_.introduction to microbiology .level 1med_٠٢٣٢١٩.pdf

Introduction to Medical Microbiology Level 1 Dr/ Samira Hameed Hanash professor in medical microbiology and immunology faculty of medicine & health sciences Taiz university 2024 1 Introduction to Mi...

Introduction to Medical Microbiology Level 1 Dr/ Samira Hameed Hanash professor in medical microbiology and immunology faculty of medicine & health sciences Taiz university 2024 1 Introduction to Microbiology What's microbiology? The word Bio means living organisms and logy means study of. Thus biology is the study of living organisms. Micro means very small organisms that are viewed with the help of microscope. Microbiology:- Microbiology is the science which studies very small living organisms (they are called Microorganisms or microbes). Organisms included in Microbiology are: 1) Bacteria related to Bacteriology. 2) Viruses related to Virology. 3) Fungi related to Mycology. 4) Algae related to Phycology. 5) Parasites related to Parasitology. 6) Protozoan related to Protozology. Microbiology is divided in to: 1) Medical microbiology that is subdivided into: A) Medical bacteriology, and B) Medical Virology. Definition of Medical Microbiology Medical microbiology is the science that studies the pathogens, their pathogenicity, and the body defenses against that Microbes. A) Medical Bacteriology: 2  Medical bacteriology is the science that studies the pathogenic bacteria, their morphology (Size, shape, structure, biochemical reaction, pathogenicity and epidemiology). B) Medical Virology: Medical virology is the science that studies the Pathogenic Viruses and their morphology (Size, shape, structure, pathogenicity and epidemiology). 2) Food Microbiology: (environmental Microbiology) This field of microbiology become important because of its increase concern about the environment. This field is concerned with soil, air, water, sewage, and food. For example: it is concerned in the study of microbes which play an important rule in food manufacturing as yoghurt also concerned with producing, processing, cooking, serving of food as well as the prevention of food spoilage, food poisoning and food toxicity. In addition, the biodegradation of toxic chemicals by various microorganisms is being used as a new method for cleaning up hazardous materials found in soil and water. 3) Industrial microbiology: Many industries depend on the proper growth and maintenance of certain microbes to produce bear, wine alcohol and organic materials such as enzymes, Vitamins and antibiotics. 4) Sanitary microbiology: This science includes the processing and disposal of garbage and sewage wastes. 3 Milestones of microbiology are: - Robert Hooke, UK (1665). - Proposed the Cell Theory. - Observed cork with crude microscope. - All living things are composed of cells. Anton Van Leeuwenhoek -The Father of Microbiology: Anton was the first scientist who described bacteria in 1667 by his simple microscope with one magnifying lens. In 1798 – Jenner used the first vaccine and began a process that will led to the eradication of smallpox in 1970s. Louis Pasteur (1822-1895), Chemist - Fermentation (1857) - Pasteurization: heat liquid enough to kill spoilage bacteria (1864) - Vaccine development – rabies - Proposed the germ theory of disease - Proposed aseptic techniques (prevent contamination by unwanted microbes) Pasteur noted that the beer and wine are spoilage due to microbes that produce a specific change in the substance (fermentation). Such as Yeast Ferment sugar in grape Juice to Produce ethyl alcohol (ethanol) in Wine. Some contaminating bacteria such as Acetobacter may change the alcohol into acetic acid (cause spoilage of wine taste). To eliminate the harmful contaminating bacteria. Pasteur heated them to 50 C°- and to 60 C°. This process called pasteurization. Milk can be pasteurized by heating it to 63C° for 30 Minutes. Or to 72 C° for 15 seconds. Joseph Lister, UK (1867) used: - phenol (carbolic acid) to disinfect wounds 4  - First aseptic technique in surgery Robert Koch, DE (1876): - Postulated – Germ theory (1876) - Identified microbes that cause: anthrax (1876); tuberculosis (1882); and cholera (1883). - Developed microbiological media & streak plates for pure culture (1881). Did the isolated organism actually cause the disease. This is the problem that Koch faced in 1877. Koch’s Postulated that in (1877) - the specific causative agent must be found in every case of the disease. - the organism must be isolated from the lesions of the infected case and maintained in pure culture. - the pure culture, inoculated into a susceptible or experimental animal, should produce the symptoms of the disease. - the same organism should be re-isolated in pure culture from the intentionally infected animal. Eukaryotic and Prokaryotic The difference between Eukaryotic and prokaryotic is as follows. Eukaryotes means: Eu means true while Karyon means nucleus. Eukaryotes refers of organisms or body that have true nucleus and nuclear membrane.,while Prokaryotic includes Pro which means before and Karyon which means nucleus. 5 Prokaryotic cell prokaryotic Cells are morphologically simpler than Eukaryotes they are about ten times smaller than Eukaryotic cells. Table 1: Comparison between Prokaryotic and Eukaryotic Cells: Prokaryote as Eukaryotes (as Property bacteria) Human Cell) 1- Membrane – bound No Yes nucleus D N A 2- D NA associated with No Yes histones 3- Mitochondria Absent Present 4- Chromosome Number One More than one 70 s which 80 s which is exist 5- Size of ribosome exist freely in on the surface of cytoplasm R.E.R 6- Mitotic division No Yes 7- Cell wall containing Yes NO peptidoglycan Structure of the Bacterial Cell: Bacterial cell are small prokaryotic unicellular organisms that multiply by binary fission. Shape of Bacteria: There are three basic shapes of bacteria - Bacilli or rod. - Cocci (round.or Spherical). - Spirals 6  Some bacteria are variable in shape and are pleomorphic. Shapes of bacteria The shape of bacteria is determine by its rigid cell wall as shown in the figures below. Cocci (staphylococci) Cocci (streptococci) Spirochaetes Rods or bacilli 7  Branching filaments Chain streptococci Clusters (Chain streptococci) (Fig 1) Bacterial arrangement When the bacterial cell divides, the two doughter cells may separate or may remain attached to one another by the cell membrane which may occur in:- - Clusters. (staphylococci) - Pairs or diplococcal. (pneumococci) 8  - Chain (streptococci) Size of Bacteria:- Bacteria vary in size as much as in shape Bacteria range in size from about 0.1 m to 8m. They are smaller than other cells, but larger than viruses and are measured in units of length called micrometers. One millimeters is equal to 1,000 microns. Structure of the Bacterial Cell: Fig 2: Structure of the Bacterial Cell Bacteria cell wall:- It is the outer membrane layer, it's Multiple layerd structure located external to the cytoplasmic membrane. All bacteria have cell wall except Mycoplasma haven't cell wall. 9 Main function of the cell wall:- 1) Protect the cell from the external environmental condition. 2) Responsible for the rigidity of the cell that maintains the shape of bacteria. 3) Express many of the bacterial receptors. 4) Play role in cell division. 5) According to the cell wall composition peptidoglycan layer Bacteria classified into gram positives and gram negative bacteria. Gram positive cell wall: The main component of G + ve bacterial cell wall:- - Peptidoglycan layer forms about 50% of the cell wall material. - Teichoic acid responsible for the rigidity of the cell wall also play role in the antigenicity of the bacteria. Gram negative cell wall: - Peptidoglycan layer forms only 5-10 % of the cell wall material. - Lipoprotein molecules across link LPS layer with peptidoglycan layer. - Outer layer of lipopoly saccarid (LPS) Protect the bacteria from phagocytosis which Toxic to the body, responsible for fever hypotension and shock. - Periplasmic space which contains the functional enzyme of cell wall. 10  Outer – membrane ( L. P.S ) Peptidoglycan Lipoprotein Periplasmic Teichoic acid spaces Cytoplasmic Membrane G+ Ve G- ve (Fig 3) 11 (Fig 4) 12 Cell Wall Deficient Bacteria 1- Mycoplasma 2- Protoplast and spheroplast (bacterial cell wall may be lost as a result of unfavorite environmental condition as treatment with pencillin & lysozyme). Protoplast liberate from gram positive &spheroplast from gram negative.These bacteria cannot reproduce. 