Microbiology for MD - Africa Medical College - Dec, 2024 PDF

AFRICA MEDICAL COLLEGE Microbiology for MD Dr. Alem A. (PhD, Medical Microbiology) Dec, 2024 Course content 2 1. Chapter 1: General Microbiology 2. Chapter 2: Systematic Bacteriology 3. Chapter 3: Systematic Virology 4. Chapter 4: Systematic Mycology 5. Chapter 5: System based microbial infections (Group Seminar) Assessement 3 1. Tests: 1, 2, 3 2. Lab report 3. Attendance 4. Individual presentation 5. Group presentation 6. Final Exam 7. Oral Exam What is Microbiology? 4 Microbiology is the study of all living organisms that are too small to be visible with the naked eye Bacteria Protozoa Fungi Viruses Prions Archaea Algae Collectively known as microbes or Microorganisms There are thousands of different types of microbes that live in, on, and around us—and hundreds that cause serious human diseases What is Microbiology… 5 Microbiology Microbiology Medical Microbiology Food Microbiology Bacteriology Veterinary Microbiology Virology Plant Microbiology Mycology Pharmaceutical Immunology Microbiology Parasitology Applied Microbiology Industrial Microbiology Soil Microbiology Etc. What is Microbiology… 6 The agents of human infectious diseases belong to five major groups Bacteria, Fungi, Protozoa, Helminths, Viruses Bacteria, fungi, protozoa, and helminths are cellular, whereas viruses are not → 3 criteria Structure Method of replication Nature of the nucleic acid Viruses are not cellular… 7 Structure Cells have a nucleus or nucleoid, which contains DNA surrounded by cytoplasm, where proteins are synthesized and energy is generated Viruses have an inner core of genetic material (either DNA or RNA) no cytoplasm, and so they depend on host cells to provide the machinery for protein synthesis and energy generation Viruses are not cellular… 8 Method of replication Cells replicate either by binary fission or by mitosis one parent cell divides to make two progeny cells while retaining its cellular structure Prokaryotic cells (e.g., bacteria) replicate by binary fission Eukaryotic cells replicate by mitosis Viruses disassemble, produce many copies of their nucleic acid and protein, and then reassemble into multiple progeny viruses Viruses must replicate within host cells because they lack protein-synthesizing and energy-generating systems Viruses are not cellular… 9 Nature of the nucleic acid Cells contain both DNA and RNA, Viruses contain either DNA or RNA, but not both Eukaryotes and Prokaryotes 10 Based on their structure and the complexity of their organization, cells are classified in to A. Eukaryotic B. prokaryotic Fungi, protozoa, and helminths are eukaryotic Bacteria are prokaryotic Viruses are neither eukaryotic nor prokaryotic Eukaryotes and Prokaryotes… 11 The eukaryotic cell has a true nucleus multiple chromosomes surrounded by a nuclear membrane uses a mitotic apparatus to ensure equal allocation of the chromosomes to progeny cells The prokaryotic cell has nucleoid typically consists of a single circular molecule of loosely organized DNA lacks a nuclear membrane and mitotic apparatus Eukaryotes and Prokaryotes… 12 Eukaryotic cells contain organelles, such as mitochondria and lysosomes Prokaryotes contain no organelles Eukaryotic cells contain larger ribosomes (80S) compared to prokaryotes (70S) Most prokaryotes have a rigid external cell wall that contains peptidoglycan (a polymer of amino acids and sugars) Eukaryotes do not contain peptidoglycan Eukaryotes are either bound by a flexible cell membrane, or, in the case of fungi, they have a rigid cell wall with chitin Eukaryotes and Prokaryotes… 13 The eukaryotic cell membrane contains sterols, whereas no prokaryote, except the wall-less Mycoplasma, has sterols in its membranes Most protozoa and some bacteria are motile, whereas fungi and viruses are non-motile The protozoa are a heterogeneous group that possesses three different organs of locomotion: flagella, cilia, and pseudopods The motile bacteria move only by means of flagella Eukaryotes and Prokaryotes…Summary 14 Scientific nomenclature of Microbes 15 Bacteria, fungi, protozoa, and helminths are named according to the binomial Linnean system that uses genus and species Example: Escherichia coli, Escherichia is the genus and coli is the species name Viruses typically have a single name, such as poliovirus, measles virus, or rabies virus. Some viruses have names with two words, such as herpes simplex virus, but those do not represent genus and species Microorganisms 16 Are small living things which are too small to be seen with our necked eye Includes bacteria, fungi, protozoa Viruses, which are microscopic but not cellular, are also included in this group Bacteria 17 Relatively simple in structure They are prokaryotic organisms; i.e. simple unicellular organisms with no nuclear membrane, mitochondria, Golgi bodies, endoplasmic reticulum Reproduce by asexual division; i.e. by dividing into two equal cells called binary fission Are enclosed in cell walls except Mycoplasma species Classified by size (1 to 20 µm or larger), shape (spheres, rods, spirals), and arrangement (single cells, chains, clusters) The human body is inhabited by thousands of different bacterial species: as commensal or pathogen Bacteria also exist in the environment: air, water, food Most are avirulent Fungi 18 The cellular structure of fungi is more complex compared to bacteria Fungi are eukaryotic organisms that contain a well-defined nucleus, mitochondria, Golgi bodies, and endoplasmic reticulum Fungi can exist either in a unicellular form (yeast) that can replicate asexually or in a filamentous form (mold) that can replicate asexually and sexually Most fungi exist as either yeasts or molds; however, some fungi can assume either morphology (dimorphic fungi) Fungi… 19 Parasites 20 Parasites are the most complex microbes All parasites are eukaryotic some are unicellular and others are multicellular Parasites range in size from tiny protozoa as small as 4 to 5 µm in diameter to tapeworms that can measure up to 10 meters in length and arthropods (bugs) Reproduce sexually or asexually Viruses 21 Viruses