MBY 161 Introduction to Microbiology PDF

MBY 161 Introduction to Microbiology Introduction What is microbiology? Study organisms. Too small to be seen by naked eye. Usually requires microscope. Microbial diversity and impact Microbes have both a positive and negative impact on humans. Some essential to...

MBY 161 Introduction to Microbiology Introduction What is microbiology? Study organisms. Too small to be seen by naked eye. Usually requires microscope. Microbial diversity and impact Microbes have both a positive and negative impact on humans. Some essential to humans Pathogenic (Causes disease) Bacteria Virus Fungi Protozoa Meningitis. HIV/AIDS. Ringworm. Malaria. Sinusitis. Flu. Yeast infection. Cryptosporidiosis. Pneumonia. Ebola fever. Candidiasis. Beneficial Food Chemicals Disease treatment Decompose organic Cheese. Ethanol. Insulin. waste. Yogurt. Acetone. Antibiotics. Break down Bread. Butanol. Human waste + Microbial Synthesis: microbiome incorporate N fermentation - Vitamins. (group microbes form air into (safe food - Organic live in our organic supplies). acids. bodies). compounds. Alcoholic - Enzymes. beverages. - Alcohols. - Drugs. What do microbiologists do? Studies living organisms and infectious particles can only be seen with microscope. Job opportunities Degree – Ph. D. Wages depend education + job sector + experience. Sectors: - Food production - Environmental science - Medicine - Research.  Job titles: - Biosafety officer - Professor. Microbiology careers Microbial genetics Medical micro Food micro Agricultural microbiology Microbial philosophy and biochemistry Teaching and research Taxonomy Hybrid and other. Clinical research Areas of specialization in microbiology Mycology Virology Parasitology Bacteriology Immunology rDNA Technology - Molecular biology - Genomics - Microbial genetics - Gene therapy - Biocontrol - Bioremediation - Microbial ecology - Biotechnology. Theme 1 The Microbial World and You Chapter 1 Definitions Microorganism A living organism too small to be seen with the naked eye. Includes bacteria, fungi, protozoa, microscopic algae and also viruses. Microbiome/microbiota All microorganisms in an environment. Normal microbiota The microorganisms that colonize a host without causing disease; also called normal flora. Bacteriology The scientific study of prokaryotes, including bacteria and archaea. Mycology The scientific study of fungi. Parasitology The scientific study of parasitic protozoa and worms. Virology The scientific study of viruses. Microbial genetics The mechanism by which microorganisms inherited traits. Molecular biology The science of dealing with DNA and protein synthesis of living organisms. Genomics The study of all an organism’s genes. Recombinant DNA Manufacturing and manipulating genetic material in vitro; also called genetic (rDNA) technology engineering. The microbiome Humans + animals rely on microbes maintain good health. Maintain + develop human body. Before birth – body populated bacteria. Newborn. -acquire fungi, viruses, bacteria. - E.g. E. coli (large intestine). Factors influence whether microbe colonize body as normal microbiota/fleeting member community (transient microbiota). Only colonize body sites appropriate nutrients. Factors influence types microbes: - Temperature. - pH. - Presence/absence chemical compounds. Types of microorganism Prokaryotes Eukaryotes Virusses Bacteria Archaea Fungi Protozoa Algae Acellular Cellular Cellular Cellular Cellular Cellular  Prokaryotes Genetic material not enclosed by nuclear membrane. Bacteria Archaea Peptidoglycan cell walls Lack peptidoglycan. - Cell wall is NOT a requirement for all Extreme environments. living bacteria. Not known to cause disease in humans. Unicellular. Include Several shapes. - Methanogens (methane waste product Reproduce respiration). - Binary fission. - Extreme halophiles (salty environments Energy – Great Salt Lake + Dead Sea). - Organic chemicals. - Extreme thermophiles (hot sulphureous - Inorganic chemicals. water – hot springs Yellowstone - Photosynthesis. National Park). Swim - Flagella. Nutrition - Organic chemicals (dead/living organisms). - Photosynthesis. - Inorganic substances. Eukaryotes Cells distinct nucleus containing genetic material – nuclear membrane. Fungi Protozoa Algae Unicellular (yeast) or Unicellular. Unicellular or multicellular (molds, No cell walls. multicellular. mushrooms). Absorb/ingest organic Cellulose cell walls. Multicellular consist chemicals or Energy masses mycelia photosynthesis. - Photosynthesis – light (filaments, hyphae). Motile + water + CO2 → Chitin cell walls. - Pseudopods (false oxygen + Energy feet). carbohydrates - Organic chemicals. - Cilia (numerous short (utilized other Reproduce appendages). organisms). - Asexually. - Flagella (long). - Do not need organic - Sexually. Parasitic or free living. compounds No photosynthesis. Reproduce environment. Nourishment: - Asexually. Produce molecular - Absorb organic - Sexually. oxygen and organic material environment. compounds. Reproduce - Asexual. - Sexually. Viruses Acellular – not cells. Coat enclosed lipid membrane - envelope. Not considered living outside host cell – Replicated only when in living – use cellular inert outside living host. machinery other organism.  Core (one type nucleic acid – DNA/RNA) Very small – electron microscope. surrounded by protein coat. DNA or RNA nucleic acid. Classification of microorganisms Three domains based on cellular organization - Bacteria (cell walls contain protein – carbohydrate complex called peptidoglycan). - Archaea (cell walls, if present, lack peptidoglycan). - Eukarya o Protists (slime molds, protozoa, algae). o Fungi (unicellular yeast, multicellular molds, mushroom). o Plants (mosses, ferns, conifers, flowering plants). o Animals (sponges, worms, insects, vertebrates). Developed by Carl Woese (1978). Nomenclature Naming of organisms. Linnaeus (1753) - System of scientific nomenclature. Each organism – 2 names - Genus. - Species epithet (Species name). Italicized (typed) or underlined (written) - Genus (capitalized). - Specific species (lowercase). “Latinized” - Used worldwide. Descriptive or honor scientist. Named once – can be abbreviated. Use of scientific names After first use, may be abbreviated - First letter genus and species epithet. Microbes and human disease Normal microbiota Microbes normally present in and on human body. Prevent growth pathogens. Produce growth factors (Vitamins B and K). Transient microbiota Microbes present in human body short period. Biofilms Microbial community forms a slimy layer on surface.  