3- L-form may be formed during active infection under the effect of antibiotics Can rproduce. 2) Plasma membrane:- Is semi permeable double layerd structure and is composed of phospholipds and protein. - Transporting nutrients into the cell and waste materials outside the cell. - It plays a role in DNA replication. - It plays a role in cell wall synthesis. - Excretion of pathogenicity protein e.g. IgA protease and some exotoxine. Mesosomes Mesosomes are invaginations of the cytoplasmic – membrane. The cytoplasm Present inside the cell membrane, gelatinous mass of protein, carbohydrate, lipids, nucleic acid, salt & inorganic ions which all dissolve in water and contain ribosomes. Intra cytoplasmic structure: 1) Ribosomes are composed of 40% protein & 60% RNA.Bacterial ribosomes are 70s in size with 30s and 50s subunits. 2) Nuclear material → single, circular, ds DNA. 13 3) Inclusion bodies → accumulation of food reserve. 4) Plasmids → extra chromosomal double stranded circular DNA (carry genes that responsible for virulence factors & antibiotic resistance). Structure Outside the Cell Wall:- 1) Capsule:- gelatinous layer outside the cell wall and is composed of polysaccharide except in the anthrax bacillus bacteria which is composed of poly peptide. - Is used as immunogens in certain vaccine. - protects bacteria from phagocytosis. - plays role in adherance of bacteria to human tissue. 2) Glycocalyx:- Some bacteria have Glycocalyx which is network of polysaccharide that surround some bacterial cell wall and allow bacteria to adhere to different structures e.g. skin, heart valves, and catheters. also help strept. mutans to adhere to the surface of the teeth leading to dental caries. 3) Flagella:- (single= Flagellum) structure of Motility or locomotion composed of long – rigid strand of protein called Flagellin attached to the cell wall cytoplasmic membrane by a basal body. Bacteria can be identified by using specific antisera and can be prepard against flagellar "H" antigen to identify some strains of bacteria. 14 Types of Flagella: One flagellum One flagella at each end Monotrichous Amphitrichous Flagella at one or both ends Lophotrichous Peritrichous (Fig 5) Pili(fambriae) - These are hair like filaments. - Shorter &thinner than flagella. - Compossed of protein called pilin. - Present mainly in gram negative bacteria. Type of pili:- a- Ordinary pili:- At all over the surface responsible for adherence of bacteria to specific receptors on human cell surface. b- Sex pili: Responsible for attachment between the male (F+ donor) &the female (F- recipient) bacteria during conjugation. 15 Bacterial growth and Metabolism:- Reproduction of bacteria:- Bacteria reproduce by binary fission, one parent cell divides to form two progeny cells. The process starts by elongation of the bacterial cell and multiplication of the chromosome and the two sister chromosomes become attached cytoplasm membranes across the bacterial cell and new cell membranes is synthesized and a septum between these two cells is formed which divides theme in to two separated cells. "Bacterial growth curve" The growth cycle of bacteria has 4 major phases:- 1- Lag phase:- It is the first phase and during this phase there is no cell division occurs. The bacteria adapt to the new environment and absorp nutrient and synthesis enzymes that needed for replication. 2- Logarithmic phase:- (log phase) Rapid cell division occurs in these phases. The number of bacterial cell increases). The growth rate is increased. 3- Stationary phase:- In this phase there is accumulation of toxin products and exhausted of nutrients in the media occurs, which leads to decrease the rate of growth. this means the number of dying cell equal the newly cell formed. 4- Decline phase:- This is the final phase and called death phase. In this phase the death rate exceeds the multiplication rate, and means the number of living bacteria decrease. 16  (Fig 6) Bacterial metabolism: Many bacteria secrete enzymes as lipases, proteinases, hydrolytic enzymes and nucleases enzymes. The function of these enzymes is to breakdown extra cellular, nutritive material in to simple molecules that transported across the cytoplasmic membrane in to the bacterial cell. This process is called metabolism. Metabolism Metabolism mean all process (chemical reaction) that occurs in the cell resulting to the production of energy. a) Catabolism: Catabolism refers to the metabolic degradation (breakdown) of organic compounds that results in products of energy. (ATP) 17 b) Anabolism Anabolism refers to those biosynthetic processes that use energy for the synthesis of protoplasmic material needed for the growth and anabolic reaction. * Fermentation: This means the breakdown of a suger to pyrovice acid and then to lactic acid. Faculitative bacteria can generate the ATP by fermentation process in the absence of oxygen. Bacterial nutrition: Bacteria, like all cells, require nutrient for their metabolism processes and for cell division as some bacteria are autotrophs and others are hetorotrophs. *Autotrophs This bacteria can utilize simple inorganic substance (co2) as a source of carbon, ammonium salt as a source of nitrogen and energy for these metabolism obtained from light. *Heterotrophs All bacteria that is medical ingredient are heterotrophs (parasitic bacteria). These bacteria require complex material or substances such as- protein, Suger derived from plant or animal source. Many pathogenic bacteria to grow required essential substance such as vitamins A, acid, purines & pyrimidines. Gaseous: oxygen & carbon dioxide (o2 - co2) Oxygen: according to their needs to oxygen bacteria are classified in to: 1- Obligate aerobic bacteria. (grow only in the presence of o2) 18  2-Obligate anaerobic bacteria (grow only in the complete absence of O2). 3-Facultative anaerobic bacteria:- These bacteria can grow in the presence or absence of o2 4-Microaerophilic bacteria:- These bacteria require low amount of o2 for their growth. 5-Aerotolerant anaerobes:-These bacteria have super oxide dismutase enzyme that help bacteria to survive in the presence of oxygen. Carbon dioxide (co2) Certain species of bacteria require high concentration of co2 from 5 % to 20% should include in the atmosphere surrounding the culture. Temperature Bacteria require temp for growth. Most medically important species grow at range between 25-40co. The optimum temp for growth is 37co. No pathogenic bacteria can grow at temp lower than 20-22co or higher than 55co. "Hydrogen Ion concentration" (PH) Most pathogenic bacteria can grow at range of PH 7.2-7.6 some species grow at alkaline PH = 8 (e.g. vibrio cholera) and others at acidic PH = 4 (e.g. lactobacilli). Classification of bacteria: Bacteria are related in to one kingdom known as a prokaryotes. This kingdom divided into 4 division: Sections and each section contains a number of orders. Each order contains a number of families. Each family contains a number of Genus. Each genus contains a number of species. Finally each species contains a number of strains. * Prokaryotic genera includs 19  1) Filamentous bacteria. 2) True bacteria. 3) Spirochetes bacteria. 4) Mycoplasma. 5) Richettsiae and Chlamydia. Bacteria are classified according to: - Phenotypic characters - Genotypic characters and - Medical classification - Phenotypic classification includes: 1- Shape of bacteria: - Cocci (spherical) - Bacilli (rods) - Cocco bacilli (oval) - Spirochetes - Filamentous 2- Staining reaction:- Is divided into gram positive and gram negative (According to the cell wall composition). Some bacteria such as T.B bacteria stain by zeihl-neelson stain (Z.N. stain) give acid fast bacilli (A.F.B) 3- Ways of respiration:- Obligate aerobic, obligate anaerobic, facultative anaerobic, microaerophilic, aero tolerant anaerobic. 4-Bacteria arrangement: This occurs during cell division (arrangement of bacteria in to single, double, chain, or clusters). Biochemical reaction:- Different bacteria have different types of biochemical reaction depends on the enzymes that present in these bacteria. 