are the smallest infectious particles, ranging in diameter from 18 to 600 nanometers Viruses typically contain either DNA or RNA but not both − some viral-like particles do not contain any nucleic acids; e.g., prions − the recently discovered Mimivirus contains both RNA and DNA Viruses are made up of nucleic acids enclosed in a protein shell with or without a lipid membrane coat Viruses are true parasites, requiring host cells for replication − Obligate intracellular More than 2000 species of viruses have been described, with approximately 650 infecting humans and animals Comparison of Medically Important Organisms 22 History of Microbiology 23 A. Robert Hooke, an Englishman, in 1665 Observed a thin slice of cork using relatively crude microscope life's smallest structural units: "little boxes“ or "cells” Using his improved version of a compound microscope Hooke was able to see individual cells His discovery marked the beginning of the cell theory Though Hooke's microscope was capable of showing large cells, it lacked the resolution to see microbes History… 24 A. Antoni van Leeuwenhoek in 1673 Using his microscope he observed small creatures what he called ‘animalcules’ Leeuwenhoek’s microscopes could magnify up to 300x From where did these small creatures come? Arguments about the origin of living things a: lens b: mounting pin Spontaneous generation theory c and d: focusing screws The theory of biogenesis History… 25 Spontaneous generation theory Living organisms could arise spontaneously from non-living matter snakes and mice could be born of moist soil Flies could emerge from manure maggots, the larvae of flies, could arise from decaying corpses The theory of biogenesis life arises only from already existing life Francesco Redi (1626–1697) in 1668 26 larvae found on putrefying meat arose from eggs deposited by flies the beginning of the end for the spontaneous generation theory Experiment a) Redi filled three jars with decaying meat b) The first left unsealed flies laid eggs on the meat, and eggs developed into larvae c) The second jar was sealed flies could not lay their eggs on the meat, no maggots appeared d) The third jar was covered with fine net (gauze) flies kept away, and no maggots appeared on the meat Scientists began to doubt spontaneous generation theory and adopt the view that animals come only from other animals John T. Needham (1713–1781), 1745, British investigator 27 He boiled beef gravy and infusions of plant material in vials, which he then tightly sealed with corks Some days later, Needham observed that the vials were cloudy, and examination revealed an abundance of “microscopical animals of most dimensions” As he explained it, there must be a “life force” that causes inanimate matter to spontaneously come to life because he had heated the vials sufficiently to kill everything Lazzaro Spallanzani (1729–1799), in 1799, Italian 28 reported results that contradicted Needham’s findings Spallanzani boiled infusions for almost an hour and sealed the vials by melting their slender necks closed His infusions remained clear unless he broke the seal and exposed the infusion to air, after which they became cloudy with microorganisms Criticisms of Spallanzani’s work his sealed vials did not allow enough air for organisms to thrive his prolonged heating destroyed the “life force.” The theory of biogenesis… Louis Pasteur (1822–1895) He disproved the theory of spontaneous generation once and for all Done a series of experiments that led to the acceptance of biogenesis experiment using his swan-necked flasks in 1861 29 Louis Pasteur’s experiments 30 Pasteur demonstrated that microorganisms are present in the air and can contaminate sterile solutions, but air itself does not create microbes He filled several short-necked flasks with beef broth and then boiled their contents. Some were then left open and allowed to cool In a few days, the open flasks were found to be contaminated with microbes whereas the sealed flasks were free of microorganisms Pasteur’s swan naked flasks 31 The Germ Theory of Disease 32 Pasteur’s observation on wine spoilage when lactic acid was produced in wine (spoiled), rod- shaped bacteria were always present, as well as the expected yeast cells This led him to believe “while the yeast produced the alcohol the bacteria were responsible for the spoilage” This led to “microorganisms may also be responsible for diseases in humans, animals and plants” The germ theory of disease… 33 Joseph Lister indirect, evidence on the involvement of microorganisms in infections of humans The use of heat-treated instruments and spraying phenol on dressings and over the surgical area reduced the number of fatalities following surgery The germ theory of disease… 34 Friedrich Henle in 1840 (German pathologist) proposed criteria for proving that microorganisms were responsible for causing human disease (the "germ theory" of disease) The germ theory of disease… 35 Robert Koch (1843-1910) Definitive proof of the Germ Theory of Disease in 1876 Did an experiment on cattle disease anthrax and Bacillus anthracis Koch discovered the rod-shaped bacteria Bacillus anthracis Robert Koch (1843-1910) Robert Koch’s Experiment 36 a. Koch discovered Bacillus anthracis in the blood of cattle that had died of anthrax b. He cultured the bacteria on artificial media c. He injected samples of the culture into healthy animals d. These animals became sick and died of anthrax e. He isolated the same bacteria in their blood Robert Koch’s Experiment… 2018 37 Koch’s postulates 38 1. The microorganism must be present in every instance of the disease and absent from healthy individuals 2. The microorganism must be capable of being isolated and grown in pure culture 3. When the microorganism is inoculated into a healthy host, the same disease condition must result 4. The same microorganism must be re-isolated from the experimentally infected host Exceptions to Koch’s postulates 39 Many healthy people carry pathogens but do not exhibit symptoms of the disease Some microbes are very difficult or impossible to grow in artificial media. E.g. Treponema pallidum Many