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Microbiology Notes To accompany Foundations in Microbiology by K. Talaro Updated Summer 2007 By, Kevin Young, Utah State University Table of Contents Unit 1. Unit 2. Unit 3. Unit 4. Unit 5. Unit 6. Unit 7. Unit 8. Unit 9. Unit 10. Unit 11. Unit 12. Unit 13. Unit 14. Unit 15. Unit 16. Unit 17. Unit 18. Unit 19. Unit 20. Main Themes of Microbiology (Ch 1) ............................................................. 2 Tools of the Laboratory (Ch 3) ......................................................................... 6 Procaryotic Profiles (Ch 4) ............................................................................. 11 Eucaryotic Cells and Microorganisms (Ch 5) ................................................ 22 Elements of Microbial Nutrition, Ecology, and Growth (Ch 7) ..................... 26 Microbial Metabolism: The Chemical Crossroads of Life (Ch 8).................. 30 Microbial Genetics (Ch 9) .............................................................................. 36 Physical and Chemical Control of Microbes (Ch 11)..................................... 42 Drugs, Microbes, Host—The Elements of Chemotherapy (Ch 12) ............... 44 Microbe-Human Interactions: Infection and Disease (Ch 13)........................ 54 The Nature of Host Defenses (Ch 14) ............................................................ 62 The Acquisition of Specific Immunity and its Applications (Ch 15) ............. 67 Cocci of Medical Importance (Ch 18) ............................................................ 68 Gram-Positive Bacilli of Medical Importance (Ch 19) .................................. 76 Gram-Negative Bacilli of Medical Importance (Ch 20) ................................. 81 Miscellaneous Bacterial Agents of Disease (Ch 21) ...................................... 87 Introduction to Viruses (Ch 6) ........................................................................ 94 DNA Viruses of Medical Importance (Ch 24) ............................................. 101 RNA Viruses of Medical Importance (Ch 25).............................................. 111 Appendix: Bacteria to Know ........................................................................ 112 Elementary Microbiology Unit 1. Main Themes of Microbiology (Ch 1) A. Introduction 1. As recently as 1967 it was believed that we would soon be rid of infectious diseases 2. 30 years later it was noted that infectious diseases were an increasing problem 3. Microbes have changed a. Some that we thought we could eradicate or control have evolved resistance b. There are a lot of emerging diseases from previously unknown viruses and bacteria 4. People have changed a. Higher densities 1) Earth’s human population increasing at about 200,000 people daily (74,000,000 yearly!) b. More contact (sexual promiscuity, drug use, etc) c. More travel d. More encroachment into natural habitats 1) May be coming into contact with previously unknown disease agents 5. Variety of factors lead to one sobering conclusion: serious epidemics WILL happen a. Flu of 1918-19 killed an estimated 50-100 million people 1) Compare to 20-40 million killed on both sides of WWI 2) Compare to AIDS killing 25 million in 25 years 3) Virus was similar to the bird flu virus we are worried about today 6. While we focus on microbes that cause disease, most of them are harmless or helpful B. The Scope of Microbiology 1. Groups of Microorganisms a. Bacteria—our main focus for this course b. Viruses—strange “rogue bits of DNA” that are not really alive (or dead) c. Fungi—includes molds, mushrooms, yeasts d. Protozoa—complex single-celled organisms; mostly free-living e. Algae—we will be ignoring these f. Helminthes—roundworms, such as pin worms, are mostly not dangerous 1) Can be problematic if you have a weakened immune system 2) Lack of roundworms may contribute to allergies and autoimmune diseases 2. Branches of Study within Microbiology a. Immunology 1) The study of the immune chemical and cells that are produced in response to infection 2) Hypersensitivity or inappropriate immune response that can be harmful to the human host (i.e. allergies) 2 Main Themes of Microbiology (Ch 1) b. Epidemiology 1) Monitor and control the spread of diseases in communities 2) CDC – Centers for Disease Control and Prevention 3) WHO – World Health Organization c. Food, Dairy, and Aquatic microbiology examine the ecological and practical roles of microbes in food and water (see chapter 26) d. Agricultural microbiology 1) Studies relationships between microbes and crops, with an emphasis on improving yields and combating plant diseases e. Biotechnology 1) The use of the metabolism of living things to arrive at a desired product (a) Bread making (b) Gene therapy f. Genetic Engineering and recombinant DNA technology 1) Techniques used to deliberately alter the genetic makeup of organisms to mass-produce human hormones and other drugs 2) Create totally novel substances 3) Develop organisms with unique methods of synthesis and adaptation (see chapter 10) C. The Impact of Microbes on Earth: Small Organisms with a Giant Effect 1. For billions of years microbes have shaped the development of the earth’s habitats and the evolution of other life forms 2. Bacterial-type organisms have been on this planet for about 3.5 billion years (prokaryotes – pre-nucleus) 3. Eucaryotes, with a nucleus, emerged 1.8 billion years ago (these cells were precursors to the cell type that eventually formed multicellular animals, including humans 4. Microbes can be found everywhere, from deep in the earth’s crust, to the polar ice caps and oceans, to the bodies of plants and animals 5. Microbial Involvement in Energy and Nutrient Flow a. Microorganisms are involved in nutrient production and energy flow. Algae and certain bacteria trap the sun’s energy to produce food through photosynthesis b. Photosynthetic microorganisms, including algae, account for more than 50% of the earth’s photosynthesis, contributing the majority of the oxygen to the atmosphere c. Other microbes are responsible for the breakdown and recycling of nutrients through decomposition. Microbes are essential to the maintenance of the air, soil, and water. 6. Human Use of Microorganisms a. Microbes have been called upon to solve environmental, agricultural, and medical problems 3 Elementary Microbiology b. Biotechnology applies the power of microbes toward the manufacture of industrial products, foods, and drugs c. Microbes form the basis of genetic engineering and recombinant DNA technology, which alter genetic material to produce new products and modified life forms. d. With bioremediation, microbes are used to clean up pollutants and wastes in natural environments 7. Infectious Diseases and the Human Condition a. Nearly 2,000 microbes are pathogens that cause infectious diseases. Infectious diseases result in high levels of mortality and morbidity. Many infections are emerging, meaning that they are newly identified pathogens gaining greater prominence. Many older diseases are also increasing D. The General Characteristics of Microorganisms 1. Cellular Organization Excluding viruses there are two types of microorganisms a. Prokaryotes – small and lack a nucleus and organelles, and b. Eucaryotes – larger and have both a nucleus and organelles 2. A Note on Viruses a. Viruses are not cellular and are therefore sometimes called particles rather than organisms. They are include din microbiology because of their small size and close relationship with cells 3. Microbial Dimensions: How Small is Small? a. Microbes are too small to be seen with the unaided eye. b. Microorganisms are measured in micrometers and sometimes nanometers and millimeters. (see figure 1.7, pg. 8) 1) Review the metric system and be comfortable using it! 2) We are putting ourselves at a disadvantage by sticking with the old British system (a) The use of two different systems was the contributing factor in the loss of the Mars Climate Orbiter in 1998. - NASA specified metric units in the contract. NASA and other organizations worked in metric units but one subcontractor, Lockheed Martin, provided thruster performance data to the team in pound force seconds instead of newton seconds. - The spacecraft was intended to orbit Mars at about 150 km altitude but the incorrect data meant that it descended to about 57 km and probably burned up in the Martian atmosphere. 3) http://www.themetricsystem.info/ is one place to review metric 4. Life-styles of Microorganisms a. Most microorganisms are harmless, free-living species that perform vital functions in both the environment and larger organisms. Few species are agents of disease. 4 Main Themes of Microbiology (Ch 1) E. The Historical Foundations of Microbiology 1. Microbiology is about 200 years old. Hundreds of contributors have provided discoveries and knowledge to help our understanding. 2. The Development of the Microscope: “Seeing is Believing” Leeuwenhock discovered organisms he called animalcules. 3. The Establishment of the Scientific Method a. The scientific method develops rational hypothesis and theories that can be tested. Theories that withstand repeated scrutiny become law in time. 4. The Development of Medical Microbiology a. Early microbiology blossomed with the conceptual developments of sterilization, aseptic techniques, and the germ theory of disease F. Taxonomy: Organizing, Classifying, and Naming Microorganisms 1. Taxonomy is a hierarchy scheme for the classification, identification, and nomenclature of organisms, which are grouped according to categories called taxa, based on features ranging from general to specific 2. The Levels of Classification (see figure 1.14, pg. 17) a. Kingdom b. Phylum (division) c. Class d. Order e. Family f. Genus g. Species 3. Assigning Specific Names a. Organisms are assigned binomial scientific names consisting of their genus and species names. 4. The origin and Evolution of Microorganisms a. Evolution states that living things change gradually through hundreds of millions of years and that these evolvements result in various types of structural and functional changes through many generations. 1) All new species originate from preexisting species 2) Closely related organisms have similar features because they evolved from common ancestral forms. 5. Systems of Presenting a Universal Tree of Life a. Evolution patterns show a treelike branching from simple, primitive life forms to complex, advanced life forms 5 Elementary Microbiology Unit 2. Tools of the Laboratory (Ch 3) A. Methods of Culturing Microorganisms – the Five I’s 1. Inoculation: Producing a Culture a. Sample is placed into a container of sterile medium that provides microbes with appropriate nutrients for growth b. Inoculation involved using a sterile tool to spread the sample on the surface of a solid medium or to introduce the sample into a flask or tube c. Selection of media with specialized functions can improve later steps of isolation and identification d. Some microbes may require a live organism (animal, egg) as the inoculation medium 2. Incubation: a. An incubator can be used to adjust the proper growth conditions of a sample b. Setting the optimum temperature and gas content promotes multiplication of the microbes over a period of hours, days, and even weeks c. Incubation produces a culture – the visible growth of the microbe in the medium 3. Isolation: Separating One Species from Another a. The end result of inoculation and incubation is isolation of the microbe in macroscopic form b. The isolated microbes may take the form of separate colonies (discrete mounds of cells) on solid media, or turbidity in broths c. Further isolation, known as subculturing, involves taking a tiny bit of growth from an isolated colony and inoculating a separate medium d. This is one way to make a pure culture that contains only a single species of microbe 4. Inspection: a. The culture is observed macroscopically for obvious growth characteristics that could be useful in analyzing the specimen contents: 1) Color 2) Texture 3) Size b. Slides are made to assess microscopic details such as: 1) Cell shape 2) Cell size 3) Cell motility c. Staining techniques may be used to gather specific information on microscopic morphology 5. Identification A major purpose of the 5 I’s is to determine the type of microbe, usually to the level of species a. Summaries of characteristics are used to develop profiles of the microbe or microbes isolated from the sample 6 Tools of the Laboratory (Ch 3) b. Information can include relevant data already taken during inspection and additional tests that further describe and differentiate the microbes c. Specialized tests include: 1) Biochemical tests to determine metabolic activities specific to the microbe 2) Immunologic tests 3) Genetic analysis 6. Media: Providing Nutrients in the Laboratory a. Types of Media 1) Physical Form (a) Liquid: water-based solutions that do not solidify at temperature above freezing and that tend to flow freely when the container is tilted. Broths, milks, or infusions, are made by dissolving various solutes in distilled water (b) Semisolid: at room temperature they exhibit a clotlike consistency because they contain an amount of solidifying agent (agar or gelatin) that thickens them but does not produce a firm substrate. This media is used to determine the motility of bacteria and to localize a reaction at a specific site (c) Solid: provide a firm surface on which cells can form discrete colonies and are advantageous for isolating and culturing bacteria and fungi Two forms: - Liquefiable solid media, reversible solid media, contain a solidifying agent that changes its physical properties in response to temperature. The most widely used media because it is solid at room temperature and melts. - Agar – complex polysaccharide isolated from the red alga Gelidium - Role of Fannie Hess - Non liquefiable solid media have less versatile applications than agar media because they do not melt 2) Chemical Composition (a) Synthetic (chemically defined) - Contain pure organic and inorganic compounds that are specified by an exact formula - Minimal media – for fungi – contain only a few essential compounds like salt and amino acids, other types of synthetic media are useful in research and cell culture when the exact nutritional needs of the test organisms are known (see table 3.2, pg. 65) (b) Nonsynthetic (not chemically defined) - Contain at least one ingredient that is not chemically definable, most of these substances are extracts of animals, plants, or yeasts. Examples include: - Blood - Serum - Meat extracts - Serums – high in vitamins, minerals, proteins and other organic nutrients - Milk - Yeast extracts - Soybean digests - Peptone – partially digested protein, rich in amino acids, a source of carbon or nitrogen 3) Functional Type (a) General purpose 7 Elementary Microbiology - (b) (c) (d) (e) (f) (g) Depending upon what is added, a microbiologist can fine-tune a medium for nearly any purpose, only a few species of bacteria or fungi cannot yet be cultivated artificially because of this. - General purpose media are designed to grow as broad a spectrum of microbes as possible - They are usually nonsynthetic and contain a mixture of nutrients that could support the growth of pathogens and nonpathogens - Examples – nutrient agar and broth, brain-heart infusion, trypticase soy agar (TSA) Enriched - Contain complex organic substances (blood, serum, hemoglobin, special growth factors) - Special growth factors are specific vitamins and amino acids that certain species must have to grow - Bacteria that require growth factors and complex nutrients – fastidious - Blood agar, made by adding sterile sheep, horse or rabbit blood, to a sterile agar base is used to grow fastidious streptococci or other pathogens Selective & Differential - These media are designed for special microbial groups, and have extensive applications in isolation and identification, they can permit a single step preliminary identification of a genus or even a species - Selective medium contain one or more agents that inhibit the growth of a certain microbe or microbes, this encourages, or selects, a certain microbe to grow - Selective media are very important in primary isolation of a specific type of microorganism from samples that contain dozens of different species - Mannitol salt agar contains a 7.5% concentration of NaCl that is inhibitory to most human pathogens except staphylococcus - See table 3.3 pg. 66 for other types of selective media - Differential media grow several types of microorganisms and are designed to display visible differences among them - Differentiation shows up as variations in colony size or color, media color changes, or formation of gas bubbles and precipitates - Dyes can be used as differential agents because many of them are pH indicators that change color in response to the production of an acid or a base - See table 3.4 on pg. 67 for other examples of differential media Anaerobic growth Specimen transport used to maintain and preserve specimens that have to be held for a period of time before clinical analysis or to sustain delicate species that die rapidly if not held under stable conditions Assay media – used to test the effectiveness of antimicrobial drugs Enumeration media are used to count the numbers of organisms in milk, water, food, soil and other samples B. The Microscope: Window on an Invisible Realm 1. Magnification and microscope design a. Microbiology did not start until microscopes were invented 8 Tools of the Laboratory (Ch 3) b. Antonie Van Leeuwenhoek, a Dutch textile maker, was the first to see living microscopic organisms c. Light is bent (refracted) as it passes through a curved glass 1) Different sizes and curvatures of lenses give different magnifications 2) Early microscopes only had a single lens that was held up to the light 3) With a typical modern light microscope the light will pass through two magnifying lenses (a) The ocular lens is what you look directly through. It typically magnifies 10X. (b) There are usually 3 or 4 objective lenses to choose from, and they typically magnify between 10X and 100 X. (c) There is a condenser lens to focus light onto the sample (this lens does not magnify the image) (d) A series of mirrors takes the image from the objective to the ocular lens. (e) Understand the path of light: light source, condenser, specimen, objective, ocular, eye 4) The total magnification is the product of the objective lens times the ocular lens (a) A 10X ocular and a 40X objective gives 400X total magnification (b) You can have high magnification but a terrible image if you have low resolution - Resolution is limited by several factors, including wavelength (smaller wavelength gives better resolution) - Placing a drop of oil between the specimen and the objective lens increases resolution by reducing refraction, because the oil and glass have the same optical properties (but air and glass are different) 5) The maximum resolution of a light microscope is 200 nm (0.2 μm). (a) An electron microscope has a maximum resolution of 0.5 nm 2. Variations of light microscopes a. Bright-field microscope is the most common type 1) Specimen is darker than the surrounding light background b. Dark-field microscope blocks all light except that entering from the sides. 1) The result is a bright image against a dark background 2) Used to see bacteria from a chancre when someone has a syphilis infection (a) Treponema pallidum, a spirochete (b) Also used for viewing live, unstained specimens to see them moving c. Phase-contrast microscope is used for viewing intracellular structures (such as internal organelles of eucaryotes or bacterial endospores) d. Fluorescence microscopy uses fluorescent dyes and a UV light 1) If the dye is attached to an antibody that sticks to a specific organism, this will greatly speed up identification (a) For example, if you are wondering if a patient has tuberculosis, then a fluorescent antibody that sticks to Mycobacterium tuberculosis is added to a sample of phlegm - If the bacteria are present the dye will stick to them and the bacteria will grow brightly under the fluorescent microscope - The dye will not stick to other bacteria that may be present (b) A different fluorescent antibody must be made for each bacteria of interest 3. Electron microscopes 9 Elementary Microbiology a. Because the electron beam makes waves 100,000 times shorter than light waves, the resolution is much greater 1) This allows magnification from 5,000X to 1,000,000X b. The image is not viewed directly through a lens—it is projected onto a viewing screen c. All specimens must be prepared with a dye or a thin coating of metal, so no live specimens can be viewed d. All images from electron microscopes are black and white, but are often colored for clarity e. Two types: scanning electron microscope (SEM) and transmission electron microscope (TEM) 1) TEM passes electrons through the specimen. This has greater resolution and up to 1,000,000X magnification 2) SEM bombards a metal-coated specimen with electrons while scanning over the surface (a) This produces detailed 3D images (b) Can magnify up to 100,000X - Not as high magnification as the TEM, but still much higher than a light microscope (which has maximum magnification of about 1,000X) 4. Preparing specimens for optical microscopes a. Hanging drop slides are used to view live specimens 1) If movement is seen, then you can assume the bacteria has flagella, even though you cannot see them b. Positive staining is when the dye sticks to the specimen 1) This is the most common form of slide preparation (a) Simple stains use only one dye (b) Differential stains use a primary dye and a counterstain to distinguish between cell types (c) Gram stain is the most universal - We will learn how to do a Gram stain and why it works the way it does - “Gram-negative” bacteria stain reddish pink, while “Gram-positive” bacteria stain purple (d) Acid-fast stain used mainly to distinguish Mycobacterium (one species causes tuberculosis, a different species causes leprosy) (e) An endospore stain helps determine if the bacterial species you are looking at can form spores - Bacillus and Clostridium are endospore formers (f) Other special staining techniques used to detect capsules and flagella c. Negative staining darkens the background but does not stick to the specimen 1) India ink is a common negative stain 2) Allows for a quick assessment of cell shape, size, and arrangement 3) Also good for viewing cell capsules when present 10 Procaryotic Profiles (Ch 4) Unit 3. Procaryotic Profiles (Ch 4) A. Procaryotic Form and Function: External Structure It is thought that the very first cells to appear on the earth were a type of archaea possibly related to modern forms that live on sulfur compounds in geothermal ocean vents. The cellular structure and function are amazingly versatile and adaptable (see Fig 4.1, pg. 88) B. The Structure of a Generalized Procaryotic Cell 1. Appendages: Cell Extensions Appendages are not present on all species and can be divided into to major groups: those that provide motility (flagella and axial filaments) and those that provide attachments (fimbriae and pili) a. Flagella – Bacterial Propellers (see Fig 4.2, pg. 89) 1) Filament is a helical structure composed of proteins, it inserts into the hook 2) Hook (sheath) is anchored to the cell by the basal body – the hook with its filament can rotate 360, rather than whipping back and forth and was once thought 3) Basal body is a stack of rings firmly anchored through the cell wall, to the cell membrane 4) All spirilla, about half of the bacilli and some cocci are flagellated. The flagella vary in both number and arrangement according to the following patterns: (a) Polar arrangement – flagella are attached at one or both ends of the cell – the fastest swimmers Monotrichous – single flagellum Lophotrichous – small bunches or tufts of flagella emerging from same site Amphitrichous – flagella at both poles of the cell (b) Peritrichous arrangement – flagella are dispersed randomly over the surface of the cell 5) Flagellar Function: (a) Motility - flagellated bacteria can detect and move in response to chemical signals (chemotaxis) - Positive chemotaxis is movement of a cell in the direction of favorable chemical stimulus (usually a nutrient) - Negative chemotaxis is movement away from a repellent (potentially harmful) compound (b) The system for detecting chemicals is linked to the mechanism that drive the flagellum, there are clusters of receptors in the cell membrane that bond specific molecules coming from the immediate environment, when these molecules attach it transmits a signal to the flagellum and sets the bacteria into rotary motion, b. Periplasmic Flagella: Internal Flagella (see Fig. 4.7, pg. 92) 11 Elementary Microbiology 1) A type of internal flagellum that is enclosed in the space between the cell wall and the cell membrane – causes a spinning and undulating pattern of locomotion c. Appendages for Attachment and Mating – these provide some type of adhesion, but no locomotion 1) Fimbriae – shorter, numerous strands, small bristlelike fibers sprouting off the surface of many bacterial cells, contain protein, responsible for the mutual clinging of cells on the surface of liquids and for the microbial colonization of inanimate solids like rocks and glass (a) Examples – gonococcus (agent of gonorrhea) and E. coli 2) Pili – longer, sparser appendages, only found on gram-negative bacteria, involved in a mating process between cells (conjugation), which involves partial transfer of DNA from one cell to another 2. The Cell Envelope: The Outer Wrapping of Bacteria a. The Bacterial Surface Coating, or Glycocalyx 1) A coating of macromolecules to protect the cell and sometimes it will help adhere to its environment 2) Glococalyces differ among bacteria in thickness, organization, and chemical composition 3) Slime layer – soluble shield that protects bacteria from loss of water and nutrients 4) Capsule – repeating polysaccharide units of protein (a) Examples of encapsulated bacteria - Streptococcus pneumonia – causes pneumonia - Haemophilus influenzae – one cause of meningitis - Bacillus anthracis – causes anthrax (b) Capsules protect the bacteria against white blood cells called phagocytes b. Structure of the Cell Wall Determines the shape of the cell, provides a strong structural support to keep the bacterium from bursting or collapsing because of changes in osmotic pressure Cell walls of most bacteria gain their relatively rigid quality from a macromolecule, peptidoglycan (PG), composed of a repeating framework of long glycan chains cross-linked by short peptide fragments Drug companies have taken advantage of the knowledge of what the cell wall is made up, specifically targeting the peptide cross-links and disrupting the integrity Some disinfectants (alcohol, detergents) kill bacterial cells by damaging the cell wall Lysosome, an enzyme found in tears and saliva, provides a natural defense against certain bacteria by hydrolyzing the bonds in the glycan chains and causing the walls to break down 1) Differences in Cell Wall Structure: gram-staining technique developed by Hans Christian Gram (see Fig 4.16, pg. 98) (a) The Gram-Positive Cell Wall 12 Procaryotic Profiles (Ch 4) - A microscopic section resembles and open-faced sandwich with 2 layers: a thick outer cell wall, composed primarily of peptidoglycan, and a cell membrane - Contain a tightly bound acidic polysaccharides, including teichoic acid (a polymer of ribitol or glycerol and phosphate embedded in the peptidoglycan sheath) and lipoteichoic acid (similar in structure but attached to lipids in the plasma membrane) (b) The Gram-Negative Cell Wall - A microscopic section resembles a complete sandwich with three layers: the cell wall, composed of an outer membrane and a thin layer of peptidoglycan, and the cell membrane - The outer membrane containing specialized types of polysaccharides and proteins - Upper most layer of the outer membrane contain lipopolysaccharide (LPS), chains of LPS extend off the surface and function as antigens and receptors - Innermost layer of the outer membrane is a phospholipid layer anchored by lipoproteins to the peptidoglycan layer below - Serves as a chemical sieve by letting only relatively small molecules to penetrate, special membrane channels formed by porin proteins span the entire outer membrane and act as a defense by blocking the entrance of harmful chemicals 2) Practical Consideration of Differences in Cell Wall Structure (a) The outer membrane contributes and extra barrier in gram-negative bacteria that makes is more difficult to inhibit or kill than the gram-positive bacteria (one exception is alcohol-based compounds, which dissolve the lipids in the outer membrane and disturb its integrity) (b) Treating infections caused by gram-negative bacteria often requires different drugs form gram-positive infections (c) The cell wall interacts with human tissue and contributes to disease (d) The lipids are referred to as endotoxins because they stimulate fever and shock reaction sin gram-negative infections like meningitis and typhoid fever (e) Proteins attached to the outer portion of the cell wall of several gram-positive species (i.e. Corynebacterium diphtheriae – causes diphtheria, Streptococcus pyogenes – causes strep throat) have toxic properties (f) Lipids in the cell wall of certain Mycobactgerium species are harmful to human cells too 3) Nontypical Cell Walls (a) Some bacterium have cell walls that contain lipids responsible for a high degree of resistance to certain chemical dyes, this is why acid fast stain is used (i.e. diagnose tuberculosis and leprosy) (b) Archaea have chemically distinct cell walls, in some, the walls are composed almost entirely of polysaccharides, and in others, the walls are pure protein; but as a group, they all lack the true peptidoglycan structure that the gramnegative and gram-positive cell walls contain (c) A few archaea and all mycoplasmas lack a cell wall, the cell membrane serves a dual function of support and transport (d) L forms or L-phase variants are bacteria that ordinarily have a cell wall and lose it during their life cycle, caused by a mutation in the wall-forming genes, this can be induced artificially by introducing lysosome or penicillin (see Fig. 4.17, pg. 100) 13 Elementary Microbiology - When gram-positive cells are exposed to either of the above chemicals, it will lose the cell