20  For example urease enzyme present in proteus. This enzyme split urea in to ammonia and carbon dioxide 5-Motility: Bacteria can be classified according to the present of flagella or absence of flagella. If present (motile bacteria) if absent of flagella called non-motile bacteria 6-Presence of capsule:- According to the presence or absence of capsule bacteria are classified in to capsulated, and non capsulated bacteria. Capsulated bacteria such as klebsiella pneumoniae, streptococcus pneumoniae and bacillus anthracis and non capsulate bacteria e.g. Shigella. 7-Spore production:- Most bacteria do not produce spores and are called non spore forming bacteria others such as clostridium, bacillus can produce spores and are called spore forming bacteria. Bacterial sporulation: This step is done by bacteria to survive in the unfavorable environment condition. 1- The bacteria DNA is divided into two DNA. 2- Each DNA is separated with cytoplasm at the end of cell. 3- Plasma membrane grows in ward separated isolating DNA and cytoplasm. 4- Cortex is formed around the spore that contains calcium. 5- Protein coat develops around the spore. 6- Mother cell is lysis and the endo spore is released. 7- Finally when this unfavorable condition disappears the spore is repeat the normal growth to bacterial cell. 21 7) Fig 7 9-The ability of bacteria to produce toxins:- Certain bacteria can produce toxins and are called toxigenic bacteria. Bacteria that not produce toxin arecalled non toxiygenic bacteria. 10-Antigenic structure:- Most bacteria are antigenic that they can stimulate the hosts immune response to produce antibodies. These antigenic are called antigenic determinants. 11-Classification of bacteria according to the place of existence: Some bacteria living outside the cell of the host are called (extra cellular bacteria) or inside the host cell and are called intracellular bacteria. 22 Medical classification 1) According to the mode of transmission: a- Air borne infection. b- Food and water borne infection. c- Sexually transmitted infection. d- Through animal bites infection. e- Arthropod borne infection. f- Direct contact infection. 2) According to the site of pathogenesis: a- Bacteria infect the urinary system. b- Bacteria infect the digestive system. c- Bacteria infect the nervous system. d- Bacteria infect the genital system. e- Bacteria infect the respiratory system. f- Bacteria infect the cardiovascular system. g- Bacteria infect the musculoskeletal system. h- Bacteria infect the blood lymph. I-Bacteria infect the skin 23 Genotypic classification Genetic characters are also used for classification of bacteria according to the analysis and sequencing of bacterial DNA. Virulence factors of bacteria Virulence is the degree of pathogenicity of an organism and the ability of an organism or pathogen to cause disease. Virulence bacteria The ability of bacteria to cause disease. This depends on the 1- Number of infecting bacteria (infection dose). 2- The route of entry in to the body. 3- Degree of damage. 4- Host defense mechanisms. Virulence factors definition Defined as the properties or characteristics that enable a microorganism to establish itself on /within a host of a particular species and enhances its potential to cause a disease. Virulence factors of bacteria include:- - Cell surface proteins (for attachment). - Cell surface carbohydrate and proteins (for protection). - Hydrolytic enzyme (for invasion). - Bacterial toxin. Types of Virulence factors: 1- Adhesion factors: for attachment or adherence of certain bacteria. Have specialized structures, help them to adhere to the host cell mucous membrane such as , a- Flagella:- (fimbriae) 24  These organs of motility help bacteria to invade the aqueous areas of the body. e.g : fimbriae help streptococci adhere to buccal mucosa. b- Pili Helps adherence of some strain of bacteria to urinary tract epithelium. e.g:- Niesseria gonorrhea and Escherichia-coli. c- Glycocalyx Some bacteria secrete a polysaccharide fibers they are called Glycocalyx network increase the ability of bacteria to adhere to the teeth and mucous membrane. e.g:- Streptococcus viridances, staphylococcus epidermidis adhere to the endothelial surface of the heart valves. 2-Protection factors (Anti phagocytosis factors) such as: a- M-proteins that are found in Pili of streptococcus pyogenes help this bacteria to adhere and act as antiphagocytic function. b- Capsule of many organism protect the bacteria from phagocytosis. c- Protein "A" in staphylococcus aurous protect this bacteria from phagocytosis. 3-Invasion factors (extra cellular enzymes): These are hydrolytic enzymes, Substances produced by some bacteria that help the bacteria to spread, invasion and establishment of Microorganisms in to the host tissue. a) Coagulase enzyme:- This enzyme is produced by staphylococcus aurous which enable this bacteria to clot plasma, deposition of a fibrin wall 25  around staphylococcus lesion and helps this bacteria to persist and protect them from phagocytsis. b) Collagenase and hyaluronidase:- Collagenase degrade collagen layer & hyaluronidase degrade hyaluronic acid in connective tissue they help bacteria to spread through subcutaneous tissue such as clostridium. c) Lecithinase:- This enzyme is produced by clostridium perfringens able to breakdown lecithin. d) Streptokinase:- (Fibrinolysin) Is produced by streptococcus pyogenes, which causes lysis of fibrin clot to facilitate the spread of bacteria in the tissue. e) Immunoglobulin A (IgA) protease:- This enzyme is produced by – Neisseria gonorrhea. – Neisseria meningitides. – Streptococcus pneumonia. – Heamophilus - influenzae. Able to degrade the secretory IgA on mucous surface and thus eliminates the microorganisms protection of the host by antibody. f) Deoxyribonuclease: Is produced by streptococcus pyogenes cause breakdown DNA→facilitate the spread of this bacteria in the tissue. 2- Bacterial toxins: Toxins are bacterial products which have a direct harmful action on tissue cell. 26 There are two groups of toxin: a) Endotoxins:- These are integral part of the cell wall of gram negative bacteria from which they are librated when the cell dies. e.g. Microorganism that producing endotoxin include: Escherichia coli, meningococci. Endotoxins are the most important causes of septic shock all endotoxin produce gene generalized non specific toxic effect of fever, hypotension, disseminated intravascular coagulation (DIC) shock these effects through activation of macrophages to produce IL1, 6, and TNF- which causes (fever, tissues damage, hypotension and shock). Complement (ca3, c5a) these due to vasodilatation, vascular permeability, hypotension and shock. b) Exotoxins:- They are protein toxins, secreted by living bacteria and diffuse freely in to the surrounding medium most exotoxins production are controlled by genes on plasmids. Exotoxins are sub classified as : - Neurotoxins. - Entertoxins. - Cytotoxins. According to their mode of action the organs are affected. - Clostridium tetani (neurotoxins) - Clostridium botulinum ,salmonella,staph.aureus.ETEC,EHEC(enterotoxins) - Corynae bacterium diphtheria,shigella,cholera(cytotoxine) 27  Tab 2: Differences Between Bacterial Endotoxins And Exotoxins m property EXOTOXIN ENDOTOXIN Chemical Lipo polysaccharide ( 1 Protein nature/composition lipid A) 2 Molecular weight 50-1000kDa 10kDa Extra cellular, 3 Relationship to cell Part of outer membrane diffusible Gets denatured at 4 Effect of boiling Denatured at 100co 60co 5 Can be toxoided Yes No Many gram positive bacteria 6 Produced by Gram negative bacteria and few gram negative bacteria 8 Antigenicity Good / strong Poor / weak In plasmide or 9 Location of gene Bacterial chromosome chromosome 10 Specificity High Low Note:- Toxoid is a toxin treated usually with formaline which loses its toxicity but still immunogenic. Infection and Epidemiology What is pathogen? Pathogen is a microorganisms that is capable of causing disease,where some organism are frequently pathogen and others cause disease rarely. Bacteria is a saprophytic or parasitic bacteria. Definition of saprophytic bacteria are those which live freely in nature ,insoil or water, and on decaying organic matter. Definition of parasitic bacteria or pathogenic bacteria are those which live in or on a living host. 28  Most human microbes are commensals which live external or internal of the surface of the body without causing disease such as normal flora. Under certain conditions these normal flora change their function and causing disease. Such as opportunistic disease caused by opportunistic bacteria. These due to: 1- Change in the nature habitat of the microorganisms. Such as Escherichia-coli which is normal flora in the intestine, if it reaches the genital system it is causing disease also lead to U.T.I. also strept-viridance-in mouth (N.F) may cause endocarditis after teeth extraction. 