Medical Microbiology and Immunology PDF

Medical microbiology and immunology OVERVIEW OF THE IMMUNE SYSTEM E IMMUNE SYSTEM Chapter (1) Overview of immune system The main function of the immune system is to prevent or limit infections, fungi, and parasi...

Medical microbiology and immunology OVERVIEW OF THE IMMUNE SYSTEM E IMMUNE SYSTEM Chapter (1) Overview of immune system The main function of the immune system is to prevent or limit infections, fungi, and parasites, such as protozoa and worms. Two arms of the Immune System: ❑ Innate Immunity ❑ Acquired Immunity Innate Immunity Acquired Immunity Present since birth Occurs along lifetime after exposure to pathogens Immediately after infection More delayed but very effective but less efficient Non-specific Specific directed against certain In all individuals against all pathogen upon exposure (adaptive), microorganisms at all time Improve with repeated exposure Immunological memory absent Immunological memory develops No life-long protection May give life-long protection Interact with acquired immunity Interact with innate immunity through (antigen presentation) through (opsonization) Main cells (neutrophils, monocytes, Main cells (B & T lymphocytes) macrophages and NKs) 1 Medical microbiology and immunology 2  Medical microbiology and immunology Mechanisms of Innate Immunity I. Mechanical barriers and surface secretions: these are as, intact skin, mucous membranes, cilia of respiratory tract, blinking, sneezing, and coughing reflexes. II. Normal Bacterial flora: that competes with pathogenic bacteria for essential nutrients. III.Humoral defense mechanisms: this presented in lysozyme, complement, acute phase proteins and cytokines. IV. Cellular defense mechanisms: as phagocytic cells (neutrophils, macrophages), natural killer cells and eosinophils. V. Inflammation Mechanisms of acquired Immunity: 1- T- Cell Mediated Immunity (consist of helper T cells and cytotoxic T cells). 2- Humoral immune system (consists of B -lymphocytes that differentiate to plasma cells that will secret immunoglobulin The Lymphoid Organs: Organized tissues where the following immune functions occur: 1. Lymphoid cells interact with other cells. 2. Maturation of immune cells 3. starting acquired immune response There are two types of lymphoid organs: 1. Primary lymphoid Organs: Where lymphocytes are formed & their complete maturation Bone marrow: origin of all immune cells and maturation of B cells 3  Medical microbiology and immunology Thymus :maturation of T cells 1. Secondary lymphoid organs: Where lymphocytes meet antigen and undergo activation. Lymph nodes Spleen MALT (mucosal associated lymphoid tissues) GALT (gut associated lymphoid tissues) BALT (bronchial associated lymphoid tissues) 4  Medical microbiology and immunology Role of Cells of the Immune System: Granulocytes: Neutrophils: Phagocytic, Most numerous (60-70%), Most important cells of innate IS, Short-lived, Absent from normal tissue ,Attracted by chemotactic factors to site of infection, Dead neutrophils form pus Eosinophils: Important against helminthes by release of toxic chemicals from granules. , Phagocytic, Important in allergy, Mast cells, Found in tissues (not blood), Around blood vessels or in submucosa, Release mediators from granules → allergy and inflammation Basophils: Found in low concentrations in blood, Action similar to mast cells Monocytes/macrophages: monocytes in blood leave to tissues where it becomes macrophages in tissue. special macrophages include kupffer cells(in liver) and alveolar macrophages ( in lungs), functions of macrophages include phagocytosis and antigen presentation Lymphocytes: 1. T lymphocytes: they originates in bone marrow and complete maturation in thymus, they represent 75% of peripheral blood lymphocytes, there are two types: T helper : its main function is secretion of cytokines 2. T cytotoxic: its main function is killing target cells Ratio: T helper: T cytotoxic = 2:1 B Lymphocytes: they originate in bone marrow and complete maturation in 5  Medical microbiology and immunology bone marrow, they represent 10% of peripheral blood lymphocytes, their main function is to change into plasma cells that produce antibodies Natural killer Cells: they are large granular lymphocyte, non-t, non-b, they represent 10 - 15% of peripheral blood lymphocytes, its main function is killing target cell as virus infected cells, killing mechanism are similar to t cytotoxic but recognition is different (part of innate immune functions). The professional antigen presenting cells include: 1. Dendritic Cells 2. Macrophages 3. B cell Recognition and effector mechanisms of acquired immunity: Antigen Presenting Cell (APC) uptake the antigen (may be by phagocytosis), break- up that antigen into small peptides (processing) then present the antigen peptides to T cells (antigen presentation). Different pathogens have different lifestyles, and thus different mechanisms of detection, recognition and destruction: B cells (humoral immunity): secrete antibodies that combat Extracellular pathogens (most bacteria). T cells (Cell-mediated Immunity): combat Intracellular pathogens (all viruses, some bacteria) by either: 1. Cytotoxic T cell function: killing of virus infected cells. 2. Helper T cell function: production of cytokines(2 types): a. T-helper 1cells: secrete cytokines that activate macrophages to become more capable of killing bacteria inside them. 6  Medical microbiology and immunology T-helper 2 cells: secrete cytokines that activate B cells into plasma cells to produce of antibodies. Lymphocytes circulate mutually between blood and secondary lymphoid organs until a Specific Lymphocyte finds its specific pathogen thus it recognizes its antigens and undergoes: Activation: lymphocyte becomes lymphobast Proliferation: rapid multiplication differentiation: cells become effector cells (e.g. B cell becomes plasma cell, cytotoxic T cell → capable of killing, helper T cell → produces cytokines) A very important result is the development of memory cells which have: 1. More rapid response 2. More effective response Figure 37: Effector Mechanisms of Antibodies (Humoral immunity) 7  Medical microbiology and immunology CYTOKINES & CHEMOKINES: They are a diverse collection of soluble proteins made by cells that affect the behavior of other cells. They are similar to hormones but they are not produced by organised cells located in glandular tissue. They have many biological activities (pleiotropic), Some cytokines are largely associated with chemoattraction of other cells (chemokines). The balance & level of cytokines and chemokines secreted affects the outcome of the response Differences between T and B Lymphocyte Receptors: B cell receptor T cell receptor Recognition sites an immunoglobulin with has one recognition site two antigen recognition sites Secretion can be secreted always a cell-surface (Antibody) molecule Recognition recognizes antigen cannot recognize antigen directly directly Unsuitable immune responses include: 1. Failure of Host Defence Mechanisms (Immunodeficiency): Primary Immunodeficiency: can lead to death early in life (if 8  Medical microbiology and immunology severe) or characterized by repeated infections Secondary Immunodeficiency: like AIDS (acquired immunodeficiency syndrome) caused by HIV Virus which infects T helper cells 2. Harmful Immune Responses Allergy: immune response against harmless antigen (e.g. pollens, foods and drugs) Autoimmune diseases: immune response against self-antigens Graft rejection: immune response against transplant Manipulation of the Immune System: 1. Immunosuppression: used in cases of harmful immune responses like autoimmune diseases by using Immunosuppressant drugs which lead to general inhibition of immune response (including beneficial immune responses) thus can lead to infections with opportunistic pathogens which otherwise are harmless. 2. Immunostimulation (vaccination): this is an antigen-specific immunostimulation by Introducting the whole or part of a pathogen in harmless form, resulting in an immune response to that particular pathogen, on exposure to the real pathogen in a later time, the person will be protected by the resultant immune response. 9 Medical microbiology and immunology 10  Medical microbiology and immunology Chapter (2) Antigen – antibody reactions Reactions of antigens and antibodies are highly specific. An antibody will react only with the antigen that induced it or with a closely related antigen. Because of the great specificity, reactions between antigens and antibodies are suitable for identifying one by using the other. This are in vitro reactions for the diagnosis of diseases and for identification and quantitation of antigens and antibodies. Antigen: It is a substance that can stimulate the immune system to produce an immune response (humoral and/or cell-mediated) and reacts specifically with the product of this response. The immune system does not recognize the antigen molecule as a whole but reacts to structurally limited parts of the molecule called epitopes (four to five amino acids or monosaccharide residues). They determine the specificity of the antigen. Antibodies (immunoglobulins): it is glycoproteins that, bind specifically to antigens that induced their formation 11  Medical microbiology and immunology The results of many immunologic tests are expressed as a titer, which is defined as the highest dilution of the specimen (e.g., serum) that gives a positive reaction in the test. Note that a patient’s serum with an antibody titer of, for example, 1/64 contains more antibodies (i.e., is a higher titer) than a serum with a titer of, for example, 1/4. The major uses of serologic (antibody-based) tests are: 1. Diagnosis of infectious diseases 2. Diagnosis of autoimmune diseases 3. Typing of blood and tissues prior to transplantation. Microorganisms and other cells possess a variety of antigens and thus induce antisera containing many different antibodies (i.e., the antisera are polyclonal). Monoclonal antibodies excel in the identification of antigens because cross-reacting antibodies are absent (i.e., monoclonal antibodies are highly specific). TYPES OF DIAGNOSTIC TESTS Many types of diagnostic tests are performed in the immunology laboratory. Most of these tests can be designed to determine the presence of either antigen or antibody. To do this, one of the components, either antigen or antibody, is known and the other is unknown. For example, with a known antigen such as herpes virus, a test can determine whether antibody to the virus is present in the patient’s serum. *Methods of detection of Antigen-Antibody reactions: 1) Reactions accompanied by visible phenomena 2) Reactions detected by labeled reagents 12  Medical microbiology and immunology 1) Reactions accompanied by visible phenomena Agglutination In this test, the antigen is particulate (e.g., bacteria and red blood cells) or is an inert particle (latex beads) coated with an antigen. Antibody, because it is divalent (has two antigen binding sites) or multivalent, cross-links the antigenically multivalent particles and forms a latticework, and clumping (agglutination) can be seen. One very commonly used agglutination test is the test that determines a person’s ABO blood group Precipitation (Precipitin) This form of antigen antibody reaction occurs if the antigen is soluble instead of cellular. Agar gel diffusion is a commonly used technique in which antigen antibody reaction takes place in semisolid media. 