wall completely and becomes a protoplast When gram-negative cells are exposed, they lose their peptidoglycan but still retain the outer membrane becoming a spheroplast c. Cell Membrane Structure 1) Just beneath the Cell Wall is the cell membrane, flexible sheet molded completely around the cytoplasm, composed as a lipid bylayer with proteins embedded to varying degrees (a) Phospholipids (30-40% membrane mass) (b) Proteins (60-70% membrane mass) (c) Mycoplasmas contain high amounts of sterols (rigid lipids) that stabilize and reinforce the membranes (d) Archaea contain a unique branched hydrocarbon membrane rather than fatty acids (e) Mesosomes – internal folds formed in the membrane - Prominent in gram-positive bacteria but harder to see in gram-negative bacteria - Increase the internal surface area available for membrane activities - Function in cell wall synthesis and guide the duplicated bacterial chromosomes in to the two daughter cells during cell division 2) Functions of the Cell Membrane (a) (b) (c) (d) (e) Energy reactions Nutrient processing Synthesis Regulate transport – selectively permeable Involved in secretion C. Bacterial Form and Function: Internal Structure 1. Contents of the Cell Cytoplasm – mainly composed of water (70-80%), site form many biochemical and synthetic activities a. Bacterial Chromosomes and Plasmids: The Sources of Genetic Information 1) Bacterial chromosome – hereditary material in the form of a single circular strand of DNA, and extremely long strand of DNA tightly coiled around special basic protein molecules 2) Bacteria do not have a nucleus to contain the DNA instead the DNA is aggregated in a dense area called the nucleoid 3) Plasmids –pieces of DNA not essential to growth and metabolism, but may contain protective traits such as resisting drugs and producing toxins and enzymes, they are also an important agent in modern genetic engineering techniques b. Ribosomes: Sites of Protein Synthesis (see Fig. 4.18, Pg. 101) 1) Show up as fine, spherical specks dispersed throughout the cytoplasm that occur in chains (polysomes) 2) Attach to the cell membrane 3) Combination of a special type of RNA called ribosomal RNA (rRNA) 60% and protein – 40% 14 Procaryotic Profiles (Ch 4) 4) Characterize ribosome’s by S ((Svedberg) units, which rate molecular sizes of various parts that have been spun down and separated by molecular weight and shape in a centrifuge, the heavier, more compact structures are assigned a higher the S rating (a) Prokaryiotic ribosomes have an overall rating of 70S and are composed of two smaller subunits, the 30S looks like a heart and the 50 S looks like a crown, they fit together to form a mini platform where protein synthesis is performed (see Fig. 4.19, Pg. 102) c. Inclusions, or Granules: Storage Bodies 1) Inclusions – when bacteria are exposed to an abundance of nutrients, extra nutrients are laid down intracellularly (a) They vary in size, number and content (b) When exposed to an environment where nutrients are depleted, the bacterial cell can mobilize its own storehouse as required (c) Some inclusion bodies enclose condensed, energy-rich organic substances, including glycogen and poly ß-hydroxybutyrate (PHB), within special singlelayered membranes (d) A unique type of inclusion found in some aquatic bacteria are gas vesicles that provide buoyancy and flotation 2) Granules – inclusions containing crystals of inorganic compounds, not enclosed by membranes (a) Examples: sulfur granules of photosynthetic bacteria and polyphosphate granules of Corynebacterium and Mycobacterium (b) Magnetotactic bacteria contain crystalline particles of iron oxide that contain magnetic properties, these granules are use to be pulled by the polar and gravitational fields into deeper habitats with a lower oxygen count 2. Bacterial Endospores: An Extremely Resistant Stage a. Endospore – dormant bodies produced by the gram-positive genera Bacillus, Clostridium, and Sprosarcina, these bacteria have a two-phase life cycle vegetative cell and an endospore 1) Vegetative Cell - metabolically active and growing entity that can be induced by environmental conditions to undergo spore formation, once the spore is formed, it exists in an inert, resting condition that shows up prominently in a spore or Gram stain (see Fig. 4.21b, pg. 103) b. Endospore Formation and Resistance 1) Depletion of nutrients, especially carbon and nitrogen, stimulate a vegetative cell to begin spore formation 2) Once stimulated, the cell undergoes a conversion to a committed sporulating cell (sporangium) 3) The vegetative cell requires 6-8 hrs to completely transform from a to a sporangium, and then to a spore (see Table 4.2, pg. 104) 4) Bacterial endospores are the hardiest of all life forms, capable of withstanding extremes in heat, drying, freezing, radiation, and chemicals that would readily kill vegetative cell; their survival is due to several factors: (a) High calcium and dipicolinic acid helps spores resist high heat (b) Thick, impervious cortex and spore coats protect against radiation and chemicals 15 Elementary Microbiology c. The Germination of Endospores 1) Happens in the presence of water and a specific chemical or environment stimulus (germination agent –generally it is a small organic molecule such as an amino acid or an inorganic salt) 2) Takes about 1 ½ hours 3) Germination agent stimulates the formation of hydrolytic (digestive) enzymes by the spore membranes – these enzymes digest the cortex and expose the core to water 4) As the core rehydrates and takes up nutrients, it begins to grow out of the spore coats, in time it reverts to a fully active vegetative cell d. Medical Significance of Bacterial Spores 1) The majority of spore-forming bacteria are relatively harmless, several bacterial pathogens are sporeformers, aspects of the diseases they cause are related to the persistence and resistance for their spores (a) Bacillus anthracis – agent of anthrax (b) Clostridium includes even more pathogens that embed their spores in wounds that contain dead tissue, they germinate, grow, and release potent toxins - C. . tetani – cause of tetanus (lockjaw - C. perfringens – causes gas gangrene 2) Because they inhabit the soil and dust, spores are a constant intruder where sterility and cleanliness are important 3) Spores resist ordinary cleaning methods 4) Hospital and clinics take precautions to guard against the potential harmful effects of spores in wounds 5) Several endospore-forming species cause food spoilage or poisoning (a) Ordinary boiling (100º C) will not destroy such spores, so canning is carried out in pressurized steam at 120º C for 20-30 min, this will ensure that the food is sterile and free from viable bacteria D. Bacterial Shapes, Arrangements, and Sizes (see Fig. 4.22, pg. 105) 1. Most bacteria have one of three general shapes based on the configuration of the cell wall and range in size from the smallest rickettsias to the largest spiral forms a. Coccus (round) 1) Diplococci (cocci in pairs) 2) Neisseriae (coffee-bean shaped in pairs) 3) Tetrads (cocci in packets of 4) 4) Sarcinae (cocci in packets of 8, 16, or 32 cells) 5) Streptococci (cocci in chains) 6) Micrococci and staphylococci (large cocci in irregular clusters) b. Bacillus (rod) 1) Coccobacilli 2) Mycobacteria 3) Corynebacteria (palisades arrangement) 4) Spore-forming rods 5) Stretomycetes (mold-like, filamentous bacteria) 16 Procaryotic Profiles (Ch 4) c. Spiral 1) Vibrios (curved rods) 2) Spirilla (rigid helix, vary from 1-20 helical turns, gram negative) 3) Spirochetes (flexible helix, varies from 3-70 helical turns, gram negative) 2. Shape and arrangement of cells are key means of describing bacteria a. Arrangements of cells are based on the number of planes in which a given species divides 1) Coccus divide in many planes to form pairs, chains, packets of clusters 2) Bacillus only divide in the transverse plane, if they remain attached, they form chains or palasades E. Bacterial Identification and Classification Systems 1. Methods Used in Bacterial Identification 2. Every spec of soil, dab of saliva, and droplet of pond water is teeming with a rich variety of bacteria 3. The methods that a microbiologist uses to identify bacteria to a level of genus and species fall into the main categories of morphology (microscopic and macroscopic), bacterial physiology or biochemistry, serological analysis, and genetic techniques a. Microscopic Morphology 1) Cell shape 2) Cell size 3) Gram stain reaction 4) Acid-fast reaction 5) Special structures (a) Endospores (b) Granules (c) Capsules b. Macroscopic Morphology 1) Appearance of colonies (a) (b) (c) (d) (e) (f) Texture Size Shape Pigment Speed of growth Patterns of growth 2) Gelatin media c. Macroscopic Morphology 1) Enzymes and other biochemical properties of bacteria are fairly reliable and stable expressions of the chemical identity of each species 2) Diagnostic tests exist for determining the presence of specific enzymes and to assess nutritional and metabolic activities (a) Test for fermentation of sugars (b) Capacity to digest or metabolize complex polymers such as proteins and polysaccharides 17 Elementary Microbiology (c) Production of gas (d) Presence of enzymes such as catalase, oxidase, and decaroxylases (e) Sensitivity to antimicrobic drugs d. Chemical Analysis 1) Analyzing the types of specific structural substances that the bacterium contains, such as the chemical composition of peptides in the cell wall and lipids in the membranes e. Serological Analysis 1) Bacteria have surface and other molecules called antigens that are recognized by the immune system, because of the production of antibodies is so specific in response to foreign agents in the body, it can be used to identify bacteria in specimens and cultures f. Genetic and Molecular Analysis 1) G + C base composition – the overall % of guanine and cystonine in DNA is a general indicator of relatedness because it is a trait that does not change rapidly (a) Bacteria with a significant difference in % are likely to be genetically distinct in species or general - Escherichia has a G + C base composition of 48-52% - Pseudomonas has a composition of 58-70% (b) This technique is most applicable for clarifying the taxonomic position of a bacterium 2) DNA analysis using genetic probes (a) The exact order and arrangement of the DNA code is unique to each organism (see Fig. 2.26, pg. 109) (b) With a technique called hybridization, it is possible to identify a bacterial species by analyzing segments of its DNA (c) This test is conducted by extracting unknown test DNA from cells in specimens or cultures and binding it to special blotter paper 3) Nucleic acid sequencing and rRNA analysis (a) One of the most viable indicators of evolutionary relatedness and affiliation is comparison of the sequence of nitrogen bases in ribosomal RNA (b) Ribosomes have the same function (protein synthesis) in all cells, and they tend to remain more or less stable in their nucleic acid content over long periods (c) Any major difference in sequence, or “signature” of the rRNA is likely to indicate some distance in ancestry (d) This technique is powerful at two levels - It is effective for differentiating general group differences - It can be fine-tuned to identify at the species level 4. Classification Systems in the Procaryotae a. Taxonomic Scheme (see Table 4.4, pg. 110) b. Ribosomal RNA Scheme 1) The classification developed to analyze the similarities in base sequence rRNA revealed 12 distinct branches on the bacterial “tree” (see pg. 111) c. Diagnostic Scheme (see table 4.6, pg. 113) 1) Medical microbiologists prefer an informal working system that outlines the major families and genera, this system is more applicable for their 18 Procaryotic Profiles (Ch 4) purposes because it is restricted to bacterial disease agents and is based on readily accessible morphological and physiological tests rather than on phylogeneric relationships 2) Divides the bacteria into gram positive, gram negative, and those without cell walls and then subgroups them according to cell shape, arrangement, and certain physiological traits such as oxygen usage d. Species and Subspecies in Bacteria 1) A bacterial species is loosely defined as a collection of bacterial cells that shares an overall similar pattern of traits different from other groups of bacteria 2) Variant forms within a species (subspecies) include strains and types F. Survey of Procaryotic Groups with Unusual Characteristics 1. Unusual Forms of Medically Significant Bacteria 2. Most bacteria are free-living or parasitic forms that can metabolize and reproduce by independent means, two groups of bacteria, the rickettsias and chlamydias, have adapted to life inside their host cells, where they are considered obligate intracellular parasites a. Rickettsias – distinctive, very tiny, gram-negative bacteria 1) Atypical in their life cycle and other adaptations 2) Most are pathogens that alternate between a mammalian host and bloodsucking arthropods, such as fleas, lice, or ticks 3) Can’t survive or multiply outside a host cell and can’t carry out metabolism completely on their own 4) Examples: (a) Rickettsia rickettsii – causes Rocky Mountain spotted fever – transmitted by ticks (b) Rickettsia prowazekii – causes epidemic typhus – transmitted by lice (c) Coxiella burnetti – causes Q fever – transmitted in air and dust by arthropods b. Chlamydias 1) Similar to Rickettsias in that they require host cells for growth and metabolism, but they are not transmitted by arthropods 2) They were considered a virus at one time because of their tiny size and obligately parasitic lifestyle 3) Examples: (a) Chlamydia trachomatis – causes a severe eye infection (trachoma) that can lead to blindness and is the cause of the most common sexually transmitted disease (b) Chlamydia psittaci – causes ornithosis (parrot fever) a disease that can be transmitted to humans (c) Chlamydia pneumoniae – causes lung infections 3. Mycoplasmas and Other Cell-Wall-Deficient Bacteria a. Bacteria that naturally lack a cell wall b. Considered the smallest cells, ranging from 0.1-0.5 µm in size c. Range in shape from filamentous to coccus or doughnut shaped 19 Elementary Microbiology d. Not obligate parasites and can grow on artificial media e. Found in many habitats (plants, soil, and animals) f. Most important medical species is Mycoplasma pneumoniae – cause an atypical form of pneumonia in humans by adhering to the epithelial cells in the lung 4. Free-Living Nonpathogenic Bacteria a. Photosynthetic Bacteria – independent cells that contain special light-trapping pigments and can use the energy of sunlight to synthesize all required nutrients from simple inorganic compounds 1) The two types of photosynthetic bacteria are: (a) Those that produce oxygen during photosynthesis (b) Those that produce some other substance, such as sulfur granules or sulfates b. Cyanobacteria|: Blue-Green Bacteria 1) Gram-negative cell wall 2) Can be unicellular or can occur in colonial or filamentous groupings 3) Some species occur in packets surrounded by a gelatinous sheath 4) Thylakoids – extensive internal membranes of a specialized adaptation of cyanobacteria which contain granules of chlorophyll a and other photosynthetic pigments, they also have gas inclusions that allow them to float c. Green and Purple Sulfur Bacteria 1) Differ from the cyanobacteria in have a different type of chlorophyll call bacteriochlorophyll and by not giving off oxygen as a product of photosynthesis 2) Live in sulfur springs, freshwater lakes and swamps that are deep enough for the anaerobic conditions they require 3) Names for their predominant colors, but can also develop brown, pink, purple, blue and orange coloration 4) Exist in single cells of many different shapes and frequently are motile 5) Some can deposit intracellular granules of sulfur or sulfates d. Gliding, Fruiting Bacteria 1) A mixed collection of gram-negative bacteria that live in water and soil 2) Do not have a flagella 3) Morphological forms – slender rods, long filaments, cocci and some miniature, tree-shaped fruiting bodies e. Appendaged Bacteria 1) Produce an extended process of the cell wall in the form of a: (a) Bud - Reproduce entirely by budding - Form a tiny bulb (bud) at the end of a thread - The bud breaks off, enlarges, develops a flagellum, and swarms to another area to start its own cycle (b) Stalk - Live attached to the surface of objects in aquatic environments - One type can grown in distilled water or tap water - The stalks help them trap minute amounts of organic materials (c) Long thread 20 Procaryotic Profiles (Ch 4) 5. Archaea: The Other Procaryotes a. Single-celled, simple organism b. Prokaryotic in structure and share many bacterial characteristics c. Eucaryotes and archae share a number of rRNA sequences, ribosomal subunits structures, and protein synthesis d. Genetic sequence are found only in the rRNA e. Have unique membrane lipids and cell wall construction f. Most primitive of all life forms and most closely related to the first cells that originated on the earth g. Live in a hot anaerobic “soup” with sulfuric gases and salts h. Adapt to multiple combination of heat, salt, acid, pH, pressure, and atmosphere i. Examples: 1) Methanogens (methane producers) (a) Convert CO2 and H2 into methane gas (CH4) through unusual and complex pathways (b) Inhabit anaerobic swamp mud, the bottom sediments of lakes and oceans, and the digestive systems of animals 2) Hyperthermophiles (a) Adapted to grow at very high temperatures, flourish at temperatures between 80ºC and 105ºC, and can’t grow at 50ºC (b) Live in volcanic waters and soils and submarine vents (c) Also salt and acid tolerant 3) Extreme halophiles (a) Require salt to grow and may have such a high salt tolerance that they can multiply in sodium chloride solutions (36% NaCl) that would destroy most cells (b) Inhabit the saltiest places on earth, inland seas, salt lakes, salt mines and salted fish (c) Many use a red pigment to synthesize ATP in the presence of light, these pigments are responsible for “red herring” like those found in the Red Sea 4) Sulfur reducers 21 Elementary Microbiology Unit 4. Eucaryotic Cells and Microorganisms (Ch 5) A. Nature of eucaryotes 1. endosymbiont theory a. Lyn Margulis 1) proposed that a larger procaryote engulfed a smaller one 2) the smaller cell survived in the larger one, and benefited it 3) mitochondria and chloroplasts were once free-living bacteria b. bacterial traits of mitochondria 1) independent division 2) bacterial-like circular chromosome (a) only inherited maternally 3) bacterial-like ribosomes (a) 70S ribosomes (compared to 80S made in eucaryotic nucleus) 4) inhibited by drugs that target bacterial cell membranes B. External Cell Structures 1. cilia & flagella a. eucaryotes may have one, both, or neither b. eucaryote flagella structurally different but functionally similar to procaryotes 1) in vertebrates only the sperm cells have flagella c. many protozoans are covered with cilia and can move quickly 1) cells of our trachea have cilia to move debris away from lungs 2. glycocalyx a. sometimes a distinct layer in eucaryotes, but often just projections from cell membrane b. usually polysaccharides (carbohydrate chains) c. cell surface markers (antigens) are part of the glycocalyx 1) how the immune system distinguishes self and nonself 3. cell wall a. found in fungi, plants, and algae, but not protozoans or animals 1) cell wall of fungi typically made from chitin 2) plants use cellulose for cell wall b. no peptidoglycan in eucaryotes 4. cell membrane a. surrounds the cytoplasm and organelles b. phospholipid bilayer 1) sterols (e.g. cholesterol) stabilize the membrane c. selectively permeable 1) gases and water and really small molecules pass through 2) transport proteins for larger or charged particles d. a very similar membrane surrounds each organelle 22 Eucaryotic Cells and Microorganisms (Ch 5) C. internal cell structures a. nucleus 1) largest, most prominent organelle (a) the cell's "control center" 2) contains the cell's chromosomes (a) not visible until they coil tightly during mitosis 3) ribosomes manufactured in nucleolus (dark region) 4) pores in membranes allow passage of ribosomes and mRNA 5) membrane continuous with membrane of endoplasmic reticulum b. endoplasmic reticulum 1) the cell's "workbench" for protein manufacture 2) rough ER has ribosomes embedded in it 3) mRNA passes from nucleus and is translated by ribosomes on the ER 4) as proteins are made the polypeptide chains elongate within cavity of the ER (a) First step in secretion of cell products c. golgi apparatus 1) the cell's "packaging and mailing center" (a) receives products from the ER - when vesicle from ER touches membrane of Golgi, it fuses and dumps its contents 2) final protein modification occurs here (a) two smaller proteins may be combined into one larger (quaternary structure) 3) vesicles containing finished products pinch off (a) these can be transported to different places in the cell or be released at the cell membrane - e.g. insulin being secreted from a pancreatic cell (b) a lysosome is a vesicle filled with digestive enzymes - your white blood cells use these to tear apart bacteria they engulf d. mitochondria 1) the cell's "power house" (a) Site of Kreb's cycle (TCA cycle) and electron transport chain - enzymes arranged in the mitochondrial membrane 2) believed to have once been a free-living bacteria (a) See "endosymbiont theory" 3) the higher the cell's energy needs, the more mitochondria it has e. chloroplasts 1) organelle for converting light energy into chemical energy (photosynthesis) (a) CO2 + H2O (with energy from sunlight) gets converted into glucose (b) Oxygen is an important byproduct 2) primary producers of the energy for life on earth 3) found in algae and plants 4) believed to have once been cyanobacteria (a) See "endosymbiont theory" f. cytoskeleton 1) microfilaments and microtubules crisscross throughout the cytoplasm 23 Elementary Microbiology (a) thin strands that can contract and move materials through cell (b) involved in ameboid movement (c) maintain shape of cell in eucaryotes that have no cell wall g. ribosomes 1) the cell's "factory workers" for making proteins 2) many of them on the rough ER, but many also distributed throughout cytoplasm 3) large and small subunits come together on mRNA at beginning of translation 4) 80 S ribosomes on ER and in cytoplasm (a) 70S ribosomes in chloroplasts & mitochondria - Same size and type as found in procaryotes 5) made in nucleolus from rRNA and protein D. survey of eucaryotes 1. fungi a. cell wall, typically made of chitin b. includes yeast, molds and mushrooms 1) most are unicellular; some multicellular c. microscopic spores d. always heterotrophic (no photosynthesis) e. don't need to know about reproductive strategies f. Roles 1) many are saprobes (feed off of dead material) (a) important decomposers in the ecosystem (b) sometimes parasitic 2) many antibiotics made from fungal products 3) Yeast fermentation useful in making alcoholic beverages & bread 2. protists a. Algae 1) All are photosynthetic 2) May be microscopic & unicellular or large colonies or multicellular 3) Base of food web in aquatic systems 4) Important producers of oxygen b. Protozoa 1) Mostly unicellular & all heterotrophic (no chloroplasts) 2) Most have flagella or cilia 3) Most are free-living, but a few cause disease (a) Giardia & amebic dysentery are protozoan illnesses (b) some parasitic forms spread by insect vectors - malaria c. helminths 1) In the kingdom Animalia; all are multicellular (a) As animals, they have no cell wall (b) Can be difficult to kill since their cells are similar to ours 2) Mostly free-living, but some parasitic 24 Eucaryotic Cells and Microorganisms (Ch 5) (a) round worms - pin worms or other intestinal round worms (b) flat worms - tapeworms - flukes (c) produce massive quantities of eggs - microscopic eggs are ingested through fecal-contaminated water or food 25 Elementary Microbiology Unit 5. Elements of Microbial Nutrition, Ecology, and Growth (Ch 7) A. Microbial nutrition, ecology, and growth 1. Source of nutrients a. Nutrition consists of taking in chemical substances (nutrients) and assimilating and extracting energy from them. b. Substances required for survival are essential nutrients 1) Carbon (C), hydrogen (H), nitrogen (N), oxygen (O), phosphorus (P), sulfur (S), sodium (Na), chlorine (Cl), potassium (K), calcium (Ca), iron (Fe), and magnesium (Mg) are considered essential 2) Macronutrients are required in fairly large amounts (a) For example, proteins, carbohydrates 3) Micronutrients are required in trace amounts (a) Such as zinc, manganese, and nickel (b) Often micronutrients are needed as coenzymes or maintenance of protein structure 4) Nutrients can be classed as organic or inorganic (a) Inorganic molecules lack carbon and hydrogen - Mineral deposits, atmospheric air, etc. - Some microbes can obtain all their nutrients from inorganic sources (b) Organic molecules contain carbon and hydrogen and are usually made by living things - Methane, carbohydrates, lipids, proteins, nucleic acids, etc. c. A growth factor is an organic nutrient that cannot be synthesized and must be provided in the diet 1) Vitamins and some amino acids B. Nutritional Categories 1. Microorganisms are classified both by the chemical form of their nutrients and the energy source they utilize. 2. Nutrient requirements of microorganisms determine their respective niches in the food webs of major ecosystems. 3. There are 2 major Nutritional categories. a. “Autotroph” depends on carbon dioxide for its carbon need; they derive their energy from sunlight or chemical reactions. 1) “Photoautotrophic” is photosynthetic; energy from light rays is transformed into energy for cell metabolism. 2) “Chemoautotroph” extracts energy from inorganic substances. “Methanogens” a chemoautotroph that produces methane. b. “Heterotroph” acquires carbon from organic molecules. 1) “Saprobe” is a decomposer that feeds on dead, organic matter. 2) “Parasite” feeds from a live host and usually causes harm. (a) Disease causing parasites are pathogens. 26 Elements of Microbial Nutrition, Ecology, and Growth (Ch C. Transport Mechanisms 1. A microbial cell must take on nutrients from it surrounding by transporting them across the cell membrane. 2. The molecular size and concentration of a nutrient determine which method of transport is used. a. Passive Transport (diffusion): involves the natural movement of substances down a concentration gradient and requires not additional energy. 1) Facilitated diffusion: a form a passive transport that can move specific substances. b. Active transport: Transport requiring carrier proteins and the use of energy. 1) Substances are taken into the cell by a process that requires energy 2) Group translocation: molecules are altered during transport. 3) Phagocytosis & pinocytosis: active transport in which bulk quantities of solid and fluid material are taken into the cell. c. Osmosis: is diffusion of water through a selectively permeable membrane. 3. Osmotic changes affect cells. a. Hypotonic solution: has a lower osmotic pressure/solute concentration that the reference solution. b. Hypertonic solution: has a higher osmotic pressure/solute concentration that the reference solution. 1) a halophile thrives in hypertonic surroundings. 2) an obligate halophile requires a slat concentration of at least 15%, and grows optimally at 25%. c. Isotonic solution: has an equal osmotic pressure/solute concentration to the inside of a cell; there is not net movement of solvent. D. Environmental influences on microbes 1. Every organism adapts to a particular habitat or niche a. Temperature: An organism exhibits optimum, minimum, and maximum temperatures. 1) Psychrophiles: Organisms that cannot grow above 20 degrees Celsius but thrive below 15 degrees Celsius, and continue growth even at 0 degrees Celsius. 2) Mesophiles: Organisms that grow from 10 – 50 degrees Celsius, having an optimum temperature from 2040 degrees Celsius. 3) Thermophiles: 45-80 degrees Celsius. b. Oxygen requirements: The ecological need for free oxygen is based on whether a cell can handle toxic by-products such as super oxide and peroxide. c. Most eukaryotic microorganisms are aerobic, but bacteria vary widely in their oxygen requirements. 1) Aerobes grow in normal atmospheric oxygen and have enzymes to handle toxic oxygen by products. (a) Facultative anaerobe: capable of living without oxygen. 27 Elementary Microbiology (b) Microaerophile: prefers a small amount of oxygen, but does not grow under anaerobic conditions. 2) Strict “Obligate” Anaerobes: do not use free oxygen and cannot produce enzymes to dismantle reactive oxides; they are damaged or killed by oxygen. (a) Aero tolerant anaerobe: cannot use oxygen but is not harmed by it. d. Effects of pH: Acidity and alkalinity affect the activity and integrity of enzymes and the structural components of a cell. Optimum pH range for microbes is 6 to 8. 1) Acidophiles: Prefer a lower, more acidic pH. 2) Alkalinophiles: Prefer a higher, more alkaline pH. e. Other environmental factors: Radiation and barometric pressure affect microbial growth 1) Barophile: microbe adapted to live under high pressure. (Ocean bottom dwellers) E. Microbial Interactions 1. Microbes coexist in various relationships in nature. a. Symbiosis (mutualism): reciprocal, obligatory, mad beneficial, relationship between two organisms. 1) Commensalism: an organism receiving benefits from another without harming the other organism. (a) Ex. Satellitism 2) Parasitism: occurs between a host and an infectious agent. b. Synergism: mutually beneficial but not obligatory coexistence. c. Antagonism: competition, inhibition, and injury directed against the opposing organism. 1) Ex. Antibiotic production. F. Microbial growth 1. Microbial growth refers both to increase in cell size and increase in the number of cells in a population. 2. Binary(Transverse) Fission: the splitting of a parent bacterial cell to form a pair of similar sized daughter cells. 3. Generation (doubling time): duration of each division. a. Exponential growth rate: population theoretically doubles within each generation; each cycle increases in geometric progression. 4. Microbial cell populations show a distinct phase of growth in response to changing nutrient and waste conditions. a. Growth curve: graphic representation of closed population over time. b. Viable count: estimate of live cells. c. Lag phase: initial flat period of the curve. d. Exponential growth phase: viable cells increase in logarithmic progression. 