species are species specific. E.g. Brucella abortus cause abortion in animals but not in humans Certain diseases develop only when an opportunistic pathogen invades immuno-compromised host Pathogen: a microorganism capable of causing disease Normal flora: microorganisms that inhabit the skin and mucous membranes of healthy normal persons Importance: defense against microbial pathogens → by competing for attachment and nutrition 40 41 General Bacteriology Bacteria Are prokaryotic unicellular organisms Are relatively simple in structure Have no membrane bound organelles: mitochondria, Golgi bodies, or endoplasmic reticulum Most of them fall within a range of 0.2-2 μm The smallest bacteria (Chlamydia and Rickettsia) are 0.1-0.2 μm in diameter 42 Introduction to bacterial classification 43 Classification is the categorization of Kingdom organisms into taxonomic groups Linnaeus’s system Phylum By Swedish botanist Carolus Linnaeus Class (1707–1778) Strains with 97% similarity are grouped Order in to the same species Classification criteria Family Morphology Genus Biochemical characteristics Physiologic Characteristics Species Genetic analysis Strain 44 Introduction to bacterial classification… 45 Bacterial morphology, structures and function Bacterial Morphology 46 Bacterial Structure 47 Cytoplasmic components 48 The cytoplasm of the bacterial cell contains the DNA chromosome Plasmid Ribosomes mRNA Proteins Metabolites Cytoplasmic components 49 Bacterial chromosome Single, circular, double-stranded DNA Contained in a discrete area called nucleoid No nuclear membrane → simplifies synthesis of proteins No histones to maintain the conformation of the DNA Cytoplasmic components… 50 Plasmids extrachromosomal, double-stranded, circular DNA molecules that are capable of replicating independently are usually extrachromosomal, but can be integrated into the bacterial chromosome may occur in both gram-positive and gram-negative Transmissible vs Non-transmissible plasmids not usually essential for cellular survival, often provide a selective advantage Antibiotic resistance Resistance to ultraviolet light Exotoxins Bacteriocins Pili (fimbriae) Resistance to heavy metals, such as mercury Cytoplasmic components… 51 Transposons are pieces of DNA that move readily from one site to another either within or between the DNAs of bacteria, plasmids, and bacteriophages → nicknamed “jumping genes” Replicative transposition − move by replicating their DNA and inserting the new copy into another site Direct transposition − excised from the site without replicating and then inserted into the new site Transposons can code for − drug-resistant enzymes, toxins, or a variety of metabolic enzymes − can either cause mutations in the gene into which they insert or alter the expression of nearby genes Cytoplasmic components… 52 Ribosome Site of protein synthesis Consists of 30S + 50S subunits, forming a 70S ribosome S=Svedberg units(rate of sedimentation in a centrifuge) The proteins and RNA of the bacterial ribosome are significantly different from those of eukaryotic ribosomes major targets for antibacterial drugs Cytoplasmic components… 53 Cytoplasmic inclusion (Granule) The cytoplasm of bacteria contains several different types of granules that serve as storage areas for nutrients Cytoplasmic membrane (CM) 54 lipid bilayer structure similar to the structure of the eukaryotic membranes but it contains no sterols (e.g., cholesterol) except the mycoplasmas The membrane has four important functions a. active transport of molecules into the cell b. energy generation by oxidative phosphorylation c. synthesis of precursors of the cell wall d. secretion of enzymes and toxins Cell wall 55 All bacteria except Mycoplasma posses a thick, rigid cell wall external to the cytoplasmic membrane Bacteria possess Peptidoglycan (murein) as major cell wall component → unique to bacteria Functions maintain the shape of a bacterium serves as a point of anchorage for flagella Protects the interior of the cell from adverse changes in the outside environment Cell wall of Gram-positive bacteria 56 Thick, multilayered mainly consisting of peptidoglycan Other components: proteins, teichoic and lipoteichoic acids, and complex polysaccharides (C polysaccharides) Teichoic acids are water-soluble, covalently linked to peptidoglycan important virulence factors Lipoteichoic acids have a fatty acid and are anchored in the cytoplasmic membrane important in serotyping promote attachment to other bacteria and to specific receptors Cell wall of Gram-negative bacteria 57 more complex than gram- positive cell walls, both structurally and chemically Thin peptidoglycane (5-10% of the gram-negative cell wall by weight) Possess outer membrane (unique to gram-negative bacteria) Lipopolysaccharide (endotoxin), Porin proteins Periplasmic space Site for variety of hydrolytic enzymes Cell wall of Gram-negative bacteria 58 Porins allow diffusion of hydrophilic molecules less than 700 Da in mass through the membrane restricts entry of large and hydrophobic molecules including many antimicrobials Lipoprotein covalently attached to the peptidoglycan and is anchored in the outer membrane provide a membranous route for the delivery of newly synthesized outer membrane components to the outer membrane Cell Walls of Acid-Fast Bacteria 59 Have an unusual cell wall containing high concentration of lipids, called mycolic acids. E.g. Mycobacteria, Nocardia i. Glycocalyx External structures ii. Flagella iii. Pili and Fimbriae 60 Glycocalyx Viscous, gelatinous polymer external to the cell wall For the most part, it is made inside and secreted to cell surface If organized and is firmly attached to the cell wall → capsule If unorganized and loosely attached to the cell wall → slime layer Capsule important bacterial virulence factor prevent phagocytosis Slime layer adherence of bacteria to other bacteria and surfaces in their environment → Biofilm Flagella 61 are ropelike propellers composed of helically coiled protein subunits (flagellin) are anchored in the bacterial membranes provide motility for bacteria, allowing the cell to swim (chemotaxis) toward food and away from poisons express antigenic and strain determinants and are a ligand for a pathogen PRRs four types of arrangement are known a) monotrichous (single polar flagellum) b) lophotrichous (multiple polar flagella) c) amphitrichous (flagella at both poles of the cell d) peritrichous (flagella distributed over the entire cell) Flagella… 62 Fimbriae and Pili 63 Fimbriae (pili) (Latin for “fringe”) are hairlike structures on the outside of bacteria composed of protein subunits (pilin) morphologically distinguished from flagella are smaller in diameter (3-8 nm versus 15-20 nm) usually are not coiled in structure Fimbriae promote adherence to other bacteria or to the host F pili (sex pili) bind to other bacteria & are a tube for transfer of DNA between bacteria are encoded by a plasmid (F) Bacterial spores (endospores) 64 Spores have a thick, keratin-like coat that allows them to survive for many years, especially in the soil Spores are formed when nutrients are in short supply When nutrients are restored, spores germinate to form bacteria that can cause disease Spores are metabolically inactive but contain DNA, ribosomes, and other essential components Spores are medically important because they are highly heat resistant and are not killed by many disinfectants Formed by certain gram-positive rods, especially Bacillus and Clostridium species Bacterial spores (endospores) 65 C. tetani Bacterial exceptions 66 Mycobacteria have a peptidoglycan layer (slightly different structure) surrounded by a wax like lipid coat of mycolic acid Mycoplasma have no peptidoglycan cell wall incorporate sterols from the host into their membranes Bacterial growth 67 Growth in bacteria refers to increase in number of cells Bacteria reproduce by binary fission, a process by which one parent cell divides to form two progeny cells An increase in number of microbes from a single parent cell, gives to a single colony of cells Requirements for microbial growth 2. Chemical requirements 1. Physical requirements sources of carbon, nitrogen, Temperature Sulfur, Phosphorus, Oxygen pH trace elements osmotic pressure organic growth factors Generation Time (G) 68 It is the time required for a bacterium to double in number Physical and chemical conditions determine a bacteria’s generation time Many bacteria have generation times of 1– 3 hours ₋ E. coli and S. aureus → 20 minutes ₋ Mycobacterium species → up to 10 days Because one cell gives rise to two progeny cells, bacteria are said to undergo exponential growth (logarithmic growth) Growth Curve 69 If a fixed volume of liquid medium is inoculated with bacterial cells the number of viable cells per milliliter is determined and plotted as follows 1. Lag phase – The cells are adapting to their new niche – Enzymes are synthesized to utilize broth nutrients – According to the bacteria being grown, this phase can take from less than an hour to several days 2. Log/exponential phase – There is rapid replication and reproduction – The bacteria are dividing at their greatest rate – Metabolic activity peaked high – The new cells are young, delicate and immature – These cells are susceptible to antibiotics and UV radiation 70 3. Stationary phase – number of cells being replicated equals number of cells dying – Nutrients are depleting → growth rate slows – Waste products are building up, causing an acidic pH – Metabolic activity is greatly reduced 4. Death/decline phase – All nutrients are totally depleted – There is tremendous buildup of metabolic waste products – Cells are dying faster than they are being replicated – Total death will occur if this culture is not transferred to a new broth tube 71 Aerobic & Anaerobic Growth 72 The natural by-products of aerobic metabolism are the reactive compounds hydrogen peroxide (H2O2) and superoxide (O2−). In the presence of iron, these two species can generate hydroxyl radicals ( OH), which can damage any biologic macromolecule Aerobic & Anaerobic Growth… 73 Aerobic can survive in the presence of oxygen by possessing an elaborate system of defenses → respiration Anaerobic Live in the absence of oxygen → fermentation Do not possess the defenses that make aerobic life possible and therefore cannot survive in air Defense mechanisms to the reactive compounds a. 2O2- + 2H+ superoxide dismutase H2O2 + O2 b. 2H2O2 Catalase 2H2O + O2 Aerobic & Anaerobic Growth… 74 Obligate aerobes: require oxygen to grow because their ATP- generating system is dependent on oxygen as the hydrogen acceptor, E.g. M. tuberculosis Facultative anaerobes: use oxygen, if it is present, but they can use the fermentation pathway in the absence of oxygen Obligate anaerobes: cannot grow in the presence of oxygen because they lack either superoxide dismutase or catalase, or both. E.g. Clostridium tetani Microaerophiles: require small amounts of oxygen (2–10%) for aerobic respiration. E.g. campylobacter species Aerotolerant anaerobes: can grow in the presence of oxygen presence, but they do not use it as a hydrogen acceptor Aerobic & Anaerobic Growth… 75 76 Cultivation of Bacteria Cultivation 77 Cultivation is the process of propagating organisms by providing proper environmental conditions Bacteria divide by binary fission, asexual reproduction where a single cell divides giving rise to two cells → Those two cells give rise to four cells and so on Because one cell gives rise to two progeny cells, bacteria are said to undergo exponential (logarithmic) growth → 2n E.g. How many bacteria will a single bacterium produce after 4 generations? 