microbial antagonism, its mechanisms. Antibiotic-producing microorganisms.

6. Classification of antibiotics based on chemical structure, origin, mechanisms and spectrum of antimicrobial action, methods of receipt.

7. Methods of determination of susceptibility of bacteria.

8. Side effects of antibiotic therapy.

To be 1. To determine fermentative activity of bacteria.

capable: 2. To determine susceptibility of bacteria to antimicrobial agents

Methodical guidelines Stability of fermentative systems of bacteria allows using biochemical features of bacteria in combination with their morphological, cultural and other properties for identification. For this purpose, culture of bacteria is inoculated

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in differential media which depending on composition and purpose can be divided on 4 groups:

• Protein-containing media (gelatin, milk, serum, etc. ) for detection of proteolytic enzymes.

• Sugar- and polyatomic alcohol containing media for detection of saccharolytic activities.

• Media with chemicals, which are changing under the action of oxidizing or reducing enzymes produced by bacteria.

• Media containing indifferent chemicals, which can be used a nutrient source by some microorganisms, but not by others.

Differential media usually contain dye, which indicates presence or absence of fermentation, oxidation or reduction of particular compound(s).

Saccharolytic properties (capabilities of fermenting sugars and polyatomic alcohols with formation of acid or gas and acid) are studied on Hiss serum water sugars, which contain different carbohydrates and indicator (so called 'many-colored row’). Under fermentation of carbohydrates with formation of acids and aldehydes, color of medium is changing due to changes of color of Indicator. In case of production of gases, they are accumulated in ‘floating tube’.

In addition, saccharolytic activity can be studied on Endo, Levine, Ploskirev and other media. In case of fermentation of lactose, colonies are becoming colored (color depends of indicator added to medium). Those bacteria, which cannot ferment lactose, remain colorless.

Another example is Wilson-Blair medium which is a meat-peptone agar with added glucose, Na₂SO₃ and FeCl₂ Growth of Clostridium petfringens leads to blackening and ruptures of agar.

Presence of proteolytic enzymes in bacteria is studied on media with gelatin, milk, serum and peptone. During the stab inoculation of media some bacteria (e.g. Vibrio cholerae, Bacillus anthracis and others) at room temperature ( +20+22'C) are liquefying gelatin. In addition, different bacteria give specific type of liquefaction (e.g. in a form of nail, Christmas tree, etc. ). During the inoculation in milk, there is splitting of casein clot with formation of peptone, leading to yellow discoloration. After inoculation in serum, there are depressions formed around the colonies due to liquefaction.

Indicators of deeper splitting of protein are formation of ammonia, indole and hydrogen sulphide. For detection of indole, which is formed in case of presence of tryptophanase, by Morel method, filter paper strips are washed with hot fat solution of oxalic acid and allowed to dry off. Indicator paper is placed between tube wall with meat-peptone agar and plug. If indole is produced on 2- 3 day, lower part of strip is becoming pink. Other, more sensitive method, allowed to concentrate indole on the surface of medium by xylene or ether, followed by detection with Kovacz’ reagent (para-dimethylaminobenzaldehyde). In case indole is produced, red ring is formed.

Presence of ammonia is detected by changing color of pink litmus paper,

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placed between the tube wall and plug, into blue.

Presence of urease is detected on urea-containing media with indicator (phenyl red). Initial color of the medium is yellow, but in case the urea is splitting into ammonia and CO₂, concentration of the former is growing, leading to alkalinization of medium and changing of color of the indicator to red.

Detection of production of acetone is done using Voges-Proskauer reaction. Addition to the culture of 40% solution of KOH and 5% of α-naphtho leads to formation of red color due to formation of acetylmethylcarbinol.

Citrate utilization (capability of bacteria to use citrate as a carbon source) can be detected on Simmons medium. Initial color of medium is green and in case of presence of enzyme citrate permease, color is changing to blue.

Russian system — System of Indicator Paper Disks (SIB) allows detecting various enzymes and includes from 10 to 30 different tests.

Presence of catalase in aerobes and facultative anaerobes is detected by placing one loop of bacteria into 1 drop of 3% hydrogen peroxide. In case of presence of catalase, gas (O₂) bubbles are formed.

Oxidase detection is done by placing and rubbing loop of culture onto the indicator paper soaked in alcohol solution of α-naphtho and 1% water solution of menthol. In case of presence of oxidase, paper is becoming blue.

Hugh-Leifson OF medium that contains bromothymol blue is used for establishment whether microorganism is oxidizing or fermenting glucose. For creation of anaerobic conditions, medium is sealed with layer of liquid paraffin. Initial color of medium is green which is changing to yellow upon fermentation or oxidation of glucose. It should be noted that obligate aerobes (e.g. Pseudomonas aeruginosa) are oxidizing glucose (aerobic conditions), anaerobes (e.g. Clostridium petfringens) - fermenting (only in anaerobic conditions), but facultative anaerobes (e.g. Escherichia coli) — can ferment and/or oxidize glucose (both in anaerobic and aerobic conditions).

Hemolytic properties of microorganisms are studied on blood agar. If broth is used, it is becoming transparent, but when solid agars are used, semi- or completely transparent zones are formed.

ANTIMICROBIAL SUSCEPTIBILITY TESTING

Antimicrobial susceptibility testing is a very important from different points of view. It allows both guiding therapy of individual patient and also, when used judiciously, provides data that are used as a basis for empiric therapy in particular hospital or community settings.

There are different methods for susceptibility testing of bacteria exist. By far, more widespread is disk diffusion testing. The major publication on this technique came up in 1966 by Drs. Bauer, Kirby, Sherris and Turck (thus sometimes this technique is called Kirby-Bauer method), which tested different media, pH, temperature of incubation, etc. Since that time, the technique changed a little bit. It should be noticed that independently on method used, the most critical step is

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obtaining a pure culture of microorganism tested. There are different steps involved in the procedure, but the preparation of inoculum is a critical step. At least 3-5 colonies of pure culture of organism should be picked up and direct colony suspension method should be used in majority of circumstances. After colonies are selected, they should be suspended in saline or broth (e.g. Mueller-Hinton or tryptic soy). Then, inoculum should be adjusted to a turbidity equivalent to 0. 5 McFarland standard (corresponds to 1.5 x 10⁸ CFU/ml). For this purpose, tubes are placed in front of a paper with lines. For disk diffusion technique, paper disks (usually commercially available from reliable manufacturers are used). In general, they are labeled with international nomenclature of antimicrobials (e.g. ERY for erythromycin) and also disk load (concentration of antimicrobial in disk) is indicated (5 means 5 g). Disks are normally stored either in refrigerator or freezer. When the container with disks is removed from refrigerator or freezer, it should be allowed to equilibrate to room temperature (for this purpose 1-2 hours are usually sufficient) to minimize condensation and reduce the possibility of moisture affecting the concentration of antimicrobial agents. A special agar (different to those used for isolation of bacteria) is used for susceptibility testing (Mueller-Hinton II agar is used more often). After the suspension is prepared, sterile cotton-tipped swab should be dipped into the inoculum. Then excess liquid should be taken out from swab by pressing it against the side of tube. Then, starting at the top, surface of Mueller- Hinton agar plate should be inoculated with the swab. The entire plate should be swabbed by streaking back and forth from edge to edge. Then plate is rotated approximately 60°, and swabbing procedure is repeated again. Then plate is rotated again approximately 60°, and swabbing of entire plate is repeated a third time to be ensured that inoculum is evenly distributed. Disks should be applied within 15 min of inoculating the plate. Typically, 5-6 disks are recommended on a 100 mm plate. Each disk should be pressed down firmly to ensure compete, level contact with the agar. After that, plates should be inverted and incubated with agar side up. Different microorganisms require different incubation time, varying from 16-18 hours at +35°C at ambient air conditions for non-fastidious organisms (e.g. Escherichia coli) to 20-24 hours at +35°C at 55 CO₂ for some fastidious (e.g. Streptococcus pneumoniae). Inhibition zones are measured from the back of the plate using reflected light with a ruler or special caliper. The results (whether strains is susceptible, intermediate resistant or resistant) are interpreted according to the special tables published by corresponding institutions. The most widely used are National Committee for Clinical Laboratory Standards (USA) standards which me reviewed and updated at every year.

However, in spite of the easiness of use of disk diffusion method and low price,

there are some limitations. The major of them that disk diffusion technique is semi- quantitative rather than quantitative. In addition there are some antimicrobials for which disk diffusion does not give reliable results (e.g. Ill generation cephalosporins and Streptococcus pneumoniae). Also this method is not suitable for slow growing microorganisms (e.g. anaerobes or Mycobacterium tuberculosis).

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Thus for many cases methods providing quantitative results (e.g. determining minimal inhibitory concentration of antimicrobials) are used in some circumstances. The minimal inhibitory concentration (MIC) of an antimicrobial agent is the lowest concentration of the antimicrobial agent required to inhibit a given bacterial isolate from multiplying and producing visible growth in a test system. The MIC is determined in the laboratory by incubating a known quantity of bacteria with specified dilution of antimicrobial agent. The results are then interpreted as susceptible, intermediate or resistant using special interpretative criteria (e.g. NCCLS being far most common). MIC tests can be performed using broth or agar media. Cation-adjusted Mueller Hinton-II broth and Mueller- Hinton II agar are used for broth and agar dilution techniques, respectively.

There is another method sometimes used in laboratory which combines simplicity of disk diffusion technique and also being quantitative as MIC testing, called Etest. Etest comprises a predefined gradient of antibiotic concentrations on a plastic strip sized 50x5 mm. After the incubation (incubation conditions depend on microorganism), site of intersection of inhibition zone with scale on the front side indicates MIC result. There are Etests with antibiotics, antifungal and antimycobacterial agents available.

Demonstrations

1. Using SIBs for interspecies identification of bacteria based on their fermentative activity.

2. Fermentative activity of Enterobacteriaceae (E. coli) on Hiss serum water sugars.

3. Detection of H₂S and indole production.

4. Influence of volatile and non-volatile fractions of garlic fitoncides on bacteria.

5. Determination of antimicrobial susceptibility of bacteria by disk diffusion, dilution and Etest methods.

6. Determination of antibiotic concentration in human serum.

7. Flasks and ampoules with different antibiotics and disks for determination of antibiotic susceptibility.

Students’ activities

1. Each student examines growth on agar slant, prepares a smear and makes a Gram stain.

2. Students in groups re-culture bacteria from agar slant into ‘many colored row’ (study of fermentative activity of bacteria).

3. Students in groups determine susceptibility of isolated bacteria to antibiotics by disk diffusion method.

Control questions

1. What are the functions of enzymes in bacteria?

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2. What is the classification of enzymes in bacteria?

3. What is the principle of identification of bacteria based on their fermentative activity?

4. How saccharolytic properties of bacteria are studied and what nutrient media are used for that purpose?

5. How proteolytic properties of bacteria are studied and what nutrient media are used for that purpose?

6. What does ‘many colored row’ mean?

7. What are SIBs stand for and their purpose of use?

8. What gases are produced upon splitting of proteins by bacteria and how they can be detected?

9. Name enzymes participating in biological oxidation and ways how they can be determined.

10. What types of symbiotic relationships between microorganisms do you know?

11. What types of competitive relationships between microorganisms do you know?

12. What is microbial antagonism and what practical aspects of its use do you know?

13. What is chemotherapy and what are its principles?

14. What chemotherapeutic agents do you know?

15. What is chemotherapeutic index?

16. What are antibiotics?

17. What classifications of antibiotics on mechanisms of the receipt, chemical structure, origin, mechanism and spectrum of activity do you know?

18. What is the mechanism of action of antineoplastic antibiotics?

19. What side effects of antimicrobial therapy do you know?

20. What are the methods of prophylaxis or decrease of intensity of side effects of antibiotics?

21. What are the mechanisms of development of antibiotic resistance in bacteria?

22. What does stimulate development of antibiotic resistance in bacteria?

23. What measures might be used for prophylaxis of development of antibiotic resistance in bacteria?

24. What methods of determination of antimicrobial susceptibility of bacteria do you know?

25. What re the problems associated with the receipt and use of antiviral drugs?

26. What are anomalous nucleosides and what is the mechanism of their antiviral action?

27. What is the mechanism of action of azidothymidine and other similar antiretroviral substances used for treatment of patients with AIDS?

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PRACTICAL SESSION No. 8

Methods of isolation and identification of pure cultures of aerobic bacteria. Determination of susceptibility to antimicrobials (continuation).

Plan of the session

1. Identification of isolated pure cultures of bacteria based on biochemical properties. Examination of results of determination of antimicrobial susceptibility.

2. Natural microbiocenoses. Ecological niches of microorganisms.

3. Sanitary indicative bacteria, their characteristics.

4. Determination of microbial number of air by Koch method and using Krotov apparatus.

5. Methods of sanitary-microbiological evaluation of water: determination of coli-titer, coli-index by zymotic and membrane filters methods. Determination of microbial number of coli-phages.

6. Determination of microbial contamination of hands and environmental objects using wash-out method.

7. Determination of microbial number and coli-titer of pasteurized milk.

Foreword notes

Microorganisms are widespread. Majority of them in natural conditions are having a particular type of relationships. Potentially pathogenic bacteria can get to environment, for example, from patients, carriers and survive there for some time. Thus sanitary-microbiological investigations are performed for study and evaluation of different objects for determination of their epidemic potential, Methods for sanitary-microbiological investigation include determination of determination of a total microbial contamination number, detection and titration of sanitary-indicative microorganisms and detection of pathogenic microorganisms and/or their metabolites.