28 Elements of Microbial Nutrition, Ecology, and Growth (Ch 7 e. Stationary growth phase (Plateau): adverse environmental factors combine to inhibit the growth rate. f. Death phase: nutrient depletion and waste buildup cause increased cell death. 5. Cell numbers can be counted directly by a microscope counting chamber, coulter counter, or flow cytometer. 6. Signs of population growth can be quantified by measuring turbidity, colony counts and direct cell counts. 29 Elementary Microbiology Unit 6. Microbial Metabolism: The Chemical Crossroads of Life (Ch 8) A. Overview: 1. Cells maintain a complex internal habitat that is highly structured and controlled 2. Cells are constantly involved in an orderly activity called metabolism that encompasses all of their chemical and energy transactions 3. Enzymes are essential metabolic participants that drive cell reactions a. Enzymes are protein catalysts that speed up chemical processes by lowering the required energy b. Enzymes have a specific shape tailored to perform their actions on a single type of molecule called a substrate c. Enzymes derive some of their special characteristics from cofactors such as vitamins, and they show sensitivity to environmental factors d. Enzymes are involved in activities that synthesize, digest, oxidize, and reduce compounds, and convert one substance to another e. Enzymes are regulated by several mechanisms that alter the structure and/or synthesis of the enzyme 4. The energy of living systems resides in the atomic structure of chemicals that can be acted upon and changed a. Cell energetics involves the release of energy that powers the formation of bonds b. The energy of electrons is transferred from one molecule to another in coupled redox reactions 1) Electrons are transferred from substrates such as glucose to coenzyme carriers, and ultimately captured in high-energy adenosine triphosphate (ATP) 5. Cell pathways involved in extracting energy from fuels are glycolysis, the tricarboxylic acid cycle (TCA or Kreb’s Cycle), and the electron transport train a. The molecules used in aerobic respiration are glucose and oxygen, and the products are CO2, H2O, and ATP 6. Microbes participate in alternate pathways such as fermentation and anaerobic respiration 7. Cells manage their metabolites through linked pathways that have numerous functions and can proceed in more than one direction B. The Metabolism of Microbes: 1. Metabolism- change pertaining to all chemical reactions and physical workings of the cell 2. Metabolism entails one of 2 general categories: a. Anabolism (biosynthesis)- any process that results in synthesis of cell molecules and structures 30 Microbial Metabolism: The Chemical Crossroads of Life (C 1) It is a building and bond making process that forms larger macromolecules from smaller ones 2) Requires input of energy b. Catabolism- any process that breaks bonds of the larger molecules into smaller molecules 1) Opposite of anabolism 2) Often produces energy 3) Reactions derogative 3. Metabolism performs many functions: a. Degrades macromolecules into smaller molecules and releases energy b. Energy that is released is converted into ATP or released as heat c. The smaller molecules are assembled to form larger macromolecules specific to the cell and in this process ATP is utilized to form bonds d. It has built-in controls for reducing or stopping a process that is not in demand and other controls for storing excess nutrients 1) See Figure 8.1: Synthesis of Cell Structures C. Enzymes: Catalyzing the Chemical Reactions of Life: 1. Enzymes- chemicals that increase the rate of a chemical reaction without becoming part of the products or being consumed in the reaction 2. An example of catalysts a. See table 8.1 3. How Do Enzymes Work: a. In order for a reaction to occur the reaction must have enough energy input, called activation energy, in order to start b. There are 3 ways for activation energy to occur: 1) Increasing thermal energy (heating) to increase molecular velocity 2) Increasing the concentration of reactants 3) Adding a catalyst c. An enzyme promotes a reaction by serving as a physical site upon which the reactant molecules, called substrates, can be positioned for various interactions 4. Enzyme Structure: a. Primary structure of all enzymes is protein and can be classified as simple or conjugated b. Conjugated enzymes, sometimes referred to as a holoenzyme, is a combination of a protein, now called apoenzyme, and one or more cofactors 1) Cofactors are organic molecules, called coenzymes, or inorganic elements 5. Apoenzymes: Specificity and the Active Site: a. Apoenzymes exhibit different levels of molecular complexity: primary, secondary, tertiary, and quaternary 1) See Figure 8.4 Levels of Structure 31 Elementary Microbiology 2) The first 3 levels of structure arise when a single polypeptide chain undergoes an automatic folding process and achieves stability by forming disulfide and other types of bonds 3) The surface and the three-dimensional features of a substrate is what defines its specificity b. The active site (catalytic site) is where the substrate binds to a crevice on the enzyme 6. Enzyme- Substrate Interactions: a. Commonly known as “lock and key” mechanism b. Induced fit is when the enzyme actually helps pull the substrate into the active site through slight changes in its shape 7. Cofactors: Supporting the Work of Enzymes: a. Metallic cofactors: iron, copper, magnesium, manganese, zinc, cobalt, selenium, etc. b. Coenzymes are organic compounds that work in conjunction with an apoenzyme to perform a necessary alteration of a substrate 1) General function of a coenzyme is to remove a functional group from one substrate molecule and add it to another substrate 2) Vitamin deficiencies prevent the complete holoenzyme from forming 8. Classification of Enzyme Functions: a. Location and Regularity of Enzyme Action: 1) Exoenzymes- transported extracellularly, where they break down (hydrolyze) large food molecules or harmful chemicals (a) Examples: cellulase, amylase, and penicillinase 2) Endoezymes- retained intracellular and function there (a) Constitutive enzymes- always present and in relatively constant amounts regardless of the amount of substrate (b) Induced enzyme- not constantly present and is produced only when its substrate is present b. Synthesis and Hydrolysis Reactions 1) Condensation reactions- anabolic reactions require enzymes (ligases) to form covalent bonds between smaller substrate molecules… synthesis reaction typically require ATP and always release one water molecule for each bond 2) Hydrolysis- another term for digestion because the breaking of bonds require the input of water c. Transfer Reactions by Enzymes 1) Oxidized- loss of electrons, called oxidation because of the loss of electrons 2) Reduced- gain of electrons, called reduction because it takes or “reduces” electrons from the other atom or molecule 3) Can remember OIL RIG (Oxidation Is Loss Reduction Is Gain) 4) Oxidoreductases- which removes electrons from one substrate and adds them to another d. The Role of Microbial Enzymes in Disease 32 Microbial Metabolism: The Chemical Crossroads of Life (C 1) Many pathogens secrete unique exoenzymes that help them avoid host defenses to promote their multiplication in tissues 9. The Sensitivity of Enzymes to Their Environment: a. in general, enzymes only operate under natural temp., pH, and osmotic pressure of an organisms habitat b. Labile- when an enzyme is unstable because of forced changes in its habitat c. Denaturing- a process by which the weak bonds that collectively maintain the native shape of the apoenzyme are broken 10. Regulation of Enzymatic Activity and Metabolic Pathways: a. Metabolic pathways 1) Metabolic reactions rarely consist of single step processes 2) Regulation of pacemaker enzymes proceeds of 2 fundamental levels: (a) The enzyme itself is directly inhibited or activated, or (b) The mount of the enzyme in the system is altered b. Direct controls of the actions of enzymes 1) Competitive inhibition- when other molecules with a structure similar to the normal substrate can occupy the enzyme’s active site 2) Negative feedback- when the process is inhibited by the product 3) Positive feedback- when the process is stimulated by the product c. Allosteric enzymes have an additional regulatory site for the attachment of molecules other than substrate. 1) Feedback inhibition- when an allosteric inhibitors can temporarily stop the action of that enzyme d. Controls of enzyme synthesis 1) Enzyme repression- a means to stop further synthesis of an enzyme somewhere along its pathway 2) Enzyme induction- enzymes appear (are induced) only when a suitable substrate is present 11. Pursuit and Utilization of Energy a. Energy commonly exists in various forms: 1) Thermal, or heat, energy form molecular movement 2) Radiant (wave) energy for visible light or other rays 3) Electrical energy for a flow of electrons 4) Mechanical energy form a physical change in position 5) Atomic energy form reactions in the nucleus of an atom 6) Chemical energy present in the bonds of molecules 12. Cell Energetics a. Exergonic- is a reaction that releases energy as it goes forward, it is considered free because it is available for doing cellular work b. Endergonic- they use energy as the reaction occurs 1) Cells do NOT make energy they simply harness the energy released and made from bonds breaking and forming 13. A Closer Look at Biological Oxidation and Reduction a. Redox reaction- always occurs in pairs with and electron donor and an electron acceptor which constitutes as a conjugate pair or a redox pair 33 Elementary Microbiology b. Phosphorylate- the storing of inorganic phosphate after being added to ADP or some other compound c. Dehydrogenation- the removal of hydrgens from a redox compound during a redox reaction d. Electron carriers- shuttles that alternately load and unload, repeatedly accepting and releasing electrons and hydrogens 14. Adenosine Triphosphate: Metabolic Money; a. ATP is a 3-part molecule 1) ATP is the primary currency of the cell 2) ATP is utilized and replaced constantly 3) ATP when broken down forms ADP and AMP b. Substrate-level phosphorylation- ATP is formed by transfer of a phosphate group from a phosphorylate compound directly to ADP 15. Pathways of Bioenergetics a. Bioenergenetics- study of the production and use of energy b. Primary catabolism of fuels that results in energy release in many organisms proceeds through a series of 3 coupled pathways: 1) Glycolysis- EMP pathway 2) Tricarboxylic Acid Cycle (TCA)- Krebs cycle 3) Respiratory chain (electron transport chain)- phosphorylation D. Aerobic Respiration 1. Important concepts concerning its reactants and products: a. Steps in the oxidation of glucose b. Involvement of coenzyme carriers and the final electron acceptor c. where and how ATP originates d. Where carbon dioxide originates e. Where oxygen is required f. Where water originates 2. Glycolysis: The Starting Lineup: a. Glucose is phosphorylate 1) Glucose-6 is converted to fructose-6-phosphate 2) Fructose -6-phosphate becomes fructose-1 and 6-biphosphate 3) Fructose-1 and 6-biphosphate are split into 3 carbon fragments 4) Each molecule in glyceraldehydes-3-phosphate becomes involved in the single oxidation-reduction reaction of glycolysis 5) One of the high-energy phosphates of bisphosphoglyceric acid is donated to ADP which makes ATP 6) 3-phosphorylic acid is converted to 2-phosphoglyceric acid 7) The removal of a water molecule from 2-phosphoglyceric acid produces phosphoenolpyruvic acid 8) A second ATP is formed and phosphoenolpyruvic acid becomes pyruvic acid 3. Tricarboxylic Acid Cycle - A Carbon Energy Wheel: a. Steps in the TCA cycle 34 Microbial Metabolism: The Chemical Crossroads of Life (C 4. 5. 6. 7. 1) Oxaloacetic acid reacts with the acetyl group on acetyl CoA, thereby forming citric acid and releasing coenzyme A so it can join with another acetyl group 2) Citric acid is converted to its isomer, isocitric acid to prepare this substrate for the decarbozilatoin and dehydrogenation of the next step 3) Isocitric acid is acted upon by and enzyme complex including NAD or NADP in a reaction that generates NADH or NADPH splits off a carbon dioxide and leaves ketoglutaric acid 4) alpha- ketoglutaric acid serves as a substrate for the last decarboxylation reaction and yet another redox reaction involving coenzyme A and yielding NADH 5) Succinyl CoA is the source of the one substrate level phosphorylation in the TCA cycle. 6) Succinic acid next becomes dehydrogenated, but in this case, the electron and H+ acceptor is FAD. 7) The addition of water it fumaric acid results in malic acid 8) Malic acid is dehydrogenated and oxaloacetic acid is formed Elements of Electron Transport: the Energy Cascade: a. The sequence of electron carriers in the respiratory chain of most aerobic organisms 1) NADH dehydrogenase, which is closely associated in a complex with the adjacent carrier 2) Adjacent carrier is flavin mononucleotide (FMN) (a) Coenzyme Q (b) Cytochrome b (c) Cytochrome c1 (d) Cytochrome c (e) Cytochromes a and a3 which are complexed together Potential Yield of ATPs from Oxidative Phosphorylation a. Table 8.4 summarizes the total of ATP and other products for the entire aerobic pathway The Importance of Fermentation: a. Fermentation- incomplete oxidation of glucose or other carbohydrates in the absence of oxygen b. Is also the formation of cid, gases, and other products by the action of various bacteria on pyruvic acid Biosynthesis and the Crossing Pathways of Metabolism: a. Amphibolism- the property of a system to integrate catabolic and anabolic pathways to improve cell efficiency b. Gluconeogenesis- the process of making glucose in the event of inadequate glucose supply 35 Elementary Microbiology Unit 7. Microbial Genetics (Ch 9) A. Introduction to Genetics and Genes: Unlocking the Secrets of Heredity 1. Genetics is the study of the inheritance, or heredity, of living things a. The transmission of biological properties (traits) from parent to offspring b. The expression and variation of those traits c. The structure and function of the genetic material d. How this material changes 2. The study of genetics (see Fig. 9.1, pg. 254) a. Organismal level – observes the heredity of the whole organism b. Cell level – examines the heredity of the cell c. Chromosome (Eukaryotes only) level – examines the characteristics and actions of chromosomes d. Molecular level – deals with the biochemistry of the genes 3. The Nature of the Genetic Material a. A species must have the capacity to self-replicate to survive b. The Levels of Structure and Function of the Genome 1) Genome – the sum total of genetic material of a cell 2) Chromosome – discrete cellular structure composed of a neatly packaged elongate DNA molecule 3) Gene (a) Fundamental unit of heredity responsible for a given trait in an organism (b) In the molecular and biochemical sense, it is a site on the chromosome that provides information for a certain cell function (c) It is a certain segment of DNA that contains the necessary code to make a protein or RNA molecule c. The Size and Packaging of Genomes 1) The smallest viruses have four or five genes: the bacteria E. coli has a single chromosome containing 4,288 genes, and a human cell packs about ten times that many into 46 chromosomes 2) Bacterial chromosomes take up 1/3 to 1/2 of the cell’s volume 4. The DNA Code: A Simple Yet Profound Message a. Nucleotide – the basic unit of DNA (deoxyribonucleic acid) structure 1) a gene in a bacteria contains several million nucleotides linked end to end b. Nucleotides are composed of a phosphate, a deoxyribose sugar, and a nitrogenous base c. Nucleotides covalently bond to form a sugar-phosphate linkage that is the backbone of each strand d. Each sugar attaches in a repetitive pattern to two phosphates e. The nitrogenous bases, purines and pyrimidines, attach by covalent bonds at the 1’ position of the sugar 1) They span the center of the molecule and pair with appropriate complementary bases from the other side of the helix 36 Microbial Genetics (Ch 9) 2) The paired bases are so aligned as to be joined by hydrogen bonds (a) These bonds are weak and easily broken, allowing the molecule to be “unzipped” into its complementary strands f. Pairing of purines and pyrimidines is not random; it is dictated by the formation of hydrogen bonds between certain bases 1) In DNA to purine Adenine (A) pairs with the pyrimidine Thymine (T) and the purine Guanine (G) pairs with the pyrimidine Cytosine (C) (a) Research has shown that bases are attracted to each other in this pattern because each has a complimentary 3D shape that matches its pair g. The helix runs in an antiparallel arrangement 5. The Significance of DNA Structure a. Maintenance of the code during reproduction 1) The constancy of base-pairing guarantees that the code will be retained during cell growth and division 2) When the two strands are separated, each one provides a template for the replication of a new molecule 3) The sequence of one strand automatically gives the sequence of the partner b. Providing variety 1) The order of bases along the length of the DNA strand constitutes the genetic program, or the language, of the DNA code 2) The message present in a gene is the precise arrangement of bases 3) The genome is the collection of all DNA bases that, in an ordered combination, are responsible for the unique qualities of each organism 6. DNA Replication: Preserving the Code and passing It On (see Fig. 9.5, pg. 258) a. The overall replication process 1) Uncoil the parent DNA molecule 2) Unzip the hydrogen bonds between the base pairs, this separates the two strands and exposes the nucleotide sequence of each strand to serve as a template 3) Synthesize two new strands by attachment of the correct complementary nucleotides to each single-stranded template b. Refinements and details of replication 1) Replicon – the circular bacterial DNA molecule replicates by means of a special configuration 2) Synthesizing a new daughter strand of DNA using the parental strand as a template is carried out by the enzyme polymerase III 3) The process of replication depends on several enzymes and can be understood by keeping in mind a few points concerning both the structure of the DNA molecule and the limitations of DNA polymerase III (a) The nucleotides that need to be read by DNA polymerase III are buried deep within the double helix - Accessing these nucleotides requires both that the DNA molecule be unwound and that the two strands of the helix be separated from one another (b) DNA polymerase III is unable to begin synthesizing a chain of nucleotides but can only continue to add nucleotides to an already existing chain 37 Elementary Microbiology (c) DNA polymerase III can only add nucleotides in one direction, so the new strand is always synthesized 5’ to 3’ B. Applications of the DNA Code: Transcription and Translation 1. The Gene-Protection Connection a. A protein’s primary structure – the order and type of amino acids in the chain – determines its characteristic shape and function b. Proteins ultimately determine phenotype, the expression of all aspects of cell function and structure 1) Living things are what their proteins make them c. DNA is mainly a blueprint that tells the cell which kinds of proteins to make and how to make them 2. The Major Participants in Transcription and Translation a. Transcription-the formation of mRNA using DNA as a template b. Translation-the synthesis of proteins using mRNA as a template c. RNA is encoded like DNA, but its general structure is different in several ways 1) Single-stranded molecule that exists in helical form, it can assume secondary and tertiary levels of complexity due to bonds within the molecule 2) Contains Uracil (U), instead of Thymine (T), as the complementary basepairing mate for Adenine (A) 3) The sugar in RNA is ribose (ribonucleic acid) d. Messenger RNA (mRNA) – a copy of a structural gene or genes complementary DNA, synthesized by a process similar to the synthesis of the leading strand during DNA replication and the complimentary base-pairing rules ensure that the code will be faithfully copied in the mRNA transcript 1) Codons – a series of triplets that reads the message transcribed on the mRNA strand e. Transfer RNA (tRNA) – a transcript of DNA that converts RNA language into protein language (a) Anticodon – the trinucleotide sequence of tRNA that is complimentary to the trinucleotide sequence of the mRNA (b) For each of the 20 amino acids, there is at least one specialized type of tRNA to carry it f. The Ribosome – the interactions of proteins and rRNA create the two subunits of the ribosome that engage in final translation of the genetic code 3. Transcription: The First Stage of Gene Expression (see Fig. 9.12, pg. 265) a. RNA polymerase – an enzyme process that translates the code of DNA to RNA 1) Template strand – one strand of DNA that contains meaningful instructions for synthesis of a functioning polypeptide 2) Coding strand – nontranscribed strand 38 Microbial Genetics (Ch 9) b. Transcription begins when RNA polymerase recognizes a segment of the DNA called the promoter region, consisting of two sequences of DNA just prior to the beginning of the gene to be transcribed 4. Translation: The Second Stage of Gene Expression – all of the elements needed to synthesize a protein, from the mRNA to the amino acids, are brought together on the ribosome, the entire process proceeds through the stages of initiation, elongation, termination, and protein folding and processing a. Initiation of translation (see Fig. 9.13, pg. 266) b. The master genetic code: the message in mRNA – the master genetic code is represented by the mRNA codons and the amino acids they specify, this code is universal for prokaryotes, eukaryotes and viruses (there are a few exceptions) c. The beginning of protein synthesis – the correct tRNA enters the P site and binds to the start codon (AUG) presented by the mRNA, pairing is done in a way that the anticodon of the tRNA must be complimentary to the codon of the mRNA 5. Eucaryotic Transcription and Translation: Similar Yet Different a. Both eukaryotes and prokaryotes have a start codon of AUG b. Eukaryotes code for a different form of methionine c. Eukaryotes mRNAs code for just one protein, bacterial mRNA contain several genes in series d. Eukaryotic genes are composed of exons (expressed sequences) and introns (intervening sequences), the introns must be removed and the exons spliced together to create the final mRNA 6. The Genetics of Animal Viruses a. Viruses replicate by utilizing the transcription and translation processes of cellular organisms, they invade the host cells and “force” them to transcribe and translate viral genes into new virus particles 1) In some case, viruses connect themselves to the host genome and are passed on to its progeny, some of which will become virus factories many generations later b. The genetic material of viruses can be either DNA or RNA. 1) Occurring in single, double, positive or negative sense strands, viral DNA contains just enough information to force a cell to become a virus factory c. Genomes of viruses are found in many physical forms not seen in cells, including dsDNA, ssDNA, dsRNA, and ssRNA d. DNA viruses tend to replicate in the nucleus while RNA viruses replicate in the cytoplasm, retroviruses synthesize dsDNA from the ssRNA e. Some viruses integrate their DNA into the host cell genome 1) Integration of oncogenic viruses can lead to transformation of the host cell into an immortal cancerous cell C. Genetic Regulation of Protein Synthesis and Metabolism 1. Gene expression must be orchestrated to coordinate the organism’s needs with nutritional resources 39 Elementary Microbiology a. Genes can be turned “on” and “off” by specific molecules, which expose or hide their nucleotide codes for transcribing proteins 1) Most, but not all, of these proteins are enzymes 2. Operons are collections of genes in bacteria that code for products with a coordinated function a. They include genes for regulatory, operational, and structural components of the cell 1) Nutrients can combine with regulator gene products to turn a set of structural genes on (inducible genes) or off (repressible genes) b. The lac (lactose) operon is an example of an inducible operon c. The arg (arginine) operon is an example of a repressible operon 3. The rifamyicins, tetracyclines, and aminoglycosides are classes of antibiotics that are effective because they interfere with transcription and translation processes in microorganisms D. Mutations: Changes in the Genetic Code 1. Permanent changes in the genome of a microbe are known as mutations a. They may be spontaneous or induced 2. Point mutations entail a change in one or a few bases and are categorized as the following based on the effect of the change on the nucleotide(s): a. Missense mutations – a mutation in which a change in the DNA sequence results in a different amino acid being incorporated into a protein, with varying results (sickle cell anemia in humans is caused by a missense mutation) b. Nonsense mutations – a mutation that changes an amino acid-producing codon into a stop codon, leading to premature termination of a protein (cystic fibrosis in humans is caused by a nonsense mutation) c. Silent mutations – a mutation that, because of the degeneracy of the genetic code, results in a nucleotide change in both the DNA and the mRNA but not the resultant amino acid and thus, not the protein d. Back mutations – a mutation that counteracts an earlier mutation, resulting in the restoration of the original DNA sequence 3. Many mutations, particularly those involving mismatched bases or damage form ultraviolet light, can be corrected using enzymes found in the cell 4. The Ames test measures the mutagenicity of chemicals by determining the ability of a chemical to induce mutations in bacteria E. DNA Recombination Events 1. Transmission of Genetic Material in Bacteria a. Intermicrobial transfer and genetic recombination permit gene sharing between bacteria. Major types of recombination include: 1) Conjugation – remember plasmids 2) Transformation – the transfer of genetic material contained in “naked” DNA fragments from a donor cell to a competent recipient cell 40 Microbial Genetics (Ch 9) 3) Transduction – the transfer of genetic material from one bacterium to another by means of a bacteriophage vector b. Transposons are DNA sequences that regularly copy themselves to different places within the genome of a cell, as a consequence generating mutations and variations in chromosome structure (about 45% of human DNA consists of transposons! Transposons have been used to successfully insert new genes into fruit fly embryos) 41 Elementary Microbiology Unit 8. Physical and Chemical Control of Microbes (Ch 11) A. Physical Methods for Controlling Microorganisms 1. Moist heat denatures proteins and DNA while destroying membranes a. Sterilization: Autoclaves utilize steam under pressure to sterilize heat resistant materials while intermittent sterilization can be used to sterilize more delicate items b. Disinfection: Pasteurization subjects liquids to temperatures below 100C and is used to lower the microbial load in liquids, boiling water can be used to destroy vegetative pathogens in the home 2. Dry heat, using higher temperatures than moist heat, can also be used to sterilize a. Incineration can be carried out using a Bunsen burner or incinerator, temperatures range between 600C and 1800C b. Dry ovens coagulate proteins at temperatures of 15C to 180C 3. Cold temperatures are microblastic, with refrigeration (0 to 15C) and freezing (below 0C) commonly used to preserve food, media and cultures 4. Drying and desiccation lead to (often temporary) metabolic inhibition by reducing water in the cell 5. Radiation: energy in the form of radiation is a method of cold sterilization, which works by introducing mutations into the DNA of target cells a. Ionizing radiation, such as Gamma rays and X-rays, ahs deep penetrating power and works by causing breaks in the DNA of target organisms b. Nonionizing radiation uses ultraviolet waves with very little penetrating power and works by creating dimmers between adjacent pyrimidines, which interferes with replicaton 6. Filtration involves the physical removal of microbes by passing a gas or liquid through a fine filter and can be used to sterilize air as well as heat sensitive liquids B. Chemical Control of Microorganisms 1. Chemicals are divided into the following based on their level of effectiveness and the surfaces to which they are applied 2. Antimicrobic chemicals are found as solids, gases and liquids, liquids can be either aqueous (water based) or tinctures (alcohol bases) 3. Halogens are chemicals based on elements from group VII of the periodic table a. Chlorine is used as chlorine gas, hypochlorites and chloramines, all work by disrupting disulfide bonds and, given adequate time, are sporicidal b. Iodine is found both as free iodine (I2) and iodophors (iodine bound to organic polymers such as soaps), it has a mode of action similar to chlorine and is also sporicidal given enough time 42 Physical and Chemical Control of Microbes (Ch 11) 4. Phenolics are chemicals based on phenol that work by disrupting cell membranes and precipitating proteins, they are bactericidal, fungicidal and viricidal, but not sporicidal a. Although phenol is now considered too toxic to be used in most circumstances, phenolic compounds (Lysol, Triclosan) are commonly used as, or added to, home and hospital disinfectants 5. Chlorhexidine (hibiclens, hibitane) is a surfactant and protein denaturant with broad microbial properties, although it is not sporicidal, solutions of chlorhexidine are used as skin degerming agents for preoperative scrubs, skin cleaning and burns 6. Ethyl and isopropyl alcohol, in concentrations of 50% to 90%, are useful for microbial control, alcohols act as surfactants, dissolving membrane lipids and coagulating proteins of vegetative bacterial cells and fungi, they are not sporicidal 7. Hydrogen peroxide produces highly reactive hydroxyl-free radicals that damage protein and DNA while also decomposing to O2 gas, which is toxic to anaerobes, strong solutions of H2O2 are sporicidal 8. Detergents and soaps a. Cationic detergents known as quarternary ammonium compounds (quats) act