13  Medical microbiology and immunology 2) Antigen -Antibody Reactions detected by Labelled Reagents Radioimmunoassay (RIA) This method is used for the quantitation of antigens or haptens that can be radioactively labeled and the amount of radioactivity measured. RIA is a highly sensitive method and is commonly used to assay hormones or drugs in serum. The radioallergosorbent test (RAST) is a specialized RIA that is used to measure the amount of serum IgE antibody that reacts with a known allergen (antigen). Enzyme-Linked Immunosorbent Assay (ELISA) This method can be used for the quantitation of either antigens or antibodies in patient specimens. It is based on covalently linking an enzyme to a known antigen or antibody, reacting the enzyme-linked material with the patient’s specimen, and then assaying for enzyme activity by adding the substrate of the enzyme. The method is nearly as sensitive as RIA yet requires no special equipment or radioactive labels. For measurement of antibody, known antigens are fixed to a surface (e.g., the bottom of small wells on a plastic plate), incubated with dilutions of the patient’s serum, washed, and then reincubated with antibody to human IgG labeled with an enzyme (e.g., horseradish peroxidase). Enzyme activity is measured by adding the substrate for the enzyme and estimating the color reaction in a spectrophotometer. The amount of antibody bound is proportional to the enzyme activity. The titer of antibody in the patient’s serum is the highest dilution of serum that gives a positive color reaction. 14  Medical microbiology and immunology Chapter (3) Hypersensitivity Hypersensitivity refers to undesirable reactions produced by the normal immune system. Hypersensitivity reactions require a pre-sensitized (immune) state of the host. Hypersensitivity reactions can be divided into four types: type I, type II, type III and type IV, based on the mechanisms involved and time taken for the reaction. Frequently, a particular clinical condition (disease) may involve more than one type of reaction. Type I Hypersensitivity: It is also known as immediate or anaphylactic hypersensitivity. The reaction may involve skin (urticaria and eczema), eyes (conjunctivitis), nasopharynx (rhinorrhea, rhinitis), bronchopulmonary tissues (asthma) and gastrointestinal tract (gastroenteritis). The reaction may cause from minor inconvenience to death. The reaction takes 15-30 minutes from the time of exposure to the antigen. Sometimes the reaction may have a delayed onset (10-12hours). Immediate hypersensitivity is mediated by IgE. The primary cellular component in this hypersensitivity is mast cell or basophil. The reaction is amplified and/or modified by platelets, neutrophils and eosinophils. A biopsy of the reaction site demonstrates mainly mast cells and eosinophils. The mechanism of reaction involves preferential production of IgE, in response to certain antigens, allergens. 15  Medical microbiology and immunology Table 5: Pharmacologic Mediators of Immediate Hypersensitivity Mediator Preformed mediators in granules Histamine bronchoconstriction, mucus secretion, vasodialatation, vascular permeability, proteolysis Tryptase kinins and vasodialatation, vascular Kininogenase permeability, edema attract eosinophil ECF-A and neutrophils (tetrapeptides) Newly formed mediators Leukotriene B4 Basophil attractant same as histamine but 1000x more potent leukotriene C4, D4 edema and pain Prostaglandins D2 Platelet aggregation and heparin release: microthrombi PAF Type II Hypersensitivity It is also known as cytotoxic hypersensitivity and may affect a variety of organs and tissues. The antigens are normally endogenous, although exogenous chemicals (haptens) which can attach to cell membranes can also lead to type II hypersensitivity. Drug- induced hemolytic anemia, granulocytopenia and thrombocytopenia are such examples. The reaction time is minutes to hours. It is primarily mediated by antibodies of IgM or IgG class and complement. Phagocytes and NK cells may also play a role (ADCC). The lesion contains antibody, complement and neutrophils. 16  Medical microbiology and immunology Figure: Type II hypersensitivity mechanisms Clinical Conditions e.g 1) Transfusion reaction due to ABO incompatibility 2) Rh-incompatability (Haemolytic disease of the newborn) 3) Autoimmune diseases; the mechanism of tissue damage is cytotoxic reactions 4) Drug reaction: penicillin may attach as haptens to RBCs and induce antibodies, which are cytotoxic for the cell-drug complex leading to haemolysis Quinine may attach to platelets and the antibodies cause platelets destruction and thrombocytopenic purpura Type III Hypersensitivity It is also known as immune complex hypersensitivity. The reaction may be general (e.g., serum sickness) or may involve individual organs including skin (e.g., systemic lupus erythematosus, Arthus reaction), kidneys (e.g., lupus nephritis), lungs (e.g., aspergillosis), blood vessels (e.g., polyarteritis), joints (e.g., rheumatoid arthritis) or other organs. This reaction may be the pathogenic mechanism of diseases caused by many microorganisms. 17  Medical microbiology and immunology The reaction may take 3-10 hours after exposure to the antigen (as in Arthus reaction). It is mediated by soluble immune complexes. They are mostly of IgG class, although IgM may also be involved. Type IV Hypersensitivity It is also known as cell mediated or delayed type hypersensitivity. The classical example of this hypersensitivity is tuberculin (Montoux) reaction which peaks 48 hours after the injection of antigen (PPD or old tuberculin). The lesion is characterized by induration and erythema. Type IV hypersensitivity is involved in the pathogenesis of many autoimmune and infectious diseases (tuberculosis, leprosy, blastomycosis, histoplasmosis, toxoplasmosis, leishmaniasis, etc.) and granulomas due to infections and foreign antigens. Another form of delayed hypersensitivity is contact dermatitis (poison ivy, chemicals, heavy metals, etc.) in which the lesions are more papular. Figure: Mechanisms of damage in delayed hypersensitivity 18  Medical microbiology and immunology Table 7: Comparison of Different Types of hypersensitivity Characteristic type-I type-II type-III type-IV (anaphylactic) (cytotoxic) (immune (delayed type) complex Antibody IgE IgG, IgM IgG, IgM None Antigen Exogenous cell surface soluble tissues & organs response time 15-30 minutes minutes-hours 3-8 hours 48-72 hours Appearance weal & flare lysis and necrosis erythema and erythema and edema, induration necrosis histology basophils and antibody and complement monocytes and eosinophil complement and lymphocytes neutrophils transferred Antibody Antibody antibody T-cells with examples allergic Erythroblastosis SLE, farmer’s tuberculin test, asthma, hay fetalis, lung disease poison ivy, fever Goodpasture’s granuloma nephritis 19  Medical microbiology and immunology Chapter (4) Autoimmune disease Autoimmune disease is a disorder of the body’s defense mechanism in which an immune response is generated against component or products of its own tissues treating them as foreign material and attacking them. The disorder caused by inflammation and destruction of tissues by the body’s immune response as a result of autoimmunity is known as autoimmune disease. Proposed mechanism for induction of autoimmunity: A variety of mechanisms have been proposed to account for the T-cell mediated generation of autoimmune disease. And it is likely that autoimmune disease does not develop from a single event rather from a number of different events. Some of the proposed mechanisms are: 1. Forbidden clone 2. Altered antigen 3. Sequestered antigen 4. Immunological deficiency theory 5. Genetic influence 20  Medical microbiology and immunology 1. Forbidden clone Mutation in the lymphocytes may result in the formation of changed or altered clone. These altered clones may recognize host as foreign and lead to development of autoimmunity. 2. Altered antigen Some of the antigens on the host cell get altered by chemical, biological or physical means. Thus formed new antigenic determinants which may be recognized as foreign by the host. 3. Sequestered antigen Some of the antigens in the body are hidden from cells of immune system. If the organs containing such antigen are damaged, it causes exposure of sequestered antigen thus an immune reaction to these antigens may occur. 4. Immunological deficiency theory According to this theory, mutation or loss of immune regulatory power. i.e. deficiency in immune system results in a condition in which self- antigen behaves as foreign. 5. Genetic influence This was determined by family studies. It is well recognized that certain immune disorder predominate in females and families. This has strongly supported the role of genetic influence in autoimmunity. 21 Genetic links have occurred between disease and HLA antigens.  