Direct detection of pathogenic microorganisms, in general, is complicated because of their small quantity. Thus indirect methods of detection of microbial contamination number on the basis of quantification of concomitant sanitary- indicative microorganisms are used. A total microbial contamination is used as indicator of intensity of contamination by organic substances.

Sanitary-indicative microorganisms are those, which are used for indirect evaluation of possible presence of pathogens in the environment. For example, presence of Escherichia coli and Enterococcus faecalis on environmental objects is indicative of fecal contamination. Simultaneous isolation of Staphylococcus aureus and hemolytic streptococci indicates possible contamination by oral droplets. Presence of sanitary-indicative microorganisms is measured by titer and index.

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Main objectives of the session

1. To study methods and indicators which are necessary for sanitary- microbiological evaluation of environmental objects.

Educational tasks

To know: 1- Natural microbiocenoses. Ecological relationships in

microbiocenoses.

2. Ecological niches of microorganisms: microflora of soil, water, air, food products, domestic and industrial objects and its role in infectious diseases.

3. Principles of sanitary-microbiological studies. Detection of pathogenic microorganisms in environmental objects by indirect methods. Detection of a total microbial contamination number and sanitary-indicative microorganisms: Enterobacte- riaceae, Clostridium spp., Streptococcus spp., Enterococcus spp., and Staphylococcus spp.

To be 1. To determine sanitary-microbiological contamination of air,

capable: water, soil, food products and wash-outs by microbiological

tests.

Methodical guidelines

Sanitary-microbiological evaluation of environmental objects

Sanitary-microbiological evaluation of soil is performed on the basis of comparison of number of thermophilic bacteria and microorganisms indicative of faecal contamination. Soils with predominance of sanitary-indicative bacteria are considered to be as sanitary-non-satisfactory, contaminated with human or animal faeces. Presence of E. coci/E. faecalis, Citrobacter spp. /Enterobacter spp. and Clostridium perfringens in the soil indicates the presence of recent, non- recent and bygone fecal contamination, respectively. More accurate evaluation is performed using coli-index — number of Enterobacteriaceae (so called coliform bacteria) found in 1 g of soil; perfringens-titer - mass of soil in which 1 C. perfringens is found; a total quantity of saprophytes, thermophilic and nitrifying bacteria in 1 g of soil.

Sanitary-microbiological evaluation of water is performed on the basis of: 1) microbial number — number of mesophiiic chemoorganotrophic bacteria per 1 ml of water; 2) coli-titer — minimal volume of water in ml in which Enterobacteriaceae are found; 3) coli-index — number of Enterobacteriaceae in 1 L of water; 4) presence of spores of sulphite-reducing bacteria and cysts of Giardia lamblia is also detected.

Sanitary-microbiological evaluation of air of closed premises is performed on the basis of microbial number - number of bacteria found in 1 m³ of air, presence of sanitary-indicative bacteria — hemolytic streptococci, S. aureus.

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Sanitary-microbiological evaluation of food products includes determination of microbial number and sanitary-indicative (Enterobacteriaceae) and pathogenic microorganisms.

Indicators of microflora of air: clean air in winter time (a total microbial number < 4, 500; hemolytic streptococci - up to 35); contaminated air in winter time (a total microbial number >7, 000; hemolytic streptococci — >70).

Indicators of quality of air for hospital premises are indicated in Table 13.

Table 13. NORMAL LEVELS OF MICROBIAL CONTAMINATION OF AIR IN HOSPITAL PREMISES

Premises	Total microbial number	S. aureus in 250 L
Operation rooms before work	<500	Not present
Operation rooms before work	<1, 000	Not present
Delivery rooms	<1, 000	Not present
Wards for prematurely bom babies	<750	Not present

Sanitary-microbiological study of air is performed by sedimentation and aspiration methods.

When sedimentation method by Koch is used, Petri dishes with nutrient media are left opened for 5-10 min. to detect a total microbial number and at least for 40 min. for detection of cocci. Then incubation for 24 h at +37°C is performed, followed by 24 h at room temperature. Number of grown colonies indicates degree of air contamination. During 5 min. number of bacteria accumulated at 100 sm² is indicative of their concentration in 10 L of air.

Aspiration method is more accurate and employs use of Krotov apparatus. It uses a principle that air with pre-set speed is sucked via narrow slot of plate covering Petri dish with nutrient media. During this process, aerosol particles which contain microorganisms are evenly fixed on the surface of medium because of permanent rotation of Petri dish under entrance slot.

Microbiological indicators for drinking water are indicated in Table 14.

Table 14. NORMAL LEVELS OF MICROBIAL CONTAMINATION OF AIR IN HOSPITAL PREMISES

Indicator	Units of measurement	Normal levels
Thermotolerant coliform bacteria (+44°C) Total number of coliform bacteria (+37°C) Total microbial number Coli-phages Spores of sulphite-reducing clostridia Cysts of Giardia lamblia	No. of bacteria in 100 ml No. of bacteria in 100 ml No. of colony forming units (CFU) in l ml No. of plaque-forming units No. of spores in 20 ml No. of cysts in 50 ml	Not present Not present <50 Not present Not present Not present

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When membrane filters method is used, filter is placed into Zeits funnel, incorporated into Bunzen retort, which is connected to vacuum pump. Then water in volume of 333 ml is filtrated, followed by placement of filters on the surface of Endo medium. After 24 h incubation at +37°C, number of colonies typical of Enterobacteriaceae is counted. From 2 to 3 red-colored colonies are used for preparation of smear and Gram stain, followed by oxidase test allowing to distinguish Escherichia spp. Citrobacter spp, Enterobacter spp. and other Enterobacteriaceae from Pseudomonas spp. and other oxidase-positive non-fermenters which might be present in water. For that purpose, filter with grown colonies (do not turn over! ) is transferred with forceps to filter paper disk wetted with dimethyl—n-phenyldiamine. Presence of oxidase will lead to development of blue coloration of colony. After that, 2 or 3 colonies, which did not change color, are inoculated into semi-solid medium with 0.5% of glucose, followed by 24 h incubation at +37°C. In case of presence of formation of gas, number of red colonies is counted and coli-index is determined.

Zymotic test

1^st day. A total of 100 ml of water is taken. A total of 10 ml (3 samples) is inoculated onto 10 ml or 1 ml of concentrated glucose peptone medium (GPM) or lactose peptone medium (LPM), respectively. A 1 ml of water (3 samples) is inoculated onto 10 ml of diluted GPM with “float”. Samples are incubated at 37°C for 24 hours.

2^nd day. Each volume of water, regardless of the results (acid — gas), is re- cultured on 2 plates with Endo agar (first - supplemented with basic fuchsine solution or rosolic acid, second — supplemented with milk or gelatin — in order to detect proteolysis). Samples are incubated at +37°C for 18 hours.

3^rd day. 2 to 3 (from each plate) lactose-positive, proteolysis-negative, oxidase-negative Gram-negative colonies are taken and re-cultured on semi- solid agar supplemented with glucose and Andrade indicator or bromthymol blue (stub culture). Samples are incubated at +37°C for 20 hours.

4^th day. Culture evaluation. The volume of water in which acid and gas are present is determined and coli titer and coli index are calculated according to tables provided in State Standard (GOST 48963-73).

Microbial number is determined by culturing of 1 ml of water. Oxidase test is used to exclude oxidase-positive bacteria (Pseudomonas spp., Aeromonas spp., Vibrio spp., etc). Microscopic examination of smears is used to exclude the presence of Gram-positive flora and cocci. Glucose and lactose oxidation with the production of acid and gas confirms that bacteria belong to coliform group.

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Microflora of foods

Normal values: milk and drinks — total microbial number (TMN) <75, 000 per 1 ml, coli titer—i3; meat, sausages and meat products — no coliform bacteria per 1 g of product, no Salmonella spp. per 25 g of product, no Proteus spp. per

0. 1 g of product, no coagulase-positive staphylococci per 0. 1 g, no sulfite- reducing clostridia per 0. 1 g of product; canned products should not contain botulinum toxin and Clostridium botulinum

Phases of study.

To determine TMN, pasteurized milk is diluted by sterile isotonic saline solution in proportion of 1: 10, 1: 100, 1: 1000 and 1 ml of each dilution is poured in sterile plates and melted cool agar is added to plates. Then samples are cultured at + 37°C for 24 hours.

To evaluate coli titer, whole pasteurized milk is distributed to 6 tubes with Kessler medium: 3 tubes — 1 ml, rest tubes - 0. 1 ml (diluted by water 1: 10). Samples are incubated at +43°C for 24 hours. Samples with the presence of zymosis are cultured on Endo medium and then coliform bacteria are identified. In case of presence of Gram-negative bacilli, oxidase test is performed and colonies are cultured on medium with glucose and Koser medium. In case of presence of acid and gas in the medium with glucose, and the absence of bacterial growth on Koser medium (1 L of distilled water, 10 g of monosubstituted phosphate potassium, 0. 2 g of magnesium sulfate, 2. 5-3. 0 g of sodium citrate, 10 ml of 0. 5% bromthymol blue alcoholic solution). According to State Standard (GOST 9225-68), citrate-negative E. coli strains are only considered when coli- titer is determined. Coli-titer is calculated according to special table (L. B. Borisov et al. 1993. p. 69).

For drinks testing. TMN and coli titer are determined by methods used for water testing. Lemonade is neutralized by 10% sodium hydrocarbonate solution (previously tested by pH litmus paper). Coli titer is calculated by membrane filters method.

When meat is examined microscopically, a number of bacteria in fingerprints of meat (2x1.5x2.5 cm). Gram staining. Meat is considered fresh if a number of bacterial cells are <10 per microscopic field.

To determine TMN for coliform bacteria, Salmonella spp., Proteus spp., coagulase-positive staphylococci and clostridia, 20 g of deep parts of meat is taken under sterile conditions and 80 ml of sterile 0. 9% saline solution is added, then mixture is homogenized using electric mixer. A total of 0.1 and 0. 01 g of sample are inoculated onto meat peptone agar (MPA), incubated for 48 hours, and then number of colonies is counted.

For determination of coliform bacteria in 1 g of product, 5 ml of suspension is inoculated into Kod medium (nutrient broth, sulphanol, lactose, and bromthymol blue, followed by 18 h incubation. Lactose-positive coliforms will change the original blue color of medium into dark-green or bright- yellow.

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Detection of Salmonella spp. is performed by inoculation of Mueller, Kaufman medium or selenitic broth, followed by inoculation of pure culture.

Proteus spp. is isolated by Shukevich method, Clostridium spp. — on Wilson- Blair medium and coagulase-negative staphylococci — on yolk-salt agar.

For detection of botulinum toxin, canned products are filtrated and filtrate is used in the reaction of toxin neutralization assay with antitoxic sera of types A, B, C, E and F.

Microflora of hands and environmental objects is studied by collection of wash-outs from hands, crockery, desks, etc. Sterile cotton swabs wetted in Kessler medium are used for collection of wash-outs from hands. Wash-outs are made from both hands, thoroughly rubbing palms, interdigital spaces and hyponycheal areas, starting from the left hand. Wash-outs from surface of objects are made using wire template with are of 25 sm². Swabs them placed in the tubes with Kessler medium, followed by 24 h incubation at +43°C. In case of zymosis, inoculation onto Endo medium is performed, followed by identification of E. coli look-like colonies.

Demonstrations

0. Determination of microbial number of air using Koch method and Krotov’ apparatus. Determination of microbial number of tap water.

1. Determination of a total number of coliform bacteria by membrane filters method.

2. Detection of faecal contamination of crockery and hands of personnel.

3. Sanitary-microbiological investigation of milk and milk products (only for students of pediatric faculty).

Students’ activities

1. Students in groups are examining results of determination of biochemical tests and susceptibility to antimicrobials of previously isolated culture.

2. Students in groups are examining results of inoculation of air by Koch method, determining microbial number and studying cultural, biochemical, morphological and tinctorial properties of grown cultures of bacteria.

3. Each student is inoculating finger-prints into meat-peptone agar.

4. One student in the group is inoculating hands wash-outs into Kod medium.

5. Two groups of students are inoculating throat swabs into glucose broth, yolk-salt and blood agars.

6. Two groups of students are inoculating nose swabs into glucose broth, yolk-salt and blood agars.

7. Each student is evaluating results of sanitary-microbiological investigation of milk and milk products (only for students of pediatric faculty).

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Control questions

1. What are the population and biotope?

2. What is the ecosystem?

3. What is the microbiocenosis?

4. What ecological niches for microorganisms do you know and which ones are considered to be anthropogenic?

5. What is the role of ecological media in the spread of infectious diseases?

6. What criteria are used for evaluation of sanitary-microbiological conditions of water, air and soil?

7. What microorganisms are considered to be sanitary-indicative for water, air and soil?

8. What indicators are used for evaluation of sanitary-microbiological condition of soil?

9. What indicators are used for evaluation of sanitary-microbiological condition of air?

10. What indicators are used for evaluation of sanitary-microbiological condition of water?

11. What indicators are used for evaluation of sanitary-microbiological condition of food products?

12. What are coli-titer and coli-index?

13. What is microbial number of water?

14. What methods are used for evaluation of faecal contamination of water?

15. How microbial number of water is determined?

16. What are normal microbiological ranges for drinking water?

17. How faecal contamination of personnel and crockery is determined?

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PRACTICAL SESSION No. 10

Genetics of microorganisms.

