Theoretical Book on Microorganisms PDF

CHAPTER 1 INTRODUCTION TO MICROORGANISMS Microorganisms are generally unicellular i.e. the whole organism is one cell. In such cases, a single microbial cell performs all the functions required to maintain itself and propagate. Microorganisms may be classified in th...

CHAPTER 1 INTRODUCTION TO MICROORGANISMS Microorganisms are generally unicellular i.e. the whole organism is one cell. In such cases, a single microbial cell performs all the functions required to maintain itself and propagate. Microorganisms may be classified in the following large biological groups: 1. Algae. 2. Protozoa. 3. Slime moulds. 4. Fungi. 5. Bacteria. 6. Archaebacteria. 7. Viruses. The algae (excluding the blue-green algae), the protozoa, slime moulds and fungi include the larger and more highly developed microorganisms; their cells have the same general type of structure and organization, described as eukaryotes, as that found in higher plants and animals. The bacteria, including organisms of the mycoplasma, rickettsia and chlamydia groups, together with the related blue-green algae, comprise the smaller microorganisms with a simpler form of cellular organization described as prokaryotes. The most outstanding character of eukaryotic cells (eu=true; karyote=nucleus) is a distinct nucleus, surrounded by a membrane that separates it from the other contents of the cell. Prokaryotic cells (pro=before), on the other hand, do not contain a membrane-bound nucleus. Instead, their hereditary material is suspended in a portion of cytoplasm called nucleoid or nuclear region. They are also devoid of mitochondria and other membrane bound organelles. The viruses are the smallest of the infective agents; they have no cell structure. Viruses are obligate intracellular parasites; they require the biological machinery of a host cell for reproduction and survival.  CHAPTER 2 BACTERIA: THEIR STRUCTURE AND ORGANIZATION BACTERIAL MORPHOLOGY Bacteria are differentiated into major categories, based on their morphological features such as shape, size, arrangement and staining characteristics. Bacterial Size: Most bacteria range in size from 0.2-1.2 µm in width and 0.4-14 µm in length. Bacterial Shape and Arrangement: (A) Cocci (singular: coccus); are spherical organisms with a wide variety of arrangements, e.g.: 1. Diplococci: pairs of cells, e.g. Neisseria. 2. Irregular grape-like clusters, e.g. staphylococci. 3. Chains of four or more, e.g. streptococci. (B) Bacilli (singular: bacillus=stick); are rod-shaped organisms. These cells may occur singly, in pairs or in chains. Some bacilli are short (coccobacilli), others are curved (vibrios). (C) Spiral bacteria; are divided into two groups; spirilla which are rigid and spirochaetes that are flexible. Staining Characteristics: There are two kinds of stains; simple and differential. Simple stains employ a single dye like methylene blue, crystal violet or fuchsin. 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’s stain is the most important differential stain in clinical microbiology. It divides bacteria into Gram-positive (violet-staining) and Gram-negative (red staining). The second important stain is Ziehl-Neelsen stain. It is used to identify the acid-fast bacilli of the genus Mycobacterium.  BACTERIAL ULTRA-STRUCTURES AND 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 have also cytoplasmic inclusions and various appendages as flagella and pili. The final details of subcellular structures are best revealed by electron microscopy (Fig. 1). Fig. (1): Schematic representation of a Gram-positive and a Gram-negative bacterium. 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. It is 1 mm long and is packed into a supercoiled state inside the cell. Plasmids: In many bacteria, additional genetic information is contained on plasmids which are small circular ds-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, usually phosphates, sulphur, carbohydrates and lipids. Energy reserves are usually stored as glycogen, starch or poly-β-hydroxybutyrate. Phosphate is stored in metachromatic or volutin granules, which are used for synthesis of ATP. Mesosomes: These are convoluted membranous bodies that develop by complex invagination of the cytoplasmic membrane into the cytoplasm. Mesosomes are involved in mechanisms of cell division and sporulation. Cytoplasmic Membrane: It is a phospholipid protein bilayer similar to that of eukaryotic cells except that, in bacteria, it lacks sterols. Functions: 1. Selective transport: In bacteria, molecules move across the cytoplasmic membrane by simple diffusion, facilitated diffusion and active transport. 2. Excretion 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. Reproduction: Specific protein in the membrane attaches to the DNA and separates the duplicated chromosomes from each other. In addition, a septum is formed by the cytoplasmic membrane to separate the cytoplasm of the two daughter cells. 5. Chemotactic system: Attractants and repellants bind to specific receptors in the cytoplasmic membrane and send signals to the cell’s interior. The cell then responds to the surface message. Cell Wall: The bacterial cell wall is the structure that immediately surrounds the cytoplasmic membrane. It is 10-25 nm thick, 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 (synonym: murein or mucopeptide). Besides peptidoglycan, additional components in the cell wall divide bacteria into Gram-positive and Gram-negative (Fig. 1). Gram-positive cell wall is composed of: Peptidoglycan: comprising up to 50% of the cell wall material. Teichoic acids: They are the polymer of ribitol or glycerol phosphate. They are found in the cell wall and cytoplasmic membrane of most Gram-positive bacteria. Teichoic acids and cell wall associated proteins are the major surface antigens of the Gram-positive bacteria. Gram-negative cell wall is composed of: Peptidoglycan: It is much thinner, comprising 5-10% of the cell wall material. Outer membrane: It is phospholipid protein bilayer present external to the peptidoglycan layer. The outer surface of the lipid bilayer is composed of molecules of lipopolysaccharide (LPS) which consists of a complex lipid called lipid A chemically linked to polysaccharides. Lipid A of the LPS forms the endotoxin of the Gram-negative bacteria. Periplasmic space: It is the space between the inner and outer membranes. It contains the peptidoglycan layer and a gel-like solution of 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 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” forms may develop from cells that normally possess 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. Some L. forms resynthesize their walls once the inducing stimulus is removed. Others, however, permanently lose the capacity to produce a cell wall. L. forms may survive antibiotic therapy; their reversion to the walled state can produce relapses of the overt infection. Capsule and Related Structures: Capsule: It is a layer that adheres to the surface of the cell and forms a well- defined halo when differentially stained, This layer is formed only inside the host (in-vivo). Slime layer: It is a surface layer that is loosely distributed around the cell. Glycocalyx: It is a loose meshwork of polysaccharide fibrils extending outwards from the cell. Functions: 1. It protects the cell wall against various kinds of antibacterial agents, e.g. bacteriophages, colicins, complement and lysozymes. 2. It protects the bacterial cell from phagocytosis. Hence, the capsule is considered an important virulence factor. 