Essential Medical Microbiology and Immunology PDF

Here is the transcription of the image into a structured markdown format. # ESSENTIAL MEDICAL MICROBIOLOGY AND IMMUNOLOGY VOLUME I ## PREFACE This book is intended to be a comprehensive and up to date guide to medical microbiology and immunology in the most reliable, attractive, illustrated mann...

Here is the transcription of the image into a structured markdown format. # ESSENTIAL MEDICAL MICROBIOLOGY AND IMMUNOLOGY VOLUME I ## PREFACE This book is intended to be a comprehensive and up to date guide to medical microbiology and immunology in the most reliable, attractive, illustrated manner for undergraduate medical students, as well as a guide for postgraduates preparing for higher degrees. It is designed to cover the four aspects of medical microbiology and immunology (Bacteriology, Virology, Mycology and Immunology) in four separate volumes in addition to a book encompassing laboratory diagnostic methods of infectious diseases, each navigates the reader to this ever-expanding science. - Vol. I: General Microbiology (Bacteriology, Virology and Mycology) - Vol. II: Systematic Bacteriology - Vol. III: Systematic Virology, Systematic Mycology and some topics of clinical relevance - Vol. IV: Laboratory diagnostic methods of infectious diseases - Vol. V: Immunology In preparing this text, the primary objective of our panel was to supply the reader with a concise updated source reflecting the tremendous progress in our knowledge in the fascinating field of microbiology and immunology; it is our sincere hope that it can fulfill this goal. The Authors ## CONTENTS | | Page | | :-------------------- | :--- | | Chapter 1: Introduction to Microorganisms | 1 | | Chapter 2: Bacterial Structure | 2 | | Chapter 3: Bacterial Growth | 9 | | Chapter 4: Bacterial Viruses (Bacteriophages) | 11 | | Chapter 5: Bacterial Genetics | 15 | | Chapter 6: Antibacterial Agents (Antibiotics) | 19 | | Chapter 7: Bacterial Pathogenesis | 26 | | Chapter 8: General Virology | 28 | | Chapter 9: General Mycology | 33 | ### Chapter 1: Introduction to Microorganisms 1 ## INTRODUCTION TO MICROORGANISMS ILOS: By the end of this chapter the student should be able to: - Recall system of microbial classification - Identify the terms eukaryotes versus prokaryotes Microorganisms are, as the name implies, microscopic organisms (seen only through a microscope). They include the following major types: algae, protozoa, fungi, bacteria, archaea, viruses and prions. Algae, protozoa and fungi are eukaryotic (eu=true; karyote=nucleus) micro-organisms, i.e., they are made from larger, more complex cells similar to plant and animal cells. Their DNA is enclosed within a nuclear membrane, forming the nucleus. Bacteria and archaea are prokaryotic microorganisms (pro=before), meaning they are single-celled organisms without a membrane-bound nucleus. Their DNA, instead of being contained in the nucleus, exists as a long, folded thread suspended in a portion of cytoplasm called nucleoid. They are also devoid of mitochondria and other membrane-bound organelles. Viruses are the smallest of the infective agents. They are obligate intracellular parasites; that is, they lack metabolic machinery of their own and depend on host cells to carry out their vital functions. Viruses are made of nucleic acid (DNA or RNA) surrounded by a protein coat. Prions are the simplest infectious agents. They are described as infectious proteins devoid of nucleic acid. They have been implicated as the cause of various diseases. ### Chapter 2: Bacterial Structure 2 ## BACTERIAL STRUCTURE ILOS: By the end of this chapter the student should be able to: - Recall different shapes and arrangements of bacterial cells - Differentiate between the two main categories of bacteria based on Gram stain - Describe intracytoplasmic structures of bacterial cells and outline their function - Describe bacterial cell membrane and its function - Recognize cell wall structure and function - Define cell wall deficient bacteria - Discuss structures outside cell wall with their functions - Discuss bacterial spores and their medical importance Bacteria are found in air, water and soil. They are also found in or on the human body, animals and plants. ### Bacterial Morphology Bacteria are differentiated into major categories, based on their morphological features such as shape, size, arrangement, staining characteristics and motility. ### Bacterial Size Bacteria are measured in µm. ### Bacterial Shape and Arrangement - Cocci (singular: coccus): are spherical organisms arranged in pairs, clusters or chains. - Bacilli (singular: bacillus=stick): are rod-shaped organisms that may occur singly, in pairs or in chains. - Spiral bacteria: are coiled