Medical microbiology and immunology Types of autoimmune diseases On the basis of pathogenic mechanism, autoimmune diseases are classified into two types: 1. Organ specific autoimmune disease: This autoimmune disease is directed against a component of one particular type of organ. The organ specific autoimmune disease can further be divided into two groups: i. Autoimmune disease mediated by direct cellular damage: This type of damage occurs when lymphocytes or antibodies bind to cell membrane antigens, causing cellular lysis or inflammatory response in affected organ. The damaged cellular structure is then replaced by connective tissue (fibrous) & it loses its function. Examples: Hasimoto’s thyroiditis, autoimmune anaemia, Good pasteur’s syndrome, Insulin dependent diabetes mellitus. ii. Autoimmune disease mediated by stimulating or blocking auto antibodies: In some cases, antibodies act as antagonist & bind to hormone receptor stimulating inappropriate activity. This usually leads to overproduction of mediators or increase cell growth. They also bind to hormone receptor function and thereby block receptor function. This causes impaired secretion of 22 mediators and gradual atrophy of the affected organ. Examples: Grave’s disease, Myasthenia gravis  Medical microbiology and immunology 2. Systematic autoimmune disease: It is the type of autoimmune disease which is directed against an antigen that is present in many different sites and can include involvement of several organs and tissues. These diseases reflect a general defect in immune regulation that result in hyperactive T-cells and B-cells. Examples: Rheumatoid arthritis, Systematic lupus erythematosus (SLE), multiple sclerosis. 23  Medical microbiology and immunology Chapter (5) INTRODUCTION TO MICROORGANISMS The human infectious diseases caused mainly by five major groups of organisms: bacteria, viruses, fungi, protozoa and helminthes. Microorganisms are either prokaryotes e.g. bacteria or eukaryotes e.g. fungi. Bacteria Bacteria (singular: bacterium) are relatively simple, single-celled (unicellular) organisms. Because their genetic material is not enclosed in a special nuclear membrane, bacterial cells are called prokaryotes, from Greek words meaning prenucleus. Prokaryotes include both bacteria and archaea. Viruses They are so small that can be seen with an electron microscope, and they are acellular (not cellular). Structurally very simple, a virus particle contains a core made of only one type of nucleic acid, either DNA or RNA. This core is surrounded by a protein coat, which is sometimes encased by a lipid membrane called an envelope. Fungi (singular: fungus) are eukaryotes (yū-kar_ē-ōts), organisms whose cells have a distinct nucleus containing the cell’s genetic material (DNA), surrounded by a special envelope called the nuclear membrane. True fungi have cell walls composed primarily of a substance called chitin. 24 Medical microbiology and immunology 25  Medical microbiology and immunology STRUCTURE OF BACTERIA SHAPE & SIZE OF BACTERIA Figure 1: different shapes of bacteria Shape: Bacteria are classified by shape into three basic groups as shown in fig. 1: 1- Cocci (round -shaped) but can be oval, elongated, or flattened on one side. 2- Bacilli (rod -shaped), 3- Spirochetes (spiral-shaped). 4- Pleomorphic: some bacteria are variable in shape and are said to be (many- shaped). The shape of a bacterium is determined by its rigid cell wall. Size: Most of bacteria range from 0.2 to 2.0 μm in diameter and from 2 to 8 μm in length. 26  Medical microbiology and immunology Figure 2: Different bacterial arrangement Arrangement: The arrangement of bacteria is important for identification of bacteria. The arrangement of bacteria is determined by the planes of division. For example, if cocci divide and remain attached to one another and remain in pairs are called (diplococci), those that divide and remain attached in chain-like patterns are called (Streptococci), and those that divide in multiple planes and form grape-like clusters called (Staphylococci). Bacilli divide only across their short axis. Most bacilli appear as single rods, called single bacilli. Diplobacilli appear in pairs after division and Streptobacilli occur in chains. Some have tapered ends, like cigars, others are oval and look so much like cocci that they are called coccobacilli. Spiral bacteria have one or more twists; they are never straight. Bacteria that look like curved rods are called vibrios as shown in fig 2. 27  Medical microbiology and immunology The External Structures to the Cell Wall Figure 3: External and internal structures of bacterial cell Among the possible structures external to the prokaryotic cell wall are the glycocalyx, flagella, axial filaments, fimbriae, and pili. a) Glycocalyx (meaning sugar coat): it is general term used for substances that surround cells. It is a viscous (sticky), gelatinous polymer that is external to the cell wall and composed of polysaccharide, polypeptide, or both. It is made inside the cell and secreted to the cell surface. Types: 1-If it is organized and firmly attached to the cell wall, the glycocalyx is described as a capsule as in fig.4. 2- If it is unorganized and only loosely attached to the cell wall, the glycocalyx is described as a slime layer fig.4. 28  Medical microbiology and immunology Figure 4: slime layer and capsule. Functions: A) It is one of the most important virulence factors for the organism. It protects pathogenic bacteria from phagocytosis by the cells of the host. Also it protects bacteria from antibacterial agents as complement, lysozymes and bacteriophages. B) The glycocalyx is a very important component of biofilms. An extracellular polymeric substance (EPS) that allow attachment of bacteria to human teeth, medical implants, water pipes, and even other bacteria. For example, Streptococcus mutans, an important cause of dental caries, attaches itself to the surface of teeth by a glycocalyx and use its capsule as a source of nutrition by breaking it down and utilizing the sugars when energy stores are low. b) Flagella Definition: They are long filamentous appendages that propel bacteria. 29  Medical microbiology and immunology Figure 5: Different distribution of flagella. Composition: A single type of protein called flagellin that differs in different bacterial species. The flagellins are highly antigenic (H-antigen). So, it is important for differentiation between serovars, or variations within a species. Functions: 1-It is one of the most important virulence factors for the organism. 2- It is important for motility towards nutrition and away from disinfectant. c) Axial Filaments - Spirochetes are a group of bacteria that have unique structure and motility. Spirochetes move by means of axial filaments, or endoflagella, which are bundles of fibrils that arise at the ends of the cell beneath an outer sheath and spiral around the cell. -Axial filaments have a structure similar to that of flagella. The rotation of the filaments produces a movement of the outer sheath that propels the spirochetes in a spiral motion. This type of movement is similar to the way a corkscrew moves through a cork as shown in fig.6. 30  Medical microbiology and immunology Figure 6: Axial filament. d) Fimbriae and Pili Definition: they are hair-like appendages that are shorter, straighter, and thinner than flagella as shown in fig. 3. Composition: These are consisting of a protein called pilin arranged helically around a central core, are divided into two types, fimbriae and pili, having very different functions. Functions: 1-They are used for attachment, by helping the bacteria to adhere to epithelial surfaces of the body. 2- Allowing the transfer of DNA from one31cell to another, a process called conjugation.  Medical microbiology and immunology The Cell Wall Definition: The cell wall of the bacterial cell is a complex, semi-rigid structure responsible for the shape of the cell. Functions: 1-The cell wall surrounds the underlying, fragile plasma (cytoplasmic) membrane and protects it and the interior of the cell from adverse changes in the outside environment. 2-To prevent bacterial cells from rupturing due to high internal osmotic pressure. 2- It also helps maintain the shape of a bacterium. 3- It is important for cell division. 4- It is responsible for the staining affinity of the bacteria. Composition and Characteristics The bacterial cell wall is composed of a macromolecular network called peptidoglycan. Peptidoglycan consists of a repeating disaccharide attached by polypeptides to form a lattice that surrounds and protects the entire cell. The disaccharide portion is made up of monosaccharides called N- acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). Alternating NAM and NAG molecules are linked in rows of 10 to 65 sugars to form a carbohydrate “backbone” (the glycan portion of peptidoglycan). Adjacent rows are linked by polypeptides (the peptide portion of peptidoglycan). 32  Medical microbiology and immunology Gram-Positive Cell Walls In most gram-positive bacteria, the cell wall consists of many layers of peptidoglycan, forming a thick, rigid structure. In addition, the cell walls of gram-positive bacteria contain teichoic acids, which consist primarily of an alcohol (such as glycerol or ribitol) and phosphate as shown in fig.7. The teichoic acids have several specialized functions a. Because of their negative charge (from the phosphate groups), teichoic acids may bind and regulate the movement of cations (positive ions) into and out of the cell. b. Has a role in cell growth, preventing extensive wall breakdown and possible cell lysis. c. Finally, teichoic acids provide much of the wall’s antigenic specificity and thus make it possible to identify gram-positive bacteria by certain laboratory tests 33  Medical microbiology and immunology Figure 7: Differences between Gram positive and Gram negative cell wall. Gram-Negative Cell Walls The cell walls of gram-negative bacteria consist of one or a very few layers of peptidoglycan and an outer membrane. The peptidoglycan is bonded to lipoproteins (lipids covalently linked to proteins) in the outer membrane and is in the periplasm, a gel-like fluid between the outer membrane and the plasma membrane. The periplasm contains a high concentration of degradative enzymes and transport proteins. The outer membrane of the gram-negative cell consists of lipopolysaccharides (LPS), lipoproteins, and phospholipids as shown in fig.7. 