Plan of the session

1. Organization of genetic apparatus in bacteria and viruses. Genotype and phenotype.

2. Recombination in bacteria: transformation, transduction and conjugation.

3. Modifications in bacteria and viruses.

4. Mutations in bacteria and viruses. Dissociations.

5. Identification of nucleic acids. Polymerase chain reaction, molecular hybridization.

6. Genetic engineering.

Foreword notes

Studies of genetics of microorganisms led to the prominent discoveries, including establishment of role of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), gene structure, genetic code. In addition, new direction in genetic studies — genetic engineering — was developed, which allows gene transfer from one cell to another, influencing hereditary information of microorganisms.

Genetic information in microorganisms

Genetic information in bacteria and many viruses is encoded in DNA, but some viruses use RNA. Replication of the genome is essential for inheritance of genetically determined traits. Gene expression usually involves transcription of DNA into messenger RNA and translation of mRNA into protein.

Genome organization

The bacterial chromosome is a circular molecule of DNA that functions as a self-replicating genetic element (replicon). Extrachromosomal genetic elements such as plasmids and bacteriophages are nonessential replicons which often determine resistance to antimicrobial agents, production of virulence factors, or other functions. The chromosome replicates semiconservatively; each

DNA strand serves as template for synthesis of its complementary strand.

Chromosomal DNA

Bacterial genomes vary in size from about 0.4 x 10⁹ to 8.6 x 10⁹ Daltons

(Da), some of the smallest being obligate parasites (Mycoplasma spp. ) and the largest belonging to bacteria capable of complex differentiation such as Myxococcus spp. The amount of DNA in the genome determines the maximum amount of information that it can encode. Most bacteria have a haptoid genome, a single chromosome consisting of a circular, double-stranded DNA molecule.

The single chromosome of the common intestinal bacterium Escherichia coli is 3 x 10⁹ Da (4, 500 kilobase pairs [kbp]) in size, accounting for about 2 to 3 percent of the dry weight of the cell. The E. coli genome is only about 0.1 % as

large as the human genome, but it is sufficient to code for several thousand polypeptides of average size (40 kDa or 360 amino acids). The chromosome of E. coli has a contour length of approximately 1. 35 mm, several hundred times longer than the bacterial cell, but the DNA is supercoiled and tightly packaged in the bacterial nucleoid. The time required for replication of the entire chromosome is about 40 minutes, which is approximately twice the shortest division time for this bacterium. DNA replication must be initiated as often as the cells divide, so in rapidly growing bacteria a new round of chromosomal replication begins before an earlier round is completed. At rapid growth rates there may be four chromosomes replicating to form eight at the time of cell division, which is coupled with completion of a round of chromosomal replication. Thus, the chromosome in rapidly growing bacteria is replicating at more than one point. The replication of chromosomal DNA in bacteria is complex and involves many different proteins.

Plasmids

Plasmids are replicons that are maintained as discrete, extrachromosomal genetic elements in bacteria. They are usually much smaller than the bacterial chromosome, varying from less than 5 to more than several hundred kbp, though plasmids as large as 2 Mbp occur in some bacteria. Plasmids usually encode traits that are not essential for bacterial viability, and replicate independently of the chromosome. Most plasmids are supercoiled, circular, double-stranded DNA molecules, but linear plasmids have also been demonstrated in Borrelia spp. and Streptomyces spp. Closely related or identical plasmids demonstrate incompatibility; they cannot be stably maintained in the same bacterial host. Classification of plasmids is based on incompatibility or on use of specific DNA probes in hybridization tests to identify nucleotide sequences that are characteristic of specific plasmid replicons. Some hybrid plasmids contain more than one replicon. Conjugative plasmids code for functions that promote transfer of the plasmid from the donor bacterium to other recipient bacteria, but nonconjugative plasmids do not. Conjugative plasmids that also promote transfer of the bacterial chromosome from the donor bacterium to other recipient bacteria are called fertility plasmids. The average number of molecules of a given plasmid per bacterial chromosome is called its copy number. Large plasmids (>40 kilobase pairs) are often conjugative, have small copy numbers (1 to several per chromosome), code for all functions required for their replication, and partition themselves among daughter cells during cell division in a manner similar to the bacterial chromosome. Plasmids smaller than 7. 5 kilobase pairs usually are nonconjugative, have high copy numbers (typically 10-20 per chromosome), rely on their bacterial host to provide some functions required for replication, and are distributed randomly between daughter cells at division.

Many plasmids control medically important properties of pathogenic bacteria, including resistance to one or several antibiotics, production of toxins, and synthesis of cell surface structures required for adherence or colonization.

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Plasmids that determine resistance to antibiotics are often called R plasmids (or R factors). Representative toxins encoded by plasmids include heat-labile and heat-stable enterotoxins of E. coli, exfoliative toxin of Staphylococcus aureus, and tetanus toxin of Clostridium tetani. Some plasmids are cryptic and have no recognizable effects on the bacterial cells that harbor them. Comparing plasmid profiles is a useful method for assessing possible relatedness of individual clinical isolates of a particular bacterial species for epidemiological studies.

Bacteriophages

Bacteriophages (bacterial viruses, phages) are infectious agents that replicate as obligate intracellular parasites in bacteria. Extracellular phage particles are metabolically inert and consist principally of proteins plus nucleic acid (DNA or RNA, but not both). The proteins of the phage particle form a protective shell (capsid) surrounding the tightly packaged nucleic acid genome. Phage genomes vary in size from approximately 2 to 200 kilobases per strand of nucleic acid and consist of double-stranded DNA, single-stranded DNA, or RNA. Phage genomes, like plasmids, encode functions required for replication in bacteria, but unlike plasmids they also encode capsid proteins and nonstructural proteins required for phage assembly. Several morphologically distinct types of phage have been described, including polyhedral, filamentous, and complex. Phages are classified into two major groups: virulent and temperate. Growth of virulent phages in susceptible bacteria destroys the host cells. Infection of susceptible bacteria by temperate phages can have either of two outcomes: lytic growth or lysogeny. Lytic growth of temperate and virulent bacteriophages is similar, leading to production of phage progeny and death of the host bacteria. Lysogeny is a specific type of latent viral infection in which the phage genome replicates as a prophage in the bacterial cell. In most lysogenic bacteria the genes required for lytic phage development are not expressed, and production of infectious phage does not occur. Furthermore, the lysogenic cells are immune to superinfection by the vims which they harbor as a prophage. The physical state of the prophage is not identical for all temperate viruses. For example, the prophage of bacteriophage I in E. coli is integrated into the bacterial chromosome at a specific site and replicates as part of the bacterial chromosome, whereas the prophage of bacteriophage P1 in E. coli replicates as an extrachromosomal plasmid.

Lytic phage growth occurs spontaneously in a small fraction of lysogenic cells, and a few extracellular phages are present in cultures of lysogenic bacteria. For some lysogenic bacteria, synchronous induction of lytic phage development occurs in the entire population of lysogenic bacteria when they are treated with agents that damage DNA, such as ultraviolet light or mitomycin C. The loss of prophage from a lysogenic bacterium, converting it to the nonlysogenic state and restoring susceptibility to infection by the phage that was originally present as prophage, is called curing.

Some temperate phages contain genes for bacterial characteristics that are unrelated to lytic phage development or the lysogenic state, and expression of

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such genes is called phage conversion (or lysogenic conversion). Examples of phage conversion that is important for microbial virulence include production of diphtheria toxin by Corynebacterium diphtheriae, erythrogenic toxin by Streptococcus pyogenes (group A β-hemolytic streptococci), botulinum toxin by Clostridium botulinum, and Shiga-like toxins by E. coli. In each of these examples the gene which encodes the bacterial toxin is present in a temperate phage genome. The specificity of O antigens in Salmonella spp. can also be controlled by phage conversion. Phage typing is the testing of strains of a particular bacterial species for susceptibility to specific bacteriophages. The patterns of susceptibility to the set of typing phages provide information about the possible relatedness of individual clinical isolates. Such information is particularly useful for epidemiological investigations.

The complete set of genetic determinants of an organism constitutes its genotype, and the observable characteristics constitute its phenotype. Mutations are heritable changes in genotype that can occur spontaneously or be induced by chemical or physical treatments. Organisms selected as reference strains are called wild type, and their progeny with mutations are called mutants. Selective media distinguish between wild type and mutant strains based on growth; differential media distinguish between them based on other phenotypic properties. Phenotypic changes of one or a few characteristics are called modifications if they are not controlled by genome or accompanied by changes in primary DNA structure. They are observed in bacteria very often and can be easily lost after completion of action of acting factor. The most frequently observed in bacteria are morphological modifications, which are leading to reversible changes of bacterial shapes. For example, motile bacteria can lost their motility on formalin-containing media, rod-shaped bacteria can look as cocci in old cultures, etc. Biochemical modifications are those related to inducible synthesis of enzymes due to induction or repression of corresponding structural genes, which are controlled by regulatory genes. For example, E. coli is synthesizing enzymes fermenting lactose only in the presence of lactose. In addition, modifications include ‘switching on’ of ‘silent’ genes in bacteria, e.g. during the course of infectious diseases. The biological role of modifications can be defined as a response of bacteria to environmental changes, which allows them to survive and maintain of own population.

Another form of variability is so-called R-S dissociations of bacteria. It starts spontaneously with formation of two types bacterial cells which differ from each other by character of colonies formed on agar. One type - R (‘rough’) colonies - is characterized by irregular edges and rough surface. Second type - S (‘smooth’) colonies — is characterized by presence of round edges and smooth surface. Process of dissociation — divergence of cells forming both types of colonies - is usually observed in one direction — from S- to R-form, sometimes via intermediate forms — M (‘mucoid’) or D (‘dwarf) colonies. The reverse direction — from R- to S-form is rarely observed. Mutations which

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are leading to S-R-dissociations are called insertional, because they originate from insertion of extrachromosomal elements — temperate phages or large nonconjugative plasmids. Examples of those can be found in gram-negative (e.g. polysaccharide formation in Shigella flexneri) and gram-positive (e.g. toxin- producing Corynebacterium diphtheriae) bacteria. Biological role of S-R dissociations consists in acquiring by bacteria some properties which are advantageous for their survival in the environment, including human host.

Mutations are heritable changes in the genome. Spontaneous mutations in individual bacteria are rare. Some mutations cause changes in phenotypic characteristics; the occurrence of such mutations can be inferred from the effects they produce. In microbial genetics specific reference organisms are designated as wild-type strains, and descendants that have mutations in their genomes are called mutants. Thus, mutants are characterized by the inherited differences between them and their ancestral wild-type strains. Variant forms of a specific genetic determinant are called alleles. Genotypic symbols are lower case, italicized abbreviations that specify individual genes, with a (+) superscript indicating the wild type allele. Phenotypic symbols are capitalized and not italicized, to distinguish them from genotypic symbols. For example, the genotypic symbol for the ability to produce β-galactosidase, required to ferment lactose, is lacZ+, and mutants that cannot produce β-galactosidase are lacZ. The lactose-fermenting phenotype is designated Lac+, and inability to ferment lactose is Lac-.

The mutation rate in bacteria is determined by the accuracy of DNA replication, the occurrence of damage to DNA, and the effectiveness of mechanisms for repair of damaged DNA. For a particular bacterial strain under defined growth conditions, the mutation rate for any specific gene is constant and is expressed as the probability of mutation per cell division. In a population of bacteria grown from a small inoculum, the proportion of mutants usually increases progressively as the size of the bacterial population increases. Mutations in bacteria can occur spontaneously and independently of the experimental methods used to detect them. This principle was first demonstrated by the fluctuation test. The numbers of phage-resistant mutants of E. coli in replicate cultures grown from small inocula were measured and compared with those in multiple samples taken from a single culture. If mutations to phage resistance occurred only after exposure to phage, the variability in numbers of mutants between cultures should be similar under both sets of conditions. In contrast, if phage-resistant mutants occurred spontaneously before exposure of the bacteria to phage, the numbers of mutants should be more variable in the independently grown cultures, because differences in the size of the bacterial population when the first mutant appeared would contribute to the observed variability. The data indicated that the mutations to phage resistance in E. coli occurred spontaneously with constant probability per cell division. By repeating these procedures several times, it was possible to isolate pure cultures of phage- resistant bacterial mutants that had never been exposed to bacteriophage.

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Both environmental and genetic factors affect mutation rates. Exposure of bacteria to mutagenic agents causes mutation rates to increase, sometimes by several orders of magnitude. Many chemical and physical agents, including X-rays and ultraviolet light, have mutagenic activity. Chemicals that are carcinogenic for animals are often mutagenic for bacteria, or can be converted by animal tissues to metabolites that are mutagenic for bacteria. Standardized tests for mutagenicity in bacteria are used as screening procedures to identify environmental agents that may be carcinogenic in humans. Mutator genes in bacteria cause an increase in spontaneous mutation rates for a wide variety of other genes. Expression of these genes, induced by DNA damage, enables the repair of DNA lesions that would otherwise be lethal, but by an error-prone mechanism that increases the rate of mutation. The overall mutation rate the probability that a mutation will occur somewhere in the bacterial genome per cell divisions relatively constant for a variety of organisms with genomes of different sizes and appears to be a significant factor in determining the fitness of a bacterial strain for survival in nature. Most mutations are deleterious, and the risk of adverse mutations for individual bacteria must be balanced against the positive value of mutability as a mechanism for adaptation of bacterial populations to changing environmental conditions.