2- Decrease the host defense mechanism leads to these persons become susceptible to infection. e.g:- Diabetic patient and Immunosuppressed patient. Infectious dose (ID) It is the number of organism required to cause disease which varies greatly among the pathogenic bacteria. For example, Shigella and Salmonella both cause diarrhea by infecting the gastrointestinal tract but the ID of Shigella is < 100 organisms, whereas the ID of Salmonella is about 100,000 organisms. Colonization It is the present of a new organism that is neither a member of the normal flora nor the cause of symptomes, thus can be a difficult clinical dilemma to distinguish between a pathogen and a colonizer. Infection:- Is the invasion of the body by various agents including bacteria, fungi, viruses, protozoan, and worms and its reaction with their toxin. Types of bacterial infections. 29 Communicable infection: It is spread from person to person via airborne droplets produced by coughing as pulmonary tuberculosis. Non Communicable infection: It is spread by an exotoxin produced by an organism in contaminated food and affects only those eating that food as botulism. Epidemic infection: It occurs much more frequently than usual. Pandemic infection: It has a worldwide dtstribution. Endemic infection: It is constantly present at a low level in a specific population. In apparent or subclinical infection: It can only be detected by demonstrating a rise in antibody titre or isolating the organism. Disease Disease is defined as the destruction of host tissues by the microorganisms due to invasion of tissue, toxin production or their virulence factors. Disease to occur requires 1) A source of infection. 2) Mode of transmission. 3) A portal of entry (GIT, GUT, RT, skin, injection, insects bits). 4) Multiplication of the Microorganisms. 5) Portal of exit from the host (urine, stool, blood, respiratory secretion, and genital discharge). 30  Disease transmitted from person to person through:- 1) Direct contact skin to skin. 2) Direct contact with mucous membrane. 3) Indirect via air borne droplets or respiratory secretion. 4) Indirectly via contamination of food and water. 5) Indirectly via blood transfusion (contaminated) blood or blood products. Fig 8: methods of diseases transmission Stages of bacterial pathogenesis A generalized sequence of the stages of infection is as follows: (1) Transmission from an external source into the portal of entry. (2) Evasion of primary host defenses such as skin or stomach acid. (3) Adherence to mucous membranes, usually by bacterial pili. 31 (4) Colonization by growth of the bacteria at the site of adherence. (5) Disease symptoms caused by toxin production or invasion accompanied by inflammation. (6) Host responses, both nonspecific and specific immunity during steps 3,4,and 5. (7) Progression or recovers of the disease. The following tables give examples of some pathogenic microorganisms and their products in terms of each stage of infection. Chronic carrier It is the continous growth of an organism with or with producing symptoms in the host. 32 Asymptomatic carrier: a- Genetic disease carrier ( an individual that has inherited genetic trait or mutation but no symptoms appear). b- Infections disease carrier (person infected with pathogen but has no symptoms). Carrier is defined as healthy individuals carrying pathogenic organism without showing clinical manifestations. Types of carrier: 1- Incubatory carriers: Is a person who can transmit a pathogen during the incubation period of infection. 2- Convalescent carriers Is a person who harbor and transmit a pathogen while recovering from an infection during the convalescence period. 3- Healthy carrier harbor the pathogen but is not ill. 4- Active carrier has overt clinical case of disease. The site of carriage may be: 1- Intestinal or urinary salmonella carriers which cause entric fever. 2- blood carriers causing hepatitis B and C and HIV infections. 3- Nasal, skin, or throat carriers causing staphylococcal infections. 4- Throat carriers causing diphtheria. 5- Nasopharyngeal carriers causing epidemic cerebrospinal meningitis. 33 Normal microbial flora (N.F) Normal flora are microorganisms that are frequently found in different sites in the body of normal healthy individual except the C.S.F, (brain), muscles, blood ,upper U.T. are normally free of microorganism. Normal flora are: Resident flora, Transient flora and Opportunistic pathogens. 1- Resident flora:- Fixed type of microorganism is found in a given area typically colon, the surface of the skin, mucous membrane, digestive tract, upper respiratory tract,and distal portion of the urogenital system. 2- Transient flora These types of (N/F) derived from the environment remain for hours, days, or weeks due to: - Competition from resident microbes. - Elimination by the body's immune system. - Physical or chemical changes within the body that inhibits or discourages the growth of transient microbes. 3- Opportunistic microbes Under normal condition resident and transient microbes do not cause any harm in the body but it can change to opportunistic pathogen due to: - When the immune system is not working such as in immunocompromised patient. - When the microbes (N.F) change their habitat. - e.g:- E. coli normal flora in the digestive system but pathogenic in the urogenital system in case of catheterization,strept viridance in buccal cavity is 34  normal flora but when goto the heart causing heart valve damage. - When persons takes broad spectrum antibiotics. Function of resident flora:- 1) Help for synthesis of vit.K and producing several B vitamins 2) Help in the absorption of nutrients from the intestine. 3) Inhibit colonization of microorganisms (adherence) by occupying receptors sites in the host cells. 4) Maintain inhibitory PH in the vagina. Normal flora of the skin (1) Staphylococcus epidermidis (Predominant Organism) It is a nonpathogen on the skin but can cause disease when it reaches certain sites such as artificial heart valves. (2) Staphylococcus aureus (103 - 104 org /cm2 of skin) It is a pathogen on the skin and other parts of the body and mostly located superficially in the stratum corneum. (3) Pseudomonas aeruginosa It is also a pathogen of the skin (wounds) and other parts of the body. (4) Peptococcus and Propionibacterium (Anaerobic organisms) They are nonpathogens on the skin but can cause opportunistic infections and are situated in deeper follicles in the dermis. Normal flora of the respiratory tract A wide spectrum of organisms colonize the nose, throat, and mouth, but the lower bronchi and alveoli contain few, if there is any organisms. The nose: 35 It is colonized by a variety of streptococcal and staphylococcal species, where the most significant pathogen is S aureus (carriers). The throat: It contains a mixture of nonpathogenic viridans streptococci, Neisseria species, and Staphylococcus epidermidis which are inhibitory to the growth of the pathogenic Streptococcus pyogenes, Neisseria meningitidis, and S aureus respectively. The mouth: - It is mainly colonized by viridans streptococci which make up about 50% of the bacteria, where a member of this group called Streptococcus mutans is found in large number (1010/g) in dental plaque. - Anaerobic bacteria, such as species of Bacteroides, Fusobacterium, Clostridium, Peptostreptococcus and Actinomyces israelii, are found in the gingival crevices, where the oxygen concentration is very low. Normal flora of the intestinal tract - In normal fasting people, the stomach contains few organisms because of its low pH and its enzymes. The small intestine usually contains small numbers of streptococci, lactobacilli, and yeasts, particularly Candida albicans. Larger numbers of these organisms are found in the terminal