as surfactants that alter the membrane permeability of some bacteria and fungi, they are not sporicidal b. Soaps have little microbial activity but rather function by removing grease and soil that contain microbes 9. Heavy metals: solutions of silver and mercury kill vegetative cells (but not spores) in exceedingly low concentrations (oligodynamic action) by inactivating proteins, although heavy metal solutions are utilized in specific instances, drawbacks such as their toxicity, ability to cause allergies and neutralization by organic matter generally limits their use 10. Aldehydes such as glutaraldehyde and formaldehyde kill microbes by alkylating protein and DNA molecules, the use of aldehydes is limited by their toxicity and propensity to irritate living tissue 11. Gases and aerosols such as ethylene oxide (ETO), propylene oxide, betapropiolactone and chlorine dioxide are strong alkylating agents, all of which are sproicidal 43 Elementary Microbiology Unit 9. Drugs, Microbes, Host—The Elements of Chemotherapy (Ch 12) A. Principles of Antimicrobial Therapy (pg. 349) 1. Introduction a. Ideal drug should be: 1) Easily administered 2) Able to reach infectious agent anywhere in body 3) Absolutely toxic to infectious agent 4) Not toxic to host 5) Remain active as long as needed but be easily broken down and excreted b. The ideal drug does not exist, so compromises always made c. Chemotherapeutic agents can be organized by their origin, their range of effectiveness, and whether naturally or synthetically produced d. Terminology of chemotherapy 1) Chemotherapeutic drug 2) Prophylaxis 3) Antimicrobial chemotherapy 4) Antimicrobics 5) Antibiotics 6) Semisynthetic drugs 7) Synthetic drugs 8) Narrow spectrum 9) Broad spectrum 2. Origins of antimicrobial drugs a. Antibiotics are metabolic products, mainly from aerobic spore-forming bacteria and fungi 1) Potentially useful in the wild to reduce competitive pressure 2) Bacteria genera Streptomyces and Bacillus and fungi genera Penicillium and Cephalosporium are the main sources for antibiotics (a) Chemists create new drugs by altering these naturally occurring antibiotics 3) Looking for antimicrobial compounds in other types of organisms (a) Plants (b) Animals (frog skin secretions) B. Interactions between drug and microbe (pg. 351) 1. Introduction a. Goal is to disrupt cell processes or cause structural damage in bacteria, fungi, or protozoa or to inhibit replication in viruses 1) Most of the drugs interfere with one or more enzymes 2) Some damage existing cell structures b. Important for drugs to be toxic to microbes but not damage host tissues (selective toxicity) 44 Drugs, Microbes, Host—The Elements of Chemotherapy (C 1) Penicillins are good example because human cells have no cell wall and are not affected by these drugs 2) If a drug attacks something found both in microbes and humans it is less likely to be selectively toxic—it may instead have toxic side effects 2. Mechanisms of drug action a. Drugs that affect the bacterial cell wall 1) Peptidoglycan is part of bacterial cell walls, but is not found in humans 2) Actively growing cells must constantly make new peptidoglycan 3) Penicillins and cephalosporins (a) Also called the beta-lactams (based on a chemical ring that is part of the molecule) (b) They block peptidoglycan synthesis by reacting with enzymes involved in its production - This causes weak points in the cell wall - Cell becomes susceptible to osmotic lysis (c) Old, inactive, dormant cells not susceptible because they are not making new peptidoglycan (d) Gram-positive bacteria more susceptible than gram-negative - But there are broad-spectrum penicillins and cephalosporins 4) Cycloserine and vancomycin also affect peptidoglycan synthesis b. Drugs that affect nucleic acid synthesis 1) Many enzymes are involved in DNA and RNA synthesis—these are all potential targets 2) Sulfonamides (sulfa drugs) and trimethoprim are competitive inhibitors (occupy the binding site of enzyme and block substrate from occupying the site) (a) Competes with PABA for binding site (b) Interferes with folic acid synthesis - Folic acid needed in DNA and RNA and amino acid synthesis (c) Mammals acquire folic acid from food instead of synthesizing it, hence do not have the enzymes that are affected - Bacteria and protozoans both affected by these drugs 3) Chloroquine (antimalarial), quinolones (broad spectrum), and acyclovir (antiviral) also inhibit DNA synthesis c. Drugs that block translation (protein synthesis) 1) Inhibit translation by acting on bacterial ribosomes, which are different than eucaryotic ribosomes (a) Can target the 50S subunit or the 30S subunit (b) Possible damage to mitochondria as a side effect, since they have procaryotic type of ribosome 2) Tetracyclines, streptomycin, erythromycin and gentamicin are examples d. Drugs that disrupt cell membrane function 1) If cell membrane is damaged the cell will die 2) These antibiotics are specific to certain microbial groups based on types of lipids in their cell membranes (a) However, they are still generally toxic to humans 3) Examples include some antibacterial (polymyxins) and antifungal agents (Amphotericin B and nystatin) 45 Elementary Microbiology C. Survey of major antimicrobial drug groups (pg. 357) 1. Introduction a. Literally thousands of antimicrobial drugs for sale! b. However, only about 260 different drugs in about 20 drug families 1) Ampicillin alone is marketed under 50 different names! c. We will mainly discuss antibacterial drugs, with less emphasis on antifungal, antiviral, and antiprotozoan 2. Antibacterial drugs (note: there are really only two classes of antibiotics—groups a, b, and c are all beta-lactam drugs with similar actions; d, e, f and g are all derived from Streptomyces and have similar actions) a. Penicillins (end in suffix –cillin) 1) Derived from the mold genus Penicillium 2) Chemical structure has appearance of a house with a gazebo in the backyard (a) The “gazebo” is a variable side chain that accounts for the antimicrobial activity (b) By modifying the side-chain different properties emerge (such as making it broad spectrum or not susceptible to penicillinase) - Called “semisynthetic” when produced through microbial fermentation and then chemically altered 3) Penicillin still drug of choice for sensitive gram-positive cocci (most streptococci) and some gram-negative bacteria (Meningococcus and Treponema pallidum) 4) Semisynthetic penicillins (including Ampicillin, carbenicillin, and amoxicillin) (a) These are broader spectrum (b) Mezlocillin and azlocillin are very broad spectrum (c) Clavulanic acid increases longevity of drug in presence of penicillinaseproducing bacteria, so is often given in combination with a semisynthetic - Augmentin and Zosyn are trade names of this combination 5) All penicillins are relatively mild and well-tolerated by human cells (a) But some people develop severe allergies (b) Resistant bacteria can also be a problem b. Cephalosporins 1) Relatively new, but are the most commonly administered group of antibiotics 2) From the mold Cephalosporium 3) Similar in structure and function to penicillins, but with some advantages (a) Relatively broad spectrum (b) Resistant to penicillinases (c) Fewer allergic reactions 4) These drugs often have cef, ceph, or kef in their names 5) Some given orally but most given parenterally (not through intestine; i.e. injection) 6) Three generations, each more effective than the last 7) Third-generation examples include cephalexin (Keflex), cefotaxime 46 Drugs, Microbes, Host—The Elements of Chemotherapy (C (a) Very good against enteric bacteria (b) Ceftriaxone (rocephin) is a new semisynthetic cephalosporin used for wide variety of respiratory, skin, urinary, and nervous system infections c. Other beta-lactams 1) Beta-lactams refers to one of the rings that is found in penicillins, cephalosporins, and related antibiotics (the red square in fig. 12.7 and 12.8) 2) Imipenem is broad-spectrum, can be taken orally, effective at low concentrations, with few side effects 3) Aztreonam is from a bacterium (not a mold) (a) It is narrow-spectrum against gram-negative bacilli (b) Good for people who are allergic to penicillins and cephalosporins d. Aminoglycosides 1) Made from filamentous fungi-like soil bacteria (Streptomyces) 2) Relatively broad spectrum because they inhibit protein synthesis 3) Streptomycin & gentamicin (newer and less toxic) (a) Good against gram-negative rods including Escherichia, Pseudomonas, Salmonella, Shigella e. Tetracyclines 1) Semisynthetics derived from a species of Streptomyces (as were the Aminoglycosides) 2) Blocks protein synthesis by binding to ribosomes 3) Effective against wide range of bacteria (gram negative and positive, aerobic and anaerobic, mycoplasmas, spirochetes, . . .) 4) Used to treat several STDs as well as Rocky Mountain spotted fever, Lyme disease, typhus, cholera, acne, and some protozoan infections 5) Side effects (a) Tetracycline is toxic to growing teeth and bones and should never be given to children or pregnant women (teratogenic—causes birth defects) (b) Tetracycline can also lead to superinfections by upsetting normal flora (c) GI upset (because it kills normal flora), sensitivity to sunlight, and discoloration of tooth enamel are additional side-effects f. Chloramphenicol 1) Yet another Streptomyces antibiotic! 2) Also broad-spectrum, also blocks protein synthesis 3) Very toxic and given only when other options are not available g. Other Streptomyces antibiotics (erythromycin, clindamycin, vancomycin) 1) Erythromycin (a) Broad spectrum; fairly low toxicity (b) Blocks protein synthesis by attaching to ribosome (c) Given orally to treat Mycoplasma pneumonia, Legionellosis, Chlamydia infections, pertussis, and diphtheria (d) Given as prophylactic prior to intestinal surgery (e) Used against penicillin-resistant strains 2) Vancomycin (a) Very toxic and hard to administer (b) Used in life-threatening situations, such as treating resistant staphylococcal infections 47 Elementary Microbiology h. Bacitracin and Polymyxin 1) Not going to discuss these—narrow applications i. New classes of antibiotics 1) The only thing I want to say here is that new drugs are difficult to come by and physicians are urged to use these only as a last resort in hopes of avoiding development of resistance 3. Synthetic antibacterial drugs (these do not originate from bacteria or fungi—they were isolated from dyes or from plants and then synthesized) a. “Sulfa drugs” (sulfonamides, trimethoprim, and sulfones) 1) Originally came from dyes 2) They compete with PABA for binding sites on enzymes involved in folic acid synthesis 3) Bacteriostatic instead of bactericidal 4) Often used in UTIs because they are very soluble 5) Silver sulfadiazine ointment for burns and eye infections 6) Allergic reactions are common b. Miscellaneous antibacterial agents 1) One new class of synthetic drug inhibits initiation of protein synthesis (a) Since it is not found in nature people are hopeful bacteria will be slow to develop resistance (b) First of this class marketed as “Zyvox” - Used to treat methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) 2) Fluoroquinolones (“floxacins”)are also a new class of synthetic drug (a) Potent, broad spectrum, readily absorbed through intestine (b) Examples include norfloxacin, ciprofloxacin, sparfloxacin, and levofloxacin (c) Some serious side effects such as seizures and brain disturbances 4. Agents to treat fungal infections a. Similarity between fungal and human cells means that most anti-fungal drugs are very toxic to human tissues b. Amphotericin B is by far most versatile and effective of the antifungals 1) It attacks the cell membrane causing permeability changes which lead to cell lysis and death 2) Side effects: Amphotericin is very toxic to kidneys (nephrotoxic) c. Nystatin can be used topically d. Clotrimazole used as a topical ointment for skin, and vaginal or oral candiasis 5. Antiparasitic chemotherapy a. Antimalarial drugs 1) Quinine used for hundreds of years—comes from bark of a tree 2) Newer derivatives are less toxic 3) No drug treats all species of Plasmodium or all stages of its life cycle b. Chemotherapy for other protozoan infections 1) Flagyl used against the ameba Entamoeba histolytica and also against Giardia c. Antihelminthic drugs 1) Flukes, tapeworms, and roundworms are difficult to treat (a) They are multicellular animals (so cells and tissues are similar to ours) 48 Drugs, Microbes, Host—The Elements of Chemotherapy (C (b) Don’t need to know any specific treatments 6. Antiviral chemotherapeutic agents a. Tough because viruses do not have their own metabolism—they rely on the host cell for metabolic functions 1) So, selective toxicity is not really an option b. Prevention by vaccination is effective for many viral diseases 1) But influenza, AIDS, and other diseases indicate need for better drugs (a) Available drugs have limited applicability and are expensive (b) Can protect uninfected cells but cannot destroy extracellular or latent viruses c. Acyclovir (Zovirax) and relatives (which end in “clovir”) block DNA synthesis 1) mainly good against herpes viruses d. AZT (Azidothymidine or zidovudine) used in all stages of HIV infection 1) Anti-HIV drugs target the reverse transcriptase enzyme since HIV is a retrovirus e. Human interferon has antiviral properties by putting uninfected cells “on the alert” 1) We will discuss this again when we talk about immunity next week 2) Human interferon being produced by bacteria thanks to recombinant DNA technology 3) Effective against herpes viruses, papilloma viruses (warts and genital warts) as well as some cancers 7. Acquisition of drug resistance (evolution in action) a. How it develops 1) Given that most antibiotics come from bacteria and fungi found in nature it is no surprise that some bacteria have innate resistance to these chemicals (a) Most that are innately resistant are not pathogens—they are harmless soil bacteria 2) Given that antibiotics create a hostile environment for bacteria, there is strong selective pressure to develop resistance (a) Arms race between snakes and newts as an example (b) Any mutation or novel genetic combination that confers any degree of resistance will be strongly favored in some environments - Huge populations, very short generation times, and constant rate of mutation set the stage - Remember that you have more bacteria in your mouth than all humans who have ever lived on earth? What are the odds that just one of those is resistant? (c) Remember conjugation and transfer of plasmids? These “R factors” can confer resistance from one species of bacteria to another, greatly speeding up the spread of resistance - Bacteria can also acquire these genes through transformation (picking up random DNA) or transduction (via bacteriophage vector) b. Specific mechanisms of drug resistance 1) Enzymes that inactivate the drug (a) Penicillinase or cephalosporinase - Many strains of Staphylococcus aureus produce penicillinase 49 Elementary Microbiology - Neisseria gonorrhoeae also starting to produce it 2) Make the cell less permeable to the drug or pump it out of the cell (a) Gram-negative cells naturally resistant to some penicillin drugs because they are not permeable (b) Special proteins may pump tetracycline out of the cell - Some multidrug resistant (MDR) pumps actively transport a variety of drugs, detergents, and other toxins out of the cell 3) Change of shape or decrease in number of drug receptor sites (a) Drugs usually act on a specific target site (b) If the shape of this target changes then the drug is no longer effective - Back to the snakes: modified sodium-ion channels that the newt toxin cannot bind to - Some drug resistance due to changes in ribosomal shape or cell-wall protein shape 4) Modification of metabolic pathway targeted by drug (a) Similar to changing shape of enzyme, but in this case an alternative metabolic path is developed that uses an entirely different enzyme than the one that was being targeted - Resistance to sulfa drugs by using a different pathway to synthesize folic acid c. Natural selection and drug resistance 1) This section just explains natural selection 2) One point I want to make is that resistance is more fit when antibiotics are present but resistant strains may be at a disadvantage under normal conditions (a) Snake resistance comes at a cost—greater resistance means slower crawl speed (b) Explains why you don’t get a resistant infection in the home, but you do in the hospital d. The rise of drug resistance (Spotlight pg 370) 1) Bacteria have ruled the world for 3 billion years (a) We are not about to eliminate them now! (b) Incredibly versatile and adaptive 2) Viruses can mutate even faster! (a) HIV can become drug resistant within weeks of starting therapy 3) 60% of hospital infections are caused by drug-resistant microbes! (a) Gee, why did we switch to homebirths? 4) MRSA (methicillin-resistant Staphylococcus aureus) can tolerate nearly all antibiotics, but is sensitive to vancomycin (a) In 2002 the first case of vancomycin-resistant Staph aureus (VRSA) emerged (b) Strains of MRSA now showing up outside of hospital settings 5) Antibiotics are a standard supplement to livestock feed! (a) Hello?! This is a ruminant animal! It relies on microbes for digestion! It is a walking vat of microbial fermentation. . . so let’s expose all those bacteria to a constant dose of antibiotics to encourage all of them to develop resistance! (b) A Salmonella outbreak in Denmark which jumped from cows to humans was found to be resistant to seven different antibiotics - Denmark has since outlawed use of ANY antibiotics in animal feed - U.S. and Europe have banned use of human antibiotics in feeds 50 Drugs, Microbes, Host—The Elements of Chemotherapy (C (c) VRE (Vancomycin-resistant Enterococcus) is a nasty nosocomial infection - It is thanks to putting a vancomycin-like antibiotic in cattle feed 6) Cholera, gonorrhea, malaria, tuberculosis, typhoid fever, . . . all becoming more and more resistant 7) Antibiotics are unregulated in much of the world and taken like vitamins (a) Even here they are WAY overprescribed and a shotgun approach is taken instead of identifying the cause before deciding on treatment D. Characteristics of host-drug reactions 1. Toxicity to organs a. Certain drugs may affect liver (hepatotoxic), kidneys (nephrotoxic), GI tract, cardiovascular system, blood-forming tissue (hemotoxic), nervous system (neurotoxic), respiratory tract, skin, bones, and teeth b. Most common complaint is diarrhea 1) Not usually due to irritating the intestinal lining 2) Usually due to disrupting normal intestinal microflora c. Do not need to know the specifics—refer to table 12.6 on pg 373 for your own information 2. Allergic responses to drugs a. Have been reported for every type of antimicrobic drug 1) Penicillins are most common cause of allergic responses 2) Sulfonamides are next most common b. We will discuss allergic responses when we talk about our immune system 3. Suppression and alteration of the microflora by antimicrobics a. When you wipe out much of your normal flora it is like moving the people out of Chicago 1) Suddenly everything is vacant 2) Guess who moves in? This is called a “superinfection!” (a) Yeast infections following treatment for a UTI (b) Antibiotic-associated colitis thanks to Clostridium difficile E. Considerations in selecting an antimicrobic drug (these are often ignored!) 1. Identifying the agent a. Sample should be taken before antimicrobic drugs are given 1) Can directly examine sample from stool, body fluids, sputum, etc under microscope 2) May be able to identify bacteria or fungi from this method alone, or at least learn enough to start therapy (a) Informed best guess (e.g. Streptococcus from sore throat swab) - Epidemiologic statistics help make an informed best guess 2. Testing for the drug susceptibility of microorganisms a. Important for groups of bacteria that show resistance, such as Staphylococcus, Neisseria gonorrhoeae, Streptococcus pneumoniae, and Enterococcus faecalis 51 Elementary Microbiology b. Not always necessary (e.g. anaerobes are uniformly susceptible to penicillin G) c. Testing is done by exposing a pure culture of the pathogen to several different drugs in a standardized fashion 1) Kirby-Bauer technique (a) Agar diffusion test (b) Small discs with premeasured amounts of antimicrobics (c) Compare the zone of inhibition (measure in mm) to a standard table to determine if susceptible or resistant 2) E-test (a) Has a strip with a gradient of the drug (b) Measures MIC (Minimum Inhibitory Concentration) for each drug - MIC can also be obtained with tube dilution test 3. The MIC and therapeutic index a. The in vitro results do not always correspond to in vivo results, so it is important to observe patient’s clinical response b. Treatment may fail in vivo due to 1) Inability of drug to enter the target site 2) Presence of resistant cells that were not noted in the susceptibility testing 3) Infection due to mixed pathogens, at least one of which is resistant c. If therapy fails then a different drug, different method of drug administration, or combined drug therapy are options d. Keep in mind that factors other than bacteria susceptibility influence drug choice 1) Potential side effects 2) Condition of the patient 3) The spectrum of the drug (a) Generally want the most narrow-spectrum drug that will do the job - Reduces risk of superinfections e. Therapeutic index (TI) 1) The ratio of the dose of the drug that is toxic to humans compared to the minimum effective dose (a) If this ratio is close to 1, then there is great risk for toxic drug reaction (b) If this ratio is large then there is a greater margin of safety 2) If you have a choice between drugs with similar MIC, then the one with the highest TI is chosen f. Patient condition 1) Known allergies to classes of drugs 2) Liver or kidney problems (a) These organs responsible for metabolizing and excreting the drug 3) If patient is an infant, elderly, or pregnant 4) If patient is taking other drugs (a) Interaction may cause increased toxicity or drug failure (b) Drugs can also work synergistically so lower doses of each are needed 5) Other factors such as genetic or metabolic abnormalities, site of infection, cost of drug g. Art and Science of Choosing an Antimicrobial Drug 52 Drugs, Microbes, Host—The Elements of Chemotherapy (C 1) Example of elderly alcoholic with pneumonia 2) Example of cancer patient with systemic Candida infection (a) Choices are reduced and priorities are different in a life-threatening situation 4. An antimicrobic drug dilemma a. 75% of antimicrobial prescriptions given for respiratory infections 1) Most of these are viral 2) Many drugs misprescribed as to type, dosage, length of therapy b. Prescriptions issued without identifying pathogen or doing susceptibility testing c. Shotgun approach instead of narrow-spectrum d. Newer, more expensive drugs routinely prescribed 1) Cephalosporins and tetracyclines are among most expensive and also most prescribed (hmmm, is there a correlation??) e. Lack of control in many parts of the world 1) Easy to self-medicate without any understanding of correct drug choice, dosage, length of treatment, etc. f. Need to educate patients and caregivers 53 Elementary Microbiology Unit 10. Microbe-Human Interactions: Infection and Disease (Ch 13) A. The human host 1. Contact, infection, disease—a continuum 2. Resident flora: the human as a habitat a. Acquiring resident flora b. Initial colonization of the newborn 3. Indigenous flora of specific regions a. Flora of the human skin b. Flora of the gastrointestinal tract c. Flora of the mouth d. Flora of the large intestine e. Flora of the respiratory tract f. Flora of the genitourinary tract 4. Life without flora (microbits 13.1) B. The progress of an infection 1. Pathogens a. Pathogenicity is an organism’s potential to cause infection or disease b. Virulence is a measure of how well a microbe can invade the host and how toxic it is (a relative degree of pathogenicity) 1) Sometimes virulence factors are clearly known (such as tetanus toxin), while other times the virulence factors are unknown c. True pathogens cause disease even in healthy individuals 1) Range from mild (cold virus) to fatal (rabies virus) d. Opportunistic pathogens are not pathogenic to a normal healthy person 1) Do not have well-developed virulence properties 2) May infect compromised host (old, already sick, stressed) 3) May be normal flora that got into the wrong place or became imbalanced e. The CDC rates microbes from on a virulence scale from 1 (not known to cause disease in humans) to 4 (highly virulent, highly contagious) 1) Remember throughout this course that host deficiencies are the most important cause of disease (a) Host deficiencies are also the most important factors relating to the eventual outcome of persons who are ill. (b) Ambulance in valley or fence on cliff? 2. The portal of entry: gateway to infection 1) Portals of entry are typically the sites that support normal flora 2) Most pathogens are adapted to entering via a specific portal (e.g. STD’s enter the urogenital tract) (a) Cannot get the flu through the skin 3) Some can enter via multiple portals 54 Microbe-Human Interactions: Infection and Disease (Ch 13) (a) Streptococcus can cause an infection through the respiratory system or on the skin (b) Effects of infection by the same pathogen can differ depending on portal (inhalation anthrax versus cutaneous anthrax) 4) Note: in lectures 7, 8, and 9 I think I will deviate from the book by looking at diseases for different body systems instead of going by organization of microbes. (a) The entry portal is often the place where the disease is manifest, but not always (b) In talking about portals we will mention several diseases in passing, but not in great detail - This might give you ideas for your poster! b. Infectious agents that enter the skin 1) Intact skin is an excellent barrier 2) Unfortunately a very small imperfection can be enough to let bacteria through (a) Staphylococcus aureus causes boils (b) Streptococcus pyogenes (or Staph. aureus) can cause impetigo (c) Tetanus and gangrene are both caused by anaerobic bacteria that infect dead tissues surrounding a wound 3) Sometimes entry is facilitated by a vector (an animal that transmits the microbe) (a) Mosquito bites and malarial protozoa (b) Ticks and Lyme’s disease (a bacterial infection) 4) Some microbes can gain access through sweat glands and hair follicles. 5) "Parenteral route" is when microbes enter the body by means other than the digestive tract. (a) Examples are bites, wounds, surgery, cuts, injections, etc. 6) Eyes can be infected by Chlamydia trachomatis (trachoma, the leading cause of blindness) or Neisseria gonorrhoeae (neonatal ophthalmic gonorrhea, which is why they put antibacterial drops in newborn eyes) c. The gastrointestinal tract as portal 1) Tough place to enter since there are digestive enzymes and stomach acid 2) Pathogens are swallowed in contaminated food or water (usually fecal contamination) (a) Sand filtration and sewage treatment greatly reduce disease 3) Several gram-negative rods infect the small or large intestine, including Salmonella (food poisoning), Shigella, Vibrio (cholera), and virulent strains of E. coli 4) Poliovirus and hepatitis A virus both enter through digestive tract 5) Protozoans that infect digestive tract include Entamoeba histolytica (amebic dysentery), and Giardia (beaver fever) d. Respiratory portal of entry 1) The most common portal of entry! 2) The smaller the pathogen, the easier it is for it to get carried deep into the respiratory tree 55 Elementary Microbiology 3) Bacterial infections include strep throat, meningitis, diphtheria, bacterial pneumonia, whooping cough, and tuberculosis (Mycobacterium tuberculosis) 4) Viral infections include influenza, measles, mumps, rubella, chickenpox, and the common cold 5) e. Urogenital portals of entry 1) Sexually transmitted diseases can be bacterial (gonorrhea, syphilis), viral (genital warts, herpes, hepatitis, AIDS), protozoan (Trichomoniasis), or fungal (Candida albicans—yeast infection) 2) Bacterial infections of urinary tract, bladder, and kidneys not considered an STD, but incidence is greater in sexually active women f. Pathogens that infect during pregnancy and birth 1) There aren’t many since placenta is a good barrier 2) Syphilis caused by small spirochete (a) Who invented Salvarsan (compound 606)? (b) Now treated with penicillin 3) HIV can also cross placenta g. The size of the inoculum 1) The infectious dose is different for different pathogens (a) The smaller the ID the greater the virulence 2) ID varies widely—between 1 and a billion 3) Inoculation with less than ID generally does not cause disease (a) Why it is okay if turkey has some Salmonella on it, but not okay if you let it sit out long enough for those bacteria to multiply exponentially 4) Normal flora and state of a person’s immune system also have big effect on outcome of inoculation C. Mechanisms of invasion and establishment of the pathogen 1. Adherence a. Every part of the human body has some type of "normal flow" of materials (solids or liquids). b. Because of these flowing processes, most pathogenic microbes must have some way to attach (adhere) to the body (i.e. - fimbriae, spikes, slime layer, etc.) c. This attachment between pathogen and host is accomplished by means of surface molecules on the pathogen called “adhesins” or “ligands” that bind specifically to complementary surface receptors on the cells of certain host tissues. 1) The majority of adhesins are glycoproteins or lipoproteins. 