Microbes attach solid surface, grow into masses. Grow on - Rocks. - Pipes. - Teeth. - Medical implants. Beneficial - Protect mucus membrane harmful microbes. - In lakes – food for aquatic animals. Harmful - Clog water pipes. - Cause infection on medical implants o Endocarditis – inflammation of the heart. - Bacteria I biofilms – resistant to antibiotics (biofilm produce protective barrier). Communicate through Quorum Sensing. Emerging infectious diseases Infectious disease Resistance Pathogen invades host. Ability body ward off disease Overcomes host’s resistance. - Barrier skin. Disease. - Mucous membrane. - Stomach acid. Emerging Infectious Disease (EIDs) - Interferons. A new or changing disease that is increasing or has the potential to increase in incidence in the near future. Factors contribute to development: - Evolutionary changes existing organisms. - Spread of known disease by modern transport. - Exposure new infectious agents due to ecological changes (deforestation etc.). Middle East Respiratory Syndrome Corona Virus Avian influenza A (H5N1) (MERS-CoV) Bird flu. 2014. Influenza A virus. Same family as SARS (Severe Acute Waterfowl and poultry. Respiratory Syndrome) Human-to-human transmission not yet - Common cold. occurred. Methicillin-Resistant Staphylococcus aureus (MRSA) E. coli O157:H7 MRSA is transmissible. Toxin-producing strain E. coli. 1950s: Penicillin resistance developed. First seen 1982 1980s: Methicillin resistance. - Bloody diarrhea. 1990s: MRSA resistance to vancomycin Leading cause diarrhea. reported - VISA: Vancomycin-intermediate S. aureus. - VRSA: Vancomycin-resistant S. aureus.  Ebola hemorrhagic fever (EHF) Ebola virus. Causes - Fever. - Hemorrhaging. - Blood clotting. Transmitted - Contact infected blood. - Body fluids First identified near Ebola River, Congo 2014 outbreak, Guinea - Hundreds killed History of microbiology First Robert Hooke (Cell theory) obeservations Anton van Leeuwenhoek ("Anamalicules) Francesco Redi (jars decaying meat) Spontaneous John Needham (boiled nutrient broth in covered flasks) Lazzaro Spallanzani (boiled nutrient solutions sealed flask) generation Rudolf Virchow Louis Pasteur Agostino Bassi (silkworm disease - fungus) Louis Pateur (Silkworm disease - protozoan fungus) Ignaz Semmelweis (Handwashing prevent transmission puerperal fever) Golden era Joseph Lister (disinfectant clean surgicalwounds) Robert Koch (bacterium causes anthrax, Koch's postulates - prove specific microbe cause specific disease) Edward Jenner (Vaccination, cowpox immune smallpox, immunity) Birth of Paul Erlich ("Magic bullet", salvarsan syphilis) modern Alexander Flemming (antibiotic chemotherapy - Penicillin) Modern developments First observations Robert Hooke (1663 – 1665) Thin slice of cork through crude microscope  - Resolution allowed him to only observe large cells, not microbes. Life’s smallest structural units were “little boxes” or “cells”. Cells. Cell theory - All living things composed of cells and arise from preexisting cells. Anton van Leeuwenhoek (1623 – 1673) First to observe live microorganisms (microbes). Simple single-lens microscope he constructed (magnifying lenses). “Animalicules”. Detailed drawings organisms what he saw - Since identified as bacteria + protozoa. Importance discovery: - Brass microscope – observe living organisms too small seen naked eye. - High magnification (300x). - Drawings bacteria. Debate over Spontaneous Generation Spontaneous generation Hypothesis that life arises from nonliving matter and a “vital force” is necessary for life. Francesco Redi (1668) Filled jars with decaying meat. Demonstrate: maggots do not arise spontaneously. Condition jar Result Covered fine net No maggots Open Maggots Sealed No maggots Purpose of sealed jars: - Prevent flies from entering – maggots being born. Biogenesis: Blow to spontaneous generation theory - Many still believed small organisms simple enough to originate from nonliving material. John Needham (1745) Boiled nutrient broth into covered flasks. Cooled solutions teeming microorganisms. Conditions Result Nutrient broth heated, Microbial growth placed in covered flask Spontaneous generation: - Claimed microbes formed spontaneously from fluids. Lazzo Spallanzani (1765) Suggested microorganisms from air entered Needham’s solutions after being boiled.  Boiled nutrient solutions in sealed flasks. Conditions Result Nutrient broth placed in No microbial flask, sealed, heated growth Biogenesis: - Showed nutrient fluids heated after being sealed in flask did not develop microbial growth. Needham responded: - “Vital force” necessary for spontaneous generation destroyed by heat + kept out of flask by lids. Criticized: - Not enough O2 present support microbial growth. Biogenesis Hypothesis that living cells arise only from preexisting cells. Rudolf Virchow (1858) Louis Pasteur (1861) Microorganisms present in air + can contaminate sterile solutions BUT air itself does not create microbes. Filled several short-necked flasks beef broth + boiled contents – some left open to cool others sealed. Conditions Result Nutrient broth placed in Microbial growth flask, heated, NOT sealed Nutrient broth placed in No microbial flask, heated, then growth immediately sealed Biogenesis: - Microbes in the air – agents responsible contaminating nonliving matter. Next, broth in open-ended, long necked flasks + bent necks in S-shape curves (air pass in flask, curved neck trapped airborne microorganisms) - Contents boiled + cooled. - Broth did not decay + no signs of life for months. Principles form Pasteur’s work - Microorganisms present in nonliving matter (air, liquids and solids). - Heat destroy microorganisms. - Possible design methods block airborne contamination (standard practice laboratory + medical procedures). - Principles aseptic techniques o Prevent microbial contamination. Scientists believe that a form of spontaneous generation must have occurred in primitive Earth but agree that it does not happen under today’s environmental conditions. Golden age of microbiology 1857 – 1914. Rapid advances establish microbiology  - Agents of diseases. - Role immunity prevention + cure. - Chemical activities microorganisms. - Improved technique performing microscopy + culturing microorganisms: o Vaccines. o Surgical tehniques. Louis Pasteur + Robert Koch. Beginning Pasteur’s work discovery of - Relationship microbes and disease. - Immunity. - Vaccines. Pasteur showed - Microbes responsible fermentation. - Fermentation: microorganism (yeast) convert sugars to alcohol in absence if air. - Microbial growth responsible spoilage food and beverages. - Bacteria use air spoil wine o Wine to vinegar (acetic acid) in presence of air. o Killed by heat not hot enough evaporate alcohol. - Pasteurization: process mild heating to kill particular spoilage microorganism/pathogen. Germ theory of disease Yeast role fermentation - activity