3. Some bacteria attach to the target surface by using their capsules or glycocalyx in order to establish infection. For instance, Streptococcus mutans form glycocalyx, with which the bacteria stick to the tooth enamel. Appendages: A- Flagella: Many genera of bacteria move by means of flagella. They can be demonstrated clearly with the electron microscope. The location and number of flagella on a cell vary according to bacterial species. (Fig. 2). Flagella consist of a single type of protein called flagellin which differs in different bacterial species. The flagellins are highly antigenic (H antigen). Fig. (2): Different distribution of flagella. Axial filaments: These structures are composed of two groups of fibres that originate within the opposite ends of the cell and overlap in the middle. Spirochaetes move by means of these axial filaments. When the cell moves, it rotates around its longitudinal axis and flexes and bends along its length. B- Pili and fimbriae: Pili (singular: pilus) are shorter and thinner than flagella and can be observed only by the electron microscope. They are composed of structural protein subunits termed pilins. Functions: 1. Adherence: They enable bacteria to attach to the host surfaces, thus contributing to the establishment of infection i.e. virulence factor. 2. Conjugation between bacteria: A special long pilus called the sex pilus (F or fertile pilus) is involved in the transfer of DNA between bacteria, a process known as conjugation. Bacterial Spores (Endospores): Some bacteria, notably those of the genera Bacillus and Clostridium, develop a highly resistant resting phase or endospore that does not grow or reproduce, and exhibit absolute dormancy. Sporulation is triggered by 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). 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. The marked resistance of the spores has been attributed to: 1. Thermal resistance is provided by their high content of Ca2+ and dipicolinic acid (a compound unique to endospores). 2. The impermeability of their cortex and outer coat. 3. Their low content of water. 4. Their very low metabolic and enzymatic activity. Endospores can respond quickly to changes in the environment returning to the vegetative state within 15 min. The spores cannot be stained using Gram’s stain but can be stained by special procedures. The position and shape of spores (Fig. 3) may help in the microscopic identification of the bacterium. Fig. (3): The position of spores.  CHAPTER 3 BACTERIAL GROWTH AND PHYSIOLOGY Growth involves an increase in the size and number of organisms. Bacterial Reproduction: Bacterial multiplication takes place by simple binary fission: 1.The cell grows in size, usually elongates. 2.The bacterial chromosome acts as a template for the replication of another copy. 3.Each copy is attached to a mesosome on 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. Generation time (doubling time); is the time between two successive divisions. It may be as short as 13 min. in Vibrio cholerae and may reach 24 h. in Mycobacterium tuberculosis. Growth Requirements: 1. Nutrients: According to the means by which a particular organism obtains energy and raw material to sustain its growth, bacteria are classified into: a- Autotrophs: They can utilize simple inorganic materials, e.g. CO2 as a source of carbon and ammonium salts as a source of nitrogen. They can synthesize complex organic substances from the simple inorganic materials. The energy required for their metabolism is predominantly derived from light or simple chemical reactions. Autotrophs are of no or little medical importance. b- Heterotrophs: These bacteria, on the other hand, require organic sources for carbon, as they can not synthesize complex organic substances from simple inorganic sources. Most bacteria of medical importance are heterotrophic. 2. Oxygen requirements: According to O2 requirements, bacteria are classified into: a. Strict or obligate aerobes require oxygen for growth, e.g., Pseudomonas aeruginosa and Vibrio cholerae. b. Strict or obligate anaerobes require complete absence of oxygen, e.g., Bacteroides fragilis. c. Facultative anaerobes generally grow better in presence of oxygen but still are able to grow in its absence, e.g., Staphylococci, E.coli, …etc. d. Micro-aerophilic organisms require reduced oxygen level, e.g., Campylobacter and Helicobacter. e. Aerotolerant anaerobes have a fermentative (anaerobic) pattern of metabolism but can tolerate the presence of oxygen because they possess superoxide dismutase e.g., Clostridium perfringens. Aerobic and anaerobic bacteria differ in their oxidation-reduction reactions for energy production. Oxidation of a molecule is equivalent to removal of hydrogen or electrons.  In aerobic respiration, the final hydrogen or electron recipient in oxidation process is molecular oxygen (O2).This result in the formation of superoxide (O2-) radicals and hydrogen peroxide (H2O2) which are highly toxic. To cope with this, aerobic organisms have developed two enzymes, superoxide dismutase and catalase, which detoxify these molecules. In anaerobic conditions, i.e. in absence of O2, the final hydrogen or electron recipient is an organic molecule and the oxidation process is referred to as fermentation. Obligate anaerobes lack superoxide dismutase and catalase, and so they can not grow in the presence of O2. 3. CO2 requirements: The minute amount of CO2 present in air is sufficient for most bacteria. However, certain species require higher concentrations (5-10%) of CO2 for growth (capnophilic) e.g., Neisseria spp. and Brucella abortus. 4. Temperature: Mesophiles are organisms able to grow within a temperature range of 20-40°C (optimum temperature of 37°C). Human pathogens are mesophies. Psychrophiles (cold-loving) are capable of growth at refrigeration temperature (0-8°C) e.g., Flavobacterium spp. Thermophiles (heat-loving), grow best at high temperature (>60°C) e.g., Bacillus stearothermophilus. 5. Hydrogen ion concentration (pH): Most microorganisms of clinical significance grow best at (pH 7.2). However, some microorganisms grow better at an alkaline pH (8-9), such as V. cholerae. Others, such as lactobacilli, prefer media of acidic pH (4 or less).  GROWTH PHASES (BACTERIAL GROWTH CURVE) If a small number of an organism is placed in a suitable fluid nutrient medium under appropriate physical and chemical conditions, then the number of viable cells per millilitre are determined periodically, and plotted, a characteristic growth curve with four phases is obtained (Fig. 4): 1. Lag phase: The initial number of bacterial cells remains constant. During this period, the cells adapt to their new environment. Enzymes and intermediates are formed to permit growth. 2. Exponential (logarithmic) phase: There is marked increase in cell number and its rate is accelerated exponentially with time giving a characteristic linear plot on a logarithmic scale. In this phase, the organism shows typical morphology. 3. Stationary phase: Exhaustion of nutrients and accumulation of toxic products cause growth to decrease. There is slow loss of cells through death which is just balanced by formation of new cells through growth and division. The number of viable bacteria remains constant. 4. Decline phase: At the end of the stationary phase, the death rate increases and exceeds the multiplication rate due to nutrient exhaustion and accumulation of toxic metabolic end products. So, the number of viable bacteria decreases. Fig. (4): Phases of bacterial growth curve.  