organisms, e.g. spirochaetes that are flexible. ### Staining Characteristics There are two kinds of stains: simple and differential: - Simple stains employ a single dye like methylene blue. Cells and structures stained with them give the same colour. Therefore, they only reveal the characteristics of size, shape and arrangement. - Differential stains require more than one dye and distinguish between different types of bacteria by giving them different colours. Gram stain is the most important differential stain in clinical microbiology. It divides bacteria into Gram-positive (violet-staining) and Gram-negative (red staining). ### Chapter 2: Bacterial Structure 3 ### Motility Motility refers to the ability of bacteria to move independently. Some bacteria are non-motile, whereas others are motile. Different modes of motility, e.g. darting, corkscrew or swarming motility, may help in the identification of an organism. ### Bacterial Ultra-Structures and their Functions All bacteria have a nucleoid, ribosomes and a cytoplasmic membrane. Most bacteria also have a cell wall, and some are further enveloped by a capsule or slime layer. Some types of bacteria also have cytoplasmic inclusions and various appendages as flagella and pili. The final details of subcellular structures are best revealed by electron microscopy (Fig. 1). The image is a schematic presentation of a bacterial cell. Shown are the pilus, the capsule, the cell wall, and the cytoplasmic membrane. Inside the cell are the nucleoid (DNA), cytoplasm and ribosomes, and flagellum is seen coming out of the cell. ### Cytoplasm Few morphologically distinct components can be found within the cytoplasm: Nucleoid: Genetic information of a bacterial cell is contained in a single circular molecule of double-stranded DNA, which constitutes the bacterial chromosome. Plasmids: In many bacteria, additional genetic information is contained on plasmids which are small circular extrachromosomal DNA molecules that can replicate independently of the chromosome. Ribosomes: They are the site of protein synthesis in the cell. Ribosomes consist of protein and RNA. Prokaryotic ribosomes have a sedimentation constant of 70S, smaller than the 80S ribosomes of eukaryotes. This difference makes bacterial ribosomes a selective target for antibiotic action. Inclusion granules: These are granules of nutrient materials, e.g. carbohydrates and lipids. ### Chapter 2: Bacterial Structure 4 ### Cytoplasmic Membrane The cytoplasm is limited externally by a thin elastic cytoplasmic membrane. It is a phospholipid protein bilayer similar to that of eukaryotic cells except that, in bacteria, it lacks sterols. It has the following functions: 1. Selective transport: In bacteria, molecules move across the cytoplasmic membrane by simple diffusion, facilitated diffusion and active transport. 2. Secretion of extracellular enzymes: a. Hydrolytic enzymes: which digest large food molecules into subunits small enough to penetrate the cytoplasmic membrane. b. Enzymes used to destroy harmful chemicals, such as antibiotics, e.g. penicillin-degrading enzymes. 3. Respiration: The respiratory enzymes are located in the cytoplasmic membrane, which is thus a functional analogue of the mitochondria in eukaryotes. 4. Cell wall biosynthesis: The cytoplasmic membrane is the site of the enzymes of cell wall biosynthesis. 5. Reproduction: A specific protein in the membrane attaches to the DNA and separates the duplicated chromosomes from each other. A septum forms by the cytoplasmic membrane to separate the cytoplasm of the two daughter cells. ### Cell Wall The bacterial cell wall is the structure that surrounds the cytoplasmic membrane. It is strong and relatively rigid, though having some elasticity. ### Structure of the cell wall The cell wall of bacteria is a complex structure. Its impressive strength is primarily due to peptidoglycan, which is a complex polymer of carbohydrates and amino acids. Besides peptidoglycan, additional components in the cell wall divide bacteria into Gram-positive and Gram-negative (Fig. 2). ### Gram-positive cell wall is composed of: Peptidoglycan: There are many sheets of peptidoglycan, comprising up to 50% of the cell wall material. Despite the thickness of peptidoglycan, chemicals can readily pass through. Teichoic acids: They are fibres that protrude outside the peptidoglycan in most Gram-positive bacteria. Teichoic acids and cell wall associated proteins are the major surface antigens of the Gram-positive bacteria. ### Chapter 2: Bacterial Structure 5 ### Gram-negative cell wall is composed of: Peptidoglycan: It is much thinner, composed of only one or two sheets comprising 5-10% of the cell wall material. Outer membrane: It is a phospholipid protein bilayer present