Mutations are classified on the basis of structural changes that occur in DNA. Some mutations are localized within short segments of DNA (for example, nucleotide substitutions, microdeletions, and microinsertions). Other mutations involve large regions of DNA and include deletions, insertions, or rearrangements of segments of DNA.

Exchange of genetic information

Genetic exchanges among bacteria occur by several mechanisms. In transformation, the recipient bacterium takes up extracellular donor DNA. In transduction, donor DNA packaged in a bacteriophage infects the recipient bacterium. In conjugation, the donor bacterium transfers DNA to the recipient by mating. Recombination is the rearrangement of donor and recipient genomes to form new, hybrid genomes. Transposons are mobile DNA segments that move from place to place within or between genomes.

The biologic significance of sexuality in microorganisms is to increase the probability that rare, independent mutations will occur together in a single microbe and be subjected to natural selection. Genetic interactions between microbes enable their genomes to evolve much more rapidly than by mutation alone. Representative phenomena of medical importance that involve exchanges of genetic information or genomic rearrangements include the rapid emergence and dissemination of antibiotic resistance plasmids, flagellar phase variation in Salmonella spp., and antigenic variation of surface antigens in Neisseria spp. and Borrelia spp.

Sexual processes in bacteria involve transfer of genetic information from a donor to a recipient and result either in substitution of donor alleles for recipient alleles or addition of donor genetic elements to the recipient genome. Transformation, transduction, and conjugation are sexual processes that use

different mechanisms to introduce donor DNA into recipient bacteria (Fig. 11). Because donor DNA cannot persist in the recipient bacterium unless it is part of a replicon, recombination between donor and recipient genomes is often required to produce stable, hybrid progeny. Recombination is most likely to occur when the donor and recipient bacteria are from the same or closely related species.

Figure 11. Exchange of genetic information in bacteria

(Source http://gsbs.utmb.edu/microbook/ch005.htm)

Transformation, transduction, and conjugation differ in means for introducing DNA from donor cell into recipient cell.

1. In transformation, fragments of DNA released from donor bacteria are taken up by competent recipient bacteria.

2. In transduction, abnormal bacteriophage particles containing DNA from donor bacteria inject their DNA into recipient bacteria,

3. Conjugation occurs by formation of cytoplasmic connections between donor and recipient bacteria, with direct transfer of newly synthesized donor DNA into the recipient cells.

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In all three cases, recombination between donor and recipient DNA molecules is required for formation of stable recombinant genomes. Bacterial genome is represented diagrammatically as a circular element in bacterial cells. Donor and recipient DNA are indicated by fine lines and heavy lines, respectively. In each recombinant genome, the a+ allele from donor strain has replaced the a allele from recipient strain, and the b+ allele is derived from recipient strain.

For a recombinant to be detected, its phenotype must be different from both parental phenotypes. Growth or cell division may be required before the recombinant phenotype is expressed. Delay in expression of a recombinant phenotype until a haploid recombinant genome has segregated is called segregation lag, and delay until synthesis of products encoded by donor genes has occurred is called phenotypic lag. Testing for linkage (nonrandom reassortment of parental alleles in recombinant progeny) is possible when the parental bacteria have different alleles for several genes. The donor allele of an unselected gene is more likely to be present in recombinants if it is linked to the selected donor gene than if it is not linked to the selected donor gene. Quantitative analysis of linkage permits construction of genetic maps. The genome of E. coli is circular (Fig. 12), as determined both by genetic linkage and direct biochemical analysis of chromosomal DNA, and the genetic map is colinear with the physical map of the chromosomal DNA. Genetic and physical mapping are also used to analyze extrachromosomal replicons such as bacteriophages and plasmids.

Figure 12. Circular genetic map of E. coli (positions of representative genes are indicated on inner circle; distances between genes are calibrated in minutes, based on times required for transfer during conjugation; from Bachman, B.M., Low,

K.B. Microbiol Rev, 1980; 44: 31)

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Recombinant DNA and gene cloning

Gene cloning is the incorporation of a foreign gene into a vector to produce a recombinant DNA molecule that replicates and expresses the foreign gene in a recipient cell. Cloned genes are detected by the phenotypes they determine or by specific nucleotide sequences that they contain. Recombinant DNA and gene cloning are essential tools for research in molecular microbiology and medicine. They have many medical applications, including development of new vaccines, biologics, diagnostic tests, and therapeutic methods.

Modern genetics is intensively concentrating on molecular aspects of pathogenicity and immunogenicity of microorganisms, mechanisms of formation of new variants of pathogens and also origin and spread of antibiotic- resistant bacteria. A special attention is also paid to the receipt of immunogenic strains with low pathogenic potential — so-called vaccine strains.

Edward Jenner was the first who obtained first live vaccine containing cow- pox virus which was successfully used in humans for vaccination against smallpox in 1796. "Vaccination, " the word Jenner invented for his treatment (from the Latin vacca, a cow), was adopted by Louis Pasteur for immunization against any disease. Pasteur himself developed a principle of the receipt of attenuated vaccine strains — selection of spontaneous mutants with non-changed immunogenicity and low virulence via passages. In Russia vaccine STI obtained from environment is used against anthrax (name is derived from the name of institution — Sanitary & Technological Institute of Leningrad, personnel of which isolated nonencapsulated strains of Bacillus anthracis from soil). Other impressive examples of the vaccines include:

Vaccine against rabies — obtained by L. Pasteur using 133 passages of street rabies virus on rabbit brains.

Vaccine against plague EV76 — obtained by G. Gerard and J. Robick using 5 years cultivation of Yersinia pestis at +16°C.

BCG vaccine is a live vaccine derived from a strain of Mycobacterium bovis that was attenuated by A. Calmette and C. Guerin on glycerol potato with bile (bile was a factor which led to decrease of virulence of the strain).

Vaccine against yellow fever was originally obtained by 238 passages on white mice.

Vaccines against influenza, measles, rubella and poliomyelitis were obtained under the influence of different factors - nitrous acid, hydroxylamine, increased temperature, decrease of pH, ultrasound, UV-radiation, nucleases, etc.

Modern biotechnology of preparation of vaccines includes some stages: accumulation of considerable amounts of microorganisms or toxin on specially designed nutrient media in optimal temperature or other condition during the constant aeration (absence of oxygen for anaerobes). Them majority of vaccines are lyophilized. Among the recent advances of gene engineering is development of recombinant vaccines against influenza, hepatitis B.

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Main objective of the session

1. To get acquainted with specific features of bacterial genome, its distinctions with genome of eukaryotic cell; types, forms and mechanisms of variability of microorganisms.

Educational tasks To know: 1. Specific features of bacterial genome.

2. Distinction of bacterial genome from eukaryotic and viral genomes.

3. Extrachromosomal determinants of heredity: plasmids and their functions.

4. Mechanisms of transfer of genetic material in bacteria: conjugation, transformation, and transduction.

5. Achievements of genetic engineering in the field of the receipt of new medical preparations.

6. Mechanisms of variability of microorganisms (adaptations, mutations, recombinations).

7. Methods of molecular diagnosis of infectious diseases (PCR, molecular hybridization).

Methodical guidelines

1. Experiment with conjugation. Group of students (3-4 persons) are using strain-donor E. coli Hfr pro+, urac+, strS and strain-recipient E. coli F- pro-, urac-, his-, strR. Mix 1 ml of culture of donor with 1 ml of culture of recipient in sterile tube and incubate at +37°C for 40 min. After that inoculate 1 ml of mixture onto minimal agar plate, containing streptomycin in concentration, which inhibits growth of donor. Thus only recombinants can grow in case of recombination occurred. As controls cultures of donor and recipient are used.

2. Experiment with transduction. Group of students (3-4 persons) are using E. coli Lac- strain as recipient and temperate phage, obtained by induction from lysogenic culture E. coli Lac+. Mix 1 ml of culture of recipient with 1 ml of transducing phage in sterile tube and incubate at +37°C for 40 min. After that inoculate 1 ml of mixture onto Endo agar plate. As a result, growth of both colorless and red colonies should occur. Controls: 1) inoculate culture of recipient on Endo medium (growth of colorless colonies is observed); 2) growth of original culture E. coli Lac+ (from which phage was obtained) (growth of red colonies).

3. Experiment with transformation. Group of students (3-4 persons) are using penicillin-susceptible Staphylococcus spp. as recipient and DNA solution, obtained from penicillin-resistant Staphylococcus spp. Mix 1 ml of culture of recipient with 1 ml of DNA solution in sterile tube

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and incubate at +37°C for 40 min. After that inoculate 1 ml of mixture onto penicillin-containing agar plate. As a result, growth of colonies of penicillin-resistant Staphylococcus spp. should occur. Controls: 1) inoculate culture of recipient on penicillin-containing agar plate (absence of growth).

4. Experiment with elimination of R-factor. Group of students (3-4 persons) are diluting broth culture of E. coli R+ up to concentration 10^-4 and transfer 0.1 ml of diluted culture into 1 ml of broth (control) and 1 ml of broth with added 50 mg/ml of acridine orange (pH in both tubes is 7. 6). After 18-24 h incubation, 10^-4 dilutions are prepared from each tube and inoculate 0,1 ml into meat-peptone agar containing 200 mg/ml of tetracycline and chloramphenicol, followed by incubation for 24 h and count of number of colonies in control and test tubes. Number of colonies grew on Petri dish from test tube is significantly lower in comparison with control tube, because acridine orange destroys plasmid R-factor.

5. Identification of nucleic acids using polymerase chain reaction (PCR) is

used when amount of DNA or RNA in specimen is very low. Principle of PCR is multiplication of defined part of DNA which is catalyzed by DNA polymerase with obtaining of detectable amount. Firstly, generally separation of double-stranded DNA is performed at +95°C, followed by cooling down and addition of primer(s), containing around 20-30 nucleotides. Then tag-polymerase is added, which starts formation of secondary copies. Process is repeated in cycles 20-30 times, which allows to obtain millions DNA copies. The nucleic acid is detected using electrophoresis in 1 % gel, followed by staining in ethidium bromide which is fluorescent in UV-light. Special equipment called thermocyclers are used which allows quickly perform heating and cooling.

Method of molecular hybridization of nucleic acids is based on the capability of nucleic acids specifically bind (hybridize) with complementary fragments of homologous artificially synthesized DNA or RNA, marked by isotopes or enzymes (alkaline phosphatase, peroxidase).

6. Each student prepares Gram stain from Proteus vulgaris which grew on regular agar or agar with added carbolic acid and compares with preparation made by neighboring student.

Demonstrations

1. Genetic map of chromosome of E. coli (scheme).

2. Action of colicins on indicator culture of E. coli.

3. Experiments with conjugation, transformation, transduction and elimination of R-factor.

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Control questions

1. What are the recombinations in bacteria?

2. What is the transformation?

3. What is the transduction?

4. What is the conjugation?

5. What is the transforming agent?

6. What plasmid elements in bacteria do you know?

7. What is the role of plasmids in bacteria?

8. How the genotype of bacteria can be changed?

9. What are the biological properties of recombinants?

10. What is the phenomenon of dissociation of bacteria?

11. What are the characteristics of S- and R-forms of bacteria?

12. What is the mutation?

13. What mutagenic factors do you know?

14. What are the modifications in bacteria?

15. What is the minimal medium and when is it used?

16. What is the auxotroph?

17. How auxotrophs can be obtained?

18. What is the ecological role of phenotypic and genotypic variability in bacteria?

19. What areas of microbiology is adaptation variability of bacteria used?

20. How mapping of bacterial chromosome is performed?

21. What achievements of genetic engineering in microbiology do you know?

22. What is the basis of molecular methods of diagnostics in infectious diseases?

23. What is the principle of polymerase chain reaction in diagnosis of infectious diseases?

24. What is the principle of nucleic acid hybridization in diagnosis of infectious diseases?

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PRACTICAL SESSION No. 11 Infection and immunity.

Plan of the session

1. Infectious disease: forms of manifestations, ways of transmission, dynamics.

2. Pathogenicity and virulence of microorganisms: methods of determination and evaluation.

3. Methods of immunization of animals and experimental infection models.

4. Bacteriological investigation of dead animals.

5. Non-specific cell and humoral factors of immunity in humans (phagocytosis, lysozyme, complement etc. ): methods of study and evaluation.

Foreword notes

A pathogen is a microorganism (or virus) that is able to produce disease. Pathogenicity is the ability of a microorganism to cause disease in another organism, namely the host for the pathogen. In general, pathogenicity is a manifestation of a host-parasite interaction. In humans, some of the normal bacterial flora (e.g. Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae) are potential pathogens that live in a commensal or parasitic relationship without producing disease. They do not cause disease in their host unless they have an opportunity brought on by some compromise or weakness in the host’s anatomical barriers, tissue resistance or immunity. Furthermore, the bacteria are in a position to be transmitted from one host to another, giving them additional opportunities to colonize or infect. There are some pathogens that do not associate with their host EXCEPT in the case of disease. These bacteria are obligate pathogens, even though some may rarely occur as normal flora, in asymptomatic or recovered carriers, or in some form where they cannot be eliminated by the host.