24 = 16 bacteria Cultivation… 78 A suitable growth medium must contain all the nutrients required by the organism to be cultivated, and such factors as pH, temperature, and aeration must be carefully controlled Requirements for growth Organic matter containing the elements carbon, hydrogen, nitrogen, oxygen, phosphorus, and sulfur inorganic ions such as potassium, sodium, iron, magnesium, calcium, and chloride are required to facilitate enzymatic catalysis and to maintain chemical gradients across the cell membrane Sources of metabolic energy Fermentation Respiration Cultivation… 79 Carbon Autotrophs: bacteria that do not require organic nutrients for growth → use photosynthetic energy to reduce carbon dioxide Heterotrophs: require organic carbon for growth, and the organic carbon must be in a form that can be assimilated Temperature Different microbial species vary widely in their optimal temperature ranges for growth Psychrophilic: grow best at low temperatures (–5 to 15°C) Psychrotrophs: prefer cooler environments, 25 °C to refrigeration temperature. E.g. Listeria monocytogenes Mesophilic: grow best at 30–37°C Thermophilic: grow best at 50–60°C Cultivation of Bacteria… 80 Culture media: artificial media containing the required nutrients for bacterial growth Culturing/cultivation: the process of growing Bacteria on a culture media Purpose of culturing: - Isolation and identification of micro-organisms - Performing anti-microbial sensitivity tests Forms of culture media 81 1. Solid culture media (1.5% w/v agar) 2. Semisolid culture media (0.4-0.5% agar) 3. Fluid culture media (no agar) Types of culture media 82 1. Basic media 2. Enriched media 3. Enrichment media 4. Selective media 5. Differential (Indicator) media 6. Transport media 7. Identification media 1. Basic media – Supports growth of bacteria that do not require special nutrients – Example: Nutrient Broth, Nutrient Agar 2. Enriched media – Media that are enriched with whole blood, lyzed blood, Serum, special extracts or vitamins to support the growth of fastidious bacteria – E.g. Blood Agar, Chocolate Agar 83 3. Enrichment media – Liquid media that increases the numbers of a pathogen by containing enrichments and/or substances that discourage the multiplication of unwanted bacteria – Example: Selenite F broth media, Alkaline peptone water 4. Selective media – Media which contain substances ( E.g. Antibiotics) that prevent or slow down the growth of unwanted bacteria – Example: Mannitol Salt Agar 84 5. Differential media 85  Media to which indicator substances are added to differentiate bacteria  E.g. TCBS Agar differentiates sucrose fermenting yellow colonies of Vibrio cholerae from non-sucrose fermenting green colonies of other Vibrio species 6. Transport media Media containing ingredients to prevent the overgrowth of commensals and ensure the survival of pathogenic bacteria when specimens can not be cultured soon after collection Example: Amies transport media, Carry-Blair transport media Bacterial growth 86 Antimicrobial sensitivity testing 87 Placing antimicrobial discs Measuring the zones of inhibition in mm 88 Bacterial Genetics 2 Cellular structure Prokaryotes vs Eukaryotic 89 3 Bacterial Genome Bacterial genome is the total collection of genes carried by a bacterium a. Chromosome b. Extra-chromosomal genetic elements, if any 90 4 Bacterial chromosome Bacterial have a single, circular chromosome Bacteria usually have only one copy of their chromosomes (haploid) alteration of a bacterial gene (mutation) will have a more obvious eﬀect 91 5 Extra-chromosomal genetic elements Can be transfered from one bacterium to another Include 1. Plasmids 2. Prophages 3. Transposons 92 Plasmids Small, circular, self-replicating pieces of DNA (separate from the bacterial chromosome) Plasmids are not required for bacterial cells to survive under normal conditions Under stress, genes on plasmids can confer advantages (e.g. drug resistance, virulence factor) may exist freely in the cytoplasm or integrated in the bacterial chromosome Plasmids that can incorporate themselves into the bacterial chromosome are called Episomes Plasmids increase genetic variation and thus the likelihood of survival in bacteria 93 8 Phages Bacteriophages are viruses that can infect bacteria Bacteriophages infect bacterial cells either replicate to large numbers and cause the cell to lyse (lytic infection) or integrate into the host genome without killing the host (the lysogenic state) Some lysogenic bacteriophages carry toxin genes (e.g., gene for the diphtheria toxin) 94 9 Transposons (jumping genes) are mobile genetic elements that can transfer DNA within a cell from one position to another in the genome, or between diﬀerent molecules of DNA (e.g., plasmid to plasmid or plasmid to chromosome) They do so by synthesizing a copy of their DNA and inserting the copy at another site in the bacterial chromosome or the plasmid 95 Bacterial Genetics… 96 Mutation: any change in the base sequence of the DNA can result in the insertion of a different amino acid or stop codon into a protein → appearance of an altered phenotype Results from three types of molecular changes a. Base substitution b. Frameshift mutation c. when transposons or insertion sequences are integrated into the DNA Bacterial Genetics… 97 Silent mutation change at the DNA level that does not result in any change of amino acid in the encoded protein This type of mutation occurs because more than one codon may encode an amino acid Bacterial Genetics… 98 A. Base substitution: occurs when one base is inserted in place of another Missense mutation: When the base substitution results in a codon that simply causes a different amino acid to be inserted Nonsense mutation: when the base substitution generates a termination codon that stops protein synthesis prematurely Bacterial Genetics… 99 B. Frameshift mutation occurs when one or more base pairs are added or deleted shifts the reading frame on the ribosome results in incorporation of the wrong amino acids “downstream” from the mutation and in the production of an inactive protein Bacterial Genetics… 100 C. Mutation occurs when transposons or insertion sequences are integrated into the DNA these newly inserted pieces of DNA can cause profound changes in the genes into which they insert and in adjacent genes Many mutations occur spontaneously in nature (e.g., by polymerase mistakes) Physical or chemical agents can also induce mutation Physical agents: heat, ultraviolet light, ionizing radiation (such as x-rays) Chemical mutagens: ethidium bromide, acridine derivatives, nitrous acid (HNO2) Transfer of DNA within bacterial cells 101 A. Transposons transfer DNA from one site on the