outside the peptidoglycan. Its outer surface carries molecules of lipopolysaccharide (LPS) which consists of: - Lipid A, which forms the endotoxin of Gram-negative bacteria - Polysaccharides, which are the major surface antigens of the Gram-negative bacterial cell (somatic or O antigen). The image is a schematic presentation of Gram-positive and Gram-negative cell walls. In the image, The Gram-positive cell wall has Teichoic acid attached to peptidoglycan layer. A similar structure is present for the Gram-negative cell wall with the following components present: LPS, Outer membrane, Peptidoglycan, cytoplasmic(inner)membrane and proteins ### Functions of the cell wall 1. It maintains the characteristic shape of the bacterium. 2. It supports the weak cytoplasmic membrane against the high internal osmotic pressure of the protoplasm. 3. It plays an important role in cell division. 4. It is responsible for the staining affinity of the organism. ### Wall deficient variants a- Mycoplasma: It is the only group of bacteria that exists naturally without a cell wall. Mycoplasmas do not assume a defined recognizable shape, because they lack a rigid cell wall. These organisms are naturally resistant to cell wall inhibitors, such as penicillins and cephalosporins. b- L-Forms: They are wall-defective or wall-deficient bacteria. - "L" stands for Lister Institute in London, where they were first discovered. - L-forms may develop from cells that normally possess a cell wall, when they are exposed to hydrolysis by lysozyme or by blocking peptidoglycan biosynthesis with antibiotics, such as penicillin, provided that they are present in an isotonic medium. ### Chapter 2: Bacterial Structure 6 - Some L-forms resynthesize their walls once the inducing stimulus is removed, resulting in relapses. Others, however, permanently lose the capacity to produce a cell wall. - L-forms may survive therapy with cell wall inhibitors. ### Glycocalyx Many bacteria secrete extracellular polymers outside of their cell walls called glycocalyx. These polymers are usually composed of polysaccharides and sometimes protein. Glycocalyx forms an additional layer that may come in one of two forms: #### 1. Slime Layer: - This is a thin glycocalyx layer that is loosely bound to the cell wall. It is involved in attachment of bacteria to other cells or inanimate surfaces to form biofilms. A biofilm is defined as an aggregate of microorganisms adhering to each other on a surface and embedded within a matrix of extracellular polysaccharide (Fig. 3). - Importance of biofilms: - Biofilms protect bacteria from host defences (e.g., antibodies) and resist penetration of antibiotics and detergents. - Biofilms facilitate the exchange of antibiotic resistance genes among bacteria. The image shows a schematic presentation of a bacterial biofilm. Image displays attachment, growth and detachment in different states of the cycle #### 2. Capsule: - This is a thick glycocalyx layer that is firmly attached to the cell wall. - Importance of capsules: - Capsules protect bacteria from phagocytic cells as well as other antibacterial agents (e.g., bacteriophages). - Capsules also provide bacterial adhesion to target surfaces in order to establish infection. - Because capsules tend to repel stains, they can be demonstrated using a negative staining technique, in which the bacterial cells and the background are stained, leaving the capsule as a clear halo around the bacterial cell (Fig. 4). ### Chapter 2: Bacterial Structure 7 The image shows a negative staining of capsules. Capsules, bacteria and the background is shown in the illustration ### Appendages Several structures project through the cell wall of bacteria to form surface appendages. The most important are flagella and pili. ### A- Flagella Many bacteria move by means of flagella. - Flagella are hair-like appendages, too small to be detected by light microscope. They can be demonstrated clearly with the electron microscope. - The location and number of flagella on a cell vary according to bacterial species. Organisms may be monotrichous (single polar flagellum), lophotrichous (multiple polar flagella) or peritrichous (flagella distributed over the entire cell surface) (Fig. 5). - Flagella consist of a protein called flagellin which differs in different bacterial species. Flagellins are highly antigenic; they constitute the H antigens. Motile bacteria tend to migrate towards regions where there is a higher concentration of nutrients and away from harmful substances. Image is show different distributions of flagella. - Monotrichous has a single flagellum on one end, - Lophotrichous has multiple flagella on the end - Peritrichous has flagella distributed over the outside of the cell ### B- Pili or fimbriae Pili (singular: pilus) are protein tubes that extend from the cells. They are shorter and thinner than flagella. They are composed of structural protein subunits termed pilins. ### Functions: 1. Adherence: It is the function of the short pili (fimbriae) that occur in great numbers around the cell. They enable bacteria to attach to the surfaces, thus contributing to the establishment of infection. ### Chapter 2: Bacterial Structure 8 2. Conjugation: A special long pilus called the sex pilus is involved in the transfer of DNA between bacteria, a process known as conjugation (Chapter 6). ## Bacterial Spores (Endospores) Some bacteria develop a highly resistant resting phase called endospore, that does not grow or reproduce, and exhibits absolute dormancy. This process is called sporulation. ### Sporulation Sporulation is triggered by the onset of unfavourable environmental conditions e.g. depletion of nutrients, accumulation of metabolites or changes in the growth requirements (e.g. moisture, temperature, pH or oxygen tension). The cytoplasmic membrane invaginates, enclosing a section of the cytoplasm that contains the bacterial chromosome, some ribosomes and other cytoplasmic materials that will be needed for germination. It acquires a thick cortex and a thin but tough outer spore coat. ### Viability and resistance Spores are much more resistant to disinfectants, drying and heating. Moist heat at 121°C for 10-20 minutes is needed to kill spores while 60°C suffices to kill vegetative forms. ### Germination Endospores respond quickly to favourable environmental conditions, returning to the vegetative state within 15 minutes. The spores absorb water and swell, the protective coat disintegrates, and a single vegetative cell emerges. ### Morphology 1. Staining:** Using Gram stain, the spore remains uncoloured and can be seen as a clear area within the stained cell. The spores can be stained using special procedures. 2. Position:** Spores may be central, terminal or subterminal (Fig. 6). 3. Shape:** Spores may be oval or rounded. 4. Size:** Spores may be large (bulging) or small (non-bulging). The position and shape of spores are characteristic of the species and may help in microscopic identification of the bacterium. The image shows the position of spores; central, terminal and subterminal ### Chapter 3: Bacterial Growth 9 ## BACTERIAL GROWTH ILOS: By the end of this chapter the student should be able to: - Describe the process of binary fission - Define generation time - Recall environmental factors affecting bacterial growth ## Bacterial Reproduction Bacteria reproduce asexually by binary fission; this is the process by which a single cell divides to form two genetically identical daughter cells. ### Steps of binary fission 1. The cell grows in size, usually elongating. 2. The two strands of the bacterial chromosome separate, and each strand acts as a template for the formation of a new complementary strand. This results in the formation of two copies of double-stranded DNA molecules, each including one "old" and one "new" strand. 3. The two copies become attached to the two opposite ends of the cytoplasmic membrane. 4. The protoplasm becomes divided into two equal parts by the growth of a transverse septum from the cytoplasmic membrane and cell wall, giving rise to two identical daughter cells. ### Generation time (doubling time) It is the time required for a population of bacteria to double in number. It may be as short as 13 minutes and may reach 24 hours. ### Environmental factors affecting bacterial growth 1. **Nutrients:** According to the means by which a particular organism obtains energy and nutritional requirement, bacteria are classified into: a. **Autotrophs:** They can synthesize complex organic substances from simple inorganic materials, e.g. $CO_2$ and ammonium salts. Autotrophs are of no or little medical importance. b. **Heterotrophs:** These bacteria, on the other hand, require organic sources for carbon, as they cannot synthesize complex organic substances from simple inorganic sources. Most bacteria of medical importance are heterotrophic. ### Chapter 3: Bacterial Growth 10 2. **Oxygen:** According to the respiratory pattern and $O_2$ requirements, bacteria are classified into: a. **Strict or obligate aerobes:** require oxygen for growth. b. **Strict or obligate anaerobes:** require complete absence of oxygen. In the presence of oxygen, highly toxic molecules (superoxide and hydrogen peroxide) are formed. Unlike aerobes, obligate anaerobes lack superoxide dismutase and catalase which break down these toxic products. c. **Facultative anaerobes:** generally grow better in presence of oxygen but still are able to grow in its absence. d. **Micro-aerophilic:** organisms require reduced oxygen level. e. **Aerotolerant anaerobes:** have an anaerobic pattern of metabolism but can tolerate the presence of oxygen because they possess superoxide dismutase. 