ileum. - The colon is the major location of bacteria in the body which contains E coli and other coliforms, Streptococcus faecalis, P aeruginosa, Clostridium species, etc. All these intestinal flora play a significant role in extraintestinal diseases. Normal flora of the urogenital tract - The vagina is located close to the anus, thus can be colonized by members of the faecal flora, where Lactobacillus species are the most common organisms, followed by C albicans, 36 Corynebacterium species, Gardnerella vaginalis, S epidermidis, E coli, etc. - In normal persons urine in the bladder is sterile, but during passage through the outermost portions of the urethra it becomes contaminated with the above mentioned organisms. In addition, the area around the urethras of women and uncircumcised men contains secretions that carry Mycobacterium smegmatis. Antimicrobial chemotherapy: Definition of an antibiotic Antibiotic is an antimicrobial substance that is produced by a living microorganism and many of these antibiotic have been chemically synthesized. Action of antibiotics Bactericidal or Bacteriostatic drugs. Bactericidal These drugs have a rapid killing action of bacteria. For example. - Pencillins. - Cephalosporin's. - Amino glycosides. Bacteriostatic These types of drugs inhibit the bacterial multiplication but do not kill them. e.g Tetracycline / chloramphenicol and sulphonamides. "Spectrum of antibiotics action" 1- Broad spectrum antibiotics. 2- Narrow spectrum antibiotics. Broad spectrum antibiotics These are active against several types of microorganisms including gram positive and gram negative bacteria such as Ampicillin, chloramphenicol. Narrow spectrum antibiotics:- 37  These types of antibiotics are active against one type of microorganism. For example. vancomycin. Definition of selective toxicity:- It is mean the antibiotics that can kill or inhibit the growth of microorganism in concentration that are not harmful to the host cells. Mechanisms of action of antimicrobial agents: 1) Inhibition of bacterial cell wall synthesis For example. Penicillin, cephalosporin, vancomycin. By blocking cross linking of the cell wall structure Beta-lactam- antibiotics inhibit the final steps in synthesis of peptidoglycan layer by binding to the receptors called penicillin-binding protien in cell wall (contain B-lactam ring). 2) Inhibition of bacterial protein synthesis:- For example Chloramphenicol, erythromycin, binding to the ribosome subunits (50s) inhibits the protein syntheses. 3) Inhibition of bacterial cytoplasm function For example Polymyxin-B Cause disruption of cytoplasmic membrane and leakage of cell contents which leads to cell death. 4) Inhibition of nucleic acid synthesis:- For example. a- Novobicocin inhibit DNA synthesis by blocking DNA gyrase. b- Rifampicin inhibit RNA synthesis by binding to RNA polymerase. Trimethoprim inhibit nucleotide synthesis. Complication of antibiotics: 38  1) Development of drug resistance strains due to prolonged treatment or usage of inadequate dose 2) Super infection due to suppression of normal flora by antibiotics lead to opportunistic infection 3) Drug toxicity may lead to deafness and depression 4) Hypersensitivity some drug act as hapten bind to tissue protein and stimulate immune response leading to tissue damage. 5) Bone marrow depression caused by some drugs such as chloramphinicol. Antimicrobial drug resistance What is drug resistance? Antimicrobial resistance is the ability of microorganism to grow in the presence of drug that would normally kill it or limit its growth. Exposure Infection occurs to bacteria Drug treatment and the bacteria occurs Spread Is used None- resistant Bacteria The bacteria The bacteria die. The Multiply Person is healthy again. Drug Resistant Bacteria The bacteria The bacteria continue Multiply 39 to spread. The person remains sick  (Fig 9) Causes of the development of drug resistance bacteria: 1- Mutation (genetic change or chromosomal mutation) 2- Gene transfer (microbes may acquire genes that code for drug resistance by a process called transposition) 3- Plasmids carry genes that code for the production of enzymes that convert active drug to inactive drug such as bacteria that produce B-lactamase-enzyme that breakdown ring of penicillin or cephalosporin. 4- Inappropriate use of antibiotics:- (Inadequate dose) 5- Some time physicians will prescribe inappropriate antimicrobial agents which leads to develop resistant strains. 6- Inadequate diagnosis:- Inadequate diagnosis leads to accelerate antimicrobial resistance strains to appear due to taking at abroad spectrum antibiotics. Types of multidrug resistance bacteria a. Methacillin resistance staph.aureus-MRSA b. Vancomycin resistance staph.aureus-VRSA c. Vancomycin resistance enterococci Preventing the spread of multidrug resistance strain 1. It is important to recognize the source of MDR-organisms to stop the cycle of infection 2. Hand hygiene 3. Isolation of patient with MDR- organisms 4. Physical barriers should be used 5. Personal protection equipment 6. Use combination of drug 40 Control of bacterial growth: By Sterilization and disinfection: - Sterilization refers to the complete destruction or elimination of all viable organisms in or on a substance being sterilized including spores. - Disinfection is the procedures that is carried out to kill most microorganism except endospores and viruses. - Decontamination refers to the procedures that reduces pathogenic microorganisms to a level where items are safe for handling, for use or disposal. - Antiseptics: microbicidal agents harmless enough to be applied to the skin and mucous membrane; should not be taken internally. Examples include alcohols, mercurials, silver nitrate, iodine solution, alcohols, detergents. - Cleaning is a process that remove foreign material usually done with soap and water, detergents or enzymatic products. Methods of Sterilization 1-Sterilization by heat (physical methods) a) direct heat is used for sterilization of loopes, necks of tubes, or bottles of culture media. For example. Bunsen flame. b) dry heat kills by destructive oxidation of essential cell constituent.used for sterilization of glassware, powders, ointment, metal instruments. It is done by using a hot air oven at 160-180 ºC for 1-2hours. c) moist heat: 41  - pasteurization (at temp.below 100Cº) heating of milk at 63Cº for 30 or at 72Cº for 15 sec. - boiling (at temp of 100Cº for 20 min.) used for sterilization of surgical instruments such as forceps, scissors, cups plates, knives etc. - steaming (at temp.above 100Cº) such as autoclave used for sterilization of surgical instruments, gowns, surgical dressing, cotton qauze, and for any culture media are not destroyed by heat.the autoclave can be used at 121Cº for 15 min.withstands pressure of 5 p.s.i. - 2-Sterilization by radiation:- a) ultraviolet light (UV) present in sun rays or artificially produce by mercury lamps used to reduce the number of bacteria in air((maximal bacteriocidal at an wave-length 250 nm - 260 nm). used in operation room, drug filling cubicals and laboratory safety cabinet. b) Ionizing radiation such as gamma-rays. They are disrupt DNA&RNA in living organisms sterilizing of catheters, gloves, surgical sutures plastic petridishes and items that cannot stand with heat. Sterilization by filtration (mechanical methods ) Filtration is used to remove microorganism from biological fluid such as serum, plasma, hormons, vitamins and from fluid culture media. Also for sterilization of air in operation room, drug filling cubicles or safety cabinets. It is done by using nitrocellulose filters of a pore size of 0.22 μm or 0.45 μm which retain all types of bacteria and spores. Types of filters - Membrane filter (Millipore) used around plastic holder which can be attached to the end of syringes. 