Opportunistic pathogens

Bacteria which cause a disease in a compromised host which typically would not occur in a healthy (noncompromised) host are acting as opportunistic pathogens. A member of the normal flora can such as Staphylococcus aureus or E. coli can cause an opportunistic infection, but so can an environmental organism such as Pseudomonas aeruginosa. When a member of the normal flora causes an infectious disease, it might be referred to as an endogenous bacterial disease, referring to a disease brought on by bacteria “from within”.

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Infection

The normal flora, as well as any «contaminating» bacteria from the environment, is all found on the body surfaces of the animal; the blood and internal tissues are sterile. If a bacterium, whether or not a component of the normal flora, breaches one of these surfaces, an infection is said to have occurred. Infection does not necessarily lead to infectious disease. In fact, infection probably rarely leads to infectious disease. Some bacteria rarely cause disease if they do infect; some bacteria will usually cause disease if they infect. But other factors, such as the route of entry, the number of infectious bacteria, and status of the host defenses, play a role in determining the outcome of infection.

Determinants of virulence

Pathogenic bacteria are able to produce disease because they possess certain structural or biochemical or genetic traits that render them pathogenic or virulent. (The term virulence is best interpreted as referring to the degree of pathogenicity. ) It can be measured by the following units: DLM (dosis letalis minima) — minimal concentration causing death of 70-80% of laboratory animals; DCL (dosis certa letalis) — minimal concentration causing death of 100% of laboratory animals; LD₅₀ — dose causing the death of 50% of infected animals. The sums of the characteristics that allow a bacterium to produce disease are the pathogen’s determinants of virulence. Some pathogens may rely on a single determinant of virulence, such as toxin production, to cause damage to their host. Thus, bacteria such as Clostridium tetani and Corynebacterium diphtheriae, which have hardly any invasive characteristics, are able to produce disease, the symptoms of which depend on a single genetic trait in the bacteria: the ability to produce a toxin. Other pathogens, such as Staphylococcus aureus, Streptococcus pyogenes and Pseudomonas aeruginosa, maintain a large repertoire of virulence determinants and consequently are able to produce a more complete range of diseases that affect different tissues in their host.

There is the following classification of factors of pathogenicity (or determinants of virulence) can be offered:

• factors of adhesion and colonization (e.g. pili, fimbriae, adhesions), which allow bacteria to recognize and attach to certain membrane cell receptors;

• factors of invasion (e.g. hyaluronidaze, coagulase, neuraminidase, DNAse, etc. ), which allow bacteria to spread into and within cells and tissues.

• antiphagocytic factors, which either ‘mask’ bacteria from phagocytosis (e.g. capsule) or suppress phagocytosis (e.g. protein M in Streptococcus pyogenes, cord-factor in Mycobacteria spp., etc. );

• endotoxins (or lipopolysaccharides) in gram-negative bacteria which are released after cell death and possess inflammatory properties;

• exotoxins, secreted proteins which have toxic properties on host cells (e.g. diphtheria, botulinum, tetanus toxins).

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Infection in animals can be mediated via different ways (subcutaneous, cutaneous, intradermal, intramuscular, oral, etc. ) depending of objectives of study and nature of laboratory animals.

Host defenses

Typically the host defense mechanisms are divided into two groups:

1. Constitutive defenses. Defenses common to all healthy animals. These defenses provide general protection against invasion by normal flora, or colonization, infection, and infectious disease caused by pathogens. The constitutive defenses have also been referred to as «natural» or «innate» resistance, since they are inherent to the host.

2. Inducible defenses. Defense mechanisms that must be induced or turned on by host exposure to a pathogen (as during an infection). Unlike the constitutive defenses, they are not immediately ready to come into play until after the host is appropriately exposed to the parasite. The inducible defenses involve the immune responses to a pathogen causing an infection.

The inducible defenses are generally quite specifically directed against an invading pathogen. The constitutive defenses are not so specific, and are directed toward general strategic defense. The constitutive defenses, by themselves, may not be sufficient to protect the host against pathogens. Such pathogens that evade or overcome the relatively nonspecific constitutive defenses are usually susceptible to the more specific inducible defenses, once they have developed.

Host defense mechanisms

(source: http: //textbookofbacteriology. net/constitutivedefense. html)

Although humans are in continuous associations with microorganisms, and some readily colonize the body surfaces it is relatively rare that these microorganisms cause damage to their host. In part, this is due to the effectiveness of the host defense mechanisms, which restrict invasion by normal flora (some of which may be potential pathogens), and which defend against non-indigenous microorganisms that are overt pathogens.

Just as the outcome of an interaction between the host and a member of the normal flora always depends on specific properties inherent to both the host and the microbe, so does the outcome of an interaction between the host and a parasite. Sometimes the host tolerates colonization by a parasite but restricts it to regions of the body where it can do no harm (e.g. Staphylococcus aureus on the nasal membranes or Streptococcus pneumoniae in the upper respiratory tract). if the parasite invades (i.e., breaches an anatomical barrier or progress beyond the point of colonization), an infection is said to have occurred. If, as a result of infection, pathological harm to the host becomes evident, this is called an infectious disease. An infectious disease is a consequence of a microbial parasite causing such a degree of harm to its host that it results in a pathological process.

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The healthy animal defends itself against pathogens different stages. The host defenses may be of such a degree that infection can be prevented entirely. Or, if infection does occur, the defenses may stop the process before disease is apparent. At other times, the defenses that are necessary to defeat a parasite may not be effective until infectious disease is well into progress.

Typically the host defense mechanisms are divided into two groups:

1. Constitutive defenses: Defenses common to all healthy animals. These defenses provide general protection against invasion by normal flora, or colonization, infection, and infectious disease caused by pathogens. The constitutive defenses have also been referred to as «natural» or «innate» resistance, since they are inherent to a specific host, but these terms are better reserved for certain types of constitutive defense (see below).

2. Inducible defenses: Defense mechanisms that must be induced or turned on by host exposure to a pathogen (as during an infection). Unlike the constitutive defenses, they are not immediately ready to come into play until after the host is appropriately exposed to the parasite. The inducible defenses involve the immune responses to a pathogen causing an infection. The inducible defenses are generally quite specifically directed against an invading pathogen. The constitutive defenses are not so specific, and are directed toward general strategic defense.

Constitutive defenses of the host

The constitutive defenses of the host can be arranged in the following categories:

• Differences in susceptibility to certain pathogens

• Anatomical defenses

• Microbial antagonism

• Tissue bactericides, including complement

• Inflammation (ability to undergo an inflammatory response)

• Phagocytosis

Each of these topics is discussed in the sections below.

Differences in susceptibility of animal hosts

to microbial pathogens

This type of resistance is also called innate and natural resistance. There are two aspects innate resistance: (1) natural (genetic) resistance among all members of a species, called species resistance and (2) individual resistance within the same animal species.

Species resistance

Certain animals are naturally resistant or nonsusceptible to certain pathogens. Certain pathogens infect only humans, not lower animals, e.g. syphilis, gonorrhea, measles, poliomyelitis. On the other hand, certain

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pathogens (e.g. canine distemper vims) do not infect humans. Shigella infects humans and baboons but not chimpanzees. Little information is available to explain these absolute differences in susceptibility to a pathogen but it could be due to:

Absence of specific tissue or cellular receptors for attachment (colonization) by the pathogen. For example, different strains of enterotoxigenic E. coli, defined by different fimbrial antigens, colonize human infants, calves and piglets, by recognizing species-specific carbohydrate receptors on enterocytes in the gastrointestinal tract.

Temperature of the host and ability of pathogen to grow. For example, birds do not normally become infected with mammalian strains of Mycobacterium tuberculosis because these strains cannot grow at the high body temperature of birds. The anthrax bacillus (Bacillus anthracis) will not grow in the cold-blooded frog (unless the frog is maintained at +37°C).

Lack of the exact nutritional requirements to support the growth of the pathogen. Naturally-requiring purine-dependent strains of Salmonella typhi grow only in hosts supplying purines. Mice and rats lack this growth factor and pur- strains are avirulent. By injecting purines into these animals, such that the growth factor requirement for the bacterium is satisfied, the organisms prove virulent.

Lack of a target site for a microbial toxin. Most toxins produced by microbial cells exert their toxic activity only after binding to susceptible cells or tissues in an animal. Certain animals may lack an appropriate target cell or specific type of cell receptor for the toxin to bind to, and may therefore be nonsusceptible to the activity of the toxin. For example, injection of diphtheria toxin fails to kill the rat. The unchanged toxin is excreted in the urine. Inject a sample of the urine (or pure diphtheria toxin) into the guinea pig, and it dies of typical lesions caused by diphtheria toxin.

Individual resistance

There are many reasons why individuals of the same animal species may exhibit greater or lesser susceptibility to the same infective agent.

Age: usually this relates to the development and status of the immune system which varies with age. May also be associated with changes in normal flora coincidental to developmental changes in the animal.

Sex: usually linked to the presence and/or development of the sex organs. For example, mastitis and infectious diseases leading to abortion will obviously occur only in the female; orchitis would occur only in males). Could also be due to anatomical structure related to sex (bladder infections are 14-times more common in females than males), and possibly the effects of sex hormones on infections.

Stress. Stress is a complex of different factors and apparently has a real influence on health. Undue exertion, shock, change in environment, climatic

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change, nervous or muscular fatigue, etc. are factors known to contribute to increases in susceptibility to infection. The best explanation is that in time of stress the output of cortisone from the adrenal cortex is increased. This suppresses the inflammatory processes of the host and the overall effect may be harmful. There are also a number of relationships between stress-related hormones and the functioning of the immune defenses.

Diet, malnutrition. Infections may be linked with vitamin and protein deficiencies and this might explain partly why many infectious diseases are more prevalent and infant mortality rates are highest in parts of the world where malnourishment is a problem. Also, overfed and obese animals are more susceptible to infection. Diets high in sucrose predispose individuals to dental caries.

Intercurrent disease or trauma. The normal defenses of an animal are impaired by organic diseases such as leukemia, Hodgkin’s disease, diabetes, AIDS, etc. Frequently, inflammatory or immune responses are delayed or suppressed. Colds or influenza may predispose an individual to pneumonia. Smoking tobacco predisposes to infections of the respiratory tract. Burned tissue is readily infected by Pseudomonas aeruginosa.

Therapy against other diseases. Modem therapeutic procedures used in some diseases can render an individual more susceptible to infection. Under these conditions, not only pathogens but organisms of the normal flora and nonpathogens in the host’s environment may be able to initiate infection. Examples of therapeutic procedures that reduce the efficiency of the host’s defenses are treatment with corticosteroids, cytotoxic drugs, antibiotics, or irradiation.

Anatomical defenses

The structural integrity of the body surfaces, i.e., the skin and mucous membranes, forms an effective barrier to initial lodgment or penetration by microorganisms. The skin is a very effective barrier to bacteria so that no bacterium by itself is known to be able to penetrate unbroken skin. Of course, a puncture, cut or scrape in the skin could introduce infectious bacteria, The mucous membranes are more vulnerable to penetration by infectious bacteria but still pose a formidable barrier of mucus and antimicrobial substances. Nonetheless, most infectious agents impinge on the skin or mucous membranes of the oral cavity, respiratory tract, GI tract or urogenital tract, and from these sites most infections occur.

Skin. The intact surface of the healthy epidermis seems to be rarely if ever penetrated by bacteria. If the integrity of the epidermis is broken (by the bite of an insect, needle stick, abrasion, cut, etc. ) invasive microbes may enter. The normal flora of the skin, which metabolize substances secreted onto the skin, produce end products (e.g. fatty acids) that discourage the colonization of skin by potential pathogens. Perspiration contains lysozyme and other antimicrobial substances.

Mucous membranes. Many are heavily colonized with bacteria in whose moist secretions they survive. These normal flora are restricted from entry and

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usually occupy any attachment sites that might otherwise be used by pathogens. The normal flora established on mucous membranes may antagonize non- indigenous species by other means, as well. Typically, mucus contains a number of types of anti-microbial compounds, including lysozyme and secretory antibodies (IgA). Sometimes phagocytes patrol mucosal surfaces (e.g. in the lower respiratory tract). Nonetheless, some pathogens are able to penetrate the mucous membranes, and this is probably the major site from which pathogens invade. Probably, damage to the epithelial cells caused by toxic products of these bacteria plays a role.

Respiratory tract. Fine hairs and baffles of the nares (nasal membranes) entrap bacteria which are inhaled. Those which pass may stick to mucosal surfaces of the trachea or be swept upward by the ciliated epithelium of the lower respiratory tract. Coughing and sneezing also eliminate bacteria. The lower respiratory tract (lung) is well protected by mucus, lysozyme, secretory antibody, and phagocytosis.

Mouth, stomach and intestinal tract. Microorganisms entering by the oral route, more than any other, have to compete with the well-adapted normal flora of the mouth and intestine. Most organisms that are swallowed are destroyed by acid and various secretions of the stomach. Alkaline pH of the lower intestine can discourage other organisms. The peristaltic action of the intestine ultimately Hushes out organisms which have not succeeded in colonization. Bile salts and lysozyme are present, which kill or inhibit many types of bacteria.