3. **Carbon dioxide ($CO_2$):** The minute amount of $CO_2$ present in air is sufficient for most bacteria. However, certain species require higher concentrations (5-10%) of $CO_2$ for growth. 4. **Temperature:** Most organisms grow within a temperature range of 20-40°C. Organisms which replicate on or in human body are able to grow within this range, with an optimum temperature of 37°C, which is the normal body temperature. However, some organisms are capable of growing at refrigeration temperature, while others grow best at high temperatures. 5. **Hydrogen ion concentration (pH):** Most microorganisms of clinical significance grow best in media whose pH is close to that of the human body (pH 7.2). However, some microorganisms grow better at an alkaline (8-9), or an acidic (4 or less) pH. ### Chapter 4: Bacterial Viruses (Bacteriophages) 11 ## BACTERIAL VIRUSES (BACTERIOPHAGES) ILOS: By the end of this chapter the student should be able to: - Recall structure of bacteriophage - Contrast lytic and lysogenic replication cycles of bacteriophage - Define "lysogenic conversion" - Compare between generalized and specialized transduction - State the importance of phage-typing Bacteriophages (or phages) are viruses that infect bacteria i.e. the bacterial cell serves as a host for the virus. ### Morphology of the Bacteriophage: (Fig. 7) In most cases, the bacteriophage consists of: 1. A head.** containing the nucleic acid core (usually DNA, rarely RNA) surrounded by a protein coat (capsid) 2. **A tail** consisting of a hollow core surrounded by a contractile sheath which ends in a base plate to which tail fibres are attached. The illustration shows the structure of a bacteriophage. Labels are Capsid/Head. Phage DNA, Core Contractile sheath, Tail fibers and Base plate ### Chapter 4: Bacterial Viruses (Bacteriophages) 12 ### Replication of Bacteriophages Two cycles for phage replication are known (Fig. 8): #### A. Lytic (vegetative) cycle: It is so-called because it ends in lysis of the bacterial host cell and release of the newly formed phages. The stages of this cycle are: 1. **Adsorption:** The phage attaches by its tail to specific receptors on the bacterial cell. The specificity of this process determines the susceptibility of bacteria to different phages. 2. **Penetration:** The tail sheath contracts, and the nucleic acid is injected into the cell. The empty head and the tail are left outside the cell. 3. **Eclipse phase:** During this phase no phage components are detected inside the cell. It takes a short time (minutes to hours) during which viral nucleic acid directs the host cell metabolism to synthesize the enzymes and proteins required for phage synthesis. 4. **Replication:** Hundreds of phage components including nucleic acids, capsids and tails are synthesized. 5. **Assembly:** The phage components combine to form complete phage particles which mature into typical infectious phages. 6. **Release:** The bacterial cell bursts, liberating many phage particles to infect new cells. The image diagrams the lytic and lysogenic cycle of bacteriophage. Shown are Phage DNA, bacterial chromosome, lytic and lysogenic cycle and Prophage ### Chapter 4: Bacterial Viruses (Bacteriophages) 13 #### B. Temperate (lysogenic) cycle In this cycle, the phage (called temperate phage) does not replicate and lyse the bacteria. Instead, the phage DNA becomes integrated within the bacterial chromosome and divides with it to pass into daughter cells. The integrated phage genome is called prophage and the bacteria carrying it are called lysogenic bacteria. Lysogenic bacteria are characterized by the following: 1. They are immune to infection by another phage. 2. They acquire new properties, e.g. toxin production or resistance to antibiotics. Acquisition of a new character coded for by a prophage DNA is called lysogenic conversion or phage conversion. When the phage is lost from the bacterium, this new characteristic is lost. ### Outcome of the temperate cycle 1. The prophage may be carried inside the bacterial cell indefinitely passing to daughter cells. 