microorganism and physical + chemical changes organic materials. Germ theory of disease: The principle that microorganisms cause disease. Agostino Bassi (1835) - Silkworm disease cause by fungi. Pasteur (1865) - Using data Agostino Bassi. - Another silkworm disease caused by protozoan parasite. - Developed method recognizing afflicted silkworm moths. - Connect microbes to animal disease. Ignaz Semmelweis (1840s) - Proved handwashing prevent transmission puerperal fever. Joseph Lister (1860s) - Based on work of Semmelweis + Pasteur. - Applied germ theory to medical procedures. - Disinfectant clean surgical wounds o Phenol (carboxylic acid) kills bacteria. o Very effective – many other surgeons adopted. - Proved microorganisms cause surgical wound infections. Robert Koch (1876) - First proof: bacteria cause disease. - Pasteur’s rival find cause Anthrax. o Rod-shaped bacteria blood cattle died. o Cultured bacteria nutrients. o Inject sample healthy animal – sick + died. o Isolated bacteria blood. o Compare originally isolated bacteria. o 2 cultures, same bacteria. o Bacterium caused anthrax. - Koch’s postulates o Criteria used to determine causative agent of infectious diseases. o Relate specific microbe to specific disease.  o Same criteria invaluable investigations proving specific microorganism cause many diseases. Vaccine Edward Jenner (1796) - Inoculate cowpox virus – immune smallpox. - Importance: o Protection from disease provided by vaccination (recovering from disease itself) – immunity. - Years after – Pasteur discovered why works. o Microorganisms with decreased virulence – induce immunity infrctions by virulent counterparts. Derived Latin - Vacca = cow. The birth of modern chemotherapy (“Magic bullet”) Medical microbiologists search substance destroy pathogenic microorganism without damage infected. Chemotherapy - Treatment of disease with chemical substances. - Prepared chemicals in lab – synthetic drugs. Antibiotics - Antimicrobial agent, usually produced naturally bacterium/fungus. First synthetic drugs Quinine tree bark – malaria. Paul Ehrlich - 1st chemotherapy revolution. - “Magic bullet” o Destroy pathogen without harm host. - 1910: Developed synthetic arsenic drug o Salvarsan – treat syphilis. - 1930: Researchers developed several synthetic drugs destroy microorganisms o Most – derivatives dyes.  o Sulfonamides synthesized. Antibiotics Alexander Fleming (1928) - Discovered by accident. o Clear area bacterial growth inhibited encircled mold. o Mold inhibited growth bacterium. o Mold: Penicillium chrysogenum. o Mold’s active inhibitor: penicillin. - Penicillin o Antibiotic produced by fungus. o Killed S. aureus. - 1940s: Penicillin clinically tested and mass-produced. Problems: - Damage infected host. - Toxicity to humans. - Viral growth depend life processes normal host cells. - Few successful antiviral drugs. - Resistance. Beneficial activities of Microorganisms 1. Recycling vital elements. 2. Sewage treatment: Using microbes to recycle water. 3. Bioremediation: Using microbes to clean up pollutants. 4. Insect pest control by microorganisms 5. Biotechnology and recombinant DNA technology Recycling vital elements Microbial ecology. Microorganisms convert C, N, O, S and P into forms that plants and animals can use. Bacteria + fungi return CO2 to atmosphere when decompose organic wastes. Bacteria convert atmospheric N. Sewage water treatment: Using microbes to recycle water Sewage treatment plant remove undesirable materials + harmful microorganisms. Physical processes with beneficial microbes. Large solids removed – left behind liquid and organic materials that bacteria covert into by products as 𝐶𝑂2 , nitrates, phosphates, sulfates, ammonia, hydrogen sulfide and methane. Bioremediation: Using microbes to clean up pollutants and toxic waste Industrial processes. Bacteria - Some use pollutants as energy sources. - Other produce enzymes break down toxins into less harmful substances. Bioremediation: Use of microbes to remove an environmental pollutant.  Toxins removed from: - Underground wells. - Chemical spills. - Toxic waste sites. - Oil spills. Bacterial enzymes drain cleaner: Remove clogs, no harmful chemicals environment. Indigenous to environment or genetically modified microbes. Insect pest control by microorganisms Important agriculture + prevention human disease. Microbial control – prevent farmers from harming environment. Biotechnology and recombinant DNA technology Biotechnology: - Industrial application of microorganisms, cells, or cell components to make a useful product. Revolution – advent recombinant DNA technology - Expand potential bacteria, viruses, yeast + other fungi as miniature biochemical factories. Application recombinant DNA - Produce natural proteins, vaccines, and enzymes. - Gene therapy – inserting missing gene/replace defective gene human cells. o Virus used carry missing/defective gene into host cell. o Gene picked up + inserted appropriate chromosome. - Agriculture. Study Unit 2 Observing Microorganisms through a Microscope (Microscopy) Pierce, Chapter 3 (p 77 - 97) Units of Measurement Metric system. 1000 nm = 1 μm Micrometers (μm) = 10−6 m. 0.001 μm = 1 nm Nanometers (nm) = 10−9 m. Abbreviation Prefix m k kilo 103 d deci 10−1 c centi 10−2 m milli 10−3 𝛍 micro 10−6 n nano 10−9 p pico 10−12 Light Microscopy (LM) Visible light. Types - Bright field LM. - Dark field LM. - Phase-contrast microscopy. - Differential interference contrast (DIC). - Fluorescence Microscopy. - Confocal Microscopy. Compound Light Microscopy Instrument with two sets of lenses that uses visible light as a source of illumination. Image from objective lens magnified again by ocular lens. Total magnification = objective lens × ocular lens Resolution - Ability lenses distinguish two points that are a specific distance apart. o Resolving power 0.4 nm – distinguish two points at least 0.4 nm apart. - Shorter wavelengths light – better resolution. o Resolve structures ≥ 0.2 μm. - Resolving power 0.2 nm – distinguish points ≥ 0.2 nm. Clear, finely detailed image – specimen contrast sharply with medium – refractive index. Refractive index - Measure light-bending ability medium. - Change refractive index specimen by staining. - Immersion oil o Placed between glass slide and immersion objective lens. o Keep light from bending. o Preserve direction of light rays highest magnification. o Light may bend air so much miss small high-magnification lens.  