2) The hosts cell receptors are typically sugars (i.e. mannose) d. If this attachment can be prevented, disease can be avoided 2. How virulence factors contribute to tissue damage (virulence factors are adaptations that facilitate invading a host) a. Extracellular enzymes break down material outside the bacteria, allowing it to get past barriers 56 Microbe-Human Interactions: Infection and Disease (Ch 13) 1) Collagenase breaks down the framework of muscles and connective tissue (collagen) and is produced by Clostridium (botulism and tetanus) 2) Hyaluronidase breaks down the “cement” that binds cells together (hyaluronic acid) and is produced by Staphylococcus, Clostridium, and Streptococcus 3) Coagulases (produced by some Staphylococcus) cause blood plasma to clot (which can provide a place for bacteria to hide from macrophages) 4) Kinases digest blood clots (making it easier for bacteria to enter bloodstream via a cut) (a) Streptokinase and treatment for heart attacks or strokes 5) IgA protease destroys IgA antibodies on mucus membranes b. Bacterial toxins: a potent source of cellular damage 1) A toxin is simply a poisonous chemical produced by an organism (a) The toxin may be almost entirely responsible for the disease 2) Toxemia is a term to describe toxins being carried through the blood (e.g. tetanus toxin) 3) Intoxication is when you ingest a toxin (e.g. botulism) 4) Classify toxins as exotoxin or endotoxin (see table 13.7) 5) "Exotoxins" are proteins (often enzymes) produced by some bacteria and released into the surrounding environment. (a) Because they are proteins, most exotoxins are destroyed by high temperature (heat labile). (b) Most bacteria that do this are Gram positive (but may be Gram negative too) (c) The genes for most of them (perhaps all) are carried on bacterial plasmids or phages. (d) Exotoxins are among the most lethal substances known (Biowarfare?) (e) Antibodies which inactivate exotoxins are called "antitoxins". These antitoxins can be produced during disease, vaccination, or produced in animals for use in humans. (f) Exotoxins which kill host cells are called “cytotoxins” (usually by disrupting the plasma membrane in some way) - Also help bacteria escape sacs after phagocytosis (g) Exotoxins which attack the nerves are called "neurotoxins" while exotoxins which attack the intestine are called "enterotoxins." (h) Examples are: - Diphtheria toxin - Erythrogenic toxins - Botulism toxin (plastic surgery in a syringe?) - Tetanus toxin - Cholera enterotoxin - Staphylococcal enterotoxin - Leukocidin - Hemolysin (blood agar plates to distinguish strains of bacteria) - Etc. 6) “Endotoxins” are lipopolysaccharides and part of the cell wall of gramnegative bacteria (a) Actually, it is the lipid portion of this material ("Lipid A") that is the active ingredient of endotoxin. (b) Endotoxin is heat stable at high temperatures. 57 Elementary Microbiology (c) Most bacteria that have this are Gram negative (d) Endotoxins exert their effects when the bacteria die and their cell walls break down. (e) Endotoxins cause: - Fever (This is why endotoxin may be also called an “exogenous pyrogen”) - Weakness - Aches - Sometimes shock is seen (hypotension) - may induce miscarriage - may cause disorder of blood coagulation - systemic infections can be fatal despite treatment! (f) The effect of endotoxins may be reduced by specific antibodies c. How microbes escape phagocytosis 1) Kill the phagocytes! (leukocidins produced by some Staphylococcus and some Streptococcus) 2) Slip away with a capsule! (several bacteria) (a) The host can make anticapsular antibodies to more easily destroy an encapsulated bacteria 3) “Just eat me!” (Mycobacterium tuberculosis and Rickettsia) 3. Classic stages of clinical infections (Microbits 13.2) a. Once a microbe overwhelms the host's defenses a certain pattern of events will follow. (See Medical Microfile 13.2 on pg 398) 1) Period of incubation = the time interval between actual infection and the first appearance of any signs or symptoms of disease (a) Length dependent upon organism, how many initially present, and resistance of the host. (b) Typically 2 to 30 days (but may be several years in leprosy) 2) Prodromal period = the period where the first vague symptoms of disease appear (stiff neck, general malaise, fatigue, etc.) 3) Period of invasion = this is when the disease is most acute (also called period of illness) (a) This is when you see signs and symptoms (e.g. fever, cough, diarrhea, etc) (b) Patient may die during this period (terminal illness) 4) Period of convalescence = the time when the signs and symptoms of disease subside and a person regains strength. b. Note that persons may be contagious in all of these disease stages! 4. Patterns of infection a. Localized infection stays in place (e.g. boils, warts) b. Focal infection when pathogen breaks loose from a local infection and is carried to other tissues (strep throat giving rise to scarlet fever) c. Systemic infection spreads to several tissues throughout the body (usually via circulatory system) 1) Chickenpox, anthrax, etc. 2) Toxemia refers to a toxin that spreads through the body (a) but it may come from a local, focal, or systemic infection d. Mixed infections include several pathogens (e.g. dental caries) e. Primary infection can be complicated by a secondary infection 58 Microbe-Human Interactions: Infection and Disease (Ch 13) f. Acute infections are swift and short-lived while chronic infections are persistent (e.g. malaria) 5. Signs and symptoms: warning signals of disease a. Signs are observable (e.g. fever) while symptoms are sensed by patient (e.g. headache) 1) Inflammatory response 2) Leukocytosis and leukopenia 3) Septicemia when microbes are multiplying in blood (bacteremia, viremia shows presence but not multiplication) 4) Development of antibodies in serum b. Some infections are “asymptomatic” or “subclinical” 1) A person who is a carrier or reservoir 6. Portal of exit: vacating the host (often same as portal of entry) a. Respiratory and salivary portals 1) Coughing and sneezing, talking and laughing 2) Tuberculosis, influenza, measles, chickenpox, colds, etc. are spread through airborne droplets (a) Some are spread through larger droplets of saliva, such as rabies and mononucleosis b. Skin scales: are you a shedder? 1) Warts, fungal infections, boils, smallpox, syphilis c. Fecal exit: particular problem in areas lacking good wastewater treatment d. Urogenital tract (STDs) e. Removal of blood or bleeding (mosquitoes or ticks; needle stick) 7. Persistence of microbes and pathologic conditions a. Some microbes go latent instead of being completely destroyed 1) Periodically become active again and produce recurrent disease 2) Herpes zoster, AIDS, Epstein-Barr, syphilis, tuberculosis, malaria 3) Infected person termed a carrier if infection is shed D. Epidemiology: the study of disease in populations 1. Who, when, and where? Tracking disease in the population a. Role of local health agencies, CDC, and WHO b. Epidemiologic statistics: frequency of cases 1) Prevalence is percentage of population with the disease 2) Incidence is number of new cases over certain time compared to number of healthy individuals (a) Also called morbidity (b) CDC publishes weekly Morbidity and Mortality Report which reports incidence per 1,000 or 100,000 3) Epidemiologists track changes in incidence seasonally, by sex, race, geographic region, etc. (a) Truck stops and spread of AIDS 4) Mortality rate has declined during past century (good ambulances!), but morbidity rate has remained high (where’s the fence?) 59 Elementary Microbiology c. Occurrence of Disease 1) Sporadic disease = occurs occasionally (i.e. - typhoid fever, tetanus). 2) Endemic disease = constantly present in a certain population (i.e. common cold). May be geographically limited (Lyme disease) 3) Epidemic disease = many people in a given area acquire a certain disease in a short period of time (i.e. – gonorrhea, cholera). 4) Pandemic disease = an epidemic disease that occurs worldwide (i.e. – AIDS, influenza). d. Epidemiologists must piece together bits of apparently unrelated data to look for epidemics and discover new diseases 2. Reservoirs: where pathogens persist a. Reservoir and source of disease may be same or different b. Living reservoirs 1) Carriers = people who can transmit the disease but who have no obvious signs of the disease (a) Example = nasal carriage of Staphylococcus aureus (b) Person may be entirely asymptomatic, or just during incubation or convalescent period (c) Typhoid Mary? (d) YOU may be a passive carrier, spreading disease from one patient to another without ever becoming infected yourself - Handwashing, aseptic techniques vital! 2) Animals as reservoirs and sources (a) Vectors are animals that transmit disease - (daycare and children as vectors ) - Fleas, mosquitoes, flies, ticks (b) Biological vectors participate in life cycle of pathogen while mechanical vectors just transport (c) Zoonoses, emerging diseases, and sentinel animals c. Nonliving reservoirs 1) Soil contains spores from Bacillus anthracis, Clostridium sp. 2) Water has Giardia, Legionella, Pseudomonas 3. How and why? The acquisition and transmission of infectious agents a. Patterns of transmission in communicable disease b. Modes of direct transmission c. Contact transmission 1) Direct contact (exit portal meets entry portal) (a) (b) (c) (d) (e) Sexual intercourse Touching Kissing Coughing, sneezing, laughing, talking Biological vector d. Routes of indirect transmission 1) Indirect transmission involves disease transmission by means of a vehicle such as food, water, blood, or a nonliving object (fomite) (a) Handkerchiefs, towels, bedding, diapers, cups, needles, doorknobs, telephones, keyboards, restroom doors, etc. (b) Oral-fecal route due to inadequate hygiene (Hepatitis A, Salmonella) 60 Microbe-Human Interactions: Infection and Disease (Ch 13) e. Indirect spread by vehicles: contaminated materials f. Indirect spread by airborne route: droplet nuclei and aerosols 1) Droplet infection is NOT direct contact because hardy pathogens may persist in the dried microscopic residues of mucus and saliva and be picked up indirectly off a surface 2) Aerosols are suspensions of fine dust that may travel far through the air. 4. Nosocomial infections: the hospital as a source of disease a. About 5% of hospital patients acquire an infection during their stay (but as high as 20% in some clinical settings) 1) About 2-4 million cases per year, and 90,000 deaths 2) Patients’ health is compromised (that’s why they are there!) 3) May come from patient’s own flora (UTI from catheter), from another patient, from a fomite, or from medical personnel, visitors, air, etc. 4) Drug-resistant microbes are selected for in hospital setting 5) Most common nosocomial infection involves urinary tract, followed by surgical sites and respiratory tract b. Medical asepsis lowers microbial load in patients, caregivers, and the hospital environment 1) Handwashing, disinfection, patient isolation (see table 13.11) (a) Which group of health care workers is the worst about following correct aseptic procedures? 2) Surgical asepsis very stringent 5. Which agent is the cause? Using Koch’s postulates to determine etiology a. Etiologic agent is the cause of the disease b. Robert Koch developed postulates to determine etiology of a disease (developed these when looking for etiology of anthrax in cows) 1) Find evidence of a particular microbe in all cases of the disease 2) Isolate the microbe from an infected subject and cultivate it in the lab 3) Inoculate a healthy subject with the lab isolate and observe resultant disease 4) Reisolate the agent from this subject c. These were used to determine cause of tuberculosis, diphtheria, plague, and most every known disease 1) Cannot use in all situations (a) Leprosy and Rickettsia cannot grow in lab culture (b) Some diseases unique to humans - Unethical to inoculate healthy subject with something life-threatening! - But accidental exposure provides the proof (needle sticks and AIDS) 61 Elementary Microbiology Unit 11. The Nature of Host Defenses (Ch 14) A. Defense Mechanisms of the Host in Perspective 1. Overview The host defenses embrace a multilevel network of innate, nonspecific protection from specific immunities, referred to as the first, second, and third lines of defense. First Line of Defense: any barrier that blocks invasion at the portal of entry. Second Line of Defense: more internalized system of protective cells and fluids that includes inflammation and phagocytosis. Acts quickly at local and systematic levels once first line has been circumvented. Third Line of Defense: acquired and specific and is dependent on the function of T and B cells. 2. Barriers at the Portal of Entry: A First Line of Defense a. Physical or Anatomical Barriers at the Body’s Surface 1) Skin and mucous membranes of the respiratory and digestive tracts have built-in defenses (a) The outermost layer (stratum corneum) of the skin is composed of epithelial cells that have become compacted, cemented together, and impregnated with an insoluble protein, keratin – the result is a thick, tough layer that is highly impervious and waterproof, few pathogens can penetrate this unbroken barrier (b) Hair follicles and skin glands - The hair shaft is periodically extruded and the follicle cells are desquamated (scaled off) - The flushing effect of sweat glands helps remove microbes (c) Damaged cells are rapidly replaced 2) Mucocutaneous membranes of the digestive, urinary, and respiratory tracts and of the eye are moist and permeable (a) The mucous coat on the free surface of some membranes impedes the entry and attachment of bacteria (b) Blinking and tear production flush the eye’s surface with tears and rid it of irritants (c) The constant flow of saliva helps carry microbes into the harsh conditions of the stomach (d) Vomiting and defecation also evacuate noxious substances of microorganisms from the body (e) The respiratory tract is constantly guarded from infection by elaborate and highly effective adaptations - Nasal hair traps larger particles - Rhinitis, the copious flow of mucus and fluids that occurs in allergy and colds, exerts a flushing action - The respiratory tree (especially the trachea & bronchi) has ciliated epithelium (mucociliary escalator) that moves foreign particles entrapped in the mucous toward the pharynx to be removed - Irritation in nasal passage reflexly initiates a sneeze, which expels a large volume of air at high velocity 62 The Nature of Host Defenses (Ch 14) - The sensitivity of the bronchi, trachea, and larynx to foreign matter triggers coughing, which ejects irritants (f) The genitourinary tract has partial protection from continuous trickle of urine through the ureters and from periodic bladder emptying that flushes the urethra b. Nonspecific Chemical Defenses 1) Sebaceous secretions exert an antimicrobial effect 2) Specialized glands, like the meibomian glands of the eyelids, lubricate the conjunctiva with an antimicrobial secretion 3) Tears and saliva contain lysozyme, an enzyme that hydrolizes the peptidoglycan in the cell wall of the bacteria 4) High lactic acid and electrolyte concentration of sweat and the skin’s acidic pH and fatty acid content are inhibitory to microbes 5) The hydrochloric acid in the stomach render protection against many pathogens that are swallowed 6) The intestine’s digestive juices and bile are destructive to microbes 7) Semen contains and antimicrobial chemical that inhibits bacteria 8) The vagina has a protective acidic pH maintained by normal flora c. Genetic Defenses 1) Some hosts are genetically immune to the diseases of other hosts 2) Some pathogens have a great specificity (a) The specificity is particularly true of viruses, which can invade only by attaching to a specific host receptor. 3) Some genetic differences exist in susceptibility B. Introducing the Immune System 1. Immunology encompasses the study of all features of the body’s immune systems 2. A healthy functioning immune system is responsible for: a. Surveillance of the body to determine differences between the host’s self identity markers and the nonself identity markers of foreign cells b. Recognition of foreign material, primarily carried out by white blood cells, and a tailored immune system response is set into motion c. Destruction of entities deemed to be foreign C. Systems Involved in Immune Defenses 1. Because infectious agents could potentially enter through a number of portals, the cells in the immune system are constantly moving through the body searching for pathogens. a. This process is carried out by white blood cells which are trained to recognize body cells, called self, or any foreign body material (known as nonself). b. To deal with foreign cells, the immune system evaluates cells by the markers on their surface. 1) When the immune system recognizes that a marker or antigen is nonself, it tailors its response specifically to each different antigen. As far as the 63 Elementary Microbiology 2. a. b. 3. 4. immune system is concerned, if an antigen is not self, it does not belong, and must be destroyed. Reticuloendothelial System is a network of connective tissue fibers & phagocytic cells that permeates the tissues of all organs. It is inhabited by macrophages ready to attack and ingest microbes who have managed to bypass the first line of defense. Provides passage within and between organs. It coexists with and helps form a niche for a collection of phagocytic cells. The spaces surrounding tissue cells that contain extracellular fluid a. These spaces have both blood and lymph capillaries, which bring all components of the second and third line of defense to attack infectious microbes The bloodstream (see Fig. 14.9, pg. 423) a. Whole blood – a liquid connective tissue b. Blood cells – formed elements c. Serum – clear fluid expressed from clotted blood that contains dissolved nutrients, antibodies, and hormones but not cells or clotting factors d. Plasma – the carrier fluid element of blood mainly composed of water (92%) e. Stem cells – pluripotential undifferentiated cells f. Leukocytes 1) Types of leukocytes (a) Neutrophils – 55-90% - lobed nuclei with lavender granules – phagocytes (b) Eosinophils – 1-3% - bilobed with orange granules – destroy eukaryotic pathogens (c) Basophils, mast cells – 0.5% - constricted nuclei with dark blue granules – release potent chemical mediators (d) Lymphocytes – 20-30% - large nucleus – B&T cells involved in the specific immune response (e) Monocytes, macrophages – 3-7% - large nucleus – phagocytic 2) Characteristics of leukocytes (a) Diapedisis – migration of cells out of blood vessels into the tissues (b) Chemotaxis – migration in response to specific chemicals at the site of injury or infection 5. The lymphatic system a. Functions: 1) Provides an auxiliary route for return of extracellular fluid to the circulatory system 2) Acts as a drain-off system for the inflammatory response 3) Renders surveillance, recognition, and protection against foreign material b. Lymphatic fluid 1) Lymph is plasmalike liquid carried by lymphatic circulation 2) Formed when blood components move out of blood vessels into extracellular spaces 3) Made up of water, dissolved salts, 2-5% proteins 4) Transports white blood cells, fats, cellular debris, and infectious agents c. Lymphoid organs and tissues 1) Lymph nodes 64 The Nature of Host Defenses (Ch 14) (a) Small, encapsulated, bean-shaped organs stationed along lymphatic channels and large blood vessels of the thoracic and abdominal cavities (b) Contains segregated populations of lymphocytes 2) Spleen – filters pathogens from blood 3) Thymus – T lymphocytes mature here 4) GALT – gut-associated lymphoid tissue – provides immune functions against intestinal pathogens and is a significant source of some types of antibodies 5) Peyer’s patches – compact aggregations of lymphocytes in the ileum of the small intestine – respond to local infectious agents D. Nonspecific Immune Reactions of the Body’s Compartments 1. The Inflammatory Response: A Complex Concert of Reactions to Injury a. Signs and symptoms 1) Rubor – redness caused by increased circulation and vasodilation in injured tissue 2) Calor – warmth, heat given off by the increased blood flow 3) Tumor – swelling caused by increased fluid escaping into the tissues 4) Dolor – pain caused by the stimulation of nerve endings b. Functions: 1) Mobilize and attract immune components tot eh site of the injury 2) Set in motion mechanisms to repair tissue damage and localize and clear away harmful substances 3) Destroy microbes and block their further invasion 2. The Stages of Inflammation (see Fig. 14.16, pg. 430) a. Blood vessels dilate in response to chemical mediators and cytokines b. Edema swells tissues, helping prevent spread of infection c. WBC’s, microbes, debris, and fluid collect to form pus d. Pyrogens may induce fever 1) Benefits of fever (a) Inhibits multiplication of temperature-sensitive microbes such as the poliovirus, cold viruses, herpes zoster virus, systemic and subcutaneous fungal pathogens (b) Impedes the nutrition of bacteria by reducing the availability of iron, macrophages stop releasing their iron stores, which could retard several enzymatic reactions needed for bacterial growth (c) Increases metabolism and stimulates immune reactions and naturally protective physiological processes, speeds up hematopoiesis, phagocytosis, and specific immune reactions e. Macrophages and neutrophils engage phagocytosis 3. Phagocytes: The Ever-Present Busybodies of Inflammation and Specific Immunity a. Functions: 1) Survey tissue compartments and discover microbes, particulate matter, and dead or injured cells 2) Infest and eliminate these materials 65 Elementary Microbiology 3) Extract immunogenic information from foreign matter b. Types of phagocytes 1) Neutrophils – general purpose phagocytes that react early in the inflammatory response to bacteria and other foreign materials 2) Monocytes 3) Macrophages – WBC derived from a monocyte that leaves the circulation and enters tissues, they are important in nonspecific phagocytosis and in regulating, stimulating, and cleaning up after immune response 4. Contributors to the Body’s Chemical Immunity a. Interferon (see Fig. 14.21, pg. 437) 1) Small protein produced by certain cells (a) Alpha interferon – lymphocytes & macrophages (b) Beta interferon – fibroblasts & epithelial cells (c) Gamma interferon – T cells 2) Produced in response to viruses, RNA, immune products, and various antigens 3) Bind to cell surfaces and induce expression of antiviral proteins 4) Inhibit expression of cancer genes b. Complement (see Fig. 14.22, pg. 438) 1) Consists of 26 blood proteins that work in concert to destroy bacteria and viruses 2) Complement proteins are activated by cleavage 3) Classical pathway 4) Alternative pathway E. Specific Immunities: The Third and Final Line of Defense 1. Immunocompetent (the ability of the body to recognize and react with multiple foreign substances) individuals possess a third line of defense that is acquired only after direct exposure to an infectious agent a. This type of immunity is mediated by T and B lymphocytes and is marked by being extremely specific and creating immunologic memory 66 The Acquisition of Specific Immunity and its Applications ( Unit 12. The Acquisition of Specific Immunity and its Applications (Ch 15) A. Further explorations into the immune system B. The dual nature of specific immune responses 1. 2. 3. 4. 5. Development of the dual lymphocyte system Entrance and presentation of antigens and clonal selection B and T activation of lymphocytes and clonal expansion Products of B lymphocytes: antibody structure and functions How T cells respond to antigen: cell-mediated immunity C. Essential preliminary concepts for understanding immune reactions of sections I-V 1. Markers on cell surfaces involved in recognition of self and nonself 2. The origin of diversity and specificity in the immune response D. The lymphocyte response system in depth 1. Development of the dual lymphocyte system: stages in origin, differentiation, and maturation 2. Entrance and processing of antigens and clonal selection 3. B Activation of B lymphocytes: clonal expansion and antibody production 4. Products of B lymphocytes: antibody structure and functions 5. T activation of T lymphocytes and how T cells respond to antigen: cell-mediated immunity 67 Elementary Microbiology Unit 13. Cocci of Medical Importance (Ch 18) A. General characteristics of the Staphylococci 1. Introduction a. 31 species in the genus Staphylococcus, but we will focus on S. aureus b. Staphylococcus aureus is a gram-positive cocci that grows in grape-like clusters 1) Optimal growth at 37 degrees (body temperature) 2) Facultative anaerobe (grows best with O2 but can do without) c. S. aureus colonies are typically golden (aureus) d. S. aureus does not form spores and does not have flagella 1) Considered the most resistant of all non-spore forming bacteria (a) (b) (c) (d) Can withstand high salt concentrations (7-10%) Can withstand heat (60 ˚C for 60 min) Can withstand drying, low pH, disinfectants, and many antibiotics Produces greater variety of virulence factors than any other pathogen e. About 80,000 deaths per year in the U.S. attributed to Staph. infections 2. Growth and physiological characteristics of Staphylococcus aureus a. The enzymes of S. aureus 1) Coagulase causes clotting (maybe to protect against phagocytosis) (a) If a Staphylococcus produces coagulase it is considered S. aureus (diagnostic test) 2) Hyaluronidase digests hyaluronic acid (the “glue” holding cells together 3) Penicillinase and other enzymes to deactivate drugs are produced by many strains b. The exotoxins of S. aureus 1) Blood cell toxins (hemolysin and leukocidin) (a) α-toxin (but it produces beta-hemolysis!) 2) Intestinal toxins (enterotoxins) 3) Exfoliative toxin 4) Toxic shock syndrome toxin c. Epidemiology and pathogenesis of S. aureus 1) 20-60% of healthy adults have nasal carriage of S. aureus (a) It comes and goes rather than stay chronically (b) These people usually have no symptoms of infection (c) Poor hygiene, tissue injury, immunodeficiency, and a primary infection all predispose a person to an infection (d) MRSA (methicillin-resistant Staph aureus) increasing in non-hospital settings, and diffiulct tto rate 3. The scope of clinical Staphylococcal disease a. Localized cutaneous infections 1) Folliculitis is a mild, superficial inflammation of a hair follicle 2) Furuncles (boils) is when an infected hair follicle or gland progresses into a large, red, tender pustule 68 Cocci of Medical Importance (Ch 18) 3) A carbuncle is made of interconnected furuncles and can be quite deep and very painful (and can lead to systemic infection) 4) Impetigo (which can also be caused by Streptococcus pyogenes) is a localized skin infection that is not confined to follicles or glands (most common in newborrn b. Miscellaneous Systemic Infections 1) Systemic infections from Staph show a focal pattern (started from a localized infection) 2) Osteomyelitis is an infection within the bone tissue (a) Abscess can lead to death or breakage of bone tissue 3) Pneumonia from aspirating Staph can cause lung abscesses, fever, chest pain, and bloody sputum 4) Circulating bacteria can cause abscesses on various organs (e.g. kidneys, liver, spleen) and let off toxins into blood 5) Endocarditis can destroy heart valves 6) A severe form of meningitis occurs if S. aureus infects meninges c. Toxigenic Staphylococcal Disease 1) Food poisoning (a) Custards, creams, processed meats, chicken salad, etc. that have been handled and left at room temp for hours (beware family reunions!) (b) The enterotoxins do not alter the food’s taste or smell (c) Remember, exotoxins are heat stable, so even if the food is reheated after being out for hours the toxin will not be destroyed (d) When you get this food poisoning your body is reacting to the toxin, not to the bacteria (which get killed in your stomach). (e) Symptoms (cramping, nausea, vomiting, diarrhea) appear in 2-6 hours and recovery is generally within 24 hours (“24 hour flu” is a misnomer) 2) Scalded skin syndrome (a) Looks like child was dropped in hot water! (Watch out for child protective services!) (b) Same toxin as impetigo, but a systemic infection 3) Toxic shock syndrome (a) Identified in 1978 and traced to new ultra-absorbent tampons - It is NOT an allergic reaction to tampons—the tampons create a habitat for S. aureus which puts out exotoxins - Can cause fever, vomiting, rash, renal and liver problems, and sometimes death - Seen also from things other than tampons (such as gauze after nasal surgery) 4. Host Defenses Against S. aureus a. You have a well-developed resistance to staph infections 1) While several hundred thousand cells introduced into a cut will not generally cause an infection, a much smaller number of cells will cause an infection if there is a suture or other foreign body 2) Main defense is phagocytic response (neutrophils & macrophages) 5. The Other Staphylococci a. Coagulase-negative staphylococci 1) Basically any species other than S. aureus does not produce coagulase 69 Elementary Microbiology 2) Not nearly as pathogenic as S. aureus, but cause opportunistic and nosocomial infections in immunocompromised patients (especially if there is a shunt, catheter, or other foreign object) b. Staphylococcus epidermidis and S. saprophyticus: Don’t need to know these 6. Identification of Staphylococcus in Clinical Samples a. Specimens collected from pus, sputum, urine, and blood b. Gram-stain can show irregular clusters of gram-positive cocci, but cannot distinguish between species c. Test for catalase will distinguish Staphylococcus from Streptococcus d. Test for coagulase will identify S. aureus from other Staph e. Rapid multi-test systems are available to distinguish between Staph. species 7. Clinical concerns in Staphylococcal Infections 1) Staph are notorious for becoming resistant to new drugs (95% of S. aureus strains are now penicillin and ampicillin resistant). 