34  Medical microbiology and immunology The outer membrane has several specialized functions: a. Its strong negative charge is an important factor in evading phagocytosis and the actions of complement (lyses cells and promotes phagocytosis), two components of the defenses of the host. b. The outer membrane also provides a barrier to certain antibiotics (for example, penicillin), digestive enzymes such as lysozyme, detergents, heavy metals, bile salts, and certain dyes. c. Part of the permeability of the outer membrane is due to proteins in the membrane, called porins that form channels. Lipid A is the lipid portion of the LPS and is embedded in the top layer of the outer membrane. When gram-negative bacteria die, they release lipid A, which functions as an endotoxin. Lipid A is responsible for the symptoms associated with infections by gram-negative bacteria such as fever, dilatation of blood vessels, shock, and blood clotting. The O polysaccharide functions as an antigen and is useful for distinguishing species of gram-negative bacteria This role is comparable to that of teichoic acids in gram-positive cells. Acid-Fast Cell Walls These bacteria contain high concentrations (60%) of a hydrophobic waxy lipid (mycolic acid) in their cell wall that prevents the uptake of dyes, including those used in the Gram stain. The mycolic acid forms a layer outside of a thin layer of peptidoglycan. Fig8. 35  Medical microbiology and immunology Figure 8: Cell wall of acid-fast bacilli. Structures Internal to the Cell Wall We will now look inside the prokaryotic cell and discuss the structures and functions of the plasma membrane and components within the cytoplasm of the cell. The Plasma (Cytoplasmic) Membrane (or inner membrane) Is a thin structure lying inside the cell wall and enclosing the cytoplasm of the cell. The plasma membrane of prokaryotes consists primarily of phospholipids. Because they lack sterols, prokaryotic plasma membranes are less rigid than eukaryotic membranes except mycoplasma that lack of cell wall Structure The phospholipid molecules are arranged in two parallel rows, called a lipid bilayer each phospholipid molecule contains a polar head, composed of a phosphate group and glycerol that is hydrophilic (water-loving) and soluble in water, and nonpolar tails, composed of fatty acids that are hydrophobic (water- fearing) and insoluble in water. The protein molecules 36 in the membrane can be arranged in a variety of ways as shown in fig.9.  Medical microbiology and immunology Figure 9: Structure of cell membrane. Functions 1-It is a selective barrier through which materials enter and exit the cell. Large molecules (such as proteins) cannot pass through the plasma membrane, possibly because these molecules are larger than the pores in integral proteins that function as channels. But smaller molecules (such as water, oxygen, carbon dioxide, and some simple sugars) usually pass through easily 2- Plasma membranes are also important to the breakdown of nutrients and the production of energy. Often appear to contain one or more large, irregular folds called mesosomes 3) Synthesis of precursors of the cell wall, 4) Secretion of enzymes and toxins. 5) Responsible for respiration through the enzymes that present on it. So, it is a functional analogue of the mitochondria in eukaryotes. Cytoplasm For a prokaryotic cell, the term cytoplasm refers to the substance of the cell inside the plasma membrane (see Figure3). Cytoplasm is about 80% water and 37 contains primarily proteins (enzymes), carbohydrates, lipids, inorganic ions, and many low molecular-weight compounds. Cytoplasm is thick, aqueous,  Medical microbiology and immunology semitransparent, and elastic. The major structures in the cytoplasm of prokaryotes are a nucleoid (containing DNA), particles called ribosomes, and reserve deposits called inclusions. The Nucleoid The nucleoid of a bacterial cell (see Figure 3) usually contains a single long, continuous, and frequently circularly arranged thread of double-stranded DNA called the bacterial chromosome. This is the cell’s genetic information, which carries all the information required for the cell’s structures and functions. Unlike the chromosomes of eukaryotic cells, bacterial chromosomes are not surrounded by a nuclear envelope (membrane) and do not include histones. The nucleoid can be spherical, elongated, or dumbbell shaped. In addition to the bacterial chromosome, bacteria often contain small usually circular, double- stranded DNA molecules called plasmids. Ribosomes The sites of protein synthesis. Cells that have high rates of protein synthesis, such as those that are actively growing, have a large number of ribosomes. Ribosomes are composed of two subunits, each of which consists of protein and a type of RNA called ribosomal RNA (rRNA). Accordingly, prokaryotic ribosomes are called 70S ribosomes and those of eukaryotic cells are known as 80S ribosomes. The subunits of a 70S ribosome are a small 30S subunit containing one molecule of rRNA and a larger 50S subunit containing two molecules of rRNA as shown in fig.3. Inclusions Cells may accumulate certain nutrients when38they are plentiful and use them when the environment is deficient as shown in fig.3.  Medical microbiology and immunology Endospores When essential nutrients are depleted or bacteria exposed to bad environmental conditions, certain gram-positive bacteria, such as those of the genera Clostridium and Bacillus, form specialized “resting” cells called endospores Medical importance of spores: they can survive extreme heat, lack of water, and exposure to many toxic chemicals and radiation. The process of endospore formation within a vegetative cell takes several hours and is known as sporulation or sporogenesis. Sporogenesis: 1-In the first observable stage of sporulation, a newly replicated bacterial chromosome and a small portion of cytoplasm are isolated by an ingrowth of the plasma membrane called a spore septum. 2-The spore septum becomes a double-layered membrane that surrounds the chromosome and cytoplasm. This structure, entirely enclosed within the original cell, is called a forespore. 3-The endospore contains a large amount of an organic acid called dipicolinic acid (DPA), which is accompanied by a large number of calcium ions. Evidence indicates that DPA protects the endospore DNA against damage. 4-The highly dehydrated endospore core contains only DNA, small amounts of RNA, ribosomes, enzymes, and a few important small molecules. 5-The spore has no metabolic activity and can remain dormant for many years as shown in fig 10. 39  Medical microbiology and immunology Figure 10: Formation of bacterial spores. - The spores are highly resistant to: Disinfectants, Drying and heating BUT only moist heat at 121 ˚C for 10 -20 minutes is able to kill spores. N.B.: The position and shape of spores are characteristic of the species and may help in the microscopic identification of the bacterium. 40 Medical microbiology and immunology Figure 11: Shape and position bacterial spores. 41  Medical microbiology and immunology BACTERIAL GROWTH AND PHYSIOLOGY Bacterial growth means an increase in bacterial numbers and size of the individual cells. Bacteria normally reproduce by binary fission. A few bacterial species reproduce by budding; they form a small initial outgrowth (a bud) that enlarges until its size approaches that of the parent cell, and then it separates. This can be detected by: 1- When bacteria cultured on a solid media many colonies can be seen by naked eyes, these colonies represent about 107- 108 cells/colony. 2- Change of clear fluid media to a turbid suspension Generation Time The time required for a cell to divide (and its population to double). It varies considerably among organisms and with environmental conditions, such as temperature. Most bacteria have a generation time of 20 min to 3 hours; others require more than 24 hours per generation. The reproduction process by binary fission occurs as follow: 1-The bacteria grows in size then elongated 2-The bacterial chromosome acts as a template to make another copy and each copy attached to a mesosome on the cytoplasmic membrane 3- A transverse septum formed across the cells from cytoplasmic membrane and cell wall that divide the cell into two equal parts. The requirements for microbial growth can be divided into two main categories: 42 1-Physical requirements include temperature, pH, and osmotic pressure.  Medical microbiology and immunology 2-Chemical requirements include sources of carbon, nitrogen, sulfur, phosphorus, oxygen, trace elements, and organic growth factors. 1. Physical Requirements a) Temperature: Most microorganisms grow well at the temperatures that human favor 37°C. However, certain bacteria are capable of growing at extremes of temperature. Microorganisms are classified into three primary groups on the basis of their preferred range of temperature as shown in fig. 12: Psychrophiles (cold-loving microbes), from 0-8 (optimum 4°C). Mesophiles (moderate-temperature–loving microbes), from10-50 (optimum 39°C). Thermophiles (heat-loving microbes) 40-70 (optimum 60°C). Figure 12: Classification of microorganisms by temperature requirements. The peak of the curve represents optimum growth (fastest reproduction). Notice that the reproductive rate drops off very quickly at temperatures only a little above the optimum. At either extreme of the temperature range, the reproductive rate is much lower than the rate at the optimum temperature b) pH: Most bacteria grow best in a narrow pH range near neutrality, between pH 6.5 and 7.5. Very few bacteria grow at 43 an acidic pH 4 or as lactobacilli. Some bacteria are growing better at an alkaline pH (8-9) as V. cholerae.  Medical microbiology and immunology c) Osmotic Pressure: Microorganisms obtain almost all their nutrients in solution from the surrounding water. Thus, they require water for growth, and their composition is 80–90% water. High osmotic pressures have the effect of removing necessary water from a cell. When a microbial cell is in a solution whose concentration of solutes is higher than in the cell (the environment is hypertonic to the cell), the cellular water passes out through the plasma membrane to the high solute concentration. This osmotic loss of water causes plasmolysis, or shrinkage of the cell’s cytoplasm. 2-Chemical Requirements A) Carbon: Besides water, one of the most important requirements for microbial growth is carbon. Carbon is the structural backbone of living matter; it is needed for all the organic compounds that make up a living cell. Some species called capnophilic require higher concentration (5-10%) of carbon more than present in air. B) Oxygen: According to O2 requirements, bacteria are classified into: a- Strict or obligate aerobes: require oxygen for growth as Pseudomonas aeruginosa. b- Strict or obligate anaerobes: require complete absence of oxygen.as Bacteroides fragillis c- Faculative anaerobes: grow better in presence of oxygen but still able to grow in its absence (most of bacteria). d-Aerotolerant anaerobes: have an anaerobic pattern of metabolism but can tolerate oxgyn as it contains superoxide dismutase as Cl. Perfringens. e-Micro-aerophilic: require reduced oxygen level as H.pylori as shown in fig. 13. 