Microscopes Light Microscopy (LM) Electron Microscopy (EM) Visible light to observe specimens. Electrons instead of light. Shorter wavelength electrons give greater resolution ( 0.22 0.3 µm. c) Low temperature Inhibits microbial growth (bacteriostatic effect). Effect depends on microbe + intensity application. Refrigeration Deep-freezing Lyophilization Temp normal Slow freezing more refrigerators – metabolic harmful to bacteria. rate most microbes so Ice crystals form + grow reduced they cannot disrupt cellular + reproduce/synthesize molecular structure toxins. bacteria. - Psychrotrophs do Thawing grow. - More damaging than - Pathogenic bacteria freeze-thaw cycle. will not grow. ▪ Listeria exception. d) High pressure Pressure high enough – alters molecular structure proteins + carbohydrates – rapid inactivation vegetative bacterial cells. Denatures proteins. Endospores relatively resistant. Advantage: - Preserve flavors, colors, and nutrient values products. e) Desiccation Removal of water. Organism cannot grow/reproduce – remain viable for years. - Water available – resume growth + division. - Prevents metabolism. - Principle underlies -lyophilization/freeze-drying. Preserve microbes. Viruses resistant. Endospores VERY resistant – survived for centuries. f) Osmotic pressure Use of salts + sugars preserve food. Hypertonic environment – water leave microbial cell - causes plasmolysis. Deny cell moisture needed for growth. g) Radiation Varying effect depending wavelength, intensity + duration. Kills microorganisms. Ionizing radiation (X rays, gamma rays, electron beams) - Ionizes water to release OH. - Kills organisms by reacting with organic cellular components - damages DNA. Nonionizing radiation (UV, 260 nm) - Longer wavelength than ionizing radiation. - Damages DNA of exposed cells – cause bonds to form between adjacent pyrimidine bases. o Thymine dimers inhibit correct replication DNA during cell reproduction.  Microwaves - Kill by heat - Not especially antimicrobial. Chemical methods of microbial growth Both living tissue + inanimate objects. Few achieve sterility. - Most merely reduce microbial populations to safe levels/remove vegetative forms pathogen obj. Principles of effective disinfection Concentration of disinfectant. Organic matter. pH. Time. Evaluating a disinfectant Use-dilution test Method determining effectiveness disinfectant using serial dilutions. Glass metal rings dipped in test bacteria are dried. Dried cultures are placed in disinfectant (specific concentration) for 10 min at 20°C. Rings transferred to culture media (incubated) to determine whether bacteria survived treatment. Number cultures grow – effectiveness disinfectant. Disc diffusion test (Kirby-Bauer test)  Agar-diffusion test to determine microbial susceptibility to chemotherapeutic agents. Disk of filter paper soaked with chemical + placed on agar plate previously inoculated + incubated with test organism. After incubation – chemical is effective, clear zone representing inhibition of growth can be seen around disk. Types of disinfectants a) Phenol and phenolics Disrupt plasma membranes. Phenol - Control surgical infections operating room. - Throat lozenges – local anesthetic effect. - Concentration above 1% - phenol has significant antibacterial effect. Phenolics - Molecule of phenol chemically altered reduce irritating qualities + increase antibacterial activity in combination with soap or detergent. - Antimicrobial activity – injuring lipid-containing plasma membranes – cellular contents leak. - Disinfecting pus, saliva and faeces. b) Halogens Broad spectrum activity. Effective antimicrobial agents. Iodine - Oldest + most effective antiseptic. - Impairs protein synthesis + alters cell membranes. - Tinctures – in aqueous alcohol. - Iodophors – In organic molecules. - Betadine. Chlorine - Used in drinking and recreational water, restaurant eating utensils, household disinfectants (bleach), water purification tablets. c) Alcohols Ethanol, isopropanol - Denatures proteins, dissolve lipids. - Require water (e.g. 70% ethanol). - Effective against bacteria and fungi but not endospores and enveloped viruses. - Alcohol hand sanitizers – how effective? o Claims to kill 99.9% of germs – this effectiveness is seldomly reached under typical user’s conditions.  o Some pathogens + viruses lack lipid envelope – resistant to alcohol-based hand sanitizers. d) Heavy metals Ag, Hg, and Cu - Silver nitrate may be used to prevent gonorrheal ophthalmia neonatorum. - Silver sulfadiazine used as tropical cream on burns. - Copper sulfate is an algicide (destroys green algae). Oligodynamic action - Ability of small amounts of a heavy metal compound to exert antimicrobial activity. - Denatures proteins. e) Surface-active agents – Surfactants Soap Degerming Acid-anionic detergents Sanitizing Quaternary ammonium compounds (cationic Bactericidal, denatures proteins, disrupts plasma detergents) membrane (also fungicidal and virocidal). f) Aldehydes Inactivates proteins by cross-linking with functional groups (-NH2, -OH, -COOH, -SH). Use: Medical equipment: - Glutaraldehyde, formaldehyde, and orthophthalaldehyde. g) Gaseous sterilants Denatures proteins. Use: Heat-sensitive material - Ethylene oxide. Resistance of microbes to chemical biocides Prions Most resistant Endospores of bacteria Mycobacteria Cysts of protozoa Vegetative protozoa Gram-negative bacteria Fungi - including most fungal spores Viruses without envelopes Gram-positive bacteria Viruses with lipid envelopes Least resistant Microbial characteristics and microbial control External lipopolysaccharide layer gram-negative bacteria - Biocides more effective against gram-positive bacteria. Porins (structural openings in wall of gram-negative bacteria) - Resistance to chemical antimicrobials. Cell wall - Waxy, lipid-rich components. Study Unit 6 Antimicrobial drugs Pierce, Chapter 20 (p 584 - 615) Antimicrobial drugs - Interfere with the growth of microbes within a host. The history of chemotherapy Paul Elrich - “magic bullet” – would selectively find and destroy pathogens without harm to the host. - Selective toxicity o The property of some antimicrobial agents to be toxic for a microorganism and nontoxic for the host. - Salvarsan. - Coined term “Chemotherapy” o Treatment is disease with chemical substances. Alexander Flemming (1928) - Discovered Penicillin. - Produced by Penicillium. - Inhibitory reactions o Mechanism of inhibition – antibiosis. - Antibiotic o A substance produced by a microbe that, in small amounts, inhibits another microbe. Antibiotic use and discovery today Microbes producing the most antibiotics - Streptomyces (> 50% of antibiotics). - Bacillus. - Penicillium. - Cephalosporium. Spectrum of antimicrobial activity Problems with chemotherapy Easier to find drugs against prokaryotes than eukaryotes. - Why? o Eukaryotes resembles the host cell and the drug causes damage. o Drug targets these pathogens usually also damage host as well. Viruses - Inside cells and used the machinery of the host cell. - Takes over, directing the human cell to make viruses rather than cellular material. - Genetic information virus is directing human cell to make viruses rather than to synthesize normal cellular materials. The spectrum of antimicrobial activity Narrow spectrum.  