2) MRSA accounts for 40% of Staph infections in hospitals 3) Now there is VRSA b. Treatment of Staph Infections 1) Drain and clean abscesses (a) Sometimes that is enough, but antibiotics generally given 2) Systemic infections require intensive, lengthy antibacterial therapy c. Prevention of Staph Infections 1) Minimize nosocomial infections by careful hygiene—handwashing, proper disposal of infectious dressings, careful attention to indwelling catheters and needles 2) Vaccines may be available soon for high-risk patients B. General Characteristics of the Streptococci and Related Genera 1. Introduction a. Streptococcus is a genus with many species that range from free-living in the environment to obligate pathogens in humans 1) Many live on or in domestic animals and can be shared between humans and their pets b. Streptococcus refers to long chains of cocci, but not always seen in this arrangement c. Non spore-forming, no flagella, but they do form capsules and slime layers d. Ferment sugars to produce lactic acid e. Does not form catalase (but Staph does) f. Quite sensitive to drying, heat, and disinfectants g. Can be very drug-resistant h. Characterized by a system developed by Rebecca Lancefield in 1930s or by what type of hemolysis they do 1) β-hemolysis is complete destruction of red blood cells 2) α-hemolysis is incomplete destruction of RBCs and turns the blood agar a greenish color (the “viridans” streptococci) 70 Cocci of Medical Importance (Ch 18) 2. β -Hemolytic Streptococci: Streptococcus Pyogenes 1) This is the main Strep. pathogen (a) Inhabits throat, nasopharynx, and sometimes skin (b) Causes a variety of disease conditions (c) We will focus mainly on this species b. Cell Surface Antigens and Virulence Factors 1) Don’t worry about this section other than knowing there are numerous surface antigens in S. pyogenes c. Major Extracellular Toxins 1) Streptolysins injure many cells and tissues 2) Pyogenic toxin causes bright red rash and fever (scarlet fever) (a) Only strains infected by a bacteriophage with the gene for this toxin can synthesize it d. Major Extracellular Enzymes: Ignore this section e. Epidemiology and Pathogenesis of S pyogenes 1) Rheumatic fever and puerperal sepsis (childbed fever) used to account for many deaths 2) 5-15% of humans are carriers who transmit the bacteria through close contact 3) The bacteria gain a hold when the host resistance is low (finals time, winter, sick already with a cold, etc) 4) Can give rise to a variety of systemic infections f. Skin Infections 1) Streptococcal impetigo (a) Highly contagious yellow crust (b) Schoolyard epidemic 2) Erysipelas (a) Similar to impetigo, but deeper (b) Means “red skin” and looks like a red mask 3) Necrotizing fasciitis (a) “Flesh-eating bacteria!” (b) Starts like impetigo but can kill large areas of tissue 4) “Strep throat” or, more technically, streptococcal pharyngitis (a) Redness, edema, tenderness (painful swallowing) (b) May multiply in the tonsils (c) Can lead to scarlet fever and/or rheumatic fever - This is why severe sore throats need to be taken seriously! - Several rapid diagnostic tests available to use with throat swabs—these rely on monoclonal antibodies and agglutination g. Systemic Infections 1) Scarlet fever: bright red, diffuse rash on face, trunk, inner arms & legs, and tongue 2) Shedding of skin after rash leaves h. Long-Term Complications of Group A Infections 1) Rheumatic fever is an inflammatory response in joints or heart (a) Thought to be caused by antibodies against Streptococcus cross-reacting with heart valves 71 Elementary Microbiology (b) May get this as a child but not have problems with valves until middle age. May need artificial valve 3. 4. 5. 6. Group B: Streptococcus agalactiae: Group D Enterococci and Groups C and G Streptococci: SKIP Laboratory Identification Techniques: SKIP (mentioned earlier) Treatment and Prevention of Streptococcal Infections a. Sensitive to penicillin and derivatives (injection or oral for 10 days) b. Prophylactic treatment for people with recurring strep throat c. Tonsillectomy not done frequently anymore d. If carriers are identified they should be kept from immunocompromised patients 7. α-Hemolytic Streptococci: The Viridans Group a. This is a group of many species found throughout the mouth, nasopharynx, genital tract, and skin b. Not highly invasive, but get into tissues following dental work 1) Blood-borne bacteria can go from mouth to heart valves, especially if the valves were previously injured by rheumatic fever or if person has a heart condition (a) Prophylactic antibiotics prior to dental work or surgery for such people 2) They form thick colonies and masses of them break off occasionally as emboli that cause problems similar to blood clot emboli (e.g. strokes, lung damage) 8. Bacteria in dental disease (still focusing on viridans streptococci) a. Streptococcus mutans causes plaque (thick slime layers) and dental caries in presence of sucrose b. Mouth is a complex microecosystem 1) Tongue, teeth, gums, palate, and cheeks all provide different habitats 2) About 400 species of aerobic, microaerophilic, and anaerobic bacteria live together in your mouth (a) Nice, warm, moist environment to which food is regularly added—what more could a bacteria hope for? c. Structure of teeth and associated tissues 1) Outer crown of hard, noncellular enamel made of calcium phosphate (a) The enamel cannot be replaced when lost 2) Under the crown is a layer of dentin, and deeper to that is the pulp cavity 3) The root is anchored to the socket by the periodontal ligament 4) The space surrounding the tooth is protected by gingival (gum) d. Dental caries 1) “The most common human disease” 2) Most commonly occurs in difficult-to-clean places, such as pits and fissures 3) A complex interaction of your diet, the bacteria and its metabolism, and your oral hygiene habits (a) Begins with biofilm formation on the enamel - Streptococcus is a good pioneer to colonize the empty surface 72 Cocci of Medical Importance (Ch 18) - The biofilm is called plaque (b) Several additional species will join the colony if plaque is allowed to stay - Streptococcus and Lactobacillus produce acids which, in heavy plaques, can erode the enamel - Usually stopped at this point with fillings (c) If it progresses to the dentin the tooth can be destroyed in little time (d) If it progresses to the pulp then you have a bad toothache and are at greater risk of losing the tooth e. Periodontal disease 1) By age 45 nearly 100% of the population has this to some degree 2) When plaque is not removed, it can calcify (calculus) 3) This pushes the gum away from the tooth and also irritates the gum, causing gingivitis (inflamed gums) (a) The new space is colonized by a variety of bacteria, particularly anaerobes (b) Your immune response results in further inflammation and tissue damage 4) If the condition progresses it is called periodontitis and can lead to loss of tooth due to loss of bone f. Factors in dental disease 1) Diets high in refined sugars (sucrose, glucose, and fructose) 2) Anatomical, physiological, and hereditary factors play a role (a) How hard your enamel is depends on genetics and environment (fluoride) (b) How many antibodies and how much lysozyme in your saliva makes a difference 3) Oral hygiene habits (a) Brushing and flossing to remove plaque (b) Regular dental visits to scrape away calcified plaque 4) Vaccine against Streptococcus mutans? 5) Inoculation with genetically modified bacteria? 9. Streptococcus pneumoniae: The Pneumococcus 1) Causes 60-70% of all bacterial pneumonias 2) Identifiable with Gram stains of sputum (diplocci) b. Epidemiology and Pathology of the Pneumococcus 1) Normal flora of nasopharynx in many people 2) Can get pneumonia by breathing in your own bacteria or someone else’s (a) Again, dependent on status of immune system c. The pathology of Pneumonia 1) Breath in a chunk of bacteria-laden mucus and have a weakened immune system (a) Normally your mucociliary escalator and phagocytes would take care of the problem 2) Overwhelming inflammatory response floods the lungs with fluid (a) These fluids may solidify, making things worse 3) Chills, shaking, rapid breathing, fever, chest pain, cyanosis, bloody sputum, abnormal breathing sounds are all signs to look for 4) Can lead to bacteremia and meningitis (a) Streptococcus pneumoniae causes meningitis and middle-ear infections (otitis media) in children (b) Vaccine now available against this 73 Elementary Microbiology 5) Laboratory cultivation and Diagnosis: SKIP 6) Treatment and Prevention of Pneumococcal Infections (a) Large daily doses of penicillin drugs or, if resistant, vancomycin or cephalosporins (b) Active immunity prevents recurring infections - Already mentioned vaccine C. The Family Neisseriaceae: Gram-Negative Cocci 1. Neisseria Gonorrhoeae: The Gonococcus 1) Looks like coffee beans 2) N. gonorrhoeae and N. meningitidis are two we will talk about (a) Gonorrhea known anciently—thought to be due to excessive semen leaking out! b. Factors Contributing to Gonococcal Pathogenicity 1) Pili allow attachment to mucus membranes and help prevent phagocytosis 2) Able to cleave IgA c. Epidemiology and Pathology of Gonorrhea 1) One of top 5 STDs, and a strictly human infection 2) Perhaps 2 million new cases annually in U.S., but many asymptomatic (a) That is why there is a reservoir d. Genital Gonorrhea in the Male 1) Urethritis, painful urination, yellowish discharge (pus, not semen!) (a) Beware the sailor with a feather in his cap! 2) Can spread to spermatic cord and cause infertility by scarring e. Genitourinary Gonorrhea in the Female 1) May be pus or bloody vaginal discharge and/or painful urination 2) Can spread to uterus and fallopian tubes (a) Salpingitis or Pelvic Inflammatory Disease (PID) - Fever, abdominal pain - Ectopic pregnancies or sterility (b) (PID can be caused by Chlamydia too) f. Extragenital Gonococcal Infections in Adults (a) Can infect rectum or throat in addition to genitals, depending on sexual practices (b) Also can infect eyes, including newborns g. Gonococcal Infections in Children 1) Neonatal gonorrheal ophthalmia 2) If gonorrhea found in children other than babies, this is strong evidence of sexual abuse by an infected adult h. Clinical Diagnosis and control of Gonococcal Infections 1) Gram-stain shows gram-negative diplococci inside of neutrophils 2) Any diagnoses must be reported to public health department (a) Sexual partners are traced and offered prophylactic antibiotic therapy 2. Neisseria meningitidis: The Meningococcus 1) Epidemic meningitis b. Epidemiology and Pathogenesis of Meningococcal Disease 74 Cocci of Medical Importance (Ch 18) 1) 3-30% of population carries it in the nasopharynx 2) Close living conditions spreads it (college dorm?) 3) Seen in infants, children, and young adults 4) Must enter bloodstream and then the meninges c. Clinical Diagnosis of Meningococcal Disease 1) Suspicion of meningitis is a medical emergency! (a) Watch for sudden fever, chills, delirium, headache, stiff neck, convulsions, vomiting (b) Subcutaneous hemorrhages anywhere on body (blotches) 2) Can become a fulminant (explosive) infection with high mortality rate (a) Death can occur within hours d. Immunity, Treatment, and Prevention of Meningococcal Infection 1) Even when treated mortality is 15%, so chemotherapy must be started quickly 2) Preventative treatment may be given to people who were in close contact with the patient 3. Differentiating Pathogenic from Nonpathogenic Neisseria a. There are nonpathogenic Neisseria that can live in the body b. There are rapid tests available 4. Other Gram-Negative Cocci and Coccobacilli: SKIP 75 Elementary Microbiology Unit 14. Gram-Positive Bacilli of Medical Importance (Ch 19) A. Medically Important Gram-positive Bacilli 1. There are more rod-shaped bacteria (bacilli) that cause disease than there are cocci. 2. Note: the organization of these bacilli is based on a traditional classification of gram-stain, shape, oxygen utilization, etc. More recent work has shown that these groups often do not reflect true lineages (in other words, the most related species are not necessarily grouped together). However, this is a convenient system of classification for our purposes. 3. The genera we will discuss today include Bacillus, Clostridium, Listeria, Corynebacterium, Propionibacterium, Mycobacterium, but not Actinomyces. Some of these genera have several species of interest to us. B. Gram-Positive Spore-forming Bacilli 1. General Characteristics of the Genus Bacillus a. Endospore-forming b. Gram-positive c. Motile d. Rod-shaped e. Aerobic f. Catalase-positive g. Not fastidious (most are saprobes) h. Common in soils 1) Spores dispersed in dust 2. Bacillus anthracis and Anthrax a. Anthrax has been known for centuries as a zoonotic disease (a disease transmissible between animals and humans) b. This is the disease and bacteria that Robert Koch used to form his postulates 1) Louis Pasteur also demonstrated the benefits of vaccination by injecting live anthrax into 25 sheep that had been vaccinated and 25 sheep that had not been vaccinated c. 89 known strains, with widely varying degrees of virulence 1) The spores used in the 2001 anthrax attacks were high-quality spores of a very virulent strain d. The results of infection depend largely upon the portal of entry 1) Cutaneous anthrax is the most common form of the disease, and the least dangerous (a) Spores enter the skin through small cuts (b) A large black lesion forms (the word “anthrax” is Greek for “coal” and refers to this dark lesion) (c) Many people are exposed to the spores but never develop the disease 76 Gram-Positive Bacilli of Medical Importance (Ch 19) 2) Pulmonary anthrax (a) Textile workers (and senators who get bad mail) are at primary risk of inhaling the endospores (b) The bacilli grow in the lungs and release exotoxins into the blood stream - These exotoxins cause clotting (thrombosis) in capillaries and shock (drop in blood pressure—in this case because capillaries are leaking fluids) (c) High mortality rate (about 95%), and death can occur within a few hours after infection - A lethal dose is considered 10,000-20,000 spores - Antibiotic therapy must be started within a day of exposure, preferably before any symptoms appear (d) Story of how the British and Americans nearly wiped out all humans for hundreds of square miles in Germany (e) Story of secret Army tests on American citizens with Bacillus subtilis 3) Gastrointestinal anthrax (a) From eating contaminated meat or otherwise ingesting spores (b) This also has a high mortality rate (typically 25-65%) (c) Severe diarrhea and vomiting of blood are some symptoms e. Methods of Anthrax Control 1) Active cases treated with penicillin or other antibiotics 2) Vaccine given to military troops and some livestock (a) Vaccine requires 6 inoculations and yearly boosters 3) Gas-sterilization of imported animal hides f. Other Bacillus Species Involved in Human Disease 1) Bacillus cereus produces a form of food poisoning when the spores contaminate grain (mostly rice) 2) Other Bacillus species cause disease in immunosuppressed patients (a) The spores are all over the place, so luckily we are usually not susceptible to infection 3. The Genus Clostridium a. Traits of the genus 1) Anaerobic and catalase-negative 2) Over 120 species in the genus 3) Common in soil, sewage, organic debris, and as commensals in humans and animals 4) Infection occurs when spores are introduced into injured skin 5) Exotoxins of some species cause disease (botulism, tetanus, gas gangrene) 6) We will discuss four species: (a) (b) (c) (d) Clostridium perfringens (gas gangrene; gastroenteritis) C. difficile (antibiotic-associated colitis) C. tetani (tetanus) C. botulinum (botulism) b. The Role of Clostridia in Infection and Disease 1) Gas Gangrene (a) Infection requires dead tissue and exposure to spores (b) Infection spreads because the bacteria releases exotoxins that kill adjacent tissue (c) Inevitably fatal if treatment is not started soon 77 Elementary Microbiology (d) Treatment and Prevention of Gangrene - Rigorous cleansing and surgical repair of deep wounds, decubitus ulcers, open fractures, and infected incisions - Debridement of diseased tissue; amputation as needed - Broad-spectrum antibiotic or penicillin - Hyperbaric oxygen therapy c. Antibiotic-Associated Colitis 1) When a person takes antibiotics, C. difficile, which is normally present in low numbers, may grow out of control (a superinfection) 2) It causes serious diarrhea, and exotoxins can cause pseudomembranous colitis (a) The colon may be perforated, causing peritonitis (b) A colectomy (removal of colon) is sometimes required 3) Patients release large numbers of spores in their stool (a) This is a common nosocomial infection 4) Prevention through taking probiotics; treatment through taking vancomycin or metronidazole d. Tetanus or Lockjaw 1) The Course of Infection and disease (a) Spores must be introduced into injured tissue where there is an anaerobic environment (b) Growing cells release tetanospasmin, a potent neurotoxin - The toxin inhibits release of a neurotransmitter that normally inhibits muscular contraction - The result is uncontrollable muscle contraction (c) First symptoms are clenching of jaw (lockjaw), followed by extreme arching of the back, flexion of arms, and extension of legs - All muscles are involved, but some muscle groups are stronger than others (d) Death is due to paralysis of respiratory muscles - 10-70% mortality depending on speed of treatment and severity of infection 2) Treatment and Prevention of Tetanus (a) Get vaccinated against tetanus toxin (b) If a person has tetanus they get passive artificial immunity (receive human tetanus immunoglobulin) - The antitoxin cannot counteract any toxin that has already bound e. Botulinum Food Poisoning 1) C. botulinum produces botulinum toxin (a) This is a neurotoxin that interferes with release of acetylcholine (b) It causes flaccid paralysis, and death is due to asphyxiation 2) The Route of Pathogenesis (a) Most commonly spores are found on foods that were not properly processed (e.g. home-canned vegetables) (b) They germinate in anaerobic environment and release the exotoxins into the food (c) If the food is consumed (even tasted) without heating and destroying the toxin then a person is poisoned, with symptoms in 12-72 hours 78 Gram-Positive Bacilli of Medical Importance (Ch 19) - Important to note that it is the toxin, not living bacteria, that causes the disease (d) Mechanical respirators have reduced the death rate to about 10% 3) Infant and Wound Botulism (a) In these cases the bacteria are actually living and dividing within the person (an infection rather than intoxication) - Immature digestive tract allows spores to germinate and release the neurotoxin - “Floppy baby syndrome” (b) This is why we don’t give infants raw honey (although the spores can be found in dust or soil too) 4) Treatment and prevention of Botulism (a) Treatment with antitoxin from horses, mechanical respirator, and cardiac support (b) Proper home-canning techniques for prevention 5) Cosmetic uses (a) Botox for looks, for muscle spasms, and for migraines (b) Treatment wears off in about 4 months C. Gram-positive Regular Non-Spore-Forming Bacilli 1. An Emerging Food-Borne Pathogen: Listeria monocytogenes a. Epidemiology and Pathology of Listeriosis b. Diagnosis and Control of Listeriosis 2. Erysipelothrix Rhusiopathiae: A Zoonotic Pathogen a. Epidemiology, Pathogenesis, and Control D. Gram-Positive Irregular Non-Spore-Forming Bacilli 1. Corynebacterium diphtheriae a. Epidemiology of Diphtheria b. Pathology of Diphtheria 1) The toxin is produced only after the bacteria is infected with a bacteriophage (virus) that gives it the gene for toxin production 2) The toxin produces inflammation, sore throat, fever, nausea, enlarged lymph nodes. Once into the bloodstream it can affect the heart and nerves 3) A pseudomembrane can develop, which can block the airway and cause asphyxiation c. Diagnostic Methods for the Corynebacteria d. Treatment and Prevention of Diphtheria 1) Vaccination! 2) Treatment with antitoxin, heart medication, tracheostomy, and antibiotics 2. The Genus Propionibacterium E. Mycobacteria: Acid-Fast Bacilli 1. Mycobacterium tuberculosis: The tubercle Bacillus 79 Elementary Microbiology a. Epidemiology and Transmission of Tuberculosis b. The course of Infection and disease 1) Primary Tuberculosis 2) Secondary Reactivation Tuberculosis 3) Extrapulmonary Tuberculosis c. Clinical Methods of Detecting Tuberculosis 1) Tuberculin Sensitivity and Testing 2) Roentgenography and Tuberculosis 3) Acid-Fast Staining 4) Laboratory cultivation and Diagnosis d. Management of Tuberculosis e. Prevention and Control of Tuberculosis 2. Mycobacterium leprae: The Leprosy Bacillus a. Epidemiology and Transmission of Leprosy b. The course of Infection and Disease 1) Tuberculoid leprosy 2) Lepromatous Leprosy c. Diagnosing Leprosy d. Treatment and Prevention of Leprosy 3. Infections by Non-Tuberculous Mycobacteria (NTM) a. Disseminated Mycobacterial Infections in AIDS 1) Non-tuberculous Lung Disease 2) Skin, Lymph, Node, and Wound Infections 80 Gram-Negative Bacilli of Medical Importance (Ch 20) Unit 15. Gram-Negative Bacilli of Medical Importance (Ch 20) A. Endotoxic shock 1. Gram-negative bacterial cell wall a. Lipopolysaccharide (LPS) 1) pyrogenic and immune-stimulating 2) considered an endotoxin 3) released as side-effect of cell death 2. sepsis leads to large release of LPS a. macrophages release cytokines 1) cytokines cause fever, low blood pressure (a) organ & tissue damage 3. symptoms a. nausea, tachycardia, low blood pressure, fever b. can progress to respiratory failure, coma, heart failure, and death 4. antibiotic treatment can compound effects a. causes release of more LPS b. Drug companies developing drugs against endotoxins 5. Treatment for endotoxins? B. Pseudomonas aeruginosa 1. Pseudomonads are large group, mainly free living a. adaptable: soil, seawater, fresh water, plants, animals, etc 1) Can get energy from miniscule amounts of nutrient 2) single flagellum (monotrichous) 3) Useful in bioremediation (a) Can degrade oils, pesticides, etc b. common in soil, water, on plants, and in some people c. Resistant to soaps, disinfectants, drugs, drying, and heating 1) contaminates soap dishes, ventilators, IV solutions, etc 2) contaminates contact lens solutions d. skin rashes, UTIs, otitis externa e. corneal ulcers f. Unlikely to infect healthy host with intact skin g. Likely to infect someone with burns, cuts, cystic fibrosis, or indwelling catheter h. grapelike odor, bluish pus i. pigment fluoresces under UV light j. multidrug resistance makes sensitivity testing imperative k. Vaccine being tested for cystic fibrosis patients 81 Elementary Microbiology C. Brucella 1. brucellosis (undulant fever) a. a zoonosis (transmitted from animal to human—not human to human) 1) contact with blood, urine, unpasteurized milk of infected animals b. common disease of bison and elk 1) causes abortion in cattle c. fluctuating pattern of fever, chills, sweating, aches, fatigue 1) “Undulant fever” (a) Can last for weeks or months D. Skip tularemia E. Bordetella pertussis 1. Causes whooping cough 2. binds to ciliated epithelial cells in trachea a. releases toxins that destroy ciliated cells 3. buildup of mucus and blockage of airway lead to coughing a. catarrhal stage: nasal drainage, congestion, sneezing, some coughing b. paroxysmal stage: includes recurrent fits of several abrupt hacking coughs followed by a "whoop" 1) life-threatening in babies and little children 4. Transmission through direct contact with droplets or inhalation of aerosols a. Common in older children and adults but generally mild in them b. Vaccination is standard control measure 1) DTaP vaccine (a) accelular pertussis: contains toxoid and antigens 2) The vaccination does not give lasting protection c. upsurge in infections due to worry over the vaccine 1) correlation to autism?? (a) The old, whole-cell vaccine contained thimerosal, which contains mercury (b) The new acellular vaccine does not contain thimerosal F. Legionella 1. Named after the American Legion a. Party of old guys and 200 came down with pneumonia 1) The bacteria was in the hotel air-conditioning system 2) 29 of the legionnaires died during this 1976 outbreak 3) This was the first time the bacteria was known to cause disease (it had been described in the 1940’s) b. Males over 50 more susceptible 2. rising fever, cough, diarrhea a. progresses to impaired respiration and organ failure 3. Ironically, it is very difficult to grow in the lab 82 Gram-Negative Bacilli of Medical Importance (Ch 20) a. selective charcoal-yeast agar b. close association with amebas 4. Some cases from vegetable sprayers, hospital ventilation systems, etc a. nosocomial infections for susceptible patients b. NOT transmissible between people G. Enterobacteriaceae 1. Big family of gram-negative bacteria a. all are small, non-spore forming rods b. Also they ferment glucose and are oxidase-negative c. many biochemical tests needed to identify species 1) e.g. MacConkey agar inhibits gram-positive bacteria (a) MacConkey agar is the first of many steps (b) it allows differentiation between lactose-fermenters (red colonies) and nonlactose fermentners 2) The enterotube for faster, easier identification d. very common cause of noscocomial infections 1) Important pathogens are Salmonella, Shigella, and Escherichia 2) Opportunists include E. coli, Klebsiella, Proteus, Enterobacter, Serratia, and Citrobacter e. Known for sharing virulence factors 1) remember the sex pilus and transfer of plasmids? 2) E. coli O157:H7 produces a Shigella toxin that it obtained on a plasmid f. Found in many habitats, including human large intestines H. Diarrheal disease 1. Members of Enterobacteriaceae are most common cause of diarrhea 2. 4 billion infections worldwide yearly a. 3 million deaths annually, largely in children b. Leading cause of childhood death worldwide 1) In many countries 15-25% of children die by age 5 due to diarrhea 2) Mixing formula with contaminated water inoculates babies with pathogens (a) mothers in 3rd world countries urged to nurse instead--much safer c. diarrhea a symptom of gastroenteritis 1) usually NOT caused by normal flora 2) enterotoxins cause lining of intestines to secrete fluids more pain, blood in stool, ulceration of lining 3) in more severe forms the bacteria invades the lining of the intestine 4) severe dehydration can result (a) especially dangerous to infants d. 18% of human deaths worldwide caused by diarrhea and dehydration (note: we need to check this figure) e. Oral rehydration therapy with electrolyte drink saves lives 1) pedialyte, or make your own (pg 642) 83 Elementary Microbiology I. Escherichia coli: most important member of Enterobacteriaceae 1. Well known for living in colon, but not the most common inhabitant 2. You have a lot more Bacteriodes, Lactobacillus, and Bifidobacterium a. However, the others are strict anaerobes, so have more difficulty causing infections 3. aerobic and non-fastidious 4. 150 strains, many of them not infectious (and many are) a. E. coli O157:H7 as an emerging disease 1) H, K, and O are different antigens (a) different variations of these antigens are given numbers 2) The strain developed in cattle intestines and began causing infections in 1982 (a) This strain acquired a virulence plasmid and it makes Shigella toxin 3) Associated with fast-food restaurants and meat recalls 4) This is why there are now warning labels on meat products 5) About 10% of patients with this strain of E. coli develop hemolytic uremic syndrome (a) This can cause kidney failure (b) Antibiotics do not seem to help—avoiding infection is about the only option 6) Most common agent of nosocomial infections (a) Causes 50-80% of UTIs (b) Can cause neonatal meningitis (c) wound infection and septicemia 7) most common cause of traveler's diarrhea (not just referring to O157:H7, but to many strains) (a) Pepto-Bismol counteracts the enterotoxin 8) Used as an indicator of fecal contamination (a) If E. coli is present, then Salmonella or other enteric pathogens may be present too (b) There are tests for fast, easy detection of E. coli and it survives outside the human host well J. Other coliforms 1. Klebsiella a. Frequent cause of bacterial pneumonia b. Also common in UTIs and wound infections 2. Serratia a. Intense red pigment when grown in lab b. Can infect compromised hosts 1) Used to think it was harmless and used it as a "tracer" 3. Let's ignore Enterobacter and Citrobacter 84 Gram-Negative Bacilli of Medical Importance (Ch 20) K. Proteus 1. These swarm on the surface of moist agar and make a cool pattern 2. While common, it is usually harmless (except in immunocompromised patients) 3. Ignore Morganella and Providencia L. Salmonella typhi 1. There are many species of Salmonella a. Difficult to distinguish--specimens often sent to CDC for I.D. 2. Typhoid fever a. Not the same as typhus 1) Typhus is spread by a louse b. About 50-100 cases annually in the U.S. 1) About half of these are imported from abroad 2) Millions of cases and 25,000 deaths annually worldwide c. fecal-oral route (contaminated food & water) d. Humans are the ONLY host 1) reservoir of asymptomatic carriers e. Some people carry the bacteria for life in their gall bladder 1) "Typhoid Mary" (a) May require removing the gallbladder f. S. typhi infects mucosa of small intestine 1) Fever, diarrhea, abdominal pain are symptoms 2) Can lead to ulcerations and peritonitis 3) Moves through lymph system to liver and spleen 4) New vaccines provide temporary protection for travelers & military 3. Salmonella food poisoning a. Several species of Salmonella can cause this b. About 40,000-50,000 cases reported per year in U.S. 1) CDC estimates true number is likely 1.5 million cases c. Comes from normal flora of cattle, poultry, rodents, and reptiles 1) Wash hands after playing with pets 2) Cook meat thoroughly as preventative 3) Concern over raw eggs (a) 1 in 3 chickens is a carrier (b) Preempt is a probiotic for poultry 4) Food contamination with rodent feces d. Drug resistance rising due to antibiotics in animal feed e. Vomiting and diarrhea for 2-5 days is typical M. Shigella 1. Causes bad diarrhea (dysentery) a. watery stool with mucus and blood b. Various species cause the disease 85 Elementary Microbiology 2. Poor sanitation, malnutrition, and crowding foster outbreaks a. Invades large intestine, not small intestine (Salmonella) b. Also not as invasive as Salmonella 1) No perforations of intestinal wall c. Diagnosis difficult due to many other possible causes of bloody diarrhea N. Yersinia pestis 1. Plague a. 100 MILLION people killed in sixth century! b. Y. pestis is a small bacteria spread by flea-infested rats 1) It produces coagulase, which clots blood and blocks esophagus of fleas 2) Hungry fleas look for new hosts 3) Several vertebrate hosts, and the flea as a vector c. In bubonic plague the bacteria multiplies in the flea bite 1) It enters the lymphatic system and is stopped at lymph nodes (a) The infection causes swelling and necrosis of the node: a bubo (b) May progress to septicemic plague - Skin darkens due to subcutaneous hemorrhaging - "Black death" (c) Death can occur within 2-4 days of symptoms (d) 90-95% survival rate with antibiotic treatment d. Pneumonic plague enters the lungs and is highly contagious 1) Without treatment it is invariably fatal 2) Death can occur in only 2-6 hours 2. Haemophilus a. H. influenzae 1) For 40 years we mistakenly thought it caused the flu 2) Actually causes bacterial meningitis (a) Most common form of meningitis in 3-5 month old babies 3) Also causes epiglottitis 4) Also causes