2. The prophage may be induced to detach from the bacterial chromosome and start a lytic cycle. Induction may be spontaneous or achieved by an inducer, e.g. U.V. light. ### Transduction Transduction is the transfer of DNA from one bacterial cell to another by means of bacteriophage. There are 2 types of transduction (Table 1 and Fig. 9): #### a. Generalized transduction: During the lytic phage cycle, the bacterial DNA is fragmented, and any fragment of DNA (whether chromosomal or plasmid) may be accidentally incorporated into the phage head in place of phage DNA. The phage can then transfer the incorporated bacterial DNA into another bacterial host. #### b. Specialized transduction: It takes place when a prophage contained in a lysogenized bacterial cell is induced to detach from the bacterial chromosome to start a lytic cycle. Such prophage may carry with it an adjacent piece of chromosomal DNA - in addition to phage DNA - and transfer it to another bacterial cell. ### Chapter 4: Bacterial Viruses (Bacteriophages) 14 | | | | | :-------------------- | :------------- | :--------------- | | | GeneralizedTransduction | Specialized Transduction | | Type of phage | Lytic (virulent) phage | Temperate (lysogenic) phage | | Replication cycle | Lytic cycle | Lysogenic cycle | | The transferred DNA fragments | Any piece of bacterial DNA (chromosomal or plasmid) | A specific piece of chromosomal DNA adjacent to the site of insertion of the prophage + phage DNA | The illustration diagrams generalized and specialized Transduction. **Generalized Transduction image:** - Shows infectious phage. Next to it bacterial Chromosome. Phage DNA is floating next to it. Phage replication and fragmentation is occurring and shows fragmented DNA( a+ any baterial gene) - In the image phage DNA is floating next to the phage and is in Lysis **Specialized Transduction image:** - Shows infectious phage. Next to it bacterial Chromosome with integrated prophage. UV induction is occuring. - An image display rare abnormal ecision of prophage - Last part of drawing shows Lysis with phage dna and "st" special bacteria gene. ### Chapter 5: Bacterial Genetics 15 ## BACTERIAL GENETICS ILOS: By the end of this chapter the student should be able to: - List components of the bacterial genome - Describe the bacterial chromosome - Describe plasmid structure and function - Define transposable genetic elements - Compare between phenotypic and genotypic variation - Define mutation and list its types - Describe methods of gene transfer The bacterial genome is the total set of genes present inside the bacterial cell. It comprises the bacterial chromosome that carries genes necessary for bacterial growth. Additional genes may be carried on plasmids, bacteriophage DNA (prophage) and transposable genetic elements. 1. **Bacterial Chromosome** - The bacterial chromosome is a single, circular, supercoiled, double-stranded DNA molecule. - Being a prokaryote, the bacterial cell lacks a nuclear membrane; instead, the DNA is concentrated in a region in the cytoplasm called nucleoid. - The bacterial chromosome has the general structure of any DNA molecule. - It follows the same rules of gene expression and protein synthesis (i.e. transcription and translation) as higher organisms. - It replicates as previously described (see chapter 3). 2. **Plasmids** - Plasmids are extra-chromosomal, circular, double-stranded DNA molecules dispersed in the cytoplasm. - They are much smaller than the bacterial chromosome, as they carry a smaller number of genes. These genes encode properties that, although beneficial for the host cell, are not essential for its life; therefore, plasmids are considered dispensable. - Plasmids are capable of replicating independently of the bacterial chromosome. Thus, multiple copies of the same plasmid may exist in the same cell (Fig. 10). Image illustrates chromosomal DNA and plasmids ### Chapter 5: Bacterial Genetics 16 - According to their different functions, plasmids are classified into types (traits); the most important are: a. **Fertility (F) plasmids:** These are plasmids that carry fertility (F) factors coding for the formation of a sex pilus which mediates the process of gene transfer during conjugation. For this reason, such plasmids are also known as conjugative plasmids.** Non-conjugative plasmids lacking these F factors are incapable of initiating conjugation and can only be transferred with the help of a conjugative plasmid. b. **Resistance (R) plasmids:** These are plasmids that carry genes for resistance (R-factors) to one or several antimicrobial drugs. R plasmids are usually conjugative plasmids that can be transferred among bacteria by conjugation. This results in the rapid spread of drug-resistance among bacterial populations. c. **Virulence plasmids:** These are plasmids that may code for exotoxins, adhesins or invasion factors, rendering the organism pathogenic. - It is possible for plasmids of different types to coexist in a single cell. In addition, the same plasmid can belong to more than one of these functional groups; for example, F plasmids are major carriers of antibiotic resistance genes, especially among Gram-negative bacteria. 