CHAPTER 4 BACTERIAL VIRUSES (BACTERIOPHAGES) Bacteriophages (or phages) are viruses that parasitize bacteria. Morphology of the Bacteriophage: (Fig. 5) 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; consists of a hollow core surrounded by a contractile sheath which ends in a base plate to which are attached tail fibres. Fig. (5): Structure of a bacteriophage. Replication (Propagation) of Bacteriophages: Two cycles for phage replication are known: A- Lytic (vegetative) cycle: (Fig. 6) It is so-called because it ends in lysis of the bacterial host cell and release of the new formed phages. The stages of this cycle are: 1. Adsorption: The phage attaches, by its tail, to specific receptors on the bacterial cell. 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; in which 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. Intracellular synthesis; of phage nucleic acids, capsids and tails. 5. Assembly: The phage components aggregate to form complete phage particles. 6. Release: The bacterial cell bursts liberating a large number of phage particles to infect new cells. Fig. (6): The lytic cycle of bacteriophage. B- Temperate (lysogenic) cycle: In this cycle, the phage (temperate phage) does not replicate and lyse the bacteria but the phage DNA is integrated with the bacterial chromosome and divide with it to pass into daughter cells. The integrated phage genome is called “prophage” and the bacteria carrying it are called “lysogenic” bacteria. The presence of the prophage in the lysogenic bacterium renders it: 1. Immune to infection by another phage. 2. Acquire new properties, e.g. diphtheria bacilli can produce toxin only when lysogenized. 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: (Fig. 7) 1. The prophage is 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. 3. During the process of induction, the prophage may carry with it few genes of the bacterial chromosome. When it infects another bacterium, it passes this fragment to it giving it new characters. This is known as “specialized transduction” Fig. (7): Outcome of the temperate cycle Bacteriophage and Transduction Bacteriophages can mediate gene transfer by: 1- Generalized Transduction: During the lytic phage cycle, the bacterial DNA is fragmented and any fragment of DNA (whether chromosomal or plasmid) may be incorporated into the phage head instead of the phage DNA. The phage particle can then transfer the incorporated bacterial DNA into another bacterial host. Fig (7') Fig (7') generalized transduction  2- Specialized Transduction: During the process of detachment and induction of lytic cycle , the prophage may carry with it few genes of the bacterial chromosome. When it infects another bacterium, it passes this fragment to it giving it new characters. This is known as "specialized transduction". Fig (7") Fig (7") Specialized Transduction Practical Uses of Bacteriophages: 1. Phages are used as cloning vectors in recombinant DNA technology. They carry and introduce foreign DNA fragments into a host cell. 2. Phage typing: Since bacteria differ in their sensitivity to different phages, phages are used to identify and type strains of bacteria that are biochemically and antigenically indistinguishable. This phage typing is important in epidemiologic studies, e.g. to trace the source of infection in outbreaks of post-operative wound sepsis caused by Staphylococcus aureus. 3. Phages are used as research elements in some biological and genetic studies.  CHAPTER 5 BACTERIAL GENETICS Genetics is the science, which defines and analyzes heredity. The unit of heredity is the gene; a segment of DNA that carries information for a specific biochemical or physiologic property. The bacterial genome is the total set of genes present inside the bacterial cell. It comprises: 1. The bacterial chromosome; that can encode up to 4000 separate genes necessary for bacterial growth and propagation. Additional genes may be carried on: 2. Plasmids, 3. Transposable genetic elements, and 4. Bacteriophage DNA (prophage). 1. The Bacterial Chromosome: (Fig. 8) Being a prokaryote, the bacterial cell lacks a nuclear membrane; instead, the DNA is concentrated in the cytoplasm as a nucleoid. The nucleoid consists of a single chromosome, which is a circular, supercoiled, double- stranded DNA molecule, associated at one point with a mesosome. This attachment plays a role in the separation of the two sister chromosomes following chromosomal replication. Fig. (8): Structure of DNA. 2. Plasmids: Plasmids are extra-chromosomal, circular, double-stranded DNA molecules dispersed in the cytoplasm. They are much smaller than the bacterial chromosome (from several to 100 Kbp). Plasmids are capable of replicating independently of the bacterial chromosome. Functions (traits) exhibited by plasmids: 1. Sex pilus formation (F-factors): Some plasmids carry fertility (F) factors that code for the formation of a sex pilus which mediates the process of conjugation. 2. Antibiotic resistance (R-factors): Some plasmids carry genes for resistance to one or several antimicrobial drugs. They often control the formation of enzymes capable of destroying the antimicrobial drugs, e.g., β-lactamase enzyme which determine resistance to penicillin and cephalosporins which can be transferred among bacteria by conjugation. 3. Virulence plasmids; may code for exotoxins, adhesins or invasion factors. 4. Bacteriocin production: Bacteriocins are bactericidal substances produced by certain bacterial strains and are active against other strains of the same or closely related species, e.g., colicin produced by E.coli. 3. Transposable Genetic Elements: These are non-replicating DNA segments (units) that are capable of inserting themselves into other DNA molecules. They are also capable of mediating their own transfer from one location to another on the same chromosome or between chromosomes and plasmids. 4. 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 as a part of the genome of such bacteria.  CHAPTER 6 BACTERIAL VARIATION Bacterial variations are changes in the bacterial characters. They may be phenotypic or genotypic (Table 1). Table (1): Comparison between phenotypic and genotypic variations: Phenotypic variation Genotypic variation - It occurs in response to - It occurs as a result of a change in the changes in the underlying genetic constitution. environmental conditions without change in the genetic constitution. - Reversible (transient). - Irreversible (permanent). - Not-heritable. - Heritable. - Examples: - Examples; It occurs through: 1. L-forms of bacteria. 1. Mutation. 2. Loss of flagella upon 2. Gene transfer: exposure to phenol. a. Transformation. b. Transduction. c. Conjugation. Mutation: It results from a change in the nucleotide sequence of DNA that may occur spontaneously as a replication error (at a rate of once every 106-107 cells), or may be induced by radiation or chemical agents (at a higher rate of once every 103-104 cells). Mutation can be classified according to nucleotide substitution, insertion or deletion into: 1. Single-base (point) mutations; involve the replacement (substitution) of a single nucleotide in the coding sequence. This may result in: a. Same sense (silent) mutations; occur when the resulting base triplet (codon) codes for the same amino acid as the original triplet. b. Missense mutations; occur when the mutant base changes the coding sequence so that a different amino acid is produced. The resulting protein may be functioning or not, depending on the importance of the area affected by the mutation. 