otitis media, sinusitis, pneumonia b. H. aegyptius 1) Cause of conjunctivitis (pinkeye) 2) Spread through contaminated fingers as well as gnats and flies 3) Can ignore other Haemophilus species 86 Miscellaneous Bacterial Agents of Disease (Ch 21) Unit 16. Miscellaneous Bacterial Agents of Disease (Ch 21) A. Introduction 1. In the past few chapters we have discussed bacteria of medical significance that were organized as either gram-positive or gram-negative and either cocci or bacilli 2. This chapter lumps together “everything else,” namely spirochetes (e.g. Treponema pallidum), curviform bacteria (e.g. Vibrio), obligate intracellular parasites (Rickettsia, Chlamydia), and Mycoplasma (different than Mycobacteria) 3. The last section of the chapter discusses the complex ecosystem that is your mouth, and the mixed colonies that contribute to plaque, dental caries, gingivitis, periodontitis, and tooth loss B. The Spirochetes 1. Reminder of their form 2. Treponema pallidum and syphilis a. All members of the genus Treponema are strict parasites (and if you want to cultivate them in the lab you need to do so in live cells) b. The name means pale turning thread 1) The “pallidum” refers to how poorly it stains 2) Do you remember what type of microscopy is used to detect this bacteria? c. There are different subspecies of T. pallidum that cause different diseases, but our focus will be on T. pallidum pallidum and the disease it causes—syphilis 1) Humans are the only natural host of this bacteria (a) A very fastidious bacteria (b) Also very sensitive—cannot survive long outside the host - Easily destroyed by heat, drying, disinfectants, soap, high oxygen, and pH changes - Survives a few minutes to hours in bodily secretions - Survives 36 hours in stored blood 2) Mainly spread through sexual contact (a) Risk of infection from an infected sexual partner is 12-30% (b) Another mode of transmission is passage to the fetus in utero (c) Medical personnel sometimes get syphilis from contact with bodily fluids 3) Most cases concentrated in large metropolitan areas among prostitutes, their contacts, and IV drug users (a) Remember Paul Ehrlich and Compound 606 (Salvarsan)? (b) Worldwide, particularly in Africa and Asia, it is a more widespread problem - Coinfection with the AIDS virus is a particularly bad combination and rapidly fatal d. Infection and Incubation 1) When T. pallidum contacts mucus membranes or abraded skin it binds via its hooked tip 87 Elementary Microbiology (a) Using human volunteers (??), it was established that 57 organisms were needed to cause infection 2) At the initial point of contact the bacteria multiplies, penetrates the capillaries, and enters circulation (a) The body is now a large incubation chamber for this bacteria and virtually any tissue is a potential target (b) It produces no toxins and does not kill cells directly (c) Despite action of phagocytes and antitreponemal antibodies, the body’s immune system is unable to contain it (d) During this incubation stage there are no symptoms (this lasts about 3 weeks) e. The infection proceeds in three distinct stages (primary, secondary, and tertiary), with latent periods of varying duration in between 1) Primary Stage (lasts 2-6 weeks) (a) Chancre appears at inoculation site - Begins as small, hard, red bump - Breaks down and leaves a shallow crater with firm margins - Usually on genitals, but 20% elsewhere (e.g. lips, oral cavity, nipples, fingers, rectum) - The chancre is normally painless and sometimes goes unnoticed (b) Intense treponemal activity in body as the bacteria moves through the circulatory system (c) Ends by chancre disappearing (d) During this stage or the primary latency is when it is easiest to treat the infection (with penicillins) 2) Primary latency (lasts 2-8 weeks) (a) Chancre heals, leaving little scarring (b) Symptoms go away, but the bacteria is still busy 3) Secondary Stage (lasts weeks to months) (a) Fever, headache, and sore throat followed by breaking out in a full-body rash (including palms and soles) (b) Lesions appear on skin and mucus membranes - Beware—these are full of spirochetes! - This is the stage when the patient is the most infectious (c) Bones, hair follicles, liver, joints, eyes, and brain can all be damaged during this stage (d) Treatment is more difficult and dosages of penicillin are double than what they would have been if treated during primary stage 4) Secondary latency (lasts 6 months up to 20 years or more) 5) Tertiary Stage (a) This stage is rarely seen today thanks to effectiveness of antibiotic treatment (b) Oddly enough, the disease is largely noncommunicable during this stage (c) Many body systems are damaged (in potentially fatal ways) by this stage - Aortic wall is weakened and may rupture - Gummas (soft tumorous mass) develop in tissues such as liver, skin, and bone - Brain, cranial nerves, and spinal cord may be affected in diverse ways f. Congenital syphilis (passage through placenta) 1) Can occur in any trimester 2) Fetal growth and development are impaired to varying degrees 88 Miscellaneous Bacterial Agents of Disease (Ch 21) 3) Early symptoms in the newborn include profuse nasal discharge, bone deformation, and nervous system problems 4) Hutchinson’s teeth (notched, barrel-shaped incisors) is another symptom g. Clinical and lab diagnosis 1) The chancre and secondary lesions can look like other bacterial, fungal, or parasitic infections (or allergic reactions) 2) Often there is another STD, such as gonorrhea that is present and complicates diagnosis 3) Darkfield microscopy of serous fluid from a suspected lesion gives a quick diagnosis (a) A single negative test is not enough, since patient may have removed bacteria while washing 4) Immunofluorescence staining with monoclonal antibodies also gives rapid diagnosis 5) If microscopy tests are negative, serological tests looking for presence of antibodies can be diagnostic (but false positive results are possible) (a) This is tested in premarital blood tests (b) This is suggested for people in high risk groups (c) If a patient tests positive, there are more specific tests that can verify an active infection h. Treatment 1) Penicillin the wonder drug (a) Other antibiotics are less effective and used only when there is an allergy to penicillin (b) Important to complete the course of antibiotics (at least 7 days) 2) Health personnel must question patient, trace their sexual contacts, and give prophylactic penicillin to all at-risk individuals 3) Condoms provide good protection 4) Vaccine may be available in the future i. Bejel, Yaws, and Pinta 1) These are diseases caused by other species of Treponema 2) They are not spread sexually, but mainly through direct contact 3) Symptoms are similar to syphilis, as is treatment 4) You will not be tested on any of these 3. Leptospira and leptospirosis a. Leptospirosis is a zoonosis 1) Mainly spread through association with wild animals such as rodents, skunks, raccoons, and foxes, but sometimes spread by horses, dogs, cattle, and pigs (a) The bacteria are shed in urine and can survive several months in soil or urine (b) Infection is through contact with urine or urine-contaminated soil or water - Infection enters through skin abrasions or mucus membranes 2) Mainly a tropical disease, but 50-60 cases annually in U.S. 3) Sudden high fever, chills, headache, vomiting are early symptoms 4) Damage to kidney, liver, brain, and eyes if left untreated b. Don’t swim in cattle ponds and wear shoes in the jungle 4. Borrelia and Lyme disease 89 Elementary Microbiology a. Borrelia is a spirochete that causes transmitted by ticks (and sometimes lice) b. A couple species of Borrelia causes relapsing fever due to changing surface antigens c. Much more common in the U.S. is Lyme disease 1) Mainly in northeastern U.S., but becoming more common in the Midwest and the West (a) Hikers, backpackers, and people living nears forested areas are at highest risk (b) About 20,000 cases per year 2) The tick feeds on mice as a nymph and on deer as an adult, so there are lots of natural reservoirs of the bacteria (a) The tick becomes infected with Borrelia from the mice (b) Infected nymphs then feed on humans 3) Bull’s-eye rash at tick bite is a common symptom (a) Other symptoms include fever, headache, stiff neck, and dizziness (b) If left untreated there can be damage to cardiac or nervous systems, as well as a crippling form of arthritis 4) Protective clothing, insect repellant, and searching for ticks after outdoor activities are recommended 5) Dogs can get this too, and there is a vaccine for them C. Curviform bacteria of medical importance 1. Vibrio cholerae and cholera a. This disease has caused massive epidemics throughout history 1) Millions of infections annually on a worldwide basis (particularly Asia & Africa) b. Vibrio cholerae reservoirs are asymptomatic carriers, plus it survives a long time in the environment 1) Basic fecal-oral route of contamination 2) Outbreaks are sometimes caused by cargo ships taking in ballast water at one port and emptying it in other ports around the world 3) Sometimes isolated in shellfish c. Infection through contaminated food or water 1) Large infectious dose needed (108 cells) because stomach acid kills most of them 2) Once into the small intestine the bacteria penetrate the mucus layer with their flagella (a) They stick to the microvilli, but do not invade the cells 3) Produce an enterotoxin that disrupts the normal physiology of intestinal cells (a) The cells begin to shed large amounts of electrolytes (chloride and bicarbonate ions) - Profuse water loss from the cells - In healthy adults most cases of the disease self-limiting but in children or weakened individuals it can be a rapid, violent, fatal disease (b) Copious watery diarrhea (rice-water stool) - Can lose one liter per hour of fluids 90 Miscellaneous Bacterial Agents of Disease (Ch 21) (c) Diarrhea results in loss of blood volume, acidosis (from bicarbonate loss), and potassium depletion (d) Other symptoms include muscle cramps, severe thirst, flaccid skin, sunken eyes, hypotension, tachycardia, and cyanosis - Left untreated there is general system failure due to shock - Death can occur in less than 48 hours, with mortality rates of nearly 55% 4) Diagnosis and treatment (a) During an epidemic the clinical evidence is sufficient to make a diagnosis (b) You can also see rapidly moving curved cells with dark-field microscopy of a stool sample (c) Treatment is to replace water and electrolytes - See Medical Microfile on Oral Rehydration Therapy on page 642 - IV therapy needed for unconscious patients or patients with severe dehydration - Oral antibiotics stop the diarrhea in about 48 hours (d) Prevention is proper sewage treatment and water purification (e) Vaccine is available (remember the tsunami?) 2. Helicobacter pylori and peptic ulcers a. This bacteria thrives in the acidic environment of the stomach b. 90% of stomach and duodenal ulcers are caused by H. pylori 1) Not detected until 1979, but it is common in the population (a) 25% of healthy middle-age adults carry this (b) 60% of adults over 60 have it (c) Transmission is probably oral-oral and fecal-oral - Might also be a zoonosis from cats and dogs (d) Usually carried asymptomatically 2) It bores through the mucus and attaches to cells (a) People with type O blood are twice as likely to get ulcers from this bacteria 3) It produces alkaline compounds to neutralize the stomach acid around it (a) It converts urea into ammonium and bicarbonate - Because of this, you can test for its presence by swallowing radioactively-labeled urea - If H. pylori is present then you will exhale radioactive CO2, hence diagnosis can be made with a breath test - It can also be isolated from stomach biopsies c. Appears to be a cofactor in stomach cancer d. Treatment is 2-4 weeks of clarithromycin to eliminate the infection 3. Campylobacter jejuni and gastroenteritis a. Recently considered one of the most important causes of gastroenteritis worldwide b. Transmitted through contaminated food and drink c. Symptoms are similar to cholera, as is treatment (oral rehydration therapy) D. Medically important bacteria of unique morphology and biology 1. Rickettsia prowazekii and epidemic typhus a. War, poverty, and famine are usually accompanied by this louse-borne typhus 1) Note: unrelated to typhoid fever, which is caused by Salmonella typhi 91 Elementary Microbiology 2) All Rickettsia are obligate parasites b. Named after the two men (Howard Ricketts and Stanislas von Prowazek) that discovered the vector and the bacteria, and who both died of the disease c. Humans are the only host of the louse and the only reservoir of the bacteria d. Infection spreads when an infected louse defecates into a bite or other break in the skin. 1) Infection of eye or respiratory tract can also occur from louse feces in dust, but this is uncommon e. Crowded, unsanitary conditions spread lice 1) Disease is uncommon in developed nations f. Disease manifestation 1) 10-14 days of incubation followed by high fever, chills, frontal headache, and muscle pain 2) Within 7 days a generalized rash starting on trunk and spreading to extremities 3) Personality changes may occur (the word typhus refers to this) 4) Mortality lowest in children, highest in people over 50 (40-60% mortality in this group) g. A related bacteria spread by rat fleas causes a form of typhus that shows up in people who work in rat-infested industrial areas h. Prevention by eliminating lice. A vaccine is also available 2. Rickettsia rickettsii and Rocky Mountain spotted fever a. Despite the name it occurs more frequently in the Southeast and East than in the western U.S. (only 3 cases in Utah over a 4-year period) b. Spread by wood ticks and dog ticks c. Symptoms include sustained fever, chills, headache, and muscle pain and a distinctive spotted rash (hence the name) 1) Lesions form from rash, and these may merge and become necrotic 2) May progress to hypotension, hemorrhage, delirium, convulsions, and coma 3) 20% fatal untreated; 5-10% fatality when treated d. If it is suspected then antimicrobic therapy is started immediately, with tetracycline as the drug of choice e. Preventive measures are similar to those for Lyme disease, plus a new vaccine is available if you are at high risk 3. Chlamydia trachomatis and eye disease a. Chlamydia is an obligate intracellular parasite, similar to Rickettsia b. An ancient disease and a major cause of blindness 1) Several million cases worldwide each year 2) Transmission by contaminated fingers, flies, and fomites 3) Damage to the cornea takes a while, and early treatment prevents any complications 4) Newborns can acquire this as they pass through the birth canal (a) Causes conjunctivitis (b) 100,000 cases annually in U.S. 92 Miscellaneous Bacterial Agents of Disease (Ch 21) (c) Another reason for erythromycin in eyes of newborns (do you recall the other reason?) 4. Chlamydia trachomatis as an STD a. The bacteria is carried in reproductive tract of about 10% of all people (and much higher rates among promiscuous people) 1) Women are asymptomatic 70% of time, but men only 10% of time 2) Causes urethritis in males, similar to gonorrhea 3) Can cause salpingitis (pelvic inflammatory disease) in women, contributing to infertility 4) Millions of cases (mostly unreported) in the U.S. each year (a) It is difficult to detect (b) Due to how common it is and how difficult to detect, preventative measures (e.g. condom) are recommended 5) A particularly virulent strain of the bacteria causes buboes to form 6) Often accompanies other STDs and makes one mores susceptible to these 5. Mycoplasma pneumoniae and atypical pneumonia a. Mycoplasma is unique in that it lacks a cell wall, plus it is tiny 1) Lack of cell wall results in extreme polymorphism 2) Considered the smallest self-replicating organism b. M. pneumoniae causes atypical pneumonia, sometimes nicknamed “walking pneumonia” due to lack of acute symptoms 1) Diagnosis is difficult, and largely a product of ruling out other causative agents 2) Only 3-10% of those exposed become infected, and fatalities are rare E. Bacteria in dental disease 1. Covered this in Chapter 18 sufficiently, I think 93 Elementary Microbiology Unit 17. Introduction to Viruses (Ch 6) A. Introduction & history 1. Viruses have ravaged humans throughout time 2. We have had some remarkable success in controlling viruses a. Small pox b. Polio 3. New viruses continually emerging a. HIV b. Ebola c. SARS d. Bird flu e. West Nile virus (old, but new for us) 4. Viruses are much too small to see with a light microscope a. Even though they could not be seen, Louis Pasteur hypothesized that there were “living things” smaller than bacteria causing small pox, rabies, etc. 1) He proposed the name “virus” (Latin for “poison”) b. Tobacco mosaic virus was the first to be shown to be so small that it would pass through a ceramic filter c. Most viruses require an electron microscope to see them B. Viruses in the biological spectrum 1. For every known living thing (be it bacteria, plant, human, nematode, grasshopper, whale, turkey, . . . ) there is at least one virus that infects it 2. Should viruses be considered “alive?” Why or why not? a. They are unable to exist independent of the host cell b. Even though they lack cellular machinery, they have the means to direct cell processes c. They evolve rapidly to changing conditions d. Most people call them “infectious particles” rather than “organisms” and “active or inactive” rather than “alive or dead” 3. How can something so small and simple cause disease and death? C. General structure of viruses 1. Viruses are tiny! a. >2,000 bacteriophages can fit inside a bacterial cell b. >50,000,000 polio viruses can fit inside a human cell 1) This is not much bigger than a hemoglobin molecule c. Range in size from 20 nm (0.02 micrometers) to 450 nm (0.45 micrometers) d. There are some “giant” viruses, and there are some tiny bacteria (such as Rickettsia) 2. Viruses do not have a cell membrane, a cell wall, or any cell constituents 94 Introduction to Viruses (Ch 6) 3. A typical virus has an outer covering (a capsid), sometimes an envelope outside of that, and nucleic acid (DNA or RNA) on the inside a. A few enzymes may also be present b. If it has an envelope around the capsid it is said to be an “enveloped virus” and if it only has a capsid it is called a “naked virus” 1) The envelope is nothing more than a piece of the host cell membrane that was “stolen” as the virus exited the cell D. Capsids 1. The outer covering of a virus, the capsid, is composed of repeating subunits made of protein called capsomers a. Capsomers self assemble in an organized pattern 1) Either form cylinders (helical pattern) or 20-sided icosahedrons b. Helical capsids are simpler 1) If naked, they are skinny, rigid cylinders 2) If enveloped the cylinder may be coiled (a) Influenza, measles, and rabies are all enveloped helical viruses c. Icosahedron is like the 20-sided dice found in Dungeons and Dragons 1) It may be constructed from a single capsid type, or sometimes a different capsid is used for the 12 corners 2) The number and shape of the capsomers can vary between viruses even though the overall 20-sided shape of the capsid will be consistent 3) May be naked or enveloped (a) Papillomavirus is a naked icosahedron (b) Herpes simplex virus is an enveloped icosahedron d. Complex viruses are neither helical nor icosahedron 1) Pox viruses and bacteriophages are considered complex (a) Bacteriophages are very different from animal and plant viruses, and look like little lunar landers E. Viral envelope 1. The virus can take a piece of phospholipid bilayer membrane from the nuclear membrane, the endoplasmic reticulum, or the outer cell membrane (it just depends on the type of virus and where it matures) and coat themselves in it a. These are mostly animal viruses that do this (humans get infected with both naked and enveloped viruses) 2. Even though the virus took the membrane from the host’s cell, some or all of the regular proteins within the membrane are replaced with viral proteins a. Some of these proteins help bind the envelope to the capsid b. Some of these proteins extend out from the envelope 1) These are called spikes, and are what allow the virus to bind with the next host cell (a) Spikes are what the body recognizes as antigens to make antibodies against 95 Elementary Microbiology F. Inside the capsid: nucleic acid and enzymes 1. Viruses contain either DNA or RNA, but not both a. As opposed to living cells, which have DNA in the chromosome(s) and RNA for protein translation 2. Viruses have few genes a. As few as 4 (Hepatitis B) to several hundred (herpes virus) 1) E. coli has about 4,000 genes 2) Humans have 25,000-40,000 genes 3) The largest known virus, called Mimivirus (discovered in 2003), has thousands of genes and may qualify as a living organism 3. Viral DNA is not always double-stranded, and viral RNA is not always singlestranded a. Most viruses have double-stranded DNA 1) A few viruses have single-stranded DNA b. Some viruses have single-stranded RNA 1) A few viruses have double-stranded RNA 4. Viruses may come with a few enzymes intact, or just with the genes for those enzymes a. Polymerases for copying nucleic acid are commonly intact b. HIV has reverse transcriptase to make DNA from RNA 5. Viruses lack enzymes for metabolic processes, as they are totally dependent on the host cell a. This is why they are not affected by antibiotics 1) Most antibiotics target a metabolic process, so if there is no viral metabolic process, there is no target G. How viruses are classified and named 1. Virus taxonomy is messy and in its early stages a. Classification based on structural and chemical composition b. In the future genetics will play a larger role in classification c. There are 19 families of animal viruses, each ending in –viridae 2. We will focus just on common names (poliovirus or rabies virus) and not worry about classification (except maybe grouping them based on nucleic acid) a. See table 6.2 H. The multiplication cycle in bacteriophages 1. We are not going to discuss this cycle in detail, as we will not be focusing on bacteriophages 2. Some points to remember, however are: a. Some viruses will go through a lytic cycle where they break out of the host cell, causing cell death as the viruses are released 96 Introduction to Viruses (Ch 6) b. Some viruses will go into what is called a lysogenic phase, where their DNA gets incorporated into the bacterial DNA 1) We have already talked about the fact that viruses can introduce new genes into bacteria, which may give them new traits (a) Diphtheria toxin is not produced by Corynebacterium until it receives the gene from a virus (b) Sometimes antibiotic resistance is acquired by a virus 2) This can be used as a biotechnology technique for inserting new genes into bacteria I. The multiplication cycle in animal viruses 1. The multiplication cycle can be divided into adsorption, penetration, uncoating, replication, maturation (assembly), and release 2. Adsorption a. Each virus can bind to a specific cell receptor 1) Rabies binds to acetylcholine receptor on nerve cells 2) HIV binds to CD4 protein on helper T cells b. The virus binds to the receptor either by its spikes (if it is enveloped) or by surface molecules attached to the capsid (if it is naked) c. Most viruses are very specific (e.g. hepatitis viruses only bind to primate liver cells) 1) In the lab you can induce viruses to infect cells that they normally would not infect, which makes studying them easier 3. Penetration/Uncoating a. The entire virus may be engulfed by the cell in a vacuole 1) Enzymes in the vacuole will dissolve the envelope and capsid, thus uncoating the virus b. The virus may fuse with the host cell membrane, thus releasing the nucleocapsid into the cell 4. Replication/Maturation a. Most DNA viruses enter the host cell nucleus and are replicated there b. Most RNA viruses are replicated and assembled in the cytoplasm c. Take-home message: the nucleic acid of the virus commands the cell to make viral proteins (for capsids), and to replicate the viral nucleic acid 1) The capsids self-assemble and the nucleic acid enters in 2) In enveloped viruses there are also genes for the spikes, and these proteins are embedded into the host cell membrane so they can be picked up as the viruses leave 5. Release a. Naked viruses are released when the cell gets so full of viruses that it bursts (lyses) b. Enveloped viruses bud off by exocytosis 1) The nucleocapsid binds to the membrane, which curves completely around it to form a pouch, then pinches off 97 Elementary Microbiology 2) With budding, the cell is not destroyed immediately, but it will die eventually in most cases c. Depending on the virus, a single infected cell will release anywhere from a few thousand to a hundred thousand viruses (mature virus particles are also called “virions”) 1) These are now free to infect new host cells and start the cycle over again 2) Ultimately some of them must leave the host for the cycle to continue in new hosts (a) Next time you give someone your cold, you can know that you made the viruses yourself J. Damage to host cell and persistent infection 1. Cytopathic effects are damages to the host cell a. Sometimes changes in size b. Inclusion bodies are common (compacted masses of viruses) c. Table 6.4 on page 177 lists some cytopathic effects which can be seen under the microscope and used in diagnosis 2. In a persistent infection the host cell does not die and can harbor the virus for weeks or years (the rest of your life) a. Herpes simplex virus may lie latent for years and occasionally become active to cause cold sores b. Herpes zoster virus (chickenpox) may be latent for decades and then reactivate as shingles 1) Part of why these can be inactive for decades is because nerve cells also live for your entire life 3. Some viruses will become part of your DNA, just like lysogenic bacteriophages a. Cells are “transformed” by the viral DNA and may be cancerous (oncogenic) 1) Papillomaviruses and cervical cancer 2) Herpes virus and Burkitt’s lymphoma b. HIV is a retrovirus 1) It starts out as single-stranded RNA 2) Using reverse-transcriptase, DNA is made using RNA as the template (a) This DNA may insert into the host cell and remain there for the life of the cell (b) One reason that HIV cannot be eliminated from the body c. Viruses may be involved in many types of cancers K. Techniques in cultivating and identifying animal viruses 1. Unlike bacteria, viruses cannot be grown in the lab on a nutrient media (because they have no cellular machinery of their own a. Being able to culture viruses outside of their natural host was a big jump 1) May be cultured in vivo such as in fertilized chicken eggs, or in vitro in tissue cultures 98 Introduction to Viruses (Ch 6) b. Purposes of virus cultivation include identification, vaccine preparation, and general research 2. Using live animal inoculation a. Specific strains of mice, rats, hamsters, guinea pigs, or rabbits may be bred in the lab and inoculated with various viruses b. Using bird embryos is even better, and used whenever possible 1) A bird egg is a self-supporting, sterile environment (a) Don’t need to feed it 2) A small hole is drilled through the shell and, using careful sterile techniques to avoid contamination with bacteria or fungi, a virus is injected into the egg (often into a specific embryonic tissue) 3. Using cell culture inoculation a. Growing a line of cells in the lab was a huge step forward b. Most viruses are propagated in cell cultures, and the tricky part of the work may be keeping your cell line alive 1) There are cell lines of kidney cells, heart muscle cells, bone marrow cells, etc. 2) Most of these are not the original cell line, but rather mutated forms that grow continuously (cancerous) c. Virus infection is seen as plaques in the monolayer of cells (areas where cells have been destroyed) L. Medical importance of viruses 1. Viruses are the most common cause of infection (several billion infections annually) 2. Most are self-limiting infections that do not require hospitalization a. Others lead to death, such as rabies, AIDS, and Ebola or long-term disability (such as polio) b. Chronic viral infections may be involved in type I diabetes, multiple sclerosis, and many cancers, but we are not sure M. Prions 1. Not viruses, but rather misshapen proteins a. Cause other nearby proteins to also become misshapen b. Creutzfeldt-Jakob disease and mad cow disease N. Detection & Treatment 1. Various options for diagnosis a. Look at clinical signs of the patient b. Look at tissue sample for cytopathic changes c. Screen samples for viral DNA or RNA using polymerase chain reaction (PCR) d. Screen for antibodies that the person has developed against the virus 99 Elementary Microbiology 1) This is how the HIV test works 2. Treatment is difficult a. Antibiotics do not work since the virus has no metabolism of its own b. Antiviral drugs target the host cell machinery so can have severe side effects 1) AZT (AIDS drug) causes immunosupression and anemia c. Interferon is produced by our cells and can be given to slow an infection (HepC interferon) d. Vaccines are the most successful treatment (best offense is a good defense) 1) Unfortunately no vaccines for most viral infections O. Viruses in the news 1. For a fascinating look at new ideas on viruses and more about Mimivirus, read http://www.discover.com/issues/mar-06/cover/ 2. Most new emerging diseases are viral a. West Nile virus, AIDS, SARS, bird flu (H5N1), etc. 100 DNA Viruses of Medical Importance (Ch 24) Unit 18. DNA Viruses of Medical Importance (Ch 24) A. Viruses in Infection and Disease 1. Viruses are probably the most common infectious agents. 2. They are all obligate parasites and infect plants, animals and also other microorganisms. 3. They are classified as enveloped or non-enveloped (naked). a. Viruses are particles of parasitic DNA or RNA encased in a protein capsid, sometimes with an additional envelope of cell membrane. 