Broad spectrum antibiotic. - An antibiotic that is effective against a wide range of both gram-positive and gram-negative bacteria. Primary factor involved in selective toxicity of antibacterial action: - Lipopolysaccharide outer layer gram-negative bacteria. - Porins form water-filled channels across this layer. Identity pathogen not immediately known – use broad spectrum: - Advantage: o Treat disease by saving time. - Disadvantage: o Destroy normal microbiota. o Opportunistic pathogens - Superinfection ▪ The growth of a pathogen that has developed resistance to an antimicrobial drug being used; the growth of an opportunistic pathogen. The action of antimicrobial drugs Antimicrobial drugs Based on mode of action Based on spectrum Bacteriostatic Bacteriocidal Broad spectrum Narrow spectrum Prevent microbes from growing. Host's own defebces (phagocytosis Kills microbes directly. and antibody production) destroys microorganism.  Inhibition of protein synthesis The 5 modes of action of antimicrobial drugs Protein synthesis common feature all cells (prokaryotic and eukaryotic). Difference prokaryotic and eukaryotic – structure of ribosomes. - Eukaryotic cells: 80S. Inhibitors of cell wall synthesis - Prokaryotic cells: 70S. Call wall bacteria – peptidoglycan. - Accounts selective toxicity antibiotics affect protein synthesis. Penicillin + other antibiotics prevent synthesis intact - Mitochondria (eukaryotic organelle) also has 70S ribosomes. peptidoglycan. o Antibiotics targeting 70S ribosomes – adverse effect on - Cell wall weakened. host cells. - Cell undergoes lysis. Antibiotics: - Only actively growing cells affected. - Chloramphenicol, erythromycin, streptomycin, and the - Little toxicity human cells (no peptidoglycan). tetracyclines Inhibiting the synthesis of essential metabolites/Competitive inhibitors Enzymatic activity microorganisms can be completely inhibited by substance (antimetabolite) closely resemble normal substrate enzyme. Sulfonamides (sulfa drugs) - Inhibit folic acid synthesis. - Broad spectrum. Inhibiting nucleic acid synthesis - Structurally similar to folic acid Antibiotics interfere with processes precursor – PABA (para-aminobenzoic of DNA replication and acid). transcription in microorganisms. - Competes to bind enzyme meant for Block bacterial topoisomerase or PABA. Injuring the plasma membrane RNA polymerase. Certain antibiotics (especially polypeptide antibiotics) brings about changes in Rifamycin permeability of plasma membrane. - Inhibits mRNA synthesis. - Loss of important metabolites microbial cell. - Antituberculosis and leprosy. - Disrupt both inner and outer membranes gram-negative bacteria. Quinolones and fluoroquinolones Lipopeptides - Inhibits DNA gyrase - DNA Know mechanism and antibiotic. - Structural changes in the membrane. replication. - Gram positive. - Urinary tract infections, Polymyxin B pneumonia. - Effective against Gram negative bacteria. - Tropical (non-prescription antiseptic ointments). - Combined with Bacitracin and Neomycin in over-the-counter preparation. Common antimicrobial drugs Antibacterial antibiotics: Inhibitors of cell wall synthesis Magic bullet - Must target microbial structures/functions not shared mammalian structure/function. - Usually cell wall. 1. Penicillin Penicillin - A group of antibiotics produced either by Penicillium (natural penicillin) or by adding side chains to the -lactam ring (semisynthetic penicillins). All penicillins have common core structure: - Contain -lactam ring – nucleus. - Types differentiated by chemical side chains attached to nuclei. Prevent cross-linkage of peptidoglycans. - Interfere final stage synthesis. - Gram-positive bacteria. Produces naturally or semisynthetically. Natural penicillins Synthetic penicillins Penicillins extracted from cultures of Developed in an attempt to overcome Penicillium fungi. disadvantages natural penicillins. Narrow spectrum. Scientists develop in one of two ways: Staphylococci, streptococci and 1. Interrupt synthesis molecule by spirochetes. Penicillium and obtain only common Disadvantages: penicillin nucleus for use. - Narrow spectrum of activity. 2. Remove side chains from completed - Susceptible to penicillinases. natural molecule and chemically add - Penicillinases are enzymes produced other side chains to make more resistant by bacteria – cleave -lactam ring. to penicillinase. Term “synthetic” - Part of penicillin is produced by mold, other part is added synthetically. Penicillin G Penicillin V Oxacillin Ampicillin Requires injection. Can be taken orally – Narrow spectrum. Extended spectrum. Easily excreted from stable in stomach acid. Only gram-positive Many gram-negatives. body (3-6 hours) Narrow spectrum. Resistant to (TEXTBOOK VALUES, p Susceptible to penicillinase. 593). penicillases. Stomach acidity reduces effectiveness – not taken orally. Narrow spectrum.  2. Cephalosporins Structure – nuclei cephalosporins resemble penicillin. Inhibit cell wall synthesis same way as penicillin. Grouped according to generation reflecting continued development. 1st generation: - Narrow spectrum. - Act against gram-positive bacteria. 2nd generation: - Extended spectrum. - Includes gram-negative bacteria. 3rd generation: - Includes pseudomonads. - Injected. 4th generation: - Oral. 3. Polypeptide antibiotics Vancomycin - Very narrow spectrum of activity. - Inhibition of cell wall synthesis. - Glycopeptide. - Streptomyces. - Important “last line: against antibiotic-resistant Staphylococcus aureus (MRSA). o Widespread use – vancomycin-resistant enterococci (VRE). o Medical emergency. 4. Antimycobacterial antibiotics Cell wall Mycobacterium differs cell wall most other bacteria. - Incorporates mycolic acids (staining properties – acid-fast). - Pathogens: Leprosy and tuberculosis. Isoniazid (INH) - Inhibits mycolic acid synthesis. - Very effective synthetic antimicrobial drug against Mycobacterium tuberculosis. - Little effect nonmycobacteria. - Treat tuberculosis o Administered with other drugs – minimize development drug resistance. Tests to guide chemotherapy Different microbial species and stains have different degrees of susceptibility to chemotherapeutic agents. Susceptibility changes over time. Know sensitivity pathogen before treatment can start. Several tests can be used to indicate which chemotherapeutic agent most likely combat specific pathogen. - Organism identified – drugs can be selected without specific testing for susceptibility.  - Tests only necessary when susceptibility not predictable or antibiotic resistance problems develop. Practical, Textbook p 604 Diffusion methods Disk diffusion (Kirby-Bauer test) E-test (MIC: Minimal inhibitory concentration) Disk-diffusion method (Kirby-Bauer test) More advanced method. - An agar-diffusion test to determine E-test microbial susceptibility to - An agar diffusion test to determine chemotherapeutic agents. antibiotic sensitivity using a plastic strip Most widely used. impregnated with varying Not necessarily the best. concentrations of an antibiotic. Petri plate containing agar medium Enables lab technician to estimate the inoculated uniformly over entire surface minimal inhibitory concentration (MIC) – with standardized amount of test organism. lowest antibiotic concentration that Filter paper disk impregnated with known prevents visible bacterial growth. concentration chemotherapeutic agent Plastic strip contains gradients of antibiotic placed solidified agar surface. concentrations and MIC can be read from Incubation – chemotherapeutic agent scale printed on the strip. diffuse from disk to agar. Further agent diffuses from disk, lower concentration. Chemotherapeutic agent effective – zone of inhibition forms around disk after standardized incubation. Diameter zone measured – larger zone, more sensitive microbe is to antibiotic. - Drug with poor solubility – zone of inhibition is smaller than for drug more soluble and diffused more widely. Diameter compared standard table. Results inadequate clinical purposes. Test simple and inexpensive. Zone of inhibition - The area of no bacterial growth around a antimicrobial agent in the disk-diffusion method. Minimal inhibitory concentration (MIC) - The lowest concentration of a chemotherapeutic agent that will prevent growth of the test microorganism. Broth dilution tests Weakness diffusion method - Doesn’t determine whether a drug is bactericidal and not bacteriostatic. Broth dilution test - Method for determining the minimal inhibitory concentration by using serial dilutions of an antimicrobial drug. Useful for determining - MIC o Determined by making a sequence of decreasing concentrations of the drug in the broth which is then inoculated with test bacteria. - Minimal bactericidal concentration (MBC) o The lowest concentration of chemotherapeutic agent that will kill test microorganisms. Wells don’t show growth (higher concentration than MIC) – cultured in broth/agar plate free of drug. Growth in broth – drug not bactericidal – MBC determined. Determining MIC and MBC important - Avoids excessive/erroneous use antibiotics. - Minimize chance toxic reactions large doses. Highly automated. Resistance to antimicrobial drugs Mechanisms of resistance Bacteria become resistant to chemotherapeutic agents. Variations mechanisms occurs. - Concern: Resistant mutants replace susceptible normal populations.  Mechanisms of drug resistance Prevention of penetration to the target site within the microbe Gram-negative bacteria. - Cell wall restricts absorption many molecules. -lactamase present in periplasmic space – antibiotic enters is degraded in periplasmic space before entering the cell. Enzymatic destruction or inactivation of the drug Antibiotics that are natural drugs. - Synthetic – less likely to be affected. Alteration of the drug’s target site Synthesis proteins involves movement ribosomes Rapid efflux (Ejection) of the antibiotic along strand messenger RNA. Proteins plasma membrane gram-negative Antibiotics inhibits protein synthesis. bacteria Modifications at this site, neutralize effect of - Pumps that expel antibiotics. antibiotics without altering cellular function. - Prevent reaching effective concentration. Resistance among all major classes antibiotics. Antibiotic misuse Misuse of antibiotics selected for resistance mutants. Misuse includes: - Using outdates and weakened antibiotics. - Using antibiotics for the common cold and other inappropriate conditions. - Using antibiotics in animal feed. - Failing to complete the prescribes regimen. - Using someone else’s leftover prescription. Prevention of antibiotic resistance Always finish the prescribes regimen. Never use leftover antibiotics. Avoid unnecessary prescription of antibiotics. Choice and dosage should be correct. Use specific antibiotics instead of broad spectrum. Effects of combinations of drugs Synergism - Occurs when the effect of two drugs together is greater than the effect of either alone. Antagonism – Active opposition - Occurs when the effect of two drugs together is less than the effect of either alone. - Competition among microbes. Future of chemotherapeutic agents Antimicrobial peptides. Phage therapy. Increased knowledge of the basic genetic structure of microbes. Study Unit 7 Classification of microorganisms Pierce, Chapter 10 (p 295 - 320) Taxonomy - The science of classification living organisms. - Objective: Establish relationship between one group of organisms and another and to differentiate them. - Provides common reference identifying organisms already classified. - Provides universal language of communication. Rapid sequencing of DNA - New insight into classification and evolution. - Third Golden Age of Microbiology. The study of phylogenetic relationships All species inventory (2001 – 2025) - Aim: Identify and record every species of life on Earth. - Have identified 1.7 million species. - Estimated that number of living species range from 10 to 100 million. Diverse organisms – many similarities. - Result of evolution, decent of common ancestor or natural selection (Charles Darwin). Taxonomy Taxonomy. - The science of the classification of organisms. - Provides universal names for organisms. - Provides reference for identifying organisms. o Protologue (detailed description). o Type material / Type strain. Put organisms into categories or taxa. - Taxa (singular: Taxon) o Subdivisions used to classify organisms (e.g. Domain, kingdom, phylum etc.). o Categories we put organisms in. Show degree of similarity. - Similarity due to relatedness – all organisms related through evolution. Systematics - The science of organizing groups of organisms into a hierarchy. Phylogeny - The evolutionary history of a group of organisms; phylogenetic relationships are evolutionary relationships. - Phylogenetics: o Grouping organisms according to common properties implies that a group of organisms evolved from a common ancestor. o Each species retains some characteristics of ancestor: ▪ Anatomy. ▪ Fossils. ▪ rRNA. Systematics and phylogeny is the study of the evolutionary history of organisms. - Hierarchy taxa reflect evolutionary/phylogenetic relationship.  Understanding microbial diversity ~ 350 BC Plants and animals. Aristotle. 1735 Kingdoms Plantae and Animalia. Carolus Linnaeus. 