2. Frame-shift mutations; occur when a nucleotide is inserted to or deleted from the coding sequence, resulting in a shift of the reading frame, e.g. insertion of a transposable element. 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: 1. Transformation: (Fig. 9) Dying bacteria release DNA which can be taken up by other bacteria. Such DNA may be chromosomal or plasmid in origin, and may carry genes that “transform” the recipient bacterium. Fig. (9): Gene transfer by transformation. Transformation depends on competence; which is the ability of the recipient bacterial cell to take up DNA. Competence depends on the presence of proteins in the cell membrane that have a special affinity to bind DNA and transport it into the cytoplasm. Artificial competence can be induced during recombinant DNA techniques by treating the recipient bacteria with calcium chloride, which alters cell membrane permeability, enabling the uptake of DNA. 2. Transduction: It is the transfer of DNA from one cell to another by means of a bacteriophage. (page 13 &14) 3. Conjugation: (Fig. 10) It is the most frequently observed mechanism of DNA transfer. 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 F factor is transferred from the donor to the recipient cell. Then a complementary strand is formed thus, the recipient cell acquires a copy of the F plasmid and becomes an F+ cell. Fig. (10): Gene transfer by conjugation.  CHAPTER 7 GENETIC RECOMBINATION (GENE CLONING) The recent technology which is called genetic recombination, recombinant DNA technology, genetic engineering or gene cloning involves the in vitro modification and recombination of genetic material from different organisms to construct new gene combinations. Recombinant DNA Technique: (Fig. 11) A typical cloning experiment requires: 1. DNA of interest, sometimes called foreign, passenger, target or insert DNA. It is obtained by extracting the DNA from a given source and cleaving such DNA by a specific restriction endonuclease enzyme. 2. A cloning vector; usually derived from an extrachromosomal replicon such as plasmids, cosmids, bacteriophages, or animal viruses. 3. Restriction endonuclease enzymes to produce site-specific scissions in the DNA molecules. 4. DNA ligase to anneal the complementary ends of the vector and target DNA, thus producing a recombinant DNA molecule. 5. A prokaryotic or eukaryotic cell to serve as the biological host. I. Cloning Vectors: These are vehicles used to carry and introduce foreign DNA fragments into a host cell. At present, four types of cloning vectors are in common use: 1. Plasmids: Relaxed plasmids are among the best cloning vectors as they fulfil most of the requirements for ideal vectors. Recombinant plasmids can be introduced into the host cells by the process of transformation. 2. Bacteriophages: Some bacteriophages (e.g. phage M13 and lambda phage of E.coli) are used extensively as prokaryotic cloning vectors. They can introduce foreign DNA into their bacterial host by the process of transduction. 3. Cosmids: These are artificially constructed cloning vectors. 4. Animal viruses: Viral genomes have been manipulated to serve as cloning vectors for eukaryotic host cells in the trials of gene therapy e. g. adenoviruses and vaccinia virus.. II. Restriction Endonuclease Enzymes: These are enzymes that recognize specific nucleotide sequences within DNA molecules and catalyze the cleavage of both strands of DNA at internal positions within these sequences. III. Ligation of DNA Fragments: The construction of recombinant DNA molecules (i.e. the cloning vector and the integrated foreign DNA) is dependent on the ability to covalently seal single-strand breaks in DNA. This process is accomplished by the DNA ligase enzyme. IV. Host Organisms: Microorganisms commonly used as hosts for cloning experiments include the Gram-negative rod E.coli, the Gram-positive spore-forming rod Bacillus subtilis, and the yeast Saccharomyces cerevisiae.  Fig. (11): The recombinant DNA technique. Applications of Recombinant DNA Technology: 1. Extensive study of gene structure and function which enables mapping of the microbial genomes. 2. Production of biological products of medical importance, e.g. hormones (such as insulin), interferons, interleukins, monoclonal antibodies…etc. 3. Production of recombinant vaccines, e.g. hepatitis B vaccine. The gene coding for hepatitis B surface antigen (HbsAg) is cloned in yeast cells where it is expressed. The gene product (i.e. HbsAg) is extracted from the yeast cells, purified and used for immunization. 4. Preparation of genetic probes; which are short single-stranded DNA or RNA fragments, used for the diagnosis of infectious as well as genetic diseases. 5. Gene therapy; where viruses (e.g. retroviruses or adenoviruses) can be used as gene delivery vehicles to replace defective genes with new functioning genes, e.g. in case of immunodeficiency or diabetes mellitus.  CHAPTER 8 BACTERIAL PATHOGENESIS The outcome of bacterial infections depends on the mutual relationship between bacteria and host. Accordingly, bacteria could be classified into: 1. Saprophytic bacteria; are those which live freely in nature, on decaying organic matter, in soil or water. They do not require a living host. 2. Parasitic bacteria: are those which live on or in a living host. They are classified according to their relation to the host into: a. Pathogenic: Bacteria capable of causing disease. b. Non-pathogenic (commensals): Bacteria that do not cause disease, and are part of the normal flora. c. Opportunistic pathogens: These are potentially pathogenic bacteria that do not cause disease under normal conditions but can cause disease in immunocompromised patients, or when they find their way to another site other than their normal habitat. Many of these opportunistic pathogens are originally commensals. Stages of the Infectious Process: 1. Source of infection which may be man (case or carrier), animal or soil. 2. Mode of transmission e.g., droplet inhalation, ingestion, injection, insects, contact and transplacental. 3. Portal of entry e.g., respiratory tract, gastrointestinal tract, skin…etc. The organism then start to multiply within the host causing tissue damage (disease). 4. Portal of exist e.g., urine, stools, blood, respiratory or genital discharge, from which the organism is transmitted to a new host. Carriers: A carrier is an apparently healthy individual harbouring a pathogenic organism, without having clinical manifestations, and can transmit this organism to others. According to the duration of the carriage state, carries may be: (a) transient carriers e.g. during the incubation period and early convalescence, or (b) chronic carriers e.g. hepatitis-B virus. The organism may be discharged from the carrier in an intermittent or a continuous manner. Carriers are more dangerous than cases as a source of infection. This is because: 1. They are not known to public. 2. They are not easily detected. 3. They are not restricted to bed. 4. They carry the organism in the interepidemic periods. Infection: Entrance and multiplication of microorganism on/in the host. Disease: Damage of the host tissue due to invasion or toxin production by microorganisms. Only few infections end in clinical disease; this depends on interaction between virulence of the organism and the host resistance or immunity.  