4. First vaccine was using cowpox (Vaccinia virus, which means “cow”) to protect against smallpox (Variola). a. Edward Jenner and the milk maid b. Louis Pasteur later figured out that a weakened (attenuated) or killed bacteria or virus could be used to vaccinate 1) First was chicken cholera, and a mistake from his lab assistant Charles 2) Later he developed a rabies vaccine by drying out the spinal cord of a rabid rabbit and grinding it into powder, then injecting this into a patient 3) Louis was the one who coined the term “vaccine” in honor of Edward Jenner’s discovery B. Important Medical Considerations in Viral Disease 1. Target Cells: a. Different viruses infect different kinds of tissues including nervous system, liver, respiratory tract, intestine, skin, mucous membranes, and immune system. b. They must enter into a cell to cause an infection 1) DNA viruses are assembled and budded off the nucleus. 2) RNA viruses multiply in and are released from the cytoplasm 3) Most cells productively infected with viruses are destroyed. 2. Scope of Infection a. Viruses vary in severity, depending on virulence of the virus and age, health, and habit of the human host. Lifelong immunity develops to some but not all viral agents. b. Most DNA and some RNA viruses can cause chronic infections and combine with the host genome (become latent). They also have the potential to activate host oncogenes. 1) Many cancers may have a viral basis, as well as other chronic disease with unknown etiology (unknown cause) 3. Latency and Oncogenicity a. Most DNA viruses and a few RNA viruses can become permanent residents of the host cell. Some latent viruses alternate between periods of inactivity and recurrent infections. 1) They do this by splicing their DNA into the host cell’s DNA 101 Elementary Microbiology 2) The host cell may transform into a cancer cell (a) Oncogenisis is more likely in DNA viruses, but also seen in some RNA retroviruses. b. Latency makes it a problem to use live attenuated virus for vaccines. 4. Teratogenicity, Congenital Defects, & Viral Diagnosis a. Some viruses are able to cross the placenta from an infected mother to the embryo or fetus. 1) Rubella, cytomegalovirus, and adenovirus are all teratogenic (cause physical birth defects). b. Another risk to infants is infection at the time of birth, including hepatitis B and herpes simplex virus. C. Survey of DNA Virus Groups 1. Classification of viruses is based on the structure of their capsid, the presence of an envelope, and the type of nucleic acid they contain. a. DNA Viruses are divided into two groups, enveloped or non-enveloped. 1) Enveloped viruses have double stranded genome and consist of: Poxviruses, Herpesviruses, and Hepadnaviruses. 2) Non-Enveloped are divided into double stranded genome, consisting of Adenoviruses and Papoviruses, and single stranded genome, consisting of Parvoviruses. (a) Note that ALL DNA viruses have double stranded nucleic acid (DNA) except parvoviruses 2. Poxviruses: Classification and Structure a. Produce eruptive skin pustules called pox, that leave small depressed scars called pockmarks when they heal. b. Largest and most complex of animal viruses. 1) Mimivirus, which infects amebas, is much larger (not in book) c. Largest genome of all viruses & multiply in the cytoplasm in well defined sites called factory areas which appear as inclusion bodies infected cells. 1) Variola is the agent of small pox 2) Vaccinia is the agent of cow pox (a) Closely related to variola, and used in vaccinations (b) Story of Edward Jenner and Sarah Nelmes the milk maid D. Smallpox 1. Was once one of the deadliest infectious diseases, and now only exists in government laboratories. a. 300-500 million deaths during 1900’s. 1) Two forms: Variola Major- highly virulent, caused toxemia, shock, and intravascular coagulation. Variola Minor has much milder symptoms and low death rate. b. Killed majority of some tribes of Native Americans, people of Hawaii, etc (groups that had no prior exposure to the virus) 102 DNA Viruses of Medical Importance (Ch 24) c. 2 million deaths as recently as 1967 d. Russian and American labs kept samples 1) It has been weaponized, at least in the former Soviet Union 2. Exposure occurred through inhalation of droplets or skin crusts. 3. Infection included fever, malaise, prostration, and later, a rash begins in the pharynx, spread to the face, and progressed to the extremities. a. Initially the rash is macular, evolving to a papular, vesicular, and pustular before crusting over, leaving nonpigmented sites pitted with scar tissue. 4. Other Poxvirus Diseases a. Molluscum contagiosum- causes a common skin disease, primarily an infection of children, transmitted by direct contact and fomites. (can also be spread by sexual intercourse) 1) Skin lesions take the form of smooth, waxy nodules on the face, trunk and limbs. In STD form the lesions are small, smooth macules occurring on the genitals and thighs. 2) Treatment requires destruction of the virus by freezing (cryotherapy), electric cautery, and chemical agents applied directly to the lesions. (a) It is self-limiting and benign, so treatment is not a medical necessity b. Most mammalian groups host some sort of poxvirus infection. But humans are only susceptible to cowpox, smallpox (a human-only disease) and monkeypox. (Chickenpox is a herpesvirus.) 1) Monkeypox was once only zoonotic but was found spreading within communities. (a) Monkeypox is very similar to smallpox with skin pocks, fever, swollen lymph nodes, and seems to be getting more virulent. 2) Cowpox is an eruptive cutaneous disease that can develop on cows udders and teats. (a) Cows are not the only reservoirs: rodents, cats and even zoo animals can carry it. E. The Herpesviruses: Common, Persistent Human Viruses 1. Herpesvirus- named for the tendency of some herpes infections to produce a creeping rash. Common name for a large family. a. Note: a herpetologist does NOT study the herpes virus, but studies creeping animals (reptiles and amphibians) b. Virtually everyone becomes infected with a herpesvirus at sometime. 1) Particularly troublesome for AIDS patients c. They are among the larger viruses. 1) Enclosed within a loosely fitting envelope that contains glycoprotein spikes (a) prone to deactivation by organic solvents or detergents (b) unstable outside the host’s body. 2) The icosahedral capsid houses a core of double-stranded DNA. d. Replication occurs primarily within the nucleus, and viral release is usually accompanied by cell lysis. 103 Elementary Microbiology 2. Humans are susceptible to two varieties of herpes simplex viruses (HSVs) a. HSV-1: characterized by lesions on the oropharynx b. HSV-2: characterized by lesions on the genitals 1) However, type 2 can infect the mouth, and type 1 can infect the genitals c. Humans are the only natural reservoir to HSV. 3. Epidemiology of Herpes Simplex a. Herpes can infect all age groups, in all seasons, globally. Transmission is promoted by direct exposure to secretions containing the virus. b. Primary infections tend to be age-specific. 1) HSV-1 occurs in infancy and early childhood, and by adulthood most people exhibit some serological evidence of infection. 2) HSV-2 occurs most frequently between the ages of 14-29, a pattern that reflects sexual route of transmission. c. Genital herpes is one of the most common STD in the U.S. d. Nature of Latency & Recurrent Attacks 1) In 20%-50% of HSV primary infections the virus enters the distal regions of sensory neurons and travel to the dorsal ganglia. (a) HSV-1 enters primarily the trigeminal, 5th cranial nerve, which has extensive innervations in the oral region. (b) HSV-2 usually becomes latent in the ganglion of the lumbosacral spinal nerve trunk. 2) Recurrent infection is triggered by stimuli such as UV radiation (e.g. sunburn), stress, or mechanical injury. (a) Virus migrates to the surface and produces a local skin or membrane lesion, often at the same site as previous infections. e. The Spectrum of Herpes Infection & Disease 1) Herpes usually targets the mucous membranes through cuts or cracks on the membrane surface and then multiplies in the basal and epithelial cells in the immediate vicinity. (a) The result is inflammation, edema, cell lysis, and a characteristic thin-walled vesicle. (b) The main diseases of HSV are facial herpes (oral, optic, and pharyngeal), genital herpes, neonatal herpes, and disseminated disease. 4. Type 1 Herpes Simplex in Children & Adults a. Herpes labialis- otherwise known as fever blisters or cold sores, is the most common recurrent HSV-1 infection. 1) Vesicles crop up on the mucocutaneous junction of the lip and adjacent skin (a) A few hours of skin tingles and itching precede the formation of vesicles. (b) Pain is most acute as vesicles ulcerate (c) Lesions crust over in 2-3 days and heal in a week. 2) Herpetic gingivostomatitis- infection of the oropharynx strikes young children most frequently. Inflammation of the oral mucosa can involve gums, tongue, soft palate, and lips. (a) May continue into throat and cause pharyngitis with symptoms similar to strep throat 104 DNA Viruses of Medical Importance (Ch 24) b. Herpetic keratitis also called ocular herpes, infective inflammation of the eye in which latent virus travels to the ophthalmic branch of the trigeminal nerve. 1) In 25%-50% of cases are recurrent and chronic and can interfere with vision. 5. Type 2 Herpes Infection usually coincides with sexual maturation and increases with sexual encounters. a. Genital Herpes starts out with malaise, anorexia (loss of appetite), fever and bilateral swelling and tenderness in the groin, then sensitive vesicles break out in clusters on the genitalia, perineum, and buttocks. b. Urethritis, painful urination, cervicitis, and itching occur and after a few weeks the vesicles ulcerate before healing. c. Recurrent out breaks are usually less severe than the initial infection. d. Herpes of the Newborn is very destructive and can be fatal. 1) Most cases occur when infants are contaminated by the mother’s reproductive tract immediately before or during birth, but can come from lesions on the mothers hands as well. 2) Type 2 is more often involved but type 1 has similar complications. (a) The disease can be confined to the mouth, skin, or eyes with a mortality rate of 30%. (b) If the disease affects the CNS the mortality rate is 50%-80%! 3) Because of the danger it is now standard procedure to screen pregnant women for the herpesvirus (a) If no sign of recurrence is seen, they can have a vaginal delivery, but any evidence of a breakout at the time of delivery requires a caesarian section. 6. Miscellaneous Herpes Infections a. A hazard for health care workers who may handle patients or their secretions without hand protection is a disease called herpetic whitlow. b. Workers in the fields of obstetrics, gynecology, dentistry, and respiratory therapy are at greatest risk. c. Whitlow’s are deep set, itchy, painful, and usually occur on one finger. 7. Life-Threatening Complications a. Herpes simplex encephalitis is a rare complication of type 1 infection, and is most common sporadic form of viral encephalitis in the U.S. 1) Infection occurs along nerve pathways to the brain or spinal cord. 2) The effects include a headache, stiff neck, and can progress into mental disturbances and coma. 3) 70% mortality rate in untreated cases. b. Patients with compromised immune system are more prone to severe infections. 1) These include patients receiving organ grafts, cancer patients, patients with congenital immunodeficiencies, and AIDS patients. 8. Diagnosis, Treatment, & Control of Herpes Simplex a. Diagnosis includes symptoms of HSV, the presence of multinucleic cells when scrapings are stained, and lab tests of tissue specimens b. Acyclovir is the most effective therapy that is nontoxic and highly specific. Topical medication applied to genitalia and oral lesions cut the length of 105 Elementary Microbiology infection and reduce viral shedding. Valtrex (oral) has shown effective suppressive therapy preventing recurrent outbreaks. c. Control of infections include barriers 1) condoms to prevent the spread of genital infections. 2) Mothers need to take great caution when they have a cold sore and not kiss their babies on the mouth. 3) Medical and dental workers should wear gloves with their patients. 9. Varicella-Zoster Virus a. It is known by a composite name Varicella-zoster virus (VZV) because the same virus causes varicella (Chickenpox) and herpes zoster (shingles) 1) herpes zoster is a reactivation of latent varicella virus. b. Epidemiologic Patterns of VZV Infection 1) Humans are the only hosts for VZV (a) It is harbored in the respiratory tract, but is transmitted through respiratory droplets and active skin lesions. (b) It is most infectious a day or two prior to the rash. - Dried scabs are not infectious because the virus is unstable when exposed to the environment. (c) Patients with Shingles are a source of infection for nonimmune children. (d) Immunity protects against reinfection with chickenpox but not an attack of shingles. c. Varicella (Chickenpox) 1) Chief portal of entry and site of viral replication is the respiratory epithelium. 2) No symptoms appear initially, but after an incubation period of 10-20 days first symptoms appear (a) fever, abundant rash beginning on the scalp, face and trunk then radiates out to extremities. (b) Skin lesions progress from macules and papules to itchy vesicles that encrust and drop off, usually healing completely but sometimes scarring - May have only a few or several hundred lesions d. Herpes Zoster (Shingles) 1) In an unknown percentage of people, recuperation from varicella is associated with the entry of the VZV into the sensory endings that innervate dermatomes. 2) From here it becomes latent in the ganglia and may reemerge with asymmetrical distribution on skin of trunk or head. 3) Shingles develops after stimuli such as X-ray treatments, immunosuppressive and other drug therapy, surgery or developing malignancy. (a) Virus migrates down the ganglion to the skin where multiplication resumes and crops of vesicles appear. 4) Pain and tenderness can last several months and be quite severe (postherpetic neuralgia) e. Diagnosis, Treatment & Control 1) Varicella requires no treatment besides alleviation of discomfort. (a) An antibiotic ointment may be applied to prevent a secondary bacterial infection 106 DNA Viruses of Medical Importance (Ch 24) 2) Risk of Reye syndrome from aspirin 3) Vaccine available that should protect from getting shingles (since the virus will never infect and go latent) 4) Antiviral drugs (e.g., acyclovir) given early will reduce severity of a shingles attack F. Other Herpesviruses & the Cancer Connection 1. The Cytomegalovirus Group (CMV) a. Named for their tendency to produce giant cells with nuclear and cytoplasmic inclusions. b. Epidemiology of CMV Disease 1) Antibodies are developed during a prior infection, (a) 50% of childbearing age women have antibodies and 10% of newborns reveal some evidence of infection. (b) It is transmitted through saliva, respiratory mucous, milk, urine, semen, cervical secretions and feces. (c) Is commonly carried in latent state in various tissues. c. Infection & Disease 1) Most people are asymptomatic for CMV 2) Newborns may develop an enlarged liver and spleen, jaundice, and neurological problems (a) In U.S. about 5,000 babies each year suffer permanent brain damage from CMV 3) Cytomegalovirus mononucleosis is a syndrome characterized by fever and lymphocytosis. (a) Not the same as the typical mononucleosis caused by Epstein-Barr virus, but similar symptoms (b) Common opportunists of AIDS patients, where it produces many symptoms and high mortality d. Diagnosis, Treatment & Prevention 1) Must be distinguished from other diseases in newborns, and adults. 2) Drug therapy is reserved for immunosuppressed patients including ganciclovir, valacyclovir, and foscarnet - which have toxic side effects and cannot be administered for long periods. 2. Epstein-Barr Virus a. Michael Burkitt discovered in African children an unusual malignant tumor that appeared to be infectious. Later Michael Epstein and Yvonne Barr cultured a virus from tumors that showed herpesvirus morphology. b. Epidemiology of Epstein-Barr Virus 1) Also known as “mono” or “kissing disease” 2) 20% of asymptomatic people secrete this virus in their saliva 3) Worldwide most people are infected as children, but in the U.S. 70% of college age people have never had EBV and are vulnerable to infection. (a) By mid-life 90%-95% of all people show serological evidence of prior infection. c. Diseases of EBV 107 Elementary Microbiology 1) Portal of entry is epithelium of oropharynx (a) From this site the virus moves to parotid gland (b) After dormancy virus is reactivated. 2) Symptoms include, sore throat, high fever, and cervical lymphadenopathy, gray-white exudate in the pharynx, skin rash, and enlarged spleen and liver. d. Tumors & Other Complications Associated with EBV 1) Burkitt lymphoma - B-cell malignancy that develops in the jaw and grossly swells the cheek in young African children. 2) People with weakened immune systems vulnerable to EBV (a) Organ transplant patients and AIDS patients (b) Believed to cause some lymphatic tumors in kidney transplant patients e. Diagnosis, Treatment, & Prevention 1) Treatment of mononucleosis is relief of fever and sore throat, and sometimes antiviral drugs. 2) Burkitt lymphoma is treated with chemotherapy with an anticancer drug, and surgical removal of tumor. 3. Human herpesvirus 6(HHV-6) also known as human T-lymphotrophic virus a. Similar to EBV, but is genetically distinct from other herpes virus. b. It can enter and replicate it T-lymphocytes, macrophages and salivary gland tissues. c. Extremely common herpes virus, in 95% of human population. d. It is the cause of roseola- an acute febrile disease in babies between 2-12 months of age. 1) High fever followed by rash 2) Self-limiting and spontaneous recovery e. Over 70% of multiple sclerosis patients have positive signs of HHV-6 infections, and the brain lesions of many have active virus infections. 4. Relationships between HHV-6 and Hodgkin lymphoma, oral carcinoma, and Tcell leukemias. G. Hepadnaviruses: Unusual Enveloped DNA Viruses 1. General Considerations: What is Viral Hepatitis(HBV)? a. When certain viruses infect the liver they cause hepatitis, an inflammatory disease marked by necrosis of the hepatocytes and a mononuclear response that swells and disrupts the liver and architecture. The change interferes with liver excretions and causes jaundice. b. Hepatitis B Virus is a DNA virus, but Hep. A and Hep. C are RNA viruses 2. Hepatitis B Virus & Disease a. HBV is the most severe form of hepatitis, it is spread by direct contact, fomites and mosquitoes. It is potentially lethal. b. Epidemiology of Hepatitis B 1) HBV multiplies in the liver, which continually fills the blood with viruses. 2) The abundance of viruses in the blood is so high and the infectious dose is so low that sharing a toothbrush or razor can transmit infection. 108 DNA Viruses of Medical Importance (Ch 24) c. Pathogenesis of Hepatitis B Virus 1) HBV enters through a break in the skin or mucous membrane or by injection into the blood stream, and eventually reaches the liver (hepatocytes) where it multiplies and releases. 2) The incubation period is 4-24 weeks. Some experience fever, chills, anorexia, abdominal discomfort and diarrhea. 3) With more sever cases people also show jaundice, rashes, and arthritis. d. The association of HBV with hepatocellular carcinoma is based on these observations: 1) certain hepatitis B antigens are found in malignant cells and are often detected as integrated components of the host genome. 2) persistent carriers of the virus are more likely to develop this cancer. 3) people from areas of the world with a high incidence of HBV are more frequently affected. This connection is probably a result of infection early in life and the long-term carrier state. e. Diagnosis & Management of Hepatitis B 1) Mothers with HBV are likely to transmit it to her newborn during delivery. 2) Infection is managed by recombinant interferon (a) passive immunization also helps protect those that have been exposed. 3) Vaccine for dentists and health care workers (a) Also strongly encouraged as routine vaccination for children H. The Adenoviruses 1. Was isolated when seeking a cure for the common cold. 2. Infect lymphoid tissue, respiratory tract, intestinal epithelia, and the conjunctiva. 3. Epidemiology of Adenoviruses a. Spread from person to person by respiratory and ocular secretions. b. Infection usually occurs by age 15. 4. Respiratory, Ocular & Miscellaneous Diseases of Adenoviruses a. keratocunjuctivitis - an inflammation of the conjunctiva and cornea. b. Acute hemorrhagic cystitis - in children, blood in urine, painful urination, fever, bed-wetting, and supra pubic pain. Infects intestinal epithelium. I. Papovaviruses: Papilloma, Polyoma, Vacuolating Viruses 1. 2. 3. 4. This family contains two subtypes: papillomaviruses and polymaviruses. Papilloma- benign nodular epithelial tumor, also known as a wart or verruca. Polyoma- glandular tumor caused by a virus. Vacuolating- the process of forming small spaces or vacuoles in the cytoplasm of the cell. 5. Epidemiology & Pathology of the Human Papillomaviruses(HPV) a. HPV has 40 different strains. b. Some are specific to skin, others to mucous membranes 109 Elementary Microbiology c. common, or seed warts- painless, elevated, rough growths on the fingers and other body parts. d. plantar warts- deep, painful papillomas on the soles of the feet. e. genital warts- a prevalent STD and is linked to some types of cancer. f. Warts are transmissible through direct contact or contaminated fomites, and the can also spread on the same person by autoinnoculation. Incubation period is 2 weeks to a year. g. Genital Warts: An Insidious Papilloma 1) Genital warts among the most common STD in U.S. with 6 million new cases per year. Virus invades the genitalia both internal and external. Warts vary from tiny, flat, inconspicuous bumps to extensive, branching, cauliflower-like masses called condylomata acuminata. Strong association with cancer of the reproductive tract. 2) Diagnosis, Treatment & Prevention (a) Biopsies can determine which string of HPV is present. Treatment includes surgery and interferon. 6. The Polymaviruses a. Capable of inducing an assortment of tumors. b. Progressive multifocal leukoenceohalopathy (PML) - infection by JC virus. c. JC virus and BK virus are the most important. 1) JC virus- attacks accessory brain cells and gradually demyelinizes certain parts of the cerebrum. 2) BK virus- associated with renal transplants, causes complications in urinary function. d. Epidemiology & Pathology 1) When PML is diagnosed brain damage has already occurred and patient is irreversible immunocomprimised. J. Non-Enveloped Single Stranded DNA Viruses: The Parvoviruses 1. Parvoviruses (PV’s) - are unique among the viruses in having single-stranded DNA molecules. 2. Smaller in diameter and genome size, and resistant. 3. Can infect several mammalian groups a. B19 is most important human parvovirus, causes erythemia infectiosum. Occurs in children and is also known as the 5th disease, because it is the 5th childhood rash. “Slapped face” rash appears on their cheeks. 110 RNA Viruses of Medical Importance (Ch 25) Unit 19. RNA Viruses of Medical Importance (Ch 25) A. I’m not certain we will get to this chapter B. If we do, hopefully we will have some notes for it! 111 Elementary Microbiology Unit 20. Appendix: Bacteria to Know Bacillus anthracis Gram-positive rod; endospore-forming (extreme resistance to heat, acid, etc), aerobic, catalase-positive; Causes cutaneous and pulmonary anthrax; usually a zoonotic disease, but also used as a bioweapon; Bordetella pertussis Small aerobic gram negative coccobacillus. Causes whooping cough. Produces exotoxins that kills epithelial cells Brucella Campylobacter jejuni Chlamydia trachomatis Can cause pelvic inflammatory disease (PID) in women, blocking fallopian tubes Clostridium botulinum Gram-positive spore-forming rod; widely distributed in nature; anaerobic; catalasenegative; causes deadly food poisoning (flaccid paralysis by preventing release of acetylcholine); exotoxin used cosmetically (Botox) Clostridium tetani Gram-positive spore-forming rod; widely distributed in nature; anaerobic; catalasenegative; produces tetanospasmin, a potent neurotoxin which causes tetanus (prevents muscle relaxation); vaccine available Clostridium difficile Gram-positive spore-forming rod; common in large intestine; anaerobic; catalasenegative; causes antibiotic-associated colitis; probiotics as prevention Clostridium perfringens Gram-positive spore-forming rod; widely distributed in nature; anaerobic; catalasenegative; Causes gas gangrene Corynebacterium diphtheriae Gram-positive, non-spore forming bacillus. Produces toxin after infection by bacteriophages. Causes diphtheria, with a pseudomembrane and toxemia; Iditarod dog sled race and diphtheria antitoxin; DTaP vaccination Escherichia coli Haemophilus ducreyi Haemophilus influenzae Helicobacter pylori Legionella pneumophila Leptospira Listeria Gram-positive non-spore forming Coccobacilli; not fastidious, and resistant to cold, heat, salt, pH changes, and bile; can grow at refrigerator temperatures; danger to fetuses (it crosses the placenta) when pregnant woman consumes contaminated luncheon meats, hotdogs, aged cheeses 112 Appendix: Bacteria to Know Mycobacterium leprae Acid-fast bacillus; extremely slow-growing (20 days between divisions); not highly infective; causes leprosy; Mycobacterium tuberculosis Acid-fast bacillus; very slow-growing (20 hours between divisions); highly infectious, with about 1/3 of world population infected (about 15 million carriers in U.S.); tubercles form around infected macrophages; Mycoplasma pneumoniae Neisseria gonorrhoeae Gram-negative diplococci; resides in mucus membranes (attachment aided by pili); sexually transmitted, with humans as only reservoir; among top 5 STDs, with many asymptomatic carriers; can damage male urethra or cause pelvic inflammatory disease and possible infertility (due to scarring of fallopian tubes); also causes neonatal ophthalmia (prevention through antibiotics in eyes of newborns) Neisseria meningiditis Gram-negative diplococci; resides in mucus membranes (attachment aided by pili); Causes meningitis, in children and adults in close settings (e.g. daycare, college dorms). Reservoir is asymptomatic carriers (in their nasopharynx) Propionibacterium acnes Gram-positive, non-spore forming bacillus. Lives in sebaceous glands and causes acne. Pseudomonas aeruginosa Rickettsia prowazekii Rickettsia typhi Salmonella species Salmonella typhi Shigella Staphylococcus aureus Gram-positive cocci in clusters; golden colonies; catalase-positive, coagulase-positive; resistant to drying, heat, salt; facultative anaerobe; purulent; frequent drug resistance (e.g. MRSA); often in nasal cavity; opportunistic pathogen with several virulence factors (enzymes: coagulase, hyaluronidase, penicillinase, DNase, kinase; exotoxins: hemolysin causes beta hemolysis, leukocidin, enterotoxins, exfoliative toxin); causes furuncles, carbuncles, impetigo, toxic shock syndrome; common nosocomial infection Streptococcus mutans Gram-positive cocci in chains, catalase-negative; sensitive to drying, heat, and disinfectants; alpha-hemolytic (viridans group); abundant in mouth; causes dental caries due to acidic byproducts of sucrose metabolism Streptococcus pneumoniae Gram-positive cocci in chains, catalase-negative; alpha-hemolytic (viridans group); sensitive to drying, heat, and disinfectants; causes 60-70% of bacterial pneumonias, also meningitis and otitis media; vaccine available Streptococcus pyogenes 113 Elementary Microbiology Gram-positive cocci in chains, catalase-negative; sensitive to drying, heat, and disinfectants; Lancefield Group A; variety of virulence factors (surface antigens, toxins, enzymes); alpha-hemolytic (viridans group); inhabits throat, nasopharynx, and sometimes skin (humans only reservoir); can cause impetigo, erysipelas, pharyngitis (Strep throat), scarlet fever, rheumatic fever, puerperal sepsis (childbed fever), endocarditis, systemic infections Treponema pallidum Vibrio cholerae Yersinia pestis 114 Appendix: Bacteria to Know 115 'Twas a dangerous cliff, as they freely confessed, Though to walk near its crest was so pleasant; But over its terrible edge there had slipped A duke and full many a peasant. So the people said something would have to be done, But their projects did not at all tally; Some said, "Put a fence 'round the edge of the cliff," Some, "An ambulance down in the valley." But the cry for the ambulance carried the day, For it spread through the neighboring city; A fence may be useful or not, it is true, But each heart became full of pity For those who slipped over the dangerous cliff; And the dwellers in highway and alley Gave pounds and gave pence, not to put up a fence, But an ambulance down in the valley. "For the cliff is all right, if your careful," they said, "And, if folks even slip and are dropping, It isn't the slipping that hurts them so much As the shock down below when they're stopping." So day after day, as these mishaps occurred, Quick forth would those rescuers sally To pick up the victims who fell off the cliff, With their ambulance down in the valley. Then an old sage remarked: "It's a marvel to me That people give far more attention To repairing results than to stopping the cause, When they'd much better aim at prevention. Let us stop at its source all this mischief," cried he, "Come, neighbors and friends, let us rally; If the cliff we will fence, we might almost dispense With the ambulance down in the valley." "Oh he's a fanatic," the others rejoined, "Dispense with the ambulance? Never! He'd dispense with all charities, too, if he could; No! No! We'll support them forever. Aren't we picking up folks just as fast as they fall? And shall this man dictate to us? Shall he? Why should people of sense stop to put up a fence, While the ambulance works in the valley?" But the sensible few, who are practical too, Will not bear with such nonsense much longer; They believe that prevention is better than cure, And their party will soon be the stronger. Encourage them then, with your purse, voice, and pen, And while other philanthropists dally, They will scorn all pretense, and put up a stout fence On the cliff that hangs over the valley. Better guide well the young than reclaim them when old, For the voice of true wisdom is calling. "To rescue the fallen is good, but 'tis best To prevent other people from falling." Better close up the source of temptation and crime Than deliver from dungeon or galley; Better put a strong fence 'round the top of the cliff Than an ambulance down in the valley. -- Joseph Malins (1895)