Urogenital tract. The flushing mechanisms of sterile urine, and the acidity of urine, maintain the bladder and most of the urethra free of microorganisms.

The vaginal epithelium of the female maintains a high population of Doderlein’s bacillus (Lactobacillus acidophilus) whose acidic end products of metabolism (lactic acid) prevent colonization by most other types of microorganisms including potentially-pathogenic yeast (Candida albicans).

Eyes (conjunctiva). The conjunctiva of the eye is remarkably free of most microorganisms. Blinking mechanically removes microbes, the lavaging action of tears washes the surface of the eye, and lachrymal secretions (tears) contain relatively large amounts of lysozyme.

Microbial antagonism

This refers to the protection of the surfaces afforded by an intact normal flora in a healthy animal, and it has already been discussed in several contexts.

There are three main ways that the normal flora protect the surfaces where they are colonized:

Competition with non-indigenous species for binding (colonization) sites. The normal flora is highly adapted to the tissues of their host. That is why they are there!

Specific antagonism against non-indigenous species. Members of the normal flora may produce highly specific proteins called bacteriocins which kill or inhibit other (usually closely-related) species of bacteria.

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Nonspecific antagonism against non-indigenous species. The normal flora produces a variety of metabolites and end products that inhibit other microorganisms. These include fatty acids (lactate, propionate, etc. ) and peroxides.

Antimicrobial substances in host tissues

The body fluids and organized tissues of animals naturally contain a variety of antimicrobial agents that kill or inhibit the growth of microbes. The sources and activities of a variety of host antimicrobial substances are summarized in Table 15.

Complement

Complement can be considered as part of the constitutive host defense mechanisms (it is present at constitutive levels) because of its role in inflammation and phagocytosis. However, the antimicrobial activities of complement can be activated completely by reactions between antigens and antibodies and, therefore, it may play a role in the inducible (immune) defenses, as well.

Table 15. ANTIMICROBIAL SUBSTANCES OF HOST ORIGIN PRESENT IN BODY FLUIDS AND ORGANIZED TISSUES

Substance	Common Sources	Chemical Composition	Activity
Lysozyme	Serum, saliva, sweat, tears	Protein	Bacterial cell lysis
Complement	Serum	Protein- carbohydrate lipoprotein complex	Cell death or lysis of bacteria; participates in inflammation
Basic proteins and	Serum or organized	Proteins or basic	Disruption of bacterial
polypeptides (histones, β-lysins and other cationic proteins, tissue polypeptides)	tissues	peptides	plasma membrane
Lactoferrin and transferrin	Body secretions, serum, organized tissue spaces	Glycoprotein	Inhibit microbial growth by binding iron
Peroxidase	Saliva, tissues, cells (neutrophils)	Protein	Act with peroxide to cause lethal oxidations of cells
Fibronectin	Serum and mucosal surfaces	Glycoprotein	Clearance of bacteria (opsonization)
Interferons	Virus-infected cells, lymphocytes	Protein	Resistance to virus infections
Interleukins	Macrophages, lymphocytes	Protein	Cause fever; promote activation of immune system

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Complement is an enzymatic system of serum proteins made up of 9 major components (C1-C9) that are sequentially activated in many Ag-Ab reactions resulting in disruption of membranes. Therefore, complement (C’) may be involved in the lysis of certain bacteria, some viruses, and other microorganisms. In addition, some C’ components play a part in phagocytic chemotaxis, opsonization and the inflammatory response.

Complement is activated in the classical pathway by reactions between antibodies and antigens on the surface of a microbe. Some immunoglobulins (i.e., IgG and IgM) can "fix complement" because they have a complement binding site on the Fc portion of the molecule. The reaction between IgG and Ag activates the complement and initiates a "cascade reaction" on the surface of the microbe that results in the principal effects of complement which are:

1. Generation of inflammatory factors, C3a and C5a, which focus antimicrobial serum factors and leukocytes into the site of infection.

2. Attraction of phagocytes. Chemotactic factors C3a and C5a attract phagocytes to the site.

3. Enhancement of phagocytic engulfment. C3b component on Ag-Ab complex attaches to C3b receptors on phagocytes and promotes opsonization of Ab-coated cells. C3b-opsonization is important when Ab is IgM because phagocytes have receptors for Fc of IgM only when it is associated with C3b.

4. Lysis of bacterial cells (lysozyme-mediated) or virus-infected cells. When C8 and C9 are bound to the complex, a phospholipase is formed that destroys the membrane of Ag-bearing host cells (e.g. virus-infected cells) or the outer membrane of Gram-negative bacteria. Lysozyme gains access to peptidoglycan and completes destruction of the bacterial cell.

In addition to the classical pathway of complement activation an alternative pathway (sometimes called the «"properdin pathway") of complement activation exists which is independent of immunoglobulins. Insoluble polysaccharides (including bacterial LPS, peptidoglycan and teichoic acids) can activate complement. This allows antibody-independent activation of the complement cascade that may be important in initial (pre-antibody) defense against various types of infections caused by bacteria.

Inflammation

Of all the defense mechanisms in the animal host, the inflammatory response may be the most important for dealing with microbial infection. Inflammation Is necessary for the proper functioning of all the host defenses, including the immune defenses, because it focuses all circulating antimicrobial factors on the site of infection. These include phagocytes, lymphocytes, antibodies, complement and other antimicrobial components of plasma. However, inflammation is also an important aspect of bacterial pathogenesis since the

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inflammatory response induced by a microbe can result in considerable damage to the host and, therefore, is part of the pathology of microbial disease.

Inflammation is a tissue reaction to infection or injury, the characteristic symptoms of which are redness, swelling, heat and pain. These are sometimes called the cardinal signs of inflammation. The redness is due to increased blood flow to the area of injury. The swelling (edema) is due to increased extravascular fluid and phagocyte infiltration to the damaged area. The heat is due to the increased blood flow and the action of pyrogens (fever-inducing agents). The pain is caused by local tissue destruction and irritation of sensory nerve receptors.

Inflammation can be induced by certain immunological reactions, tissue damage, or the entry of an injurious agent (microbial or nonmicrobial). Certain bacterial cells and/or their products (e.g. structural components or toxins) can induce an inflammatory response. Inflammation increases the blood supply and temperature in the inflamed tissues, which favors maximal metabolic activity of the leukocytes, and lowers the pH slightly, which tends to inhibit the multiplication of many microorganisms.

The events involved in the induction and maintenance of the inflammatory response are summarized below.

1. The inflammatory response is triggered by pathogen invasion or tissue injury. Injured and dying cells release cytoplasmic constituents which lower the pH in the surrounding extracellular environment.

2. The increased acidity activates an extracellular enzyme kallikrein which in turn activates bradykinin.

(3a) Bradykinin binds to receptors on the capillary walls opening junctions between cells to allow leakage of plasma components collectively referred to as the inflammatory exudate.

Increased capillary permeability allows leukocytes to pass from the vessels into tissues (this process is called diapedisis). The first to appear, and the most dominant, are neutrophils, which are actively phagocytic. The other components of the inflammatory exudate and their functions are described in Table 16 below.

(3b) Bradykinin also binds to mast cells of the connective tissue that are associated with the small vessels of most tissues. This initiates other events that are associated with the process of inflammation.

Initially there is a rapid influx of Ca++, intracellular cAMP levels drop, and mediator-rich lysosomal granules migrate to the cell surface, fuse with the cell membrane, and discharge their contents (preformed mediators of inflammation such as histamine, heparin, etc.) to the exterior by exocytosis

The change in mast cell permeability activates an enzyme, phospholipase A2 to synthesize a substance called arachidonic acid. This compound can be acted upon subsequently by the cyclooxygenase pathways or lipooxygenase pathways of the mast cell leading to new synthesis of prostaglandins, leukotrienes, and other mediators of inflammation. These substances contribute to the

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Table 16. FUNCTION OF COMPONENTS AND CELLS IN THE INFLAMMATORY EXUDATE

Component

Function

Bradykinin, histamine, leukotrienes, serotonin, prostaglandins

Fibrin: (formed from fibrinogen in plasma)

Lysozyme

Complement

Antibodies (in immune individuals)

Pyrogens, including endogenous pyrogen (IL-1)

Neutrophils

Macrophages

Immunocompetent lymphocytes (B cells and T cells)

Inflammatory agents (1A) which act on the vascular system to produce increased blood flow and permeability

coagulates and may localize an invading pathogen

causes lysis of bacterial cell walls

various activities increase the inflammatory response

and lead to increased phagocytosis and complement-

mediated lysis of cells

block colonization by pathogens; neutralize

microbial toxins or viruses; opsonize pathogens

making them more susceptible to phagocytosis;

activate complement

cause fever acting on the thermo-regulatory control centers in the hypothalamus. (IL-1, which is produced by macrophages, also promotes activation and mitosis of B cells and T cells)

migrate to focus of infection and ingest and destroy

foreign agents by phagocytosis

engulf and destroy infective agents, process antigenic

components and convey them to lymphocytes

for direct participation in immunological responses

(AMI and CMI)

inflammatory exudate. The overall effect of an inflammatory reaction is to recruit various cells and components to the actual site of microbial invasion. Many of these cells and plasma components have a direct role in defense against the intruding microorganism. These include neutrophils (phagocytes which engulf and destroy the microbes); macrophages and lymphocytes which are the cells necessary to initiate immunological responses against the pathogen; pre-existing antibodies which can neutralize microbial pathogens or their toxins; and plasma components such as lysozyme, complement and fibrin, which have a variety of antimicrobial activities.

Phagocytic defenses

When invading parasites penetrate the tissues the inflammatory response, previously described, is immediately brought into play. Part of this response leads to the recruitment of phagocytes to the site of inflammation. Phagocytes are a class of cells which are capable of ingestion (engulfment) and destruction of microorganisms that are responsible for inciting the inflammatory response.

First to accumulate around the invaders and initiate the phagocytic process are neutrophils. Later, local and blood-borne macrophages also migrate to the tissue

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site and initiate phagocytosis. Neutrophils (also known as polymorphonuclear leucocytes or PMNs) and macrophages are sometimes referred to as professional phagocytes for their roles in this process.

Properties of neutrophils

Neutrophils have their origin in multi-potential stem cells in the bone marrow. They differentiate in the marrow and are released in a mature form, containing a full complement of bactericidal agents. They are short-lived cells which constitute 30-70% of the circulating white blood cells (leukocytes).

During differentiation in the marrow (2-3 days) the nucleus of the cell becomes multilobed (hence the name polymorphonuclear leukocyte), cell division ceases, and mitochondria and endoplasmic reticulum disappear from the cytoplasm. At the same time the cell becomes motile and actively phagocytic. Cytoplasmic granules are formed from the Golgi apparatus. These granules are called lysosomes and contain the various bactericidal and digestive enzymes which can destroy bacterial cells after engulfment. The contents of lysosomal granules include lysozyme, cationic proteins, acid hydrolases, proteases, peroxidase and lactoferrin. Neutrophils also contain large store of glycogen; since they derive most of their metabolic energy from glycolysis, they can function efficiently in anaerobic environments.

Some additional properties of neutrophils are:

• Only half the neutrophils in human circulation are detectable in the blood; the rest adhere to vessel walls.

• For every circulating neutrophil, approximately 100 near mature cells are held in reserve in the bone marrow pool.

• Once a neutrophil enters the tissues, intestinal tract or respiratory tract, it never returns to the circulation.

Properties of macrophages

Macrophages (also called mononuclear phagocytes) also arise from bone marrow stem cells which give rise to promonocytes which develop into monocytes that are released into the blood stream. Monocytes make up 3-7% of the circulating white blood cells. The monocyte is actively phagocytic and bactericidal. Within 2 days or so, the blood stream monocytes (sometimes called wondering macrophages) emigrate into the tissues where they settle down, enlarge and become fixed macrophages (tissue histiocytes), which also have phagocytic potential. Macrophages are more active in phagocytosis than monocytes and develop many more granules containing hydrolytic enzymes. New macrophages can develop by cell division under inflammatory stimuli, but most macrophages are matured blood monocytes.

The total pool of macrophages is referred to as the system of mononuclear phagocytes. The system is scattered throughout connective tissue, basement membranes of small blood vessels, liver sinusoids, the spleen, lung, bone marrow

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and lymph nodes. Monocytes from the blood migrate into virtually every organ in the body where they mature into fixed macrophages. In the lymph nodes, they function as scavengers to remove foreign material from the circulation.

Compared to neutrophils, macrophages are long-lived cells. As phagocytes, neutrophils play a more important role in the acute stages of an infection, while macrophages are principally involved in chronic types of infections. Neutrophils circulate in the blood stream, and during an acute inflammatory response they migrate through the endothelial cell junctions as part of the inflammatory exudate. They migrate to the focus of the infection and ingest or phagocytose the foreign agents, Neutrophils which have become engorged with bacteria usually die and largely make up the material of pus. Macrophages, which are also attracted to the area during an inflammatory response, are slower to arrive and become increasingly involved in chronic infections. They, too, are actively phagocytic and will engulf and destroy foreign particles such as bacteria. However, macrophages have another indispensable function in host defense: they «process» the antigenic components of infective agents and present them to lymphocytes, a process that is usually required for the initiation of the immune responses of the host. Macrophages are among an elite corps of antigen-presenting cells or APC’s.