MICROBIAL VIRULENCE While pathogenicity is a qualitative description of a species of bacteria denoting ability to produce disease, virulence is a quantitative character (degree of pathogenicity) of a strain belonging to a pathogenic species. Virulence Factors of Bacteria: A virulence factor is either a structure or a product that enables the organism to cause disease. A- Adherence factors: Certain bacteria have specialized structures (fimbriae) or produce substances (glycocalyces) that allow them to adhere to the cells, thereby enhancing their ability to cause disease. For example, the fimbriae of Neisseria gonorrhoeae and E. coli help the attachment of these organisms to the urinary tract epithelium; also the glycocalyx of Staphylococcus epidermidis and certain viridans streptococci allows the organisms to adhere strongly to the heart valves. Mutants that lack these factors are often avirulent. B- Invasion factors: Invasion of tissue followed by inflammation is one of the main mechanisms by which bacteria can cause disease. This invasion is helped by: 1. Enzymes: a. Collagenase and hyaluronidase, e.g. Streptococcus pyogenes. b. Immunoglobulin A protease which degrades IgA e.g. S. pneumoniae. c. Leukocidin which can destroy both polymorphonuclear leucocytes and macrophages. d. Deoxyribonuclease that breaks down DNA, e.g. S. pyogenes and clostridia. e. Lecithinase that breaks down lecithin of cell membrane, e.g. Cl. Perfringens. 2. Antiphagocytic factors: a. Capsule: The capsule prevents the phagocytes from adhering to the bacteria, e.g. S. pneumoniae. b. Cell wall proteins of Gram-positive cocci, such as the M protein of Strept. Pyogenes and protein A of Staph. aureus. c. Coagulase: It forms a fibrin clot from fibrinogen. This clot can protect bacteria from phagocytosis, e.g. Staph. aureus. C- Toxin production: Toxin production is another mechanism by which bacteria can produce disease. Bacterial toxins are either exotoxins or endotoxins (Table 4).  Table (4): Comparison of the main features of exotoxins and endotoxins: Exotoxins Endotoxins Nature: Protein. Lipopolysaccharide. Source: Secreted by living organisms both Integral part of the cell wall of Gram-positive (mainly) and Gram-negative organisms. Gram-Negative. Liberated upon cell disintegration. Coding genes: Encoded by plasmids or Encoded by genes on the Bacteriophages. chromosome. Toxicity: High. Low. Antigenicity: Highly antigenic. Poorly antigenic Detoxification: Can be converted into toxoid*. Can not. Specificity: Every toxin has specific action. Same generalized effect (non- specific action), all give fever and shock. Heat stability: Unstable to temp. above 60°C. Stable to temp. above 60°C for several hours. Examples: C. diphtheriae and Cl. tetani. E.coli and meningococci. * Treatment of exototoxin with formalin removes its toxicity and retains its antigenicity converting it into toxoid, that can be used for immunization.  CHAPTER 9 Mycology Characteristics of Fungi 1. They are eukaryotic organisms. 2. They reproduce by means of spores; both sexual (meiotic) and asexual (mitotic)spores may be produced. 3. Vegetative forms may be unicellular (yeasts) or hyphae. 4. Cell walls are composed mostly of chitin. 5. Fungal cell membrane contains ergosterol. 6. They are heterotrophic, not autotrophic. Classification of Fungi :A- Morphological classification 1. Filamentous fungi (moulds): These grow as hyphae (septate or non-septate). On artificial media, they form large filamentous colonies. They reproduce by the production of spores (conidia) 2. Yeasts: These grow as single cells. On artificial media, they form compact colonies. They reproduce by budding. 3. Dimorphic: i.e. have 2 forms of growth: At 22°C on artificial media, they grow as hyphae. At 37°C (body temperature) on enriched media or in tissue, they grow asyeasts. :B- Clinical classification: According to its relation to disease 1. Superficial mycoses: e.g. Pityriasis versicolor. 2. Cutaneous mycoses: e.g. candidia and dermatophytes. 3. Subcutaneous mycoses: In which fungi present in the soil are implanted in the subcutaneous tissue by trauma, e.g. mycetoma 4. Deep (systemic) mycoses: These are usually caused by fungi that live free in nature. Most infections are subclinical. However, some cases may develop severe andeven fatal infection, e.g. cryptococcosis, histoplasmosis and, candidiasis. 5. Mycotoxicosis: It is produced by consumption of food containing fungal toxins 6. Allergic disorders: from spores of free living fungi e.g. Aspergillus  Diagnosis of fungal infections 1. Microscopical examination: a.Unstained preparations to demonstrate hyphae, spores or yeast cells. Skin scales, nail clippings or hairs specimen is first mounted with 10-20% KOH to dissolve keratin. b.Stained preparations: Gram’s stain, India ink, lacto phenol cotton blue 2. Culture: The most commonly used are Sabouraud’s dextrose agar (SDA). Cycloheximide (actidione) is added to inhibit saprophytic fungi and chloramphenicol is added to inhibit bacterial growth. Growth of most fungi is better at room temperature. If deep mycotic infection is sustained, enriched media are inoculated and incubated at 37°C to allow growth of the yeasty phase of the causative fungus 3. Serodiagnosis Detection of fungal antigen in the specimen. Detection of specific antibody in the serum of patients may help in diagnosis of systemic mycoses. 4. Skin testing (delayed type hypersensitivity) e.g. candidine test. Antifungal Drugs Because fungi are eukaryotes, the range of non-toxic systemically active antifungal drugs is still limited. The most commonly used drugs are:.Amphotericin B: Used as I.V. infusion in systemic fungal infections.Griseofulvin: Taken orally for treatment of dermatophytes Fluconazole, Ketoconazole and Itraconazole: Available for oral and local use. Mycostatin (nystatin): Locally acting drug used in treatment of candidial infections ofthe skin, alimentary canal,.mouth and vagina  CUTANEOUS MYCOSES DERMATOPHYTES Three Genera of dermatophytes infect man. These are: Microsporum, Trichophyton and Epidermophyton. They affect the keratinized tissue (hair, nail and skin). Infection is transmitted by direct or indirect contact. The disease is characterized by being superficial, extends radially and heals at the centre to form circular lesions called ringworm. Clinical types Ringworm is usually referred to as tinea. According to the site there are several types of ringworm infections: 1. Tinea capitis (ringworm of the scalp). 2. Tinea pedis (athletes foot). 3. Tinea unguinum (ringworm of nails). 4. Tinea circinata (ringworm of non-hairy skin). 5. Tinea cruris (ringworm of the skin of the groin). 6. Tinea barbae (ringworm of the skin of beard). CANDIDIASIS Candida is gram positive, large (3-5µm x 5-10µm) oval, budding yeast cells with pseudohyphae. Candidiasis (moniliasis) is most frequently caused by Candida albicans and rarely due to infection by other species. Habitat and trasmission Candida albicans is present as normal flora in the oral cavity, vagina and intestine. Infection is usually endogenous, but confection may occur from mother to baby  Predisposing factors to oral candidiasis: 1. Chronic local irritation as ill-fitting appliances 2. Disturbed oral ecology or marked changes in oral microbial flora by antibiotics, corticosteroids, and xerosis 3. Malnutrition 4. Immunological and endocrine disorder as Diabetes 5. Malignancies and chronic diseases 6. Extremes of age; too young or too old 7. Heavy smoking Classification of oral candidiasis 1. Primary candidiasis Pseudomembranous ( thrush) Erythematous (acute & chronic atrophic) Hyperplastic (Leucoplakia) Examples; - Denture-induced stomatitis - Angular stomatitis/cheilitis 2. Secondary oral candidiasis Oral manifestations of systemic candidiasis due to defect in cell mediated immune response. Mostly present as hyperplasic