42  - Vacuum filter is used for sterilization of large volume of fluid - Syringe filters are used for sterilization of small volume of fluid - High efficiency particle arrest (HEPA) filter is used for sterilization of large volume of air. Sterilization by gaseous Ethylene oxide (EO) gas: EO gas is a highly lethal aklalyting agent that kills all microorgranism including spores. The gas is toxic, highly explosive and its use require special safety precaution. Its used for heating sensitive materials. Formaldhyde: Is a gas at high temperature and solid at room temperature When 37 gm of solid are suspended in 100 ml of water a solution is called formaline. Formaline is utilized for inactivation viruses in certain vaccines and producing toxoids from toxins. Formaldehyde in the gaseous form is used for sterilization of surgical equipments, hospital gowns and medical instruments. The surface must be exposed to the gas for up to 12 hours for effective sterilization. Chemical disinfectants and antiseptics: (Chemical methods) Chemical substances are mainly used as disinfectants for inanimate objects such as endoscopes, bed pans, floors, dialysis systems. They are used as antiseptics for application on living tissues, e.g wounds, other skin lesions. 1-Tincture of iodine (2 % of K iodine in ethanol) as skin antiseptic before blood culture 2-Ethyl alcohol in 70% concentration is effective,skin antiseptics. 43 3-Hydrogen peroxide (H2O2) is used as antiseptic to clean wounds. and is also used as acontact lenses disinfectant. 1- Chlorine is used disinfection of water supply to treat swimming pools. 2- Gluturaldehyde.2% and per acetic acid are highly level disinfectants for instruments in hospitals that some in contact with mucous membrane. Such as endoscopes, dialysis systems and respirators. 3- Phenol at 0.2% concentration bacteriostatic is used in the laboratory to clean spilled cultures in working areas or at concentration 1 % bactericidal and at 1.3 % concentration fungicidal. Bacterial genetics Definition of Genetics Is the study of inheritance of the different characters which are transmitted from parent to offspring's, who usually have the same characters as the parents. Definition of Genes Are the units of heredity. They are segments on the chromosome (DAN) that carry the information for a specific characters or specific structure. Prokaryotic Genomes: Bacterial genome: 1- Made of DNA (double stranded DNA molecule). 2- Genome flouts freely with in the cytoplasm. 3- Chemically is composed of a back bone of phosphate and suger (deoxy ribose) attached the purine and pyramiding bases (quinine, adenine, cytosine thymine). The two strands are together held by hydrogen bonds. The chromosome is subdivided in to segments or genes. e.g:- in E. coli there are more than 1000 genes can be mapped. 4- Genes that essential for bacterial growth are carried on the chromosome. 44  5- Few genes associated with specialized function are carried on plasmids such as a- antibiotic resistance gene. b- virulence factors gene. "Nucleic acid function" Replication, Transcription and translation Replication:- Making a copy of the genetic material= Replication (making DNA) - Replication occurs before cell division by binary fission. - The two strands of the chromosome separate. - Each strand is attached to the mesosome and acts as a template. - Complementary strand is formed by the action of DNA polymerase enzyme. - Cell division occurs after chromosomal replication by formation of a septum between the attachment sites to give two daughter cell. Each daughter cell contain an identical copy of the parent chromosome. Transcription Transcription is the process by which a DNA sequence is coped to produce mRNA. It is the transfer of genetic information from DNA in to RNA (making RNA) mRNA Two strand of DNA are separated. then one of them act as a template for synthesis complementary strand of RNA (messenger RNA) by RNA polymerase enzyme. Then mRNA is attached to the ribosome to synthesis the protein. There are 3 types of RNA:- mRNA – r RNA – t RNA 45  Bacterial cytoplasm (Fig10) Amino acids are found on the cytoplasm, by specific enzyme they are activated and transformed to tRNA. For each amino acid there is a specific tRNA. After that translation occurs. Translation: ( protein synthesis) m RNA + t RNA come together in the surface of the ribosome. Each t RNA carries specific A.A that encode by mRNA. Amino acid is linked with the adjacent one to form a polypeptide chain. Each 3 nucleotide group called (genetic code). Each 3 cordon give one amino acid e.g:- GCG, GCC …….. (Alanine, serine) Anti codon is attached to the codon. e.g:- a codon of GCC on the mRNA will attach to the anti codon CGG on the tRNA. 46  When the codon on the mRNA signals the end of the chain formation the process is stopped. The terminal code may be UAA, UAG or UGA Plasmid: Is extra chromosomal double stranded, circular DNA smaller than chromosomal DNA. - They replicate autonomously - Multiple copies of the same plasmid may be present in each bacterial cell. "Bacterial properties carried on plasmids" 1- Drug resistance:- Gene coding for drug resistance of many bacteria are carried on plasmids. R- Factor plasmids. Transferred among bacteria by conjugation. 2-virulence:- Some plasmids carry genes whose products are the virulence factor of some pathogenic organisms e.g. neurotoxin is produced by clostridium tetani. 3-production of antimicrobial agents by bacteria e.g by Streptomyces 4-Production of bacteriocins:- For example Colicins produce by E. coli present on plasmids are called col factor. 5-Sex factor; plasmids carry the fertility F- factor transfer by conjugation. "Bacterial variation" Bacterial variation may be phenotypic variation or Genotypic variation. 47 1-Phenotypic variation:- Change in bacterial characters under the influence of the environment with no genetic change such as increase pigment production by Staph aurous when grow on Room-temperature. 2- Genotypic variation:- Due to changes in genetic constitution these lead to change in bacterial characters. It occurs through mutation and Gene transfer. Mutation:- It is change in the sequence of bases in the DNA that occur due to as a result of:- - Substitution - Deletion - Insertion of new bases These types of mutation lead to alters on the genetic code that leads to formation of different amino acid and leads to appearance of new characters. "Transformation" Exogenous fragment of bacterial DNA Bacterial chromosome Donor cell recipient cell (Fig 11) 48 Transduction:- Fragment of chromosomal DNA can be transferd or transduced in to a second bacterium by bacteriophage. Definition of bacteriophages are viruses that parasites bacterial cells 49 Fig 12: Transductio 50 Conjugation It is the meeting of two bacterial cells of the same or different species. DNA (plasmids) transferre from donor cell to recipient cell. by aprocess called conjugation. This Process is controlled by an F (fertility) plasmid that code for the formation of sex pilus canal between these two bacteria. Fig 12: Conjugation 51  Nosocomial infection (NCI) Definition : It is an infection acquired in a medical setting in the course of medical treatment. Factors that favor nosocomial infection:-  Elderly  Children  Underlying illness as diabetes , burns ,cancer  Immunosuppressed patient  Instrumentation such as catheters Microorganism involved in NCI :- Staphylococcus- aureus , E coli , pseudomonas , klebsiella & proteus Source of NCI:- 1.Endogenous: Caused by the organisms that are present as part of normal flora of the patient as commensals of the skin, respiratory, GIT, GU tract, which are common 52 2. Exogenous: caused by organisms acquiring by exposure to hospital personnel, medical devices or hospital environment  Inanimate environment: Aspergillus from hospital construction, Legionella from contaminated water  Animate environment: hospital staff, visitors, other patients occurs as cross transmission Mechanisms of transmission : Contact: direct (person to person) indirect (transmission through an intermediate objects and-- contaminated instruments. Cross transmission: 1-airborne -aerosol and droplets 2- vector-borne - as mosquitoes Most common types of NCI : 1- Urinary tract 40% 2- Pneumonia 20% 3- Surgical site 17% 4- Blood stream(iv) 8% 5- GIT infection 53  Prevention measures: 1-isolation: Isolation precautions are designed to prevent transmission of microorganisms by common routes in hospitals. Because agent and host factors are more difficult to control, interruption of transfer of microorganisms is directed primarily at transmission. 