bacterial chromosome to another site or to a plasmid B. Transfer of DNA within bacteria also occurs by programmed rearrangements movement of a gene from a silent storage site where the gene is not expressed to an active site where transcription and translation occur the insertion of a new gene into the active site in a sequential, repeated programmed manner is the source of the consistent antigenic variation Transfer of DNA between bacterial cells 102 The transfer of genetic information from one cell to another can occur by three methods: Conjugation Transduction Transformation Consequences of DNA transfer antibiotic resistance genes are spread from one bacterium to another primarily by conjugation Several important exotoxins are encoded by bacteriophage genes and are transferred by transduction 11 103 Transformation process by which bacteria take up fragments of naked DNA and incorporate them into their genomes 104 Transformation…Griffith’s Experiment 105 Transduction transfer of bacterial DNA from one cell to another by means of a bacteriophage infection Two types a. generalized transduction b. specialized transduction 106 Generalized transduction Random packaging of bacterial host cell DNA in phage capsid 107 Specialized transduction When prophage genome is excised it drags adjacent bacterial genes resulting in hybrid phagebacterial genome 108 Conjugation is the mating of two bacterial cells DNA is transferred from donor (male) to recipient (female) cell through sex pilus Mating is controlled by an F (fertility) plasmid (F factor) carries the genes for the proteins required for conjugation Conjugation is unidirectional F+ → F - Conjugation occurs usually between members of the same or related species conjugation can transfer conjugative plasmid or plasmid with bacterial genes to which it is integrated 109 Conjugation…conjugative plasmid 110 Conjugation…chromosome integrated plasmid Some F+ cells have their F plasmid integrated into the bacterial DNA → can transfer part of the chromosome into another cell These cells are called Hfr (high- frequency recombination) cells the single strand of DNA that enters the recipient F– cell contains a piece of the F factor at the leading end followed by the bacterial chromosome and then by the remainder of the F factor The time required for complete transfer of the bacterial DNA is approximately 100 minutes Most matings result in the transfer of only a portion of the donor chromosome b/c the attachment b/n the two cells can break 19 Conjugation… General Virology General properties of viruses 113 Viruses are the smallest infectious agents (20-300 nm) most seen only with an electron microscope Viruses do not have cellular organization Viruses possess either DNA or RNA Viruses are obligate intracellular parasites Most lack enzymes for protein or nucleic acid synthesis ̶ depend on host living cells Viruses are known to infect all cells, including bacteria Viruses are unaffected by antibacterial agents Reproduce by assembly of individual components rather than by binary fission General properties of viruses… 114 Viruses are basically made up of nucleic acid and capsid viral nucleic acid is contained within a capsid Capsid is made up of protein molecules called capsomeres The complete unit of nucleic acid and capsid is called the nucleocapsid (Virion, for naked viruses) General properties of viruses… 115 Viral nucleic acids The viral nucleic acid (genome) is located internally and can be either single- or double-stranded DNA or single- or double-stranded RNA Only viruses have genetic material (a genome) composed of Single-stranded DNA; E.g. Parvovirus Double-stranded RNA; E.g. Rotavirus The nucleic acid can be either linear or circular General properties of viruses… 116 Viral nucleic acids… Viral DNA is always a single molecule Viral RNA can exist either as a single molecule or in several pieces For example: Influenza virus and rotavirus have a segmented RNA genome Almost all viruses contain only a single copy of their genome (i.e., they are haploid) Exception: Retrovirus family, whose members have two copies of their RNA genome (diploid) General properties of viruses… 117 Viral capsid & symmetry The shape of virus particles is determined by the arrangement of the repeating subunits that form the protein coat (capsid) of the virus The arrangement of capsomers gives the virus structure its geometric symmetry a. Icosahedral b. Helical General properties of viruses… 118 Icosahedral ̶ capsomers are arranged in triangles that form a symmetric figure with the approximate outline of a sphere ̶ can be either enveloped or naked Helical ̶ capsomers are arranged in a hollow coil that appears rod-shaped ̶ All human viruses with helical nucleocapsid are enveloped 2018 General properties of viruses… 119 The advantage of building the virus particle from identical protein subunits is twofold: a. It reduces the need for genetic information b. It promotes self-assembly (i.e., no enzyme or energy is required) Functional virus particles have been assembled in the test tube by combining purified nucleic acid with purified proteins in the absence of cells, energy source, and enzymes General properties of viruses… 120 Viral proteins Viral proteins serve several important functions Capsid proteins: protect the viral genome from degradation by nucleases Surface proteins: mediate the attachment of the virus to specific receptors on the host cell surface The interaction of the viral proteins with the cell receptor is the major determinant of species and organ specificity Outer viral proteins are also important antigens that induce neutralizing antibody and activate cytotoxic T cells The term “serotype” is used to describe a subcategory of a virus based on its surface antigens Viral classification 121 Based on their genetic material as DNA and RNA viruses Based on presence/absence of envelope, viruses are classified as 1. Necked viruses: consists of only nucleocapsid 2. Enveloped viruses: consists of nucleocapsid surrounded by an outer envelope or membrane matrix protein, mediates the interaction between the capsid proteins and the