The phagocytic process

Phagocytosis and destruction of engulfed bacteria involves the following sequence of events:

1. Delivery of phagocytic cells to the site of infection

2. Phagocytic adherence to the target

3. Ingestion or engulfment of the target particle

4. Phagolysosome formation

5. Intracellular killing

6. Intracellular digestion (and egestion, in the case of macrophages)

These steps involved in the phagocytic process in macrophages are illustrated

on Fig. 13.

A bacterium, which may or may not be opsonized, is engulfed by the process of endocytosis. The bacterium is ingested in a membranous vesicle called the phagosome. Digestive granules (lysosomes) merge with phagosome, release their contents, and form a structure called the phagolysosome. The killing and digestion of the bacterial cell takes place in the phagolysosome. The macrophage egests debris while processing the antigenic components of the bacterium, which it returns to its surface in association with MHC II for antigen presentation to TH cells.

Delivery of phagocytic cells to the site of infection

The delivery of phagocytic cells, monocytes or neutrophils, to the site of microbial infection involves two processes:

Diapedisis: the migration of cells across vascular walls which is initiated by the mediators of inflammation (kinins, histamine, prostaglandins, etc. )

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Figure 13. Phagocytosis by a macrophage

Chemotaxis. Phagocytes are motile by ameboid action. Chemotaxis is movement of the cells in response to a chemical stimulus. The eventual concentration of phagocytes at a site of injury results from chemotactic response by the phagocytes which is analogous to bacterial chemotaxis. A number of chemotactic factors (attractants) have been identified, both for neutrophils and monocytes. These include bacterial products, cell and tissue debris, and components of the inflammatory exudate such as peptides derived from complement.

Phagocytic adherence

Phagocytosis is initiated by adherence of a particle to the surface of the plasma membrane of a phagocyte. This step usually involves several types of surface receptors on the phagocyte membrane. Three major receptors on phagocytes recognize the Fc portion of IgG: one is for monomeric IgG and the others are for antigen-cross linked IgGs. Another receptor binds a complement factor C3b. Other phagocyte receptors bind fibronectin and mannose-terminated oligosaccharides. Under certain circumstances of infection, bacteria or viruses may become coated or otherwise display on their surfaces one or another of these substances (i.e., IgG, C3b, fibronectin or mannose). Such microbes are said to be opsonized and such substances as IgG or complement C3b bound to the surface of microbes are called opsonins (Opsonin comes from a Greek word meaning «sauce» or «seasoning»: they make the bacterium or virus more palatable and more easily ingested by the phagocyte.) Opsonins provide extrinsic ligands for specific receptors on the phagocyte membrane, which dramatically increases the rate of adherence and ingestion of the pathogen. Opsonized bacteria can be cleared from the blood by phagocytes; many types of non opsonized bacteria cannot be cleared.

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Less firm attachments of a phagocyte to a particle can take place in the absence of opsonization. This can be thought of as nonspecific attachment which might be due to net surface charge on the phagocyte or particle and/or hydrophobicity of the particle.

Also, a phenomenon called surface phagocytosis exists: a phagocyte can simply trap an organism against a surface and initiate ingestion. Surface phagocytosis may be an important pre-antibody defense mechanism which may determine whether an infection will become a disease and how severe the disease will become.

Ingestion

After attachment of the phagocyte to its target, some sort of signal generation, which is poorly understood, results in physical or chemical changes in the cell that triggers ingestion. Ingestion is an engulfment process that involves infolding or invagination of the cell membrane enclosing the particle and ultimately releasing it into the cytoplasm of the cell within a membrane vesicle. The end result of ingestion is entry of the particle enclosed in a vesicle derived from the plasma membrane of the cell. This structure is called the phagosome.

Formation of the phagolysosome

The phagosome migrates into the cytoplasm and collides with lysosomal granules which explosively discharge their contents into the membrane-enclosed vesicle (phagosome). Membranes of the phagosome and lysosome actually fuse resulting in a digestive vacuole called the phagolysosome. Other lysosomes will fuse with the phagolysosome. It is within the phagolysosome that killing and digestion of the engulfed microbe takes place. Some of the microbicidal constituents of the lysosomes of neutrophils and macrophages include lysozyme, cationic proteins, various proteases, hydrolyases and peroxidases. The killing processes are confined to the membranous organelles of the phagocytes (the phagolysosome) such that none of the toxic substances and lethal activities of the phagocytes are turned against themselves.

Intracellular killing of organisms

After phagolysosome formation the first detectable effect on bacterial physiology, occurring within a few minutes after engulfment, is loss of viability (ability to reproduce). The exact mechanism is unknown. Inhibition of macromolecular synthesis occurs later. By 10 to 30 minutes after ingestion many pathogenic and nonpathogenic bacteria are killed followed by lysis and digestion of the bacteria by lysosomal enzymes. The microbicidal activities of phagocytes are complex and multifarious (Fig. 14). Metabolic products, as well as lysosomal constituents, are responsible. These activities differ to some extent in neutrophils, monocytes and macrophages.

The microbicidal activities of phagocytes are usually divided into oxygen- dependent and oxygen-independent events.

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Oxygen-independent activity

Lysosomal granules contain a variety of extremely basic proteins that strongly inhibit bacteria, yeasts and even some viruses. A few molecules of any one of these cationic proteins appear able to inactivate a bacterial cell by damage to their permeability barriers, but their exact modes of action are not known. The lysosomal granules of neutrophils contain lactoferrin, an extremely powerful iron- chelating agent, which withholds potential iron needed for bacterial growth. The pH of the phagolysosome may be as low as 4. 0 due to accumulation of lactic acid, which is sufficiently acidic to prevent the growth of most pathogens. This acidic environment apparently optimizes the activity of many degradative lysosomal enzymes including lysozyme, glycosylases, phospholipases, and nucleases.

Oxygen-dependent activity

Liganding of Fc receptors (on neutrophils, monocytes or macrophages) and mannose receptors (on macrophages) increases their O₂ uptake, called the respiratory burst. These receptors activate a membrane-bound NADPH oxidase that reduces O₂ to O₂^- (superoxide). Superoxide can be reduced to OH (hydroxyl radical) or dismutated to H₂O₂ (hydrogen peroxide) by superoxide dismutase. O₂^-, OH, and H₂O₂ are activated oxygen species that are potent oxidizing agents in biological systems which adversely affect a number of cellular structures including membranes and nucleic acids. Furthermore, at least in the case of neutrophils, these reactive oxygen intermediates can act in concert with a lysosomal enzyme called myeloperoxidase to function as the myeloperoxidase system, or MPO.

Myeloperoxidase is one of the lysosomal enzymes of neutrophils which is released into the phagocytic vacuole during fusion to form the phagolysosome. Myeloperoxidase uses H₂O, generated during the respiratory burst to catalyze halogenation (mainly chlorination) of phagocytosed microbes. Such halogenations are a potent mechanism for killing cells.

When the NADPH oxidase and myeloperoxidase systems are operating in concert, a series of reactions leading to lethal oxygenation and halogenation of engulfed microbes occurs.

Intracellular digestion

Dead microbes are rapidly degraded in phagolysosomes to low molecular- weight components. Various hydrolytic enzymes are involved including lysozyme, proteases, lipases, nucleases, and glycosylases. Neutrophils die and lyse after extended phagocytosis, killing, and digestion of bacterial cells. This makes up the characteristic properties of pus.

Macrophages egest digested debris and allow insertion of microbial antigenic components into the plasma membrane for presentation to lymphocytes in the immunological response.

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Figure 14. Phagocytosis of Streptococcus pyogenes by a macrophage Bacterial defense against phagocytosis

Pathogenic bacteria have a variety of defenses against phagocytes. In fact, most successful pathogens have some mechanism(s) to contend with the phagocytic defenses of the host. These mechanisms will be discussed in detail later as part of the determinants of virulence of pathogens. However, in general, pathogens may resist phagocytosis by:

• Evading phagocytes by growing in regions of the body which are not accessible to them

• Avoiding engulfment by phagocytes after contact

• Being able to kill phagocytes either before or after engulfment

• Being able to survive inside of phagocytes (or other types of cells) and to persist as intracellular parasites.

Main objectives of the session

1. To study specific features and dynamics of infectious disease, forms of manifestations and ways of transmission.

2. To study pathogenicity and virulence of microorganisms, methods of their detection and evaluation.

3. To get acquainted with the main methods of experimental infections and bacteriological investigation of dead animals.

4. To study types and forms of immunity.

5. To study non-specific mechanisms of host defense with a particular focus on phagocytosis.

Educational tasks

To know: 1. Definition of term ‘infection’. General characteristics of

infectious disease.

1. Definitions of pathogenicity and virulence.

2. Definition of term ‘immunity’. General characteristics of immune system and its main functions.

3. Non-specific factors of host defense; cell (phagocytosis, natural killers), humoral (complement, interferons, lysozyme, etc. ).

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To be 1. To evaluate significance of infectious disease.

capable: 2. To perform bacteriological investigation of dead laboratory

animal.

3. To evaluate significance of non-specific factors of host defense.

Methodical guidelines

For evaluation of non-specific biological mechanisms of resistance to infectious disease, the following might be studied:

1. concentration of lysozyme, interferons, complement, cytokines in biological fluids (e.g. serum, saliva, CSF, tears):

2. phagocytic reaction of blood cells, lymph nodes, spleen and other lymphoid organs.

Concentration of lysozyme in saliva or serum is determined by titration using Micrococcus lysodeikticus. Lysis of bacteria could be observed by clearing up or by changing optical density of microbial suspension after incubation with lysozyme dilutions. Unit of activity of complements is defined as amount of complement which causes 50% hemolysis in standard conditions. Separate compounds of complement are determined by immunochemical methods using monoclonal sera.

Experimental infection and bacteriological investigation of dead animals.

Students should be getting acquainted with different methods of infection of laboratory animals, including cutaneous (rabbit, guinea pig), intradermal, subcutaneous, intravenous (vein of rabbit ear, retroorbital space in mice), intraperitoneal methods. They are taught with rules of fixation of animals, asepsis and antiseptics.

For determination of microorganism caused death of animal and its localization in organism, post-mortem examination of mouse infected in advance with Bacillus anthracoides.

1. Mouse is fixated by legs to wooden board using 4 pins with abdomen being up.

2. Hair of the mouse is washed with cotton pad washed in disinfected solution or ethanol.

3. Median incision of skin is made, followed by examination of peritoneum and lymph nodes.

4. Opening of peritoneum is performed followed by examination of internal organs in peritoneal cavity with a special focus of visible abnormalities.

5. Smear imprints of liver, spleen, and kidneys are made by taking organs by forceps, cutting small piece with scissors and touching slide by cut surface,

6. Opening of thoracic cavity is performed followed by examination of internal organs with a special focus of visible abnormalities.

7. Smear imprints of lung tissue is made.

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8. Using sterile Pasteur pipette after cauterization of surface of myocardium, blood from the heart is taken, followed by inoculation of sugar broth and smear on slide.

9. Smear imprints prepared on slide are allowed to dry, fixed in liquid fixating solution and stained by Gram stain.

10. After microscopy of smears, B. anthracoides should be found, conclusion on form of infectious disease and virulence of pathogen should be made. Post-mortem examination data should be recorded in workbook.

Methods of evaluation of functional activity of phagocyting cells

Phagocytosis with latex.

1. 0.1 ml of blood from finger is placed into centrifuge tube with heparin. Then 0.1 ml of 10% latex suspension with 1.5 mm particle size is added. Tube the incubated at +37°C for 30 min. Every 10 min. tube is shaken.

2. Perform centrifugation for 5 min., followed by taken out supernatant using pipette. Sediment then is used for smear preparation, allowed to air dry, fixed by ethanol and stained by Giemsa-Romanovsky technique.

3. Interpretation of results: using immersion microscope (magnification 630), quantification of percentage of phagocyting cells is counted (phagocytic index is defined as number of phagocyting cells per 100 leucocytes). Then mean number of phagocyted latex particles per 1 cell is calculated (phagocytic number). Healthy individuals have phagocytic index in a range of 40-80% and phagocytic number 6-9 per 1 phagocyte (if size of latex particle is 1. 5 mm).

Phagocytosis with staphylococci.

Reagents: stimulator of phagocytosis (pyrogenal), heparin, overnight culture of Staphylococcus spp., Giemsa-Romanovsky stain.

1. 0.2 ml of blood from finger is placed into two tubes: first with heparin and second with 0.1 ml of pyrogenal for stimulation of phagocytosis. Then 0.05 ml of overnight culture of Staphylococcus spp. (turbidity standard in 1 bln. )

2. Tube the incubated at +37°C for 30 min., then centrifuged, followed by taking out of supernatant.

3. Sediment then is used for smear preparation, allowed to air dry, fixed by ethanol and stained by Giemsa-Romanovsky technique.

4. Interpretation of results is done using immersion microscope (magnification 630).

1 tube

Spontaneous phagocytosis — results are inteipreted after 30 min, followed by determination of phagocytic index and phagocytic number.

Completed phagocytosis — results are interpreted after 120 min, followed by determination of phagocytic index and phagocytic number.

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Index of completion of phagocytosis (ICP) is defined as ration of phagocytic index after 30 min. to phagocytic index after 120 min. If ICP is more than 1, it indicates completeness of phagocytosis, if ICP is less then 1, it indicated non- completeness of phagocytosis.