44  Medical microbiology and immunology Figure 13: Classification of microorganisms by oxygen requirements. C) Nitrogen, Sulfur, and Phosphorus: In addition to carbon, microorganisms need other elements to synthesize cellular material. For example, protein synthesis, syntheses of DNA and RNA require considerable amounts of nitrogen as well as some sulfur and phosphorus. D) Organic Growth Factors: Essential organic compounds an organism is unable to synthesize are known as organic growth factors; they must be directly obtained from the environment. One group of organic growth factors for humans is vitamins. Many bacteria can synthesize all their own vitamins and do not depend on outside sources. However, some bacteria lack the enzymes needed for the synthesis of certain vitamins, and for them those vitamins are organic growth factors. Other organic growth factors required by some bacteria are amino acids, purines, and pyrimidines. Growth curve of bacteria If a small number of bacteria are placed in45 a suitable growth media under appropriate physical and chemical conditions, then the number of viable cells is  Medical microbiology and immunology measured per milliliter, a characteristic growth curve with four phases is plotted follow fig. 14: (1) The lag phase: during which vigorous metabolic activity occurs but cells do not divide and no change occur in bacterial number. The bacteria adapt to their new environment, this can last for a few minutes up to many hours. (2) The log (exponential) phase: during which rapid cell division occurs. There is marked increase in the cell number and its rate is accelerated exponentially with time giving a characteristic linear plot in a growth curve. (3) The stationary phase: occurs when nutrient depletion or toxic products cause growth to slow until the number of new cells produced balances the number of cells that die, resulting in a steady state. The number of viable cells remains constant. (4) The death phase (decline): which is marked by a decline in the number of viable bacteria due to exhaustion of nutrient and accumulation of toxic metabolities, so the number of viable bacteria decreases. Figure 14: Bacterial growth curve. 46  Medical microbiology and immunology BACTERIAL PATHOGENESIS Infection: is the invasion or colonization of the body by pathogenic microorganisms; most of infections end without occurrence of pathological changes and manifestation which is called subclinical, silent or abortive infections. Disease: is an abnormal state in which part or all of the body is not properly adjusted or incapable of performing its normal functions and this appears as a form of sign and symptoms on the individual. The outcome of infection depends on the relation between the pathogen and the host as follow: 1-Pathogenic bacteria: bacteria that is capable of causing disease. 2-Non-pathogenic (commensals): bacteria do not cause disease, and are a part of normal flora. 3-Opportunistic pathogens: potentially pathogenic, do not cause disease under normal conditions but can cause disease in immunocomporomised patients or when they find their way to another site other than their normal habitat. Stages of infectious process: A) Source of infection: man (case or carrier), animal, environment. B) Mode of transmission: ingestion, inhalation, injection, contact, vector. C) Portals of Entry: mucous membranes, skin, and direct deposition beneath the skin or membranes (the parenteral route). D) Portal of exit: urine, stool, blood, secretions fig.22. 47  Medical microbiology and immunology Figure 22: Stages of infectious process. Pathogenicity: The ability of the bacteria to produce disease. Virulence: is a quantitative measure of pathogenicity (degree of pathogenicity) and is measured by the number of organisms required to cause disease. Mechanisms of pathogenicity: 48  Medical microbiology and immunology MECHANISM EXAMPLE 1- Adherence: Almost all pathogens have some means of Glycocalyx as present in S.epidemidis and attaching themselves to host tissues at their portal of S.viridans that allow it to attach strongly to entry, this attachment, called adherence (or adhesion), heart valves it is a necessary step in pathogenicity. A 2- Invasion: as some bacteria produce extracellular Hyaluronidase and collagenase are other enzymes that allow its invasion to different tissues. enzymes secreted by Streptococci. It hydrolyzes hyaluronic acid and collagen 3- Antiphagocytic factors: Capsules: protect the bacteria from phagocytosis by phagocytic cells as S.pneumoniae that cause pneumonia and meningitis in children 4- Biofilm formation: Biofilms are sticky, surface-attached agglomerations of bacteria that are embedded in an extracellular matrix and provide protection from antibiotics and phagocytosis. 5-Toxin production: Toxins are poisonous substances that are produced by certain microorganisms. Toxins transported by the blood or lymph can cause serious, and sometimes fatal, effects as fever and shock. 49  Medical microbiology and immunology Differences between exotoxin and endotoxin N.B: exotoxin genes encoded on chromosome, plasmid, bacteriophages or Pathogenicity Island (PAI), while endotoxins genes encoded on chromosome as they are a part of cell wall. Exotoxin converted to toxoid by removing its toxicity and retains its antigenicity so can be used in immunization (diphtheria vaccine). 50  Medical microbiology and immunology Chapter (6) GENERAL MYCOLOGY The study of fungi is called mycology. A fungus is a member of a large group of eukaryotic organisms, they are normally harmless to humans, fungi can be opportunistic pathogens. Because fungi (yeasts and molds) are eukaryotic organisms, whereas bacteria are prokaryotic, they differ in several fundamental respects as follow: 51  Medical microbiology and immunology STRUCTURE & GENERAL FEATURES There are two types of fungi: 1- Yeasts: Yeasts grow as single cells that reproduce by asexual budding. 2- Molds: Molds grow as long filaments (hyphae) and form a mat (mycelium). Some hyphae form transverse walls (septate hyphae), whereas others do not (non-septate hyphae). Nonseptate hyphae are multinucleated (coenocytic). Several medically important fungi are dimorphic: ▪ They exist as molds in the environment at ambient temperature ▪ Yeasts (or other structures) in human tissues at body temperature. Most fungi are obligate aerobes; some are facultative anaerobes; but none are obligate anaerobes. All fungi require a preformed organic source of carbon. The natural habitat of most fungi is the environment. An important exception is Candida albicans, which is part of the normal human flora. Some fungi reproduce sexually by mating and forming sexual spores (e.g., zygospores, ascospores, and basidiospores). Most fungi of medical interest propagate asexually by forming conidia (asexual spores)from the sides or ends of specialized structures. 52  Medical microbiology and immunology PATHOGENESIS 1-The response to infection with many fungi is the formation of granulomas. It is a cell-mediated immune response. Activation of the cell-mediated immune system results in a delayed hypersensitivity skin test response to certain fungal antigens injected intradermally. 2-Acute suppuration, characterized by the presence of neutrophils in the exudate. CLASSIFICATION 1-Morphological classification: A) Moulds B) Yeast C) Dimorphic 53  Medical microbiology and immunology 2- Clinical classification: A) Fungal Infections (Mycotic infections): that classified according to the layer of skin affected into: Superficial mycoses: affect the keratinized part of the skin. Cutenious mycoses: affect deep layer of the skin Subcutaneous mycoses: affect the subcutaneous layer of the skin during trauma Deep(systemic)mycoses: affect internal organ, if it is a true pathogenic fungi so affect healthy individual, while in immunocompromised patient it is caused by opportunistic fungi. B) Fungal allergies: occurs by the spores of fungi causing asthma or urticarial. C) Mycotoxicosis: that produced by potent toxins produced as aflatoxin. 54  Medical microbiology and immunology Chapter (7) Sterilizations & Disinfections Decontamination It is a general term that applied to any procedures by which pathogenic micro- organisms are reduced to a level where items are safe to handle. It includes: cleaning, disinfection or sterilization. Cleaning It is a process that removes visible foreign material (e.g. soil, dust, dirt, organic material, microorganisms) from an object. Using water, soap or detergent. So this process must precede disinfection and sterilizations. Sterilization It is the process of removing or destroying all microorganisms including bacterial spores. These procedures include destruction of microorganisms by heat, certain chemicals, irradiation and filtration. Disinfection It is a process of removing or destroying of most but not all living micro- organisms excluding spores. In practice, the term disinfection usually implies the use of antimicrobial chemicals. Those used for disinfecting inanimate objects are called disinfectants, while those used on skin are called antiseptics. Antiseptics Special types of chemical disinfectants (alcohol) that not toxic not irritant so can be safely applied to the skin & mucous membranes. This disinfectants or antiseptics may be:- Bactericidal: killing the micro-organisms completely (irreversible). Bacteriostatic: inhibit multiplication of micro-organisms but not killing it (reversible). 55  Medical microbiology and immunology Disinfection *There are 3 levels of disinfectant; -HLD → Destroy all vegetating bacteria, MTB, fungi, all viruses except large number of spores. Ex. Oro-phthalaldehyde (OPA) for endoscopes, hydrogen peroxide for contact lens and chlorine for blood spills. -ILD→ Destroy all vegetating bacteria, MTB, majority of fungi, majority of viruses except spores even with prolonged exposure. Ex. Alcohol and iodophors. -LDL→ Destroy vegetating bacteria except MTB, some fungi, some viruses. EX. Quaternary ammonium. *Selection of an adequate level of decontamination Critical Semi-critical Non-critical item come in contact item come in contact item come in contact with sterile tissues, with intact mucous with skin cavities,vascular membrane system needles, implants, e.g. endoscopes, sphygmomanometers, catheters, surgical endotracheal tube, bed linens, floors instrument thermometer Sterilization High level Intermediate and low disinfectant: level disinfectants: alcohols, phenol 56  Medical microbiology and immunology Methods of disinfection 1- Chemical disinfectants 2- Boiling water 3- Pasteurization 4- Ultraviolet irradiation Used for sterilization of laboratory safety cabinets, drug factories, operating theater. 