2-Handwashing and gloving Handwashing frequently is called the single most important measure to reduce the risks of transmitting skin microorganisms from one person to another or from one site to another on the same patient 3-Education of all HCWs to protect them self's & patients 4-Vaccination. & Proper disposal of infectious hospital waste. INTRODUCTION TO VIROLOGY 54 What's the virus? Viruses are the smallest infectious agents. They can infect man, animal, insects, plants and bacteria. They are able to replicate in live susceptible cells. General properties of viruses Characters of viruses that can distinguish them from others microorganism. 1. They are very small in size. 2. They contain one kind of nucleic acid DNA or RNA. 3. They are obligate intracellular parasites i.e virus replicates only inside living cells. 4. They cannot be grown on artificial media (culture) they grow only in tissue culture or embryonated egg and in living animal. 5. They depend on the ribosome, enzymes of the infected cells for protein synthesis. 55 Discovery of viruses In 1892 a Russian Pathologist named Dmitri I Winooski described the causative agent of a disease. That effect the tobacco leafs as filterable virus because it can pass the bacterial filter, this virus called tobacco mosaic virus (TMV) and causes a disease is called tobacco mosiac disease. Virus Size & Structure Size of viruses is variable from 10nm to 500nm, as a result these are small in size: 1- They can pass through bacterial filters. 2- They require high speeds for their sedimentation (called ultra centrifugation 10.000-30.000rpm. 3- They are only seen by electron microscope. Structure of viruses Each virus particle is composed of a protein coat or capside and a nucleic acid core. The genetic material as nucleic acid with capisd is called nucleocapsid. 1. viral genome (genetic material): - Viruses contain either DNA or RNA but not both. - Most DNA viruses are double stranded while most RNA viruses are single stranded. - The nucleic acid may be liner or circular. - Some RNA viruses have segmented genome such as Rotavirus and influenza virus. All viruses have one copy of their genome except retroviruses have two copies 56 Function of Nucleic acid a. It is the infectious part of the virus. b. It carries the genetic information for replication, virulence, Antigenic specificity of the protein coat. 1. Viral capsid:- Protein coat is made up of many protein subunits called capsomeres. Function of capsid 1. Protect the viral genome against inactivation by nucleases enzyme. 2. It is responsible for the structural symmetry of virion (virion = complete virus particle) 3. Help for attachment of virion to susceptible cells. 4. Capsid proteins are important antigens that induce antibodies that neutralize virus infectivity. 5. Viral envelope:- Many viruses are surrounded by envelope which consists of proteins, glycoprotein, and host lipids-derived from host membrane, also there are a glycoprotein spike like projections, which attach to host cell receptors during the entry of the virus in to the cell. For example, Hemagglutinin (HA). Neuraminidase (NA) in influenza virus helps the virus attachment to the host cell. The envelope is derived or acquired from the host membrane during release from the cell. 57 58  1. Virus Enzymes Present in some viruses, Require for virus live cycle and not available in host. e.g: Integrase, RNA dependent – RNA polymerase, RNA dependent – DNA polymerase (R-T). Virus symmetry (shapes) a. Icosahedral symmetry shape. The virus capsid has 20 triangular faces, 12 corners e.g. Polio virus and herpes simplex virus. b. Helical shape in which the in which the capsomers are arranged in a hollow coil that appears rod-shaped. The helix can be either rigid or flexible. All human viruses that have a helical nucleocapsid are enclosed by an outer membrane called an envelope. Such as Rabies virus and tobacco mosiac virus. complex shape. The viruses are complicated in structure (in this shape). They are a combination of both a helical and icosahedral. Such as Poxvirus and bacteriophage Virus nomenclature 1. Name after disease causes , for example hepatitis virus. 2. Name of virus related to the scientist who first discovered the virus. For example.Epstein Barr virus (E.B.V). 59  3. Name related to the city-state-country where the virus was first discovered. For example. Hong Kong influenza virus. 4. Complex name such as HIVand CMV Classification of viruses: Viruses are classified according to: 1. Virion morphology including size, presence or absence of envelope, type of symmetry. 2. Mode of transmission the vectors and pathogenicity 3. Host infected (bacteria, parasite, plant, animal ….. and soon. 4. Genetic material (RNA or DNA.) single or double, circular or liner, segmented or non- segmented. 5. Type of target cell- type of cells that virus can infect (dermotopic, neurotopic, pneumotopic, lymphotopic, viscerotopic, spleen and liver). Methods of virus cultivation Viruses are obligatory intracellular parasites that grow in living cells but they can survive in certain conditions as non-replicating particles. Requirement for viral growth: Heat: there is great variability in the heat stability of different viruses. Most viruses are pathogenic viruses and are inactivated at 55-60co because their capsid protein is destroyed. 60 Cold: most viruses can be preserved at sub-freezing temperature.Some can with stand lyophlisation and can be stored in the dry state at 4co or at R.T which envelope Viruses lose their infectivity after prolonged storage at - 70co. or lower in liquid nitrogen Concentration of hydrogen ion (PH) Most viruses are stable in the PH range 5 to 7 enteroviruses that have to pass through the stomach resist the low PH. All viruses are destroyed by alkaline conditions. * Radiation Radiation such as U.V causing damaging of DNA that can cause inactivation of the virus, x-ray, gamma rays and beta particles inactivate viruses. * Stabilization by salts Magnesium chloride stabilises polioviruses, magnesium sulphate stabilises influenza viruses, sodium sulphate stabilizes herpes virus. They are Important in the preparation of vaccines. Ether susceptibility and lipid solubility:- - Enveloped viruses are inactivated by ether. Non ionic detergents solubilise lipid constituents but do not denature the proteins of the capsid. Anionic detergents solubilise the lipid constituents and disrupt the capsid in to separated polypeptides. 61   50% glycerol Many viruses remain alive in 50% glycerol for many years while bacteria are killed. Formaldehyde Destroys viral infectivity used in the production of inactivated viral vaccines. Hydrogen peroxide and hypochlorite are disinfectants for most viruses. - Non ionic detergents solubilise lipid constituents but do not denature the proteins of the capsid. Anionic detergents solubilise the lipid constituents and disrupt the capsid in to separated polypeptides Growth of viruses in the laboratory There are 3 systems for cultivation of viruses in the laboratory:- 1. Cell culture. 2. Embryonated eggs. 3. Living laboratory animals. A-Cell culture The recent method for cultivation growing of virus. - Pieces of animal or human tissues. - It is a homogenous collection of cells. - Prepared by treating animal tissue with enzyme to get separate cells. 62  - Cell grow and adhere to glass or plastic container to from a monolayer or sheet of cell within few days. (these growth media containing serum) - This media kept from microbial contamination by adding antibiotics for preventing the contamination. B-Embryonated eggs:- This method is used for virus cultivation. The virus suspension is injected in to the fluid of the egg. Viral growth is detected by death of the embryo or lesion on the membrane of the egg. It is the most common method for virus growth and isolation as well as used for preparation of virus vaccines. C-Living animals Mice, rabbit, quineapig Animal inoculation was mainly used in the past before tissue culture method were not known. This method still used for study of - Immunoresponse. - Viral pathogenesis. - Viral oncogenesis. - Viral disease. Primary isolation of viruses. 63 Type of tissue culture 1. Primary cell lines These are prepared from organ fragments, for example, monkey kidney. 2-Secondry cell lines Derived from primary cell lines by subculture. 3-Human diploid cell lines These are usually fibroblasts derived from human embryo tissue such as human embryo lung tissue. 