1857 Bacteria and Fungi put in the Kingdom Carl von Nägeli. Plantae – “Flora”. 1866 Kingdom Protista: Bacteria, protozoa, Ernst Haeckel. algae, and fungi. 1937 “Prokaryote” introduced for cells Electron microscope “without a nucleus”. 1959 Kingdom Fungi. 1968 Kingdom Prokaryotae proposed. Robert Murray. 1969 Kingdom Monera as part of 5 Robert Whittaker. kingdom. Prokaryotes placed in Kingdom Prokaryotae or Monera and eukaryotes the other four kingdoms. rRNA Sequencing 1978 Three domains: Eukarya, Bacteria and Carl Woese. Archaea. As biological sciences developed, a natural classification system (groups organisms based on ancestral relationships and allows us to see order in life) was sought. The three domains Discovery 3 cell types - Observations that ribosomes not the same in all cells. - Ribosomes present in all cells. - Comparing sequences nucleotides in ribosomal RNA different cells show 3 distinctly different cell groups. o Eukaryotes. o 2 different prokaryotes (Bacteria and Archaea). Carl Woese - Elevating 3 cell types level above Kingdom – Domain. - Archaea and Bacteria – own domain evolutionary tree. Three domains differ - rRNA. - Membrane lipid structure. - Transfer RNA molecules. - Sensitivity to antibiotics.  Archaea Bacteria Eukarya Cell type Prokaryotic. Prokaryotic. Eukaryotic. Cell wall Varies in composition. Contains peptidoglycan. Varies in composition. No peptidoglycan. Contains carbohydrates. Membrane lipids Composed of branched Composed of straight Composed of straight carbon chains attached carbon chains attached carbon chains attached to glycerol by ether to glycerol by ester to glycerol by ester linkage. linkage. linkage. First amino acid in Methionine. Formylmethionine. Methionine. protein synthesis Antibiotic sensitivity No. Yes. No. rRNA loop Lacking. Present. Lacking. Common arm of tRNA Lacking. Present. Present. Widely accepted scheme. - Domain Eukarya = kingdoms animals, plants and fungi - Domain Bacteria = pathogenic prokaryotes, nonpathogenic prokaryotes in soil and water as well as photoautotrophic prokaryotes. - Domain Archaea = prokaryotes do not have peptidoglycan in cell walls. o Extreme environments. o 3 major groups. ▪ Methanogens – strict anaerobes produce methane from carbon dioxide and hydrogen. ▪ Extreme halophiles – high concentrations salt. ▪ Hyperthermophiles – very high temperatures. Evolutionary relationships 3 domains Current research. rRNA analysis: - 3 cell lineages emerged 3.5 billion years ago. - Archaea (bacteria) and what will become nucleoplasm of eukaryotes. - 3 cell lines not isolated o Horizontal gene transfer. o Analysis complete genome – each domain shares genes all other domains. o Gene transfer also seen between eukaryotic hosts + prokaryotic symbionts. Fossils - Oldest: Prokaryotes.  - Eukaryotic cells evolved more recently. - Endosymbiotic theory: o Eukaryotic cells evolved from prokaryotic cells living inside one another as endosymbionts. o Similarities prokaryotic cells + eukaryotic organelles = evidence endosymbiotic relationships. Prokaryotic cells and eukaryotic organelles compared Prokaryotic cell Eukaryotic cell Eukaryotic organelles (Mitochondria and chloroplasts) DNA One circular; some Linear. Circular. two circular; some linear. Histones In archaea. Yes. No. First amino acid in Formylmethionine Methionine. Formylmethionine. protein synthesis (Bacteria). Methionine (Archaea). Ribosomes 70S. 80S. 70S. Growth Binary fission. Mitosis Binary fission. Original nucleoplasmic cell was prokaryotic. - Infoldings plasma membrane surrounded nuclear region. - Produce true nucleus. Nucleoplasmic cell provided original host in endosymbiotic bacteria developed into organelles. Phylogenetic tree Grouping organisms according to common properties. - Implies evolved from common ancestor. - Each species retain characteristic ancestor. Information from fossils (bones, shells, or stems). - Structures microorganisms not readily fossilized (some exceptions). - Prokaryotes – not available. o Phylogeny based other evidence. Genomes – group organisms into taxa. - Provide timeline. - Microorganisms don’t have fossil evidence. - Concept: Molecular clock o An evolutionary timeline based on nucleotide sequences in organisms. o Differences amino acids in hemoglobin among different animals. o Mutations accumulate constant rate.  ▪ Comparing between 2 organisms with expected rate of change – estimate 2 diverged from common ancestor. Classification of organisms Scientific nomenclature Nomenclature – naming of organisms. Classification – Placing organisms into groups of related species. - Lists of characteristics of organisms. Identification – Matching characteristics of an “unknown” organism to lists of known organisms. Cannot use common names - One name – many different organisms. - Misleading. - Different languages. Binomial - 2 names – Genus and species epithet. - Italicized/underlined. - Genus: Capitalized. - Species: lowercase. - Binomial nomenclature o The system of having two names (genus and species epithet) for each organism; also called scientific nomenclature. - Used worldwide. o Share knowledge effectively and accurately. - Taken from Latin. Rules of assignment International code of Zoological Nomenclature (protozoa and parasitic worms). International code of Nomenclature for algae, fungi and plants. International Committee on Systematics of Prokaryotes (Published in the Bacteriological code and a specific journal). International Committee on Taxonomy of Viruses. Taxonomic hierarchy Subdivisions. Developed by Linnaeus. Eukaryotic species - Group of closely related organisms that can interbreed. Species: Group of closely related organisms that breed among themselves. Genus: Species differ from each other but related by descent. Family: A taxonomic group between order and genus. Order: A taxonomic classification between class and family. Class: A taxonomic group between phylum and order. Phylum: A taxonomic classification between kingdom and class. Delightful King Philip Came Over Kingdom: A taxonomic classification between domain and phylum. From Greece Singing Domain: Taxonomic classification based on rRNA sequences. Classification of Prokaryotes Classification of Eukaryotes Classification of Viruses Taxonomic classification scheme 1969 Not composed of cells. - Bergey’s Manual of Systematic Bacteriology. - Simple eukaryotic organisms (unicellular) grouped - Anabolic machinery within living host cells - Classification schemes. as kingdom Protista. multiply. Divided 2 domains: - Eukaryotic organisms didn’t fit into other Viral genome direct biosynthesis inside host cell - Bacteria. kingdoms – places in Protista. - Some viral genomes incorporated into host - Archaea. Ribosomal