3. **Bacteriophage DNA** The DNA of the temperate bacteriophage that is integrated in the chromosome of a lysogenic bacterial cell (i.e. the prophage) is considered a part of the genome of such bacteria (see chapter 4). ## BACTERIAL VARIATION Bacterial variations are changes in bacterial characters. They may be phenotypic or genotypic. - **Phenotypic variation** It occurs in response to changes in environmental conditions without change in genetic constitution; examples include the formation of L-forms when bacteria are exposed to lysozyme and loss of flagella upon exposure to phenol. These changes are reversible (transient) and not heritable to daughter cells. - **Genotypic variation** It occurs as a result of a change in the underlying genetic constitution through mutation or gene transfer among bacterial cells. These changes are irreversible (permanent) and heritable to daughter cells. ### Chapter 5: Bacterial Genetics 17 ### Mutation - Mutation is a permanent, heritable change in the nucleotide base sequence of a DNA molecule. - It may occur spontaneously as a replication error or may be induced by radiation or chemical agents. Induced mutations may be used to manipulate viral genomes for vaccine production and gene therapy. ### Gene Transfer There are 3 methods for gene transfer among bacteria (Fig. 11): 1. Transformation - Transformation is the uptake of naked DNA from the environment by a bacterial cell. This occurs when dying bacteria release DNA, whether chromosomal or plasmid in origin, and this naked DNA is taken up by other bacteria, causing "transformation" of the recipient cell. - The transforming DNA may become integrated within the bacterial chromosome or may remain extra-chromosomally as a plasmid in the recipient cell. 2. Transduction - Transduction is the transfer of DNA from one cell to another by means of a bacteriophage. - There are two types of transduction, generalized and specialized (chapter 5). 3. Conjugation - Conjugation is the process by which one bacterium transfers genetic material to another through direct cell-to-cell contact. - It involves 2 cell types: donors (F+) which possess the fertility (F) factor, and recipients (F) which lack the F factor. The F factor carries the genes for the synthesis of the sex pilus which acts as a conjugation tube between the donor and recipient bacterial cells. - The 2 DNA strands of the F factor are then separated, and one strand is transferred from the donor to the recipient cell. Each strand forms a complementary strand; thus, the recipient cell acquires a copy of the F plasmid and becomes an F+ cell. - Conjugation is the most frequently observed mechanism of DNA transfer among bacteria. ### Chapter 5: Bacterial Genetics 18 An illustration depicting the methods of gene transfer. They include: - Transformation - Transduction - Congugation ### Chapter 6: Antibacterial agents (Antibiotics) 19 ## ANTIBACTERIAL AGENTS (ANTIBIOTICS) ILOS: By the end of this chapter the student should be able to: - Define the term antibiotic - Define the term selective toxicity - Describe different mechanisms of action of antibiotics - Define the term "MIC" - List methods of antimicrobial susceptibility testing - Define the term "empiric therapy" - List indications of empiric therapy - Recognize when to use combined therapy - Define the term "chemoprophylaxis" - List situations that require antimicrobial chemoprophylaxis - Recall possible complications of antimicrobial agents - Recognize origin of resistance to antimicrobial agents - List mechanisms of resistance to antimicrobial agents - Explain principles of choice of antimicrobial agents ### Antibacterial agents These are chemical substances that are able to kill bacterial cells or inhibit their growth. They include antibiotics, used specifically in medicine, in addition to antiseptic agents, antibacterial soaps, chemical disinfectants and others. Probiotics are live, non-pathogenic bacteria that may be effective in the treatment or prevention of certain diseases. They either exclude the pathogen from binding sites on the mucosa or enhance the immune response against the pathogen. ### Antibiotics - These are antibacterial substances that are produced by certain groups of microorganisms, e.g. *Streptomyces* and *Penicillium*. Although their original source was a microorganism, some antibiotics are currently made synthetically (synthetic antibiotics). - A bacteriostatic antibiotic is one that is capable of inhibiting bacterial multiplication, yet multiplication resumes upon its removal. - A bactericidal antibiotic is one that is capable of killing bacteria; multiplication cannot be resumed. ### Chapter 6: Antibacterial agents (Antibiotics) 20 - Selective toxicity is the ability of an antimicrobial agent to harm a pathogen without harming the host. The specific