lesions (Leukoplakia) ACUTE PSEUDOMEMBRANOUS CANDIDIASIS The lesions are characterized by formation of white membranes on the tongue and oral mucosa, then confluent plaques that resemble milk curds with erythema and bleeding. It mainly affects infants. Source of infection 1. Birth canal of the mother 2. Feeding bottles 3. Hands of attendants 4. In adults, the condition is associated with depression of immunity as in case of prolonged systemic antibiotic, Corticosteroids, Diabetes, Malignancies, or Radiation therapy  Acute atrophic candidiasis: is a condition that may arise as consequences of persistent acute pseudomembranous candidiasis when pseudomembranes are shed, or developde novo It is characterized by appearance of depappillated red areas on dorsum of thetongue, or on palate & buccal mucosa Chronic atrophic candidiasis: It may occur when acute atrophic candidiasis runs in chronic course. It is characterized by erythema and oedema of palatal surface Usually affect eldery people wearing ill-fitting upper denture so it is called denture stomatitis Angular stomatitis: It occurs in odontulous patients who have reduction in the vertical height of theirface due to long term wearing of denture. Saliva moistens the cmmissures and macerated skin soon became infected withcandida  CHRONIC HYPERPLASTIC CANDIDIASIS (CANDIDA LEUKOPLAKIA) Chronic, discrete, raised areas that vary from small, palpable, translucent whitish area to large, dense, opaque plaques, hard and rough to touch It is asymptomatic and usually occurs on the inside surface of one or both cheeks There is iron and foliate deficiency together with variable degree of defective Cell- mediated immunity Higher incidence of oral cancer (9 - 40% of cases). Therefore patients with hyperplastic candidal lesions who are resistant to treatment should be kept underregular supervision Laboratory diagnosis of candidiasis 1. Specimens: A swab from (skin, mouth, vagina), urine, sputum 2. Microscopic examination: Gram positive oval, budding yeast cells and pseudohyphae. 3. Culture: C. albicans can grow on most of culture media. The selective media is Sabouraud’s dextrose agar (SDA) containing actidione and chloramphenicol. Thecolonies are creamy white, pasty, with yeasty odour. 4. C. albicans is differentiated from other Candida species by - Formation of germ tubes in serum at 37°C within 1-2 hours. - Formation of chlamydospores on corn meal agar. - Biochemical reaction tests. Treatment 1. Correction of the predisposing factors 2. Improving the general health of the patient 3. Toluconazole as oral gel, or nystatin as lozengens PNEUMOCYSTIS CARINII Pneumocystis carinii is an opportunistic fungus that lives in the lungs humans. The species infecting the lungs of humans has been termed P. jiroveci, whereasthose from most other hosts are collectively termed "Pneumocystis carinii." Most people are infected with P. jiroveci by the age of four and develop no symptoms. It causes interstitial pneumonia in association with HIV infection, primary immunodeficiency, prolonged steroid treatment, organ transplantation andcancers. It is the most common opportunistic infection in AIDS patients. Organism can be detected in clinical specimens by PCR or Staining with silverstain.  CHAPTER 10 General virology VIRUSES Viruses are parasites at the genetic level, they are the smallest infectious agents. They are obligatory intracellular parasites because they have no metabolic activity. Differences between Viruses & Bacteria: 1. Viruses are very small in size, ranging from 20-300 nm. So: – They can only be seen under the electron microscope. – They can pass through bacterial filters. – They need ultracentrifugation for sedimentation. 2. Viruses contain only one type of nucleic acid (DNA or RNA), never both. 3. They are obligatory intracellular parasites, i.e. can only replicate inside livingcells. 4. They cannot be cultivated in the laboratory on artificial culture media. 5. They are not susceptible to antibacterial agents. STRUCTURE AND COMPOSITION OF VIRUSES The complete virus particle (virion) is composed of a nucleic acid core (DNA or RNA) surrounded with a protein coat (capsid). The nucleic acid and the protein coat are called nucleocapsid. Some viruses have additional lipoprotein layer called envelope, other viruses are non-enveloped (naked). Viral Capsid 1. It is formed of subunits called capsomeres. 2. It protects the nucleic acid. 2. It mediates attachment to host cell (in non-enveloped viruses). 3. It is the antigenic part of the virus. 4. It is responsible for the viral morphology (or symmetry). Viral Nucleic Acid (Genome) It is responsible for virulence, i.e. it is the infectious part of the virus. Only one type of nucleic acid is present in the virus, DNA or RNA. Most DNA viruses are double-stranded (ds) while most RNA viruses are single-stranded (ss). Viral Envelope It is formed of lipids or lipoproteins and is derived partly from the host cell membrane during release by budding. It is sensitive to lipid solvents, e.g. ether. It has glycoprotein spikes which are the organ of attachment of the enveloped virus to host cell receptors. VIRUS REPLICATION Viruses depend on living host cells for providing the virus components under the information given by the virus genome. Viral replication occurs in the following stages: 1. Attachment or adsorption Adsorption of the virus occurs to specific receptor sites on the surface of the susceptible host cell (tropism). 2. Penetration This occurs either by: a. Endocytosis in case of non-enveloped viruses or: b. Fusion of the viral envelope with the host cell membrane in case of envelopedviruses. 3. Uncoating The nucleic acid is released from the capsid by lysosome enzymes and is available for replication. 4. Eclipse This is the time from uncoating until assembly of mature viruses. During this phase, no infectious viruses can be detected in the host cell. 5. Synthesis of viral components Specific messenger RNAs are transcribed from the viral nucleic acid, and are translated in the cell ribosomes to form viral components. Transcription of mRNA varies according to the type of viral nucleic acid whether DNA or RNA 6. Assembly The nucleic acids are enclosed within the protein coats to form mature viruses (virions). This occurs either in the nucleus of host cell, e.g. herpes viruses or in the cytoplasm,e.g. polioviruses. 7. Release New viruses are released either by: a. Lysis of host cell and release of new viruses in case of non-enveloped viruses. b. Budding through the cell membrane in case of enveloped viruses.  CULTIVATION OF VIRUSES Viruses replicate only in living susceptible cells. There are three main methods for cultivation of viruses in the laboratory: I. Cell culture (tissue culture). II. Embryonated eggs. III. Laboratory animals (intact animals). Detection of virus replication in cell culture Virus growth in cell culture could be detected by: 1. Cytopathic effect (CPE) CPE are the morphological changes in the infected cells seen microscopically. This occurs with most viruses and include: a. Cell death or lysis. b. Syncytial formation (giant cells): due to fusion of membranes of adjacent cells toform multinucleated giant cells, e.g. respiratory syncytial virus (RSV), measles and mumps viruses. 2. Inclusion bodies These are either the site of virus assembly or degenerative changes in the cell. They may be: a. Intracytoplasmic: e.g. rabies (Negri bodies). b. Intranuclear: e.g. herpes viruses. c. Both: e.g. measles virus. 