1) Commonly used chemical disinfectants Disinfectant Use Alcohol 70% Thermometers, stethoscopes. (at least 5 min) Skin antiseptics Biguanides (chlorhexidine) mouth wash Combined with detergents for hand washing with alcohol as a handrub. Chlorine-active compounds Blood splashes and laboratory working surfaces. Linen bleaching. Disinfection of water for domestic use. Iodophores e.g. tincture iodine Antiseptic purposes Phenol and Quaternary ammonium Cleaning of the floors, walls, furniture. Hydrogen peroxide Disinfection of the soft contact lenses Endoscopes. Antiseptic for open wounds. Peracetic acid Disinfection of the soft contact lenses endoscopes. Glutaraldehyde High level disinfection or sterilization of instruments (endoscopes, respiratory and anaesthesia equipments). 2- Boiling Water Boiling at 100°C for 20 minutes: achieves high level disinfection. 57 It can be useful in emergencies if no sterilizer is available.  Medical microbiology and immunology 3- Pasteurization of milk: Heating at 63°C for 30 min. or at 72°C for 20 sec., followed by rapid cooling, destroys important pathogenic organisms e.g. Mycobacterium tuberculosis, Brucella, Salmonella and Coxiella (milk borne). 4-Ultraviolet Radiation (UV) low-energy, non-ionizing radiation present in sun rays or artificially produced by mercury lamps. It has extremely weak penetration power and used only for air and surface disinfection such as operating rooms, laboratory safety cabinets. Sterilization I. Dry heat: Incineration, red heat and hot air oven. II. Moist heat or steam sterilization. III. Low temperature ("Cold") sterilization methods: a- Hydrogen peroxide 6%: For: metal and surgical instrument. b- Liquid peracetic acid. c- Glutaraldehyde (contact time 10 hrs). d- Ethylene oxide gas: For plastic and rubber articles, syringes IV. Other sterilization methods: Ionizing radiation. Filtration. Microwaves; used in pharmaceutical industries. 58  Medical microbiology and immunology I. Dry Heat Sterilization Mode of action: By oxidation of essential cell constitutes. The sterilizing agent is dry hot air. 1) HOT AIR OVEN: -It is a double walled chamber that is heated by electricity. -Used for: material that can withstand high temperatures as glassware, tubes, flasks and syringes. -Temperatures: 170ºC for 60 minutes, or 160ºC for 120 minutes. -Advantages: non-toxic, non-corrosive, inexpensive. - Disadvantages: 1- Slow rate of heat penetration and time consuming 2- High temperatures not suitable for some materials 2) Red heat For: loops, tip of forceps, scalpel, mouth of tubes and points of forceps are sterilized by holding them in the flame until they are red hot. 3) Incinerations For: dead animal bodies, hospital waste as needles, surgical dressings II. Steam Sterilization 59 Autoclave: saturated water steam under high pressure.  Medical microbiology and immunology Mode of action: By coagulation and denaturation of enzymes and structural proteins. Sterilization temperature and exposure time: at 121°C for 20-30 minutes at double atmospheric pressure (2 bar), or at 134°C for 3-6 minutes (at 3 bar). Examples of Items: culture media, surgical instruments & dressings. Advantages: 1-Non-toxic 2-Inexpensive 3- Rapidly heat and Penetrate 60 Disadvantages: 1- Corrosive  Medical microbiology and immunology 2- High temperatures not suitable for some materials Monitoring of steam sterilizers (autoclaves) Mechanical indicators: using a graph that monitors the time, temperature and pressure. Chemical indicators: chemically impregnated paper strips which change their color at certain temperature e.g. 121°C or 132°C, should be used with each cycle of sterilization. Biological indicators: are paper strips containing the spores of Bacillus stearothermophilus. The biological indicators are placed in the depth of the load to be sterilized. After finishing the cycle of sterilization, spore strips are incubated in a fluid medium and checked for viability. Absence of bacterial growth indicates an efficient sterilization cycle. III. Low temperature ("Cold") sterilization methods: A- Chemical: 1-Ethylene oxide gas. 2-Liquid sterilization process: a- Hydrogen peroxide 6%. b- Liquid peracetic acid. c- Glutaraldehyde (contact time 10 hrs). B- Plasma sterilizers: Plasma describes any gas that consists of electrons, ions, or neutrons. Plasma sterilizers with the use of liquid peracetic acid, or hydrogen peroxide, or a mixture of both are commercially available. 61  Medical microbiology and immunology Advantages of cold sterilization: 1. Very effective 2. Penetrates medical packaging and many plastics 3. Cycle easy to control and monitor Disadvantages 1. Toxic (e.g ethylene oxide gas is highly toxic) 2. Time consuming 3. Expensive Monitored: B. subtilis spores, B. stearothermophilus. IV. Others: A.Sterilization by Irradiation *Two types of ionizing radiation used for sterilization: I. γ (gamma) rays: which are emitted by radioactive elements such as cobalt-60. II. (β-rays) High-energy electrons: which are produced by electron accelerators. *Ionizing radiation has a high penetrating power *USES: sterilization of pre-packed heat-sensitive items such as: bone grafts, surgical sutures, disposable plastic syringes, gloves, catheters, plastic Petri dishes & intravenous (IV) infusion sets. 62 B.Sterilization by Filtration  Medical microbiology and immunology 1-Sterilization of fluids which would not withstand heat such as: antibiotic solutions, blood products, hormones, vitamins….etc. Fluids can be rendered free of bacteria by passage through filters with a pore size of less than the smallest microbial size. 2-Filters can be used to remove microorganisms from air supplied to critical areas such as operating rooms and drug factories. E.g. High Efficiency Particulate Air (HEPA) filter. 63  Medical microbiology and immunology Chapter (8) Infection control Infection control (IC) is the practices that lead to detection and reduction of risks of acquiring and transmitting infections among patients and healthcare workers (HCWs). Who are Concerned? All who deal with patients: – Physicians, – Nurses, – Physical therapists, – Laboratory personnel, – Phlebotomists, – Radiological technologists, – Dentists High risk areas that need strict supervision by IC team: 1- All types of adult and pediatric intensive care units (ICUS). 2- Operating rooms (OR). 3- The Central Supply and Sterilizing Departments (CSSD). 4- Emergency room (ER). 5- Outpatient department (OPD). 6- Hemodialysis or renal dialysis unit (RDU). Infection control practices to prevent exposure to and transmission of infections 1) Hand hygiene is the most effective method to prevent co-transmission of HCWs 2) Isolation precautions include: A- Standard precautions B- Transmission based precautions 3) Preparation of infection control (IC) policies 4) Education and training of HCWs, patients, family regarding hand hygiene and isolation precautions 5) Use surveillance data to monitior performance of IC practices 6) Management of infection outbreaks and epidemics 64  Medical microbiology and immunology Isolation Precautions – These are barriers that are used to prevent transmission of the infectious organisms among patients & HCWs. – Isolation precautions are to segregate the organism and not to isolate the patient. Categories of the Isolation Precautions I. Standard precautions II. Transmission based precautions 1. Standard Precautions Designed to prevent the transmission of the blood-borne pathogens (HBV, HCV, HIV and other organisms, from patients to HCW & other patients. Should apply to all patients in all situations at all times irrespective of diagnosis, when contact is anticipated with blood, body fluids (secretions and excretions), non-intact skin & mucous membranes of patients Standard precautions include: 1) Hand hygiene (the single most important measure). 2) Use of personal protective equipment (PPE): gloves, masks. 3) Respiratory hygiene and cough etiquette 4) Cleaning, disinfecting and sterilization of patient care equipment. 5) Linen management and environmental cleaning. 6) Proper disposal of infectious hospital waste. 7) Prevent needle prick or injuries by other sharps. 8) Occupational safety and monitoring staff health. 65 2. Transmission based Precautions  Medical microbiology and immunology These precautions should be implemented after provisional or definitive diagnosis of the infective agent. They should be applied to patients according to the mode of transmission of their infections. In addition to the transmission based precautions, the standard precautions must be continued in all conditions. Transmission based precautions include: a. Airborne precautions. b. Droplet precautions. c. Contact precautions. Type of Indications Important precaution practices precautions Airborne Patients with Patients isolated in precautions Pulmonary TB , negative pressure single chickenpox and room measles Caring staff and visitors must use N95 mask Droplet Patients with Patient placed in precautions meningitis, mump, ordinary single room rubella Caring staff must use surgical mask upon room entry Contact Patients infected Patient placed in precautions with MRSA or ordinary single room other multidrug Caring staff must use resistant organism Gown and gloves upon Patients with room entry dirrhoea 66  Medical microbiology and immunology STANDARD PRECAUTIONS 1-Hand Hygiene Hand hygiene is the simplest & most effective measure to prevent infection. Rationale: To remove microbial contamination acquired by recent contact with infected / colonized patients or environmental sources. We should clean our hands as the most common ways of spreading diseases are the 10 fingers. Skin Flora include Resident organisms Transient organisms In deeper layers of skin In superficial layers of skin Permanent flora Temporary flora If disturbed reestablish Usually do not reestablish themselves themselves Not removed by routine Easily removed by routine hand wash but by hand wash antimicrobial agents Usually not associated Usually associated with with transmission of transmission of infection infection *Hand hygiene is done: 1. Before touching a patient 2. Before aseptic techniques 3. After touching a patient 4. After touching blood, body fluids or items contaminated with them 5. After touching the patient surrounding 6. After removing gloves Fiure12: 67  Medical microbiology and immunology 2-Personal protective equipment (PPE) e.g. gloves, masks, aprons, goggles 3-Respiratory hygiene and cough etiquette: To contain respiratory secretions and prevent transmission of respiratory pathogen. Person suffering acute respiratory illness e.g. flu is instructed to: A- Cover mouth and nose when sneezing\ coughing with tissue or mask with disposal in waste basket B- Wash hand or rub with alcohol 4-Safe Waste Disposal Types of Waste: 1. General waste (or non-hazardous waste): including waste that poses no risk of infection e.g. paper, boxes, hand towels. 