target of the drug may be found in the microbe but not in the human body (e.g. peptidoglycan), or the action of the drug may depend on inhibition of a biochemical event essential for the organism but not for the host (e.g. biosynthesis of folic acid). - Spectrum of activity is the range of microorganisms that are affected by a certain antibiotic. Accordingly, antibiotics may be: - broad spectrum, if they kill or inhibit the growth of a wide range of Gram-positive and Gram-negative bacteria, - narrow spectrum, if they are mainly effective against either Gram-positive or Gram-negative bacteria, - limited spectrum, if they are effective against a single organism or disease. ### Mechanisms of Action of Antibiotics Antibiotics may function through different mechanisms (Fig. 12): ### A. Inhibition of bacterial cell wall synthesis Agents acting by this mechanism are bactericidal with minimal toxicity. They include: 1. Drugs that inhibit the last steps of peptidoglycan biosynthesis, e.g. β-lactam antibiotics (penicillins and cephalosporins). This inhibition is initiated by binding of the drug to certain cell receptors known as penicillin-binding proteins (PBPs). 2. Drugs that inhibit early steps in peptidoglycan biosynthesis which occur inside the cytoplasmic membrane, e.g. glycopeptides (vancomycin). Therefore, vancomycin can be used in infections caused by β-lactam-resistant staphylococci. ### B. Interference with cell membrane function Some antibacterial agents, e.g. polymyxins, disrupt the cytoplasmic membrane function. These agents are microbicidal. They are highly toxic as they have a narrow margin of selective toxicity. ### C. Inhibition of bacterial protein synthesis Bacteria have 70S ribosomes (30S and 50S subunits) whereas mammalian cells have 80S ribosomes (40S and 60S subunits). This difference makes bacterial ribosomes a selective target for antibiotics. They may be: 1. Agents acting on the 30S ribosomal subunit, e.g. tetracycline and aminoglycosides (gentamicin, streptomycin). 2. Agents acting on the 50S ribosomal subunit, e.g. macrolides (erythromycin, azithromycin), clindamycin, chloramphenicol, streptogramins and linezolid. ### Chapter 6: Antibacterial agents (Antibiotics) 21 ### D. Inhibition of bacterial nucleic acid synthesis This may occur by: 1. Prevention of RNA synthesis through inhibiting RNA polymerase, e.g. rifampin 2. Prevention of DNA synthesis through blocking DNA gyrase, e.g. quinolones ### E. Inhibition of bacterial metabolic pathways Some antibiotics function as antimetabolites through competitive inhibition of bacterial metabolic enzymes. Examples are sulfonamides and trimethoprim, which block bacterial biosynthesis of folic acid required for nucleic acid synthesis. The image displays the mechanisms of action of antibiotics A. Inhibitors of Cell Wall Synthesis: Beta Lactams, Penicillins Cephalosporins, Vancomycin B. Inhibitors of cell membrane function: Polymyxins C. Inhibitors of protein synthesis: Macrolides, Clindamycin, Linezolid, Chloramphenicol, Streptogramins, 30S subunit, Tetracyclines Aminoglycosides D. Inhibitors of Nucleic Acid Synthesis: DNA Gyrase, Quinolones, RNA Polymerase, Rifampin E. Inhibitors of Metabolic Pathways: Sulfonamides, Trimethoprim ### Microbial Susceptibility to Antimicrobial Agents Microorganisms vary in their susceptibility to different chemotherapeutic agents. Ideally, the appropriate antibiotic to treat any particular infection should be determined in vitro before any antibiotic is given. - The in vivo activity** of an antimicrobial agent is not always the same as its in vitro susceptibility because it involves many host factors that are not tested in vitro. - The activity of an antimicrobial agent against an organism depends on its concentration. The lowest concentration of a drug that prevents growth of a test organism is known as the minimal inhibitory concentration (MIC). - In vitro** susceptibility testing is commonly done by one of the following methods: - Disc diffusion method - Dilution method such as tube broth dilution ### Chapter 6: Antibacterial agents (Antibiotics) 22 ### Empiric Antibiotic Therapy Empiric antibiotic therapy is a "best guess" directed against the organism expected to be the most likely cause of an infectious disease based on experience. "Best guess" treatment is not always necessarily successful as many bacteria have unpredictable susceptibilities to antimicrobial agents. ### Indications 1. In seriously ill patients, but after collecting specimens for culture 2. In closed lesions, where there is no available sample ### Combined Antibiotic Therapy The ideal rule in antibiotic therapy is mono-therapy, which means choosing one drug effective against a particular organism. However, there are conditions