3. Plaque formation Plaques are areas of virally-infected cells in a monolayer cell culture. These are seen by the naked eye as unstained areas when using a vital dye. 4. Transformation The nucleic acid of the virus gets incorporated with the genetic material of the cell making it transformed e.g. herpes virus and other oncogenic viruses. 5. No change for several weeks, e.g. rubella virus This could be detected by its ability to interfere with the growth of another CPE- producing virus added as an indicator (interference phenomenon). 6. Haemadsorption Haemagglutinating viruses, e.g. influenza viruses are detected by adding suspension of RBCs to the infected cell line. The RBCs will be clumped only on the virally-infected cells. 7. Direct fluorescent antibody staining of infected cells 8. Detection of viral antigens Soluble antigens released in the growth medium of cell culture can be detected by any serological test, e.g. complement fixation (CF), haemagglutination (HA)... etc. 9. Neutralization test This can be done by the use of specific antiviral antisera which will block or neutralize the infectivity of the virus. Defective virus particles They are small viral particles that can not replicate unless they are present with a helper virus. This is due to lack of genetic information necessary for completion of the replication cycle, e.g.: 1. Delta agent or hepatitis D virus (HDV): It requires hepatitis B virus (HBV) to replicate. 2. Adeno-associated viruses: These are particles found in adenovirus preparations. PATHOGENESIS OF VIRAL DISEASES I. Entry of Viruses Viruses enter the body either by inhalation (respiratory tract), ingestion (gastrointestinal tract), contact (urogenital system) and through skin (injections, blood transfusion , insect and animal bites). Viruses usually replicate in the primary site of entry. Some viruses produce disease at the portal of entry (local infections), others have to spread to distant organs either via the blood (viraemia), or by other means, e.g. along nerves and produce systemic or deep viral infections. Differences between local and systemic viral infections. Loc Systemic al Site of pathology Locally at the portal of entry. At distant sites e.g. Respiratory tract e.g. measles. influenza virus, GIT e.g. rota virus. Incubation period Short Long Viraemia Absent Present Duration of immunity Short Long Ig involved in Secretory IgA IgG or IgM immunity  II. Fate of Viral Infections 1. Inapparent or subclinical viral infections 2. Apparent infections (disease) This may be local or systemic with the appearance of clinical signs and symptoms. 3. Persistent viral infections (chronic) In this form, the virus is continuously detected with mild or no clinical symptoms, e.g. chronic hepatitis B. 4. Latent viral infections The virus persists in a dormant form and may flare up intermittently to produce disease, e.g. herpes viruses. 5. Slow virus infections Virus infections with long incubation periods (months or years). They are caused by two types of infectious agents: 1. Conventional viruses, e.g. a variant of measles virus which causes subacute sclerosing panencephalitis (SSPE). 2. Unconventional agents (prions).  LABORATORY DIAGNOSISOF VIRAL INFECTIONS I. Direct Detection of Viruses and/or their Antigens 1. Light microscopy The ordinary microscope can be used in examination of large viruses as poxviruses, or giant cells in herpes infection, or inclusion bodies, e.g. Negri bodies in nerve cells in rabies. 2. Fluorescent microscopy By using direct immunofluorescent antibody technique (IF), e.g. diagnosis of rabies in brain smears. 3. Electron microscopy (EM) This method is used when large number of viruses is present in the sample. It also gives an idea about the size and shape of viruses. 4. Immunoelectron microscopy (IEM) This is done by the addition of specific antibodies to the clinical sample. This will lead to aggregation of the unknown virus particles. 5. Solid-phase immunoassays For detection of the virus antigens by the use of either ELISA or RIA; e.g. hepatitis B antigens in blood. 6. Nucleic acid hybridization Specific labelled probes are added to clinical samples. It will hybridize with the complementary nucleic acid of the virus in the specimen. 7. Polymerase chain reaction (PCR) It is a recent method in which amplification of a short sequence of a target nucleic acid of the virus allow it to be easily detected by different methods, e.g. probes, ELISA... etc. II. Isolation of Viruses - Isolation of virus is done by inoculation of cell culture, embryonated egg or laboratory animals. III. Serological Diagnosis - It is an indirect method to detect antiviral antibodies. - Usually 2 serum samples (paired serum) are taken. The first in the acute phase and the second 2-3 weeks later, to demonstrate a rising titre (4 fold increase or more is diagnostic). - Only one sample may be used in the acute stage to detect IgM, e.g. in diagnosis of rubella in early pregnancy. The serological methods used are: Neutralization test (NT), complement fixation test (CF), haemagglutination inhibition test (HI), enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA) and indirect immunofluorescence (IIF). IV. Skin Tests Can be used as an indication of cell-mediated immunity (CMI) in some viral infections, e.g. mumps.  TREATMENT OF VIRAL INFECTIONS Viruses cannot be treated with antibiotics because they lack the structural targets on which antibiotics can act. Viruses are obligate intracellular parasites, so antiviral drugs must selectively inhibit viral replication without causing damage to the host cells. The number of antiviral drugs is little compared to antibacterial drugs. 1. Antiviral Drugs Amantadine: inhibits uncoating of influenza A virus, therefore used in prevention of its infection. Rimantadine: a derivative of amantadine, but less toxic. Acyclovir (Zovirax): guanine analogue. It inhibits viral DNA polymerase, active against HSV-I, II & V-Z virus. - Topical acyclovir is effective in treatment of primary genital herpes, herpetic corneal ulcer & herpetic skin lesions. - Parenteral acyclovir is effective in treatment of HBV infection. Ribavirin (Virazole): It inhibits synthesis of mRNA. Ribavirin aerosol is used to treat pneumonitis caused by RSV in infants &influenza B infection. Azidothymidine (Zidovudine, AZT): It inhibits viral RT enzyme of HIV. It is used in treatment of AIDS II. Interferons (IFNs) These are host coded proteins (cytokines) that inhibit viral replication. They are the first line of defense against viral infections.  CHAPTER 11 ANTIMICROBIAL CHEMOTHERAPY Bacteriostatic agent; is an antimicrobial agent that is capable of inhibiting bacterial multiplication. Multiplication resumes upon removal of the agent. Bactericidal agent; is an antimicrobial agent that is capable of killing bacteria. Multiplication can not be resumed. Selective toxicity; is the ability of an antimicrobial agent to harm a pathogen without harming the host. Spectrum of activity; The range of microorganisms that are affected by a certain antibiotic is expressed as its spectrum of action. Antibiotics which kill or inhibit the growth of a wide range of Gram-positive and Gram-negative bacteria are said to be broad spectrum. If effective mainly against either 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. MECHANISMS OF ACTION OF CLINICALLY USED ANTIMICROBIAL AGENTS A- Inhibition of Bacterial Cell Wall Synthesis: Agents acting by this mechanism include: 1. β-lactam antibiotics: e.g., Penicillins, cephalosporins, and others. 2. Glycopeptides: e.g., vancomycin, and teicoplanin. 