2. Infectious waste (hazardous waste) including: - e.g. Contaminated dressings, used syringes. Guidelines for Proper Waste disposal: Infectious waste should not be mixed with general waste. Bags must not be overfilled. Personnel must wear heavy duty gloves while handling waste bags. Black bags for general waste. Leak -proof red or orange bags for infectious waste. Needles / other sharps disposal: All needles, other sharp items must be disposed of immediately in appropriate puncture-resistant containers with a lid. 68 5- Linen management and environmental cleaning Linen management  Medical microbiology and immunology Careful handling and reprocessing of soiled linens prevents the transmission of infectious agents. Environmental Cleaning Environmental surfaces should be cleaned and disinfected regularly at least daily wearing heavy-duty gloves. Cleaning of walls, floors, and surfaces should be done by damp cloth or wet mop (wet cleaning). Cleaning up spills of blood and body fluids: - Always wear gloves e.g. latex or heavy duty gloves. - Whip it with a disposable cloth and then disinfect the surface area of the spill with another disposable cloth that has been saturated with a disinfectant (0.5-1 % chlorine solution). 6- Prevent needle prick or injuries by other sharps. Safe injection practices includes the following 7 steps: STEP 1: Clean work space. STEP 2: Hand hygiene. STEP 3: Using Sterile injection equipment , with re-use prevention and injury protection feature whenever possible. STEP 4: Sterile vial of medication. STEP 5: Skin disinfection. STEP 6: Appropriate collection of sharps. STEP 7: Appropriate waste management. 69 Needles / other sharps disposal:  Medical microbiology and immunology All needles, other sharp items must be disposed of immediately in appropriate puncture-resistant containers with a lid. Prevention of needle sticks and other sharps-related injuries: (1) (2) (3) Fig. (15) One hand scoop technique for recapping used needles Never recap used needles, if necessary to recap needles use either a one-handed "scoop" technique (Fig.). Do not remove used needles from disposable syringes by hand, do not bend, or break. 7- Healthcare workers occupational safety 1- Immunization of health care personnel 2- Prevent occupational exposure of healthcare workers HCWs to infectious agent mainly Bloodborne Pathogens by implanting the infection control policies and practices 3- Post exposure prophylaxis (PEP): Provide immediate post-exposure medical evaluation and follow-up to exposed employee. 1- Immunization of health care personnel (NB. For immunization revise vaccination) 2- Prevent occupational exposure of HCWs to infectious agent 70  Medical microbiology and immunology Healthcare workers have a high risk of occupational exposure, with high incidence of blood borne diseases and prevalence of unsafe practices. Among the various blood borne diseases, the most common and important ones are HIV infection, hepatitis B, and hepatitis C (Risk of infection following a needle stick or cut from a positive (infected) source: HBV: 30%, HCV: 3% , HIV: 0.3%) Prevent occupational exposure of healthcare workers HCWs to infectious agent mainly Bloodborne Pathogens by implanting the infection control policies and practices as: Using PPE (gloves , mask, …) Hands and other skin surfaces should be washed immediately and thoroughly if contaminated with blood or other body fluids. Avoiding unnecessary use of needles and other sharps Don’t bend, recap, or remove needles or other sharps Discarding needles and sharps in pucture resistant container Not holding patient tissue with fingers when suturing Cleaning blood spills by using household bleach 3- Post exposure prophylaxis (PEP): Provide immediate post-exposure medical evaluation and follow-up to exposed employee: At no cost Confidential Testing for HBV, HCV, HIV Preventive treatment when indicated 71  Medical microbiology and immunology 1- Recommended PEP for exposure to HBV Person exposed to HBs Ag positive source: give PEP according to vaccination status of exposed workers Vaccination and antibody response Source: HBsAg positive status of exposed workers Give one dose of Hepatitis B immunoglobulin HBIG Unvaccinated Initiate HB vaccine series Previously vaccinated ( known responder) No treatment - Previously vaccinated Give 2 dose of HBIG (known non-responder) Incomplete vaccination Give one dose of HBIG Complete vaccination 2- Follow-Up of HCWs Occupational Exposure to Hepatitis C Virus Perform baseline and follow-up testing for anti-HCV and alanine aminotransferase (ALT) 4 – 6 months after exposure. Perform HCV RNA at 4 – 6 weeks if earlier diagnosis of HCV infection desired. Post-exposure prophylaxis (PEP) not recommended. 3- Recommended PEP for exposure to HIV Person exposed to HIV positive source: Give anti HIV drugs according to risk of infection Regimen used 1- Basic (2 drug) regimen for low risk exposure 72 Low risk exposure: Minimal percutaneous injuries or mucosal exposure to small volume of blood from HIV patient without  Medical microbiology and immunology AIDS symptoms 2 drugs: Lamivudine and Zidovudine 2- Expanded (3drug) regimen for high risk exposure High risk exposure: A- Severe percutaneous injuries or mucosal exposure to large volume of blood from HIV patient without AIDS symptoms B- AIDS patient with any degree of percutaneous injuries or mucosal exposure 3drugs: Lamivudine , Zidovudine and Indinavir (or other protease inhibitors) Microbiologic Investigations Related to Infection Control in Hospitals 1. Routine monitoring: Spore test for monitoring the sterilization processes in the hospital (see chapter on sterilization). Cultures of haemodialysis water. 2. Occurrence of outbreaks: Examples include outbreaks of surgical site infections and MRSA outbreaks in ICU. Microbiologic sampling and cultures from patients, HCWs or hospital environment are useless and recommended to be stopped. 3. Strain typing: In the epidemiologic studies of outbreaks e.g. hospital acquired surgical site infections by: Colony morphology Biotype profiles Phage typing Plasmid analysis Ribotyping Chromosomal analysis and PCR. 73  Medical microbiology and immunology Chapter (9) STREPTOCOCCUS Important Properties 1. Gram-positive cocci, arranged in chains or pairs. 2. Catalase negative (D.D. staphylococci). 3. Growth requires enriched media containing blood or serum. Classification of Streptococci 1. Lancefield groups. Species-specific CHO cell wall antigen Specific antibodies are used for identification of different species.Lancefield group antigens are A – H, K-W (A-W except I & J) 2. Haemolytic reactions on sheep blood agar. 3. Colony size. 4. Biochemical reactions. 5. DNA- typing Diseases Streptococci cause a wide variety of infections. 1- S. pyogenes (group Astreptococcus) is the leading bacterial cause of pharyngitis and cellulitis. It is an important cause of impetigo necrotizing fasciitis, and streptococcal toxic shock syndrome. It is also the inciting factor of two important immunologic diseases, namely, rheumatic fever and acute glomerulonephritis. 2- Streptococcus agalactiae (group B streptococcus) is the leadingcause of neonatal sepsis and meningitis. 74 3- Enterococcus faecalisis an important cause of hospital-acquired urinary tract infections and endocarditis.  Medical microbiology and immunology 4- Viridans group streptococci are the most common cause of endocarditis Transmission Most streptococci are part of the normal flora of the human throat, skin, andintestines but produce disease when they gain access to tissues or blood. Acu Acute rheumatic fever Approximately 2 weeks after a group A streptococcal infection usually pharyngitis (not skin infection) by (rhumatogenic strain): M types 1, 3, 5, 6 and 18 Pathogenesis: - Rheumatic fever is due to an immunologic reaction between cross- reacting antibodies to certain streptococcal M proteins and antigens of joint, heart, and brain tissue. It is an autoimmune disease, greatly exacerbated by recurrence of streptococcal infections. - Rheumatic fever, characterized by fever, migratory polyarthritis, and carditis. Uncontrollable, spasmodic movements of the limbs or face (chorea) may also occur. - ASO titers and the erythrocyte sedimentation rate are elevated. - If streptococcal infections are treated within 8 days of onset, rheumatic fever is usually prevented. Diagnosis By modified Jones criteria which include evidence of recent S. pyogen infection with two major criteria, or one major and two minor criteria 75  Medical microbiology and immunology Evidence of recent Major criteria Minor criteria infection - History of acute - Carditis - Clinical: fever tonsillitis - Chorea - Laboratory: elvated - Elevation of ASO -Migratory polyarthritis ESR and positive C titre above 200 units - Erythema annulare reactive protein - Subcutanous nodules Symptom of rheumatic fever e.g Erythema annulare and Subcutanous nodules Prevention: Reinfection must be prevented by long-term prophylaxis by long acting penicillin 76  Medical microbiology and immunology Chapter (10) OSTEOMYELITIS Definition An infection of the bone. Osteo refers to bone, Myelo refers to the bone marrow. Classified as either acute or chronic Pathophysiology ▪ Most commonly by hematogenous spread (i.e., either bacteremia or fungemia) ▪ Acute bacterial osteomyelitis often arises from a pyogenic skin infection as a boil ▪ Mycobacterial and fungal osteomyelitis often arise from the initial site of infection in the lung. ▪ By direct extension or following trauma that results in an open fracture and direct contamination of the bone. ▪ Chronic osteomyelitis occurs in the lower extremity ▪ In diabetics who have vascular