2 tube

Stimulated phagocytosis (in presence of pyrogenal) — after the incubation and centrifugation, smears from sediment are prepared, followed by drying at air, fixation by ethanol and staining by Giemsa-Romanovsky technique.

Completed phagocytosis — results are interpreted after 120 min, followed by determination of phagocytic index and phagocytic number. Phagocytic index, phagocytic number and ICP are determined after 120 min. Stimulation by pyrogenal can lead to completion of phagocytosis, which should be taken into account during the correction of immunodeficiency syndromes.

Study of functional status of phagocytes based on metabolism of oxygen (chemiluminescence method)

Special liquid scintillation β-spectrophotometer (chemiluminometer) and special glass flasks are needed for scintillation count.

1. Suspension of leucocytes (neutrophils) is isolated from peripheral blood.

2. Henks solution and luminol are added to flasks for scintillation counting (luminol has a property of being oxidized under the action of oxygen metabolites and generate light quantum (length of wave is 425 nm).

3. Suspension of leucocytes is added to flasks, mixed and placed to counting camera for registration of background indicator of chemiluminescence.

4. After 45-60 min. zymosan or latex suspension is added to flasks, followed by measurement of chemiluminescence and counting number of impulses per 1 min. during 60 min. Then, calculation of number of impulses per 1 cell is made. Chemiluminescence is determined as impulse/min./cell. Results of chemiluminescence are evaluated by maximum value (peak A) of kinetic curve.

Laboratory work

1. Acquaintance with methods of experimental infection.

2. Bacteriological investigation of mice died from experimental infection, followed by completion of protocol of post-mortem examination.

3. Preparation of imprint smears from organs of mice, followed by Gram stain.

4. Study of factors of pathogenicity of microorganisms using pre-set demonstrations.

5. Microscopy of prepared stains with phagocytosis of latex particles.

6. Microscopy of clinical specimens from patients with gonorrhea.

7. Determination of titer of lysozyme in saliva by titration method.

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Demonstrations

1. Hemolytic activity of staphylococci on blood agar.

2. Lecithinase activity of staphylococci on yolk-salt agar.

3. Reaction of plasma coagulation.

4. DNAse activity of microorganisms.

5. Method of titration of lysozyme in saliva using Micrococcus lysodeikticus.

6. Smears of pus from patients with gonorrhea with non-completed phagocytosis.

7. Capsule stain of Klebsiella pneumoniae (Burry-Hins method).

Control questions

1. Definitions of ‘infection’ and ‘infectious disease’. Condition for development. Forms: local, systemic infections.

2. What are the relapse, re-infection and superinfection?

3. What are the mixed and secondary infections?

4. What are the bacteremia, sepsis, and toxinemia?

5. What is the carriage state?

6. Definitions of ‘pathogenicity’ and ‘virulence’. Units of measurement of virulence and methods of its determination.

7. Periods and dynamics of infectious disease.

8. Microbial toxins. Exotoxins and endotoxins, their origin, characteristics and differences between them.

9. Factors of pathogenicity in bacteria. Invasiveness, its basis. Adhesion, protection from phagocytosis.

10. Experimental infection. Aims and objectives. Methods of experimental infection.

11. Mechanisms and factors of non-specific immunity: barrier and bactericidal properties of skin, mucous membranes. Role of constant microflora.

12. Lysozyme and complement. Their properties and role in innate immunity.

13. Serum bactericidal activity and its factors: properdin, antibodies and others.

14. Phagocytosis as cell non-specific protection factor. Types of phagocytes, stages of phagocytosis. Non-completed phagocytosis.

15. Experiment with phagocytosis. Determination of activity and completion of reaction. Opsonophagocytic reaction.

16. Post-mortem examination and bacteriological investigation of animal died from experimental infection.

17. Reactivity of newborns and infants and tits differences from adult’ reactivity (for pediatric faculty only).

18. Condition of factors of non-specific immunity in infants (for pediatric faculty only).

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PRACTICAL SESSION No. 13 (COLLOQUIUM)

Immune system of humans. Antigens. Antibodies.

Specific forms of immune response.

Plan of the session

1. Central and peripheral organs of the immune system. Lymphoid and auxiliary cells: T and B lymphocytes, their subpopulations, and macrophages.

2. Specific forms of the immune response: synthesis of antibodies., cell immunity, hypersensitivity reactions, immunological memory and tolerance.

3. Intercell cooperation in immunogenesis. Mediators of immune response (cytokines, lymphokines, interleukins).

4. Antigens: complete, non-complete (haptens). Antigens of bacteria and viruses. Antigens of blood groups, autoantigens, tumor, embriospecific and transplant antigens.

5. Antibodies (immunoglobulins): chemical structure and function. Classes of immunoglobulins. Non-complete antibodies. Dynamics and mechanism of formation of antibodies.

6. Phenomenon of interaction of antigen and antibodies. Practical significance of reactions antigen-antibodies.

Foreword notes (adapted from http://textbookofbacteriology.net/ immune.html).

The immune system is comprised of the lymphoid tissues and organs of the body. Lymphoid tissues are widely distributed: they are concentrated in bone marrow, lymph nodes, spleen, liver, thymus, and Peyer’s patches scattered in linings of the gastrointestinal tract. The lymphoid system is encompassed by the system of mononuclear phagocytes. Lymphocytes are the predominant cells, but macrophages and plasma cells are present also. Lymphocytes are cells which circulate, alternating between the circulatory blood stream and the lymphatic channels.

The distribution of lymphatic tissues that make up the immune system in humans is illustrated in the Fig. 15.

The immunological system is able to recognize foreign substances (antigens) which stimulate the system to produce antibody-mediated immunity (AMI), cell- mediated immunity (CMI), or both. AMI and CMI are the two great arms of the immune system that are discussed in more detail below.

An antigen (Ag) is a substance, usually macromolecular, that induces an immunological response. Because of its complex macromolecular structure, a single microorganism consists of multiple antigens (e.g. surface structures such as cell wall components, fimbriae, flagella, etc., or extracellular proteins, such

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A. The Immune System B. The Lymph Node

Figure 15. Anatomy of the immune system. (A): The major components of the immune system are lymph nodes connected by lymph ducts, Peyer’s patches (masses of lymphocytes in the lower gastrointestinal tract), thymus, spleen, and bone marrow. (B): A lymph node. Afferent lymph ducts bring lymph-containing antigens into the lymph node. Macrophages, B cells or dendridic cells in the cortical region make contact with the antigen and process it for presentation to immunocompetent B cells and T cells, thereby initiating an immune response. As a result, B cells are stimulated to develop into antibody-secreting plasma cells, and T-cells are stimulated to develop into effector T cells of various classes. Antibodies leave the lymph node by the efferent ducts that empty into the blood stream. Lymphocytes can also leave the node by the efferent duct and travel to other sites in the lymphatic system or enter into the blood circulation. A single lymphocyte completes a circuit through the circulating blood and lymphatic systems once every 24 hours.

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as toxins or enzymes produced by the microorganism). The coat proteins and some of the envelope proteins of animal viruses are also usually antigenic. The host is able to respond specifically to each and every antigen to come into contact with the components of the immunological system.

The immune response

Immunological responses are associated with macrophages and two subpopulations of lymphocytes which are derived from primitive bone marrow cells. All of the cells involved in the immunological responses are derived from bone marrow stem cells which have differentiated under the influence of various tissues and stimuli. Macrophages develop from monocytes previously released from the bone marrow into the blood circulation. Lymphocytes responsible for AMI are processed by lymphoid tissue in the bone marrow and develop there into B lymphocytes or B cells. Lymphocytes responsible for CMI are processed by the thymus gland and mature into T lymphocytes or T cells.

Under antigenic stimulus, B-lymphocytes become transformed into antibody-secreting plasma cells. The plasma cells synthesize large amounts of immunoglobulins (antibodies) which will react stereochemically with the stimulating antigen.

Under antigenic stimulus, pre T-lymphocytes differentiate into several classes of effector T cells which are committed to various activities upon recognition of the specific antigen that induced their formation. T cells have many activities relevant to immunity including (1) mediation of the B-cell response to antigen; (2) ability to recognize and destroy cells bearing foreign Ag on their surface; and (3) production of a variety of diffusible compounds called cytokines and/or lymphokines, which include substances that are activators of macrophages, mediators of inflammation, chemotactic attractants, lymphocyte mitogens, and interferon.

Cytokines and lymphokines are molecules (peptides, proteins) produced by cells as a means of intercellular communication. Generally, they are secreted by a cell to stimulate the activity of another cell.

The overall aspects of the induction of the immune responses (AMI and CMI) are shown in the following schematic diagram (Fig. 16).

Three important features of the immunological system relevant to host defense and/or «immunity» to pathogenic microorganisms are:

1. Specificity. An antibody or reactive T cell will react specifically with the antigen that induced its formation; it will not react with other antigens. Generally, this specificity is of the same order as that of enzyme-substrate specificity or receptor-ligand specificity. However, cross-reactivity is possible. The specificity of the immune response is explained on the basis of the clonal selection hypothesis: during the primary immune response, a specific antigen selects a pre-existing clone of specific lymphocytes and stimulates exclusively its activation, proliferation and differentiation.

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Figure 16. Schematic diagram of the development of the immune responses

2. Memory. The immunological system has a «memory». Once the immunological response has reacted to produce a specific type of antibody or reactive T cell, it is capable of producing more of the antibody or activated T cell more rapidly and in larger amounts. This is sometimes referred to as a secondary, or memory response.

3. Tolerance. An animal generally does not undergo an immunological response to its own (potentially-antigenic) components. The animal is said to be tolerant, or unable to react to its own potentially-antigenic components. This ensures that under normal conditions, an immune response to «self» antigens (called an autoimmune response) does not occur. Autoimmune responses are potentially harmful to the host. Tolerance is brought about in a number of ways, but basically the immunological system is able to distinguish «self» components from «non-self» (foreign) antigens; it will respond to «non- self» but not to «self». Sometimes in an animal, tolerance can be «broken», which may result in an autoimmune disease.

The two types of the immunity: AMI and CMI

Antibody-mediated immunity (AMI) is the type of immunity that is mediated by soluble host proteins called antibodies or immunoglobulins. Because it is largely due to the presence of circulating antibody molecules in the serum, is also called circulating immunity or humoral immunity. Antibodies (Ab) are proteins (globulins) produced in response to an encounter with an antigen. There are several classes or types of antibodies (and subclasses of the types), but all of the classes of antibodies that are produced in response to a specific antigen react stereochemically with that antigen and not with other (different) antigens. The host has the genetic capacity

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to produce specific antibodies to thousands of different antigens, but does not do so until there is an appropriate (specific) antigenic stimulus. Due to clonal selection, the host produces only the homologous antibodies that will react with that antigen. These antibodies are found in the blood (plasma) and lymph and in many extravascular tissues. They have a various roles in host defense against microbial and viral pathogens as discussed below.

Cell-mediated immunity (CMI) is the type of immunity that is mediated by specific subpopulations of T-lymphocytes called effector T cells. In non immune animals precursor T cells (pT cells) exist as «resting T cells». They bear receptors for specific antigens. Stimulation with Ag results in their activation. The cells enlarge, enter into a mitotic cycle, reproduce and develop into effector T cells whose activities are responsible for this type of immunity. They also develop into clones of identical reactive T cells called memory T cells.

The biological activities of the antibody-mediated and cell-mediated immune responses are different and vary from one type of infection to another. The AMI response involves interaction of B lymphocytes with antigen and their differentiation into antibody-secreting plasma cells. The secreted antibody binds to the antigen and in some way leads to its neutralization or elimination from the body. The CMI response involves several subpopulations of T lymphocytes that recognize antigens on the surfaces of cells. TH cells respond to antigen with the production of lymphokines. The distinction between TH1 and TH2 is based on their lymphokine profiles. TH2 cells have previously been referred to as T helper cells because they provide lymphokines (e. g. IL-2 and IL-4) which activate T cells and B cells at the start of the immune response. TH1 cells were formerly known as delayed type hypersensitivity cells (TDTH) because of their role in this allergic process. TC cells or cytotoxic T lymphocytes (CTLs) are able to kill cells that are showing a new or foreign antigen on their surface (as virus- infected cells, or tumor cells, or transplanted tissue cells).

Membrane receptors on B cells and T cells

The nature of the membrane receptors for antigen on B cells and T cells is fairly well understood. Each B cell has approximately 10^s membrane-bound antibody molecules (IgD or IgM) which correspond in specificity to the antibody that the cel l is programmed to produce. Each T cell has about 10⁵ molecules of a specific antigen-binding T cell receptor (TCR) exposed on its surface. The TCR is similar, but not identical, to antibody. In addition, T cell subsets bear some distinguishing surface markers, notably CD4 or CD8. T cells bearing CD4 always recognize antigens in association with class II major histocompatability complex (class II MHC) proteins on the surfaces of other cells. CD4⁺ T lymphocytes generally function as T helper cells. T cells bearing CD8 (CD8⁺) always recognize antigen in association with class I MHC proteins and typically function as cytotoxic T cells. The important markers, actions and interactions of T cells, B cells and Antigen Presenting Cells (APC) are illustrated below (Fig. 18).

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Figure 18. Receptor interactions between B cells, T cells and antigen presenting cells (APC)

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