envelope Viral classification 122 Naked (un-enveloped) viruses Capsid is a rigid structure → withstand harsh environmental conditions → survive well in the outside world Environmentally stable to: temperature, acid, proteases, detergents and drying Released from cell by lysis Viral classification… 123 Enveloped viruses Frequently possess glycoproteins in the form of spike-like projections on the surface, which attach to host cell receptors The viral envelope is acquired as the virus exits from the cell in a process called “budding” The envelope of most viruses is derived from the cell’s outer membrane Exception: herpesviruses that derive their envelope from nuclear membrane Readily disrupted by drying, acidic conditions, detergents, heat Must remain wet and are generally transmitted in ﬂuids, respiratory droplets, blood, and tissue Atypical virus-like agents 124 Defective viruses 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 genetic material Pseudovirions contain host cell DNA instead of viral DNA within capsid Formed during infection with certain viruses when the host cell DNA is fragmented and pieces of it are incorporated within the capsid protein can infect cells, but do not replicate Atypical virus-like agents… 125 Viroids consist solely of a single molecule of circular RNA without a protein coat or envelope The RNA is quite small and apparently does not code for any protein Can replicate, but the mechanism is unclear Cause several plant diseases but are not implicated in any human disease Atypical virus-like agents… 126 Prions are infectious particles that are composed solely of protein they contain no detectable nucleic acid implicated as the cause of certain “slow” diseases called transmissible spongiform encephalopathies Different from viruses b/c posses neither DNA nor RNA Much more resistant to inactivation by ultraviolet light and heat than are viruses remarkably resistant to formaldehyde and nucleases. inactivated by hypochlorite, NaOH, and autoclaving no immune response is formed against this protein there is no inflammatory response in infected brain tissue Viral Replication 127 Viruses should infect cells in order to replicate The cell acts as a factory, providing the substrates, energy, and machinery necessary for the synthesis of viral proteins and replication of the genome Processes not provided by the cell must be encoded in the genome of the virus Each infected cell may produce as many as 100,000 particles only 1-10% of these particles may be infectious The noninfectious particles (defective particles) result from mutations and errors in the manufacture and assembly of the virion Steps in Viral Replication 128 1. Recognition of the target cell 2. Attachment 3. Penetration 4. Uncoating 5. Macromolecular synthesis a. Early mRNA and nonstructural protein synthesis: genes for enzymes and nucleic acid–binding proteins b. Replication of genome c. Late mRNA and structural protein synthesis d. Posttranslational modification of protein 6. Assembly of virus 7. Budding of enveloped viruses 8. Release of virus Viral Replication 129 Recognition of and attachment to the target cell The 1st step during virus replication The virus must recognize an appropriate target cell and attach to receptors on the cell determines which cells can be infected by a virus Viral attachment structure Naked viruses: part of capsid or a protein that extends from the capsid Enveloped viruses: glycoproteins Viral Replication 130 Penetration Interactions between multiple VAPs and cellular receptors initiate the internalization of the virus into the cell Most nonenveloped viruses enter the cell by receptor-mediated endocytosis or by viropexis Enveloped viruses fuse their membranes with cellular membranes to deliver the nucleocapsid or genome directly into the cytoplasm → neutral PH internalized by endocytosis, and fusion occurs in an endosome at acidic pH Viral Replication 131 Uncoating Once internalized nucleocapsid delivered to the site of replication within the cell and the capsid or envelope removed The genome of DNA viruses, except for poxviruses, must be delivered to the nucleus Most RNA viruses remain in the cytoplasm except for orthomyxoviruses and retroviruses Viral Replication 132 Macromolecular synthesis Once inside the cell, the genome must direct the synthesis of viral mRNA and protein generate identical copies of itself Transcription of the DNA virus genome (except for poxviruses) occurs in the nucleus, using host cell polymerases and other enzymes for viral mRNA synthesis RNA viruses must encode the necessary enzymes for transcription and replication Viral Replication 133 Positive-strand RNA viral genomes act as mRNA, bind to ribosomes, and direct protein synthesis The naked ps-RNA viral genome is sufficient to initiate infection by itself E.g. picornaviruses, caliciviruses, coronaviruses, flaviviruses, and togaviruses Viral Replication 134 Negative-strand RNA viral genomes are the templates for production of individual mRNAs The negative-strand RNA genome is not infectious nor can it bind to the ribosome a polymerase must be carried into the cell with the genome to make mRNAs Rhabdoviruses, orthomyxoviruses, paramyxoviruses, filoviruses, and bunyaviruses Viral Replication 135 Assembly The assembly process begins when the necessary pieces are synthesized Assembly of DNA viruses other than poxviruses occurs in the nucleus and requires transport of the virion proteins into the nucleus RNA virus and poxvirus assemblies occur in the cytoplasm Release Viruses can be released from cells after lysis of the cell, by exocytosis, or by budding from the plasma membrane Viral replication Recognition of the target cell ↓ Attachment/Adsorption ↓ Penetration ↓ Uncoating ↓ Macromolecular synthesis ↓ Assembly of virus ↓ Release of virus 136 Outcomes of viral infection of a cell 137 1. Death 2. Fusion of cells to form multinucleated cells 3. Malignant transformation 4. No apparent morphologic or functional change General Mycology Introduction 139 Mycology is the study of fungi There are ~80,000 species of fungi fewer than 400 are medically important less