insufficiency. ▪ Bone pain and localized tenderness Clinical Manifestations ▪ Constitutional symptoms such as fever, night sweats, and fatigue. ▪ Limited range of motion of affected extremity ▪ In acute osteomyelitis, the symptoms occur abruptly and progress rapidly ▪ In chronic osteomyelitis, the course is slower 77  Medical microbiology and immunology Pathogens Diagnosis ▪ Acute osteomyelitis: by culture of a specimen of the bone lesion. ▪ Blood cultures are positive in half of cases. ▪ Typical radiologic finding in acute osteomyelitis is a bone defect and periosteal elevation. ▪ Early in the disease, X-rays and even CT scans may be negative. ▪ Magnetic resonance imaging (MRI) scans are the most sensitive radiologic test Treatment Empiric therapy for acute osteomyelitis should include: 78  Medical microbiology and immunology ▪ Bactericidal drugs ▪ Penetrate well into bone ▪ Cover S. aureus. Vancomycin, nafcillin, or cephalexin parenterally Vancomycin is often used until culture and sensitivity results. Therapy ranges from 3 to 6 weeks or longer. Surgical debridement of chronic osteomyelitis lesions is often necessary. INFECTIOUS (SEPTIC) ARTHRITIS Definition An infection of the joints. Infectious and septic are used to distinguish it from immune-mediated arthritis, as rheumatoid arthritis. S. aureus causes the majority of cases of septic arthritis. Pathophysiology ▪ Organisms reach joint via bloodstream from a skin site. ▪ May enter joints through penetrating trauma, medical procedures such as arthroscopy, or an adjacent osteomyelitis. ▪ Patients with long-standing rheumatoid arthritis and those with prosthetic hips and knees are predisposed to infectious arthritis. Clinical picture ▪ Acute onset of an inflamed joint ▪ Large weight-bearing joint such as the hip or knee ▪ Fever On physical examination: ▪ The affected joint is red, warm, and swollen ▪ A joint effusion is typically present. ▪ Reluctance to use a joint, especially in a child, may be a sign Pathogens 79  Medical microbiology and immunology Diagnosis ▪ Gram stain of joint fluid to guide empiric therapy. ▪ Culture of joint fluid. ▪ Blood cultures are positive in less than 30% of cases. Radiology: ▪ Soft tissue swelling. ▪ Evidence of joint destruction can be seen if the infection progresses. Treatment ▪ Untreated: Joint destruction and loss of mobility ▪ Prompt antibiotic treatment is required ▪ Empiric therapy: Vancomycin, nafcillin, or cefazolin ▪ Ceftriaxone should be used if there is evidence of Neisseria gonorrhoeae ▪ Removal of joint fluid is important with antibiotics. 80  Medical microbiology and immunology Chapter (11) General virology Viruses are too small to be seen with a light microscope and cannot be cultured outside their hosts. Viruses differ from bacteria in different features as follow Structure of viruses A virion is a complete, fully developed, infectious viral particle composed of nucleic acid and surrounded by a protein coat outside of a host cell, and is a vehicle of transmission from one host cell to another. The nucleic acid genome and the capsid proteins are called the nucleocapsid as shown in fig.31. SIZE & SHAPE OF VIRUSES Size: viruses range from 20 to 300 nm in diameter. Cannot be seen by light microscope seen by electron microscope except poxviruses. Can pass through bacterial filters. Shape: The shape of virus particles is determined by the arrangement of the repeating subunits that form the protein coat (capsid) of the virus. 1- Capsid 81  Medical microbiology and immunology The nucleic acid of a virus is protected by a protein coat called the capsid (Figure 13.2a). Each capsid is composed of protein subunits called capsomeres. The arrangement of capsomers gives the virus structure its geometric symmetry as shown in fig 32. (1) Icosahedral symmetry, in which the capsomers are arranged in 20 triangles that form a symmetric figure (an icosahedron) with the approximate outline of a sphere. All DNA viruses except poxviruses (brick shaped) and some RNA are icosahedral. (2) Helical symmetry, in which the capsomers are arranged in a hollow coil that appears rod-shaped. Many RNA viruses are helical. (3) Complex symmetry, as brick shaped poxviruses and bacteriophage. Its functions are: a. Protection of nucleic acid against harmful factors b. Important for attachment to the host cell c. Responsible for viral symmetry 2- Envelope ▪ In some viruses, the capsid is covered by an envelope, which usually consists of some combination of lipids, proteins, and carbohydrates. ▪ In many cases, the envelope contains proteins determined 82  Medical microbiology and immunology by the viral nucleic acid and materials derived from normal host cell components. ▪ Envelopes may or may not be covered by spikes, which are carbohydrate-protein complexes that project from the surface of the envelope and may responsible of attachment of virus to the host cell. ▪ Viruses whose capsids are not covered by an envelope are known as non- enveloped viruses Its presence of an envelope confers instability on the virus. Enveloped viruses are more sensitive to heat, drying, detergents, and lipid solvents 3- Nucleic Acid It is responsible for virulence (it is the infectious part of the virus) In contrast to prokaryotic and eukaryotic cells, in which DNA is always the primary genetic material (and RNA plays an auxiliary role), Virus can have either DNA or RNA but never both. The nucleic acid of a virus can be single-stranded or double- stranded. The nucleic acid can be linear or circular ATYPICAL VIRUS-LIKE AGENTS (1) Defective viruses: are composed of viral nucleic acid and proteins but cannot replicate without a ―helper‖ virus, which provides the missing function. Defective viruses usually have a mutation or a deletion of part of their 83  Medical microbiology and immunology genetic material. Because some of these defective particles may interfere with the growth of the infectious particles, it has been hypothesized that the defective viruses may aid in recovery from an infection by limiting the ability of the infectious particles to grow. (2) Prions: are infectious particles that are composed of protein (i.e., they contain no detectable nucleic acid). They are implicated as the cause of certain ―slow‖ diseases called transmissible spongiform encephalopathies (e.g: Mad cow disease). Virus replication cycle Attachment The proteins on the surface of the virion attach to specific receptor proteins on the cell surface. The specificity of attachment determines the host range of the virus. Penetration The virus particle penetrates by being engulfed in a pinocytotic vesicle, within which the process of uncoating begins. Also can be fused by its envelop with the host cell membrane in enveloped viruses. Uncoating 84  Medical microbiology and immunology A low pH within the vesicle favors uncoating. Rupture of the vesicle or fusion of the outer layer of virus with the vesicle membrane deposits the inner core of the virus into the cytoplasm. Eclipse: it is the time from uncoating till assembly, during this period no infectious viruses can be detected in the host cell. In some viruses synthesis doesn't initiate and the virus remain dorminant or latent within the host cell for variable periods Synthesis of viral component The first step in viral gene expression is mRNA synthesis to synthesize the specific virus proteins. It is at this point that viruses follow different pathways depending on the nature of their nucleic acid and the part of the cell in which they replicate as shown in fig33. DNA viruses, with one exception, replicate in the nucleus and use the host cell DNA-dependent RNA polymerase to synthesize their mRNA. The poxviruses are the exception because they replicate in the cytoplasm, where they do not have access to the host cell RNA polymerase. The genome of all DNA viruses consists of double-stranded DNA, except for the parvoviruses, which have a single- stranded DNA genome. Most RNA viruses undergo their entire replicative cycle in the cytoplasm. The two principal exceptions are retroviruses and influenza viruses, both of which have an important replicative step in the nucleus. Retroviruses integrate a DNA copy of their genome into the host cell DNA, 85  Medical microbiology and immunology Influenza viruses synthesize their progeny genomes in the nucleus. In addition, the mRNA of hepatitis delta virus is also synthesized in the nucleus of hepatocytes. The genome of all RNA viruses consists of single-stranded RNA, except for members of the reovirus family, which have a double- stranded RNA genome. Rotavirus is the important human pathogen in the reovirus family. Once the viral mRNA is transcribed, it is translated using host ribosomes to synthesize viral proteins Assembly & Release The progeny particles are assembled by packaging the viral nucleic acid within the capsid proteins. This process occurs either in nucleus or cytoplasm. Virus particles are released from the cell by either of two processes. 1) Rupture of the cell membrane and release of the mature particles; this usually occurs with non-enveloped viruses. 2) The other, which occurs with enveloped viruses, is release of viruses by budding through the outer cell membrane Pathogenesis of viral disease Pathogenesis of viruses in the infected patient involves (1) Transmission of the virus and its entry into the host; 86  Medical microbiology and immunology transfer may either through inhalation, fecal contamination of water or food, contact, injection, vector born and between mother and offspring in utero across the placenta, at the time of delivery, or during breast feeding. (2) Replication of the virus and damage to cells; that may be as follow: (A) Death, (B) fusion of cells to form multinucleated cells, (C) Malignant transformation, (D) no apparent morphologic or functional change (1) Spread of the virus to other cells and organs; (2) then the virus fate differ as follow: Subclinical viral infection: in which infection occur without signs or symtoms Clinical infection or disease: there is a mild sign and symptoms appear on patients which may be local or systemic infections. Local infections Systemic infections Disease/ex Common cold Measles Site of pathology Portal of entry At distant sites 87  Medical microbiology and immunology Incubation Period Short Long Viraemia absent Present Duration of immunity Usually short Usually longer/lifelong Immunoglobulin involved secretory Ig A IgM, IgG Persistent viral infection (chronic): the virus continuously detected w