4-Continuous cell lines These are derived from tumor cells. They can be divided indefinitely such as Hela cell line derived from carcinoma of the cervix. Detection of virus replication in cell culture Many viruses kill the cells in which they replicate; 1-Cytopathogenic effects (CPE) Altered shapes (rounding and group like cluster formation. Cell lyses, cell death, detachment from glass surface. Giant cell formation (cell fusion)&Cell transformation. 2-Plaque formation Plaque are virally infected area in cell culture. 3-Inclusion bodies Result from accumulation of viruses are eosinophilic or basophilic bodies that appear within the cell as a result of infection. They are intracytoplasmic or intranuclear or both. 64  For example, Rabies-negribodies in cytoplasm of nerve cells present in the brain tissue. Diagnostic Test for rabies. 4-Neutralization test This is done by mixing the virus with specific antisera then the mixture is added to cell line. absence of CPE indicates virus neutralization. 5-Heamadsorption When RBCs are added to infected cells they will appear as rosettes shape on the area where the virus is growing. Virus replication Viruses are strict intracellular parasites, replicating only within the host cells. Steps of virus replication 1- Attachment or adsorption. 2- Penetration. 3- Uncoating. 4- Macromolecules synthesis. 5- Assembly. 6- Release. Infection to occur depends on 1) Suitable site. 2) Number of viruses (Infective dose). 3) Immune response of the host. 4) Virulence of the microbes. 65 66 1-Attachment or adsorption This step depends on the presence of specific receptors and selection of the cells by viruses and depends on the – nature of receptors which differ for different viruses (all viruses have receptors -binding protein) on their outside surface. The virus receptors on the cells are often glycoprotein or glycolipids. Examples  Hamagglutinine in influenza virus, sialic acid which is found on most epithelial cells.  Glycoprotein 120 in (HIV) → with CD4 receptor on T- helper lymphocyte. Concentration of receptors play role in attachment each 4 5 susceptible cell contain about 10 -10 receptors on the cell surface..G. protein in rabies virus with acetylecholine Penetration or entry: Viruses enter the cell by different mechanisms Membrane fusion mechanism These viruses have fusion protein – which mediate the fusion between the lipids of the viruses and the lipids of the cell membrane and viral nucleic acid enters the cell directly. (e.g. enveloped viruses). 67  Receptors-mediated endocytosis:- The virus is endocytosed by the host cell in acidification states. phagolysosome is formed. The viral envelope is digested by hydrolyzing enzyme of the phagolysosome lead to release of the capsid and uncoating occurs. Both (1) and (2) occur in enveloped viruses. Endosome lysis Non enveloped viruses lack the lipid membranes found in enveloped viruses as a result these viruses cannot enter cells via a simple process of membrane fusion between the virus envelope and the host cell membrane. Endocytosis occurs to form endosome which dissolved by virus enzyme, the virus is released in to the cytoplasm of the host cells and uncoating occurs. Uncoating This process main the hydrolysis of virus capisd protein to release the viral nucleic acid in to the host cytoplasm under the effect of hosts enzyme or viral enzymes (proteases enzymes). Macromolecules synthesis:- (synthesis of proteins) N.A. Most DNA viruses replicate in nucleus (because these viruses do not have RNA dependent RNA polymerase enzyme except pox virus replicate in cytoplasm and (have RNA dep. RNA. Poly). Most RNA viruses replicate in the cytoplasm because they have: 68  (RNA dependent-RNA polymerase) except influenza virus replicate in nucleus due to these viruses do not have (RNA dep-RNA poly) enzyme. Virus assembly Virus assembly occurs in the nucleus or in the cytoplasm which depends on the viruses. during assembly Virus nucleic acid is surrounded by the capsid proteins and all the accessory parts as the genomic enzyme and other capsid enzyme. Virus Release Release of virus depends on the type of virus some release by budding through the cell membrane. These are called enveloped viruses which acquired lipoproteins envelope (enveloped virus). Others release by a process called cytolysis, rupturing the cell membrane and Release (Non enveloped viruses). Virion = complete virus particle. prions = Infectious particle, are protein, without nucleic acid. Prions Highly resistance to heat, formaldehyde, U.V. These prions cause a disease called spongy form encephalopathies. Examples, Scrapie in sheep. 69 Mad cow disease in cattle called brain sponge form encephalopathies. Kuru, Creutzfeldt Jakob disease ( CJD) in human. Transmitted through contaminated surgical instrument and consumption of contaminated meat. Viroid. Small infection agents cause disease of plants. They are nucleic acid molecules without a protein coat (ss RNA). Pathogenesis: The ability of viruses to cause disease can be viewed on two distinct levels: (1) the changes that occur within individual cells (discussed previously) and (2) the process that takes place in the infected patient. The Infected Patient Viral infection in the person typically has four stages: incubation period, prodromal period, specific-illness period, and recovery period. Types of virus infection 1. Local virus infection: This type of infection occurs at the site of entry with no viraemia characterized by: - short incubation period. - Short lasting immunity. 70  - IgA, interferon are present e.g. human papilloma virus, rotavirus and influenza virus. 2--Systemic infection: Systemic infection occurs after primary replication at the site of entry then virus travels to the different organs via blood stream or lymphatic ducts characterized by: - Long incubation period. - Long immunity. - IgM, IgG present. Host Defenses Host defenses against viruses fall into two major categories: (1) nonspecific defense of which the most important are interferons and natural killer cells and 71  (2) specific defense including both humoral and cell- mediated immunity and Interferons which are an early, first-line defense, whereas humoral immunity and cell- mediated immunity are effective only later because it takes several days to induce an immune response. General Action of interferon's: Interferons are small proteins released by macrophages, Lymphocytes, and tissue cells infected with a virus, when a tissue cell is infected by a virus, it releases interferon. Interferon will diffuse to the surrounding cells, when it binds to receptors on the surface of those adjacent cells, It is inhibit virus replication by blocking the production of viral proteins, primarily by degrading viral mRNA. They induce the synthesis of a ribonuclease that specifically cleaves viral mRNA but not cell mRNA. Three types of interferon's: alpha, beta and gamma. Alpha and beta interferon’s have a stronger antiviral action than gamma interferon. The latter acts primarily as an interleukin that activates macrophages Other Nonspecific Defenses Natural killer (NK) cells are lymphocytes that destroy cells infected by many different viruses, i.e., they are nonspecific. NK cells do not have an antigen receptor on their surface, unlike T and B lymphocytes. Rather, NK cells recognize and destroy cells that do not display class I MHC proteins on the surface. They kill cells by the same mechanisms as do cytotoxic T cells, i.e., by secreting perforins and granzymes. Phagocytosis by macrophages and the clearance of mucus by the cilia of the respiratory tract are also important defenses. Damage to these defenses predisposes to viral infection. Specific Defenses 72  Specific Defenses Active immunity to viral infection is affected by both antibodies and cytotoxic T cells. It can be elicited either by exposure to the virus or by immunization with a viral vaccine. Passive immunity consists of antibodies preformed in another person or animal. Two specialized examples of passive immunity include the transfer of IgG from mother to fetus across the placenta and the transfer of IgA from mother to newborn in colostrum. The duration of active immunity is much longer than that of passive immunity. Active immunity is measured in years, whereas passive immunity lasts a few weeks to a few months. 73