than 50 species cause more than 90% of the fungal infections of humans and other animals Fungi are ubiquitous as free-living organisms Important commercially in baking, brewing and in pharmaceuticals Characteristics of Fungi 140 Fungi are classified into their own separate kingdom, Kingdom Fungi (Myceteae) They are eukaryotic organisms but are distinguished from other eukaryotes by possession of: rigid cell wall composed of chitin and glucan cell membrane in which ergosterol is substituted for cholesterol as the major sterol component Relative to bacteria, fungi are slow growing with cell- doubling times in terms of hours rather than minutes Fungi have emerged in the past three decades as major causes of human disease Characteristics of Fungi… 141 Most fungi are obligate aerobes; some are facultative anaerobes; but none are obligate anaerobes All fungi require a preformed organic source of carbon hence their frequent association with decaying matter The natural habitat of most fungi is, therefore, the environment An important exception is Candida albicans, which is part of the normal human flora Characteristics of Fungi… 142 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 A. Arthrospores: arise by fragmentation of the ends of hyphae and are the mode of transmission of Coccidioides immitis B. Chlamydospores: are rounded, thick-walled, and quite resistant C. Blastospores: are formed by the budding process by which yeasts reproduce asexually D. Sporangiospores: are formed within a sac (sporangium) Asexual spores Chlamydospores (Candida) Arthrospores (Coccidioides) Blastoconidia (Candida) Sporangia and sporangiospores (Mucor) Microconidia (Aspergillus) Microconidia and macroconidia (Microsporum) 143 Comparison of Fungi and Bacteria 144 Fungal classification Based on morphology a) Yeasts Unicellular w/c reproduces by budding or by fission produce round, pasty, or mucoid colonies on agar b) moulds multicellular organisms consisting of threadlike tubular structures called hyphae The hyphae form together to produce a matlike structure called a mycelium The colonies formed by moulds are often described as filamentous, hairy, or woolly 14 5 14 6 Yeast Mold 147 Fungal classification… c. Dimorphic fungi exhibit thermal dimorphism (i.e., exist as yeast at body temperature [37°C] and mould at room temperature [25°C]) Example o Histoplasma capsulatum o Blastomyces dermatitidis o Coccidioides immitis o Paracoccidioides brasiliensis 14 8 Classification of Human Mycoses 1) Superficial mycoses  Infections limited to the very superficial surfaces of the skin and hair 2) Cutaneous mycoses  infections of the keratinized layer of skin, hair, and nails  These infections may elicit a host response and become symptomatic 3) Subcutaneous mycoses  involve the deeper layers of the skin, including the cornea, muscle, and connective tissue 14 9 Classification of Human Mycoses… 4) Endemic mycoses (systemic mycoses) Are caused by the classic dimorphic fungal pathogens these organisms are true pathogens and can cause infection in healthy individuals All of these agents produce a primary infection in the lung, with subsequent dissemination to other organs and tissues 5) Opportunistic mycoses infections caused by fungi that are normally found as human commensals or in the environment 15 0 Fungal Toxins & Allergies 151 Amanita mushrooms produce five toxins amanitin and phalloidin: potent hepatotoxins amanitin inhibit cellular RNA polymerase → prevents mRNA synthesis Aflatoxins produced by Aspergillus flavus that cause liver damage and tumors in animals ingested with spoiled grains and peanuts metabolized by the liver to the epoxide, a potent carcinogen Aflatoxin B1 induces a mutation in the p53 tumor suppressor gene Fungal Toxins & Allergies 152 Allergies to fungal spores, particularly those of Aspergillus, are manifested primarily by an asthmatic reaction (rapid bronchoconstriction mediated by IgE) Eosinophilia “wheal and flare” skin test reaction These clinical findings are caused by an immediate hypersensitivity response to the fungal spores 153 Host-parasite relationship Symbiotic Associations 154 All living animals are used as habitats by other organisms None is exempt from such invasion Bacteria are invaded by viruses (bacteriophages) Amoebas are natural hosts for Legionella pneumophila infection Symbiosis Interaction between two different organisms living in close physical association Symbiotic Associations… 155 Commensalism 156 one species of organism lives harmlessly in or on the body of a larger species one species of organism uses the body of a larger species as its physical environment and may make use of that environment to acquire nutrients Mutualism 157 Mutualistic relationships provide benefits for the two organisms involved Frequently, the relationship is obligatory for at least one member, and may be for both Parasitism 158 the symbiotic relationship benefits only the parasite Parasitism is a one-sided relationship in which the benefits go only to the parasite the host provides parasites with their physicochemical environment, their food, respiratory and other metabolic needs 159 The bacterial flora of human The bacterial flora… 160 Up until the time of birth, the human fetus lives in a remarkably protected and for the most part sterile environment Then, the infant is exposed to bacteria, archaea, fungi, and viruses from the mother, other close contacts, and the environment Over the next few years, communities of organisms (microbiota or normal flora) form on the surfaces of the skin, nares, oral cavity, intestines, and genitourinary tract The bacterial flora… 161 Microbiota (normal flora) Community of microbes that live in and on an individual Microbiome Aggregate collection of microbial genomes in the microbiota The bacterial flora… 162 Core Microbiome Most individuals share a core microbiome species that are present at a specific site in 95% or more of individuals Secondary microbiome The remaining portion of the population (

Microbiology for MD - Africa Medical College - Dec, 2024 PDF

Document Details

Tags

Related

Summary

Full Transcript