3. Cycloserine and bacitracin. These antibiotics are bactericidal with minimal tissue toxicity. B- Interference with the Cell Membrane Function: Some agents disrupt the cytoplasmic membrane and interfere with its function. These include: 1. Antibacterial agents: e.g., polymyxin and colistin. 2. Antifungal agents: e.g., amphotericin B, nystatin and imidazoles. These agents are microbicidal. They are highly toxic as they have narrow margin of selective toxicity. C- Inhibition of Bacterial Protein Synthesis: whereas mammalian cells have (with 30S and 50S subunits) Bacteria have 70S ribosomes This difference makes bacterial ribosomes a selective.(40S and 60S subunits) 80S ribosomes :target for antimicrobials as follows 1. Agents acting on the 30S ribosomal subunit: e.g., aminoglycosides (gentamicin, amikacin, streptomycin) and tetracycline. 2. Agents acting on the 50S ribosomal subunit: e.g., macrolides (erythromycin), lincomycins (clindamycin), chloramphenicol and fusidic acid. D- Inhibition of Bacterial Nucleic Acid Synthesis: This may occur by: 1. Inhibition of RNA synthesis through the strong binding to DNA-dependent RNA polymerase: e.g., rifampin. 2. Inhibition of DNA synthesis through blocking DNA gyrase: e.g., quinolones & novobiocin. 3. Inhibition of dihydrofolic acid reductase leading to inhibition of folic acid synthesis.e.g. trimethoprim and pyrimethamine. E- Competitive Antagonism e.g., sulphonamides For many organisms, para-amino benzoic acid (PABA) is essential for the synthesis of folic acid. Sulphonamides are structural analogues of PABA. They compete with PABA for the active centre of the enzyme involved in folic acid synthesis. RESISTANCE TO ANTIMICROBIAL AGENTS Resistance to antimicrobial agents may be 1- Intrinsic resistance “Natural” 2- Acquired resistance due to changes in bacterial genome a- Mutation and selection b- Exchange of resistance genes between strains through; - Conjugation by plasmids - Transduction by bacteriophages - Transformation by transposos Mechanisms of Emerging Drug Resistance: 1. Production of destroying or inactivating enzymes; e.g.: - Resistance to β-lactams due to production of β-lactamases. - Chloramphenicol resistance due to production of acetyl transferase enzyme. 2. Reduction of interacellular conc. of antibiotic a-decrease influx of antibiotic by reduction in the permeability to the drug b-Efflux pumb that pumbed antibiotic out faster than it can diffuse 3. Target modification; - Resistance to β-lactams due to alterations in the pre-existing penicillin binding protein (PBP) e.g., methicillin-resistant Staphylococcus aureus (MRSA). - Aminoglycoside resistance due to alteration of the 30S ribosomal subunit. 4. Target elimination by Development of alternative metabolic pathway; that enables bacteria to bypass the reaction inhibited by the drug, e.g.: - Resistance to sulfonamides and trimethoprim. 5- Target over production e g.VISA  COMPLICATIONS OF CHEMOTHERAPY 1. Toxicity; - Tetracycline may cause staining of teeth in infants. - Streptomycin may affect the 8th cranial nerve leading to vestibular dysfunction. - Aminoglycosides may cause nephrotoxicity. - Chloramphenicol can cause bone marrow depression. 2. Allergy (hypersensitivity): - Penicillins may cause urticaria, anaphylactic shock or serum sickness. - Local application of sulphonamides may result in contact dermatitis. 3. Emergence of Resistant Strains: The abuse of antibiotics (low dosage, interrupted course, no real indication, and improper choice) encourages the emergence of resistant mutants. These mutants will overgrow and replace the originally susceptible bacteria. It is recommended that in vitro susceptibility testing should be performed to guide the selection of antibacterial drugs. 4. Superinfection: It occurs as a result of outgrowth of resistant members of normal flora when the sensitive ones are eradicated during antibiotic therapy e.g., - Pseudomembranous colitis caused by outgrowth of Clostridium difficile. - Oral thrush caused by overgrowth of the yeast Candida. Choice of an Antimicrobial Agent for Therapy: 1. In vitro culture and susceptibility testing: Whenever possible to isolate the organism from the patient, it should be tested for the most appropriate antibiotic. Therefore, collection of the appropriate specimen is essential before giving the antibiotic. 2. Empiric antimicrobial therapy (mentioned below). It is indicated: - while waiting for the results of susceptibility testing, or - in closed lesions, where there is no available sample. Empiric Therapy: In the majority of situations in which antibiotics are used, a "best guess" procedure is followed. The physician makes a provisional diagnosis that a patient has a bacterial infection which requires treatment. Depending on the type of infection, there will be a short list of bacteria most likely to be causing that infection. Depending on the type of bacteria, there will be an antibiotic most likely to successfully treat that infection. "Best guess" treatment is not always successful or appropriate as many bacteria have unpredictable susceptibilities to antimicrobial agents.  IN VITRO MICROBIAL SUSCEPTIBILITIES TO ANTIMICROBIAL AGENTS AND RELATION TO IN VIVO ACTIVITY Microorganisms vary in their susceptibility to different chemotherapeutic agents, and susceptibilities can change over time. Ideally, the appropriate antibiotic to treat any particular infection should be determined in vitro before any antibiotic is given. The activity of an antimicrobial agent against an organism is dependent on its concentration. Some idea of the effectiveness of a chemotherapeutic agent can be obtained from determining the minimal inhibitory concentration (MIC). The MIC is defined as the lowest concentration of a drug that prevents growth of a test organism. The MIC measurement forms the basis for susceptibility testing methods and determining breakpoints for defining susceptibility. The breakpoint of an antimicrobial agent is the concentration that can be achieved in the serum with optimal dose. Organisms with MICs at or below the breakpoint are considered susceptible. On the other hand, organisms with MICs above the breakpoint are considered resistant. However, these in vitro test conditions do not include the host factors that can have an influence on antimicrobial activity in vivo such as: 1) protein binding, 2) post antibiotic effect, 3) effects on organism's virulence, and 4) the pharmacokinetic changes resulting from different drug levels in blood and in the site of infection over time. These host factors can result in discrepancies between the results of in vitro susceptibility tests and the clinical response to antibiotic therapy. The routine in vitro susceptibility testing can be done by one of the following methods: 1. Disc diffusion methods. 2. Dilution methods such as tube broth dilution. 3. Gradient diffusion (E test) methods. COMBINED THERAPY There are conditions which necessitate the use of more than one antibiotic in order to achieve a successful clinical response. Possible Indications: 1. Severely ill patients suspected of having serious infections; e.g.: - Bacterial meningitis. - Sepsis in immunocompromised patients caused by Pseudomonas aeruginosa, Klebsiella spp., Enterobacter spp. or S. aureus. 2. Febrile neutropenia. 3. To delay the emergence of drug-resistant mutants e.g. in treatment of T.B. 4. To achieve bactericidal action through synergistic effect e.g. in enterococcal sepsis (endocarditis) to eradicate the pathogen. 5. Mixed infections e.g. infections following massive trauma. Effects of Combined Therapy: 1. Synergistic effect (1+1=>2): 2. Antagonistic effect (1+1=