RNA sequencing: genome. Classification based similarities rRNA nucleotide - Divide protists groups based descent common Ecological niche virus: sequences. ancestor. - Specific to host cell – more closely related to host Defined differently from eukaryotic species. - Organisms once classified as protists divided into than other viruses. - Cell division not tied to sexual conjugation. clades/genetically related groups. International Committee on Taxonomy of Viruses Prokaryotic species Fungi, plants and animals – kingdoms more complex - Viral species - A population of cells that share certain rRNA eukaryotic organisms. o A group of viruses sharing the same sequences, in conventional biochemical testing, it Kingdom: Fungi genetic information and ecological niche. is a population of cells with similar characteristics. - Unicellular yeasts, multicellular molds, Obligatory intracellular parasites. Members bacterial species indistinguishable each macroscopic species. - Viral genes carried genome other organisms. other, distinguishable other species. - Cells joined to form thin tubes – hyphae. - Record viral evolution. Culture - Develop from spores. Hypotheses origin of viruses: - Bacteria grown in a media. Kingdom: Plantae (plants) - Arose from independently replicating strands Clone - Mosses, ferns, conifers and flowering plants. nucleic acids (plasmids). - A population of cells arising from a single parent - Multicellular. - Developed from degenerative cells, through many cell. - Photosynthesis. degenerations, lost ability to survive - All cells identical. Kingdom: Animalia independently. - Some cases, not identical – strain - “Animals”: Sponges, worms, insects, vertebrates. - Coevolved with host cells. o Genetically different cells within a clone. - Multicellular. o Identified: Numbers, letter or names follow - Ingesting organic matter. species epithet. Strain - Genetically different cells within a clone. Type culture - Strain representing a species. Methods of classifying and identifying microorganisms Classification scheme - List of characteristics. - Means for comparison to aid in identification. Organism identified. - Placed in previously devised classification scheme. Microorganisms identified for practical purposes. Not necessarily identified by same techniques as classified. Identification: - Laboratory. - As few tests and procedures as possible. Identification methods Morphological characteristics - Useful for identifying eukaryotes. Differential staining - Gram staining, acid-fast staining. Biochemical tests - Determines presence of bacterial enzymes. Serological methods - Study of serum and immune responses (antibodies in serum). Molecular methods - Based on DNA, RNA or proteins present in microorganisms. - Widely used today Putting classification methods together Dichotomous keys Cladograms (phylogenetic trees) - Identification based on successive - Maps that show evolutionary questions. relationships among organisms. - Based on rRNA sequences. Study Unit 8 Principles of Disease and Epidemiology Pierce, Chapter 14 (p 419 - 448) Pathology, Infection and Disease Pathogens - A disease-causing organism. Pathology - The scientific study of disease. Etiology - The study of the cause of a disease. Pathogenesis - The manner in which a disease develops. Infection - The growth of microorganisms in the body. - Invasion or colonization of the body by pathogenic organisms. - May exist in the absence of detectable disease. - Presence of particular type of microorganism in a part of the body where it is not normally found. Disease - An abnormal state in which part or all of the body is not properly adjusted or is incapable of performing normal functions, any changes from a state of health. - Infection causes an abnormal state in which the body is not functioning normally. Few microorganisms are pathogenic. - Presence some = beneficial. Human microbiome Normal microbiota and the host Normal microbiota Transient microbiota The microorganisms that colonize a host The microorganisms that are present in an without causing disease. animal for a short time without causing a Establish more or less permanent residence. disease. Do not produce disease under normal Present several days to months then conditions. disappear. “Normal flora”. Microorganisms are localized. - Only colonize body sites that can supply appropriate nutrients. Relationship between normal microbiota and the host Normal microbiota benefit host. - Prevent overgrowth harmful microorganisms. Microbial antagonism (competitive exclusion) - Growth of some microbes prevent growth of other microbes. - Competition between microbes. - Normal microbiota protect the host through various mechanisms. o Competing for nutrients. o Producing substances harmful invading microbes. o Affect conditions (pH, available oxygen etc.). Balance upset - Disease. Symbiosis - The relationship between normal microbiota and the host. Symbiosis Symbiosis - The living together of two different organisms or populations. - At least 1 dependent of other. Commensalism - A type of symbiosis in which two organisms live in association and one is benefited while the other is neither benefited nor harmed. - One organism benefits and the other is unaffected. - E.g. o Staphylococcus epidermidis. o Corynebacterial. o Most microorganisms that make up normal microbiota. Parasitism - A symbiotic relationship in which one organism (parasite) exploits another (host) without providing any benefits in return.  - One organism benefits at the expense of the other. - Many disease causing bacteria are parasites. Mutualism - A type is symbiosis in which both organisms or populations are benefited. - Both organisms benefit. - E.g. o E. coli Relationships can change under certain conditions. Some normal microbiota are opportunistic pathogens. - A microorganism that does not ordinarily cause a disease but can become pathogenic under certain circumstances. - Don’t cause disease in normal habitat in healthy person. - Do cause disease in different environment. - E.g. o E. coli The etiology of infectious diseases How do we know a specific pathogen causes a specific disease? Diagnosis and effective treatment of an infection does not just depend on isolating an organism, but in establishing a plausible link between the laboratory findings, recognized syndromes and the patient’s clinical condition. Koch’s postulates Koch’s postulates - Criteria used to determine the causative agent of infectious diseases. - Provides framework for the study of etiology of any infectious disease. 1. The same pathogen must be present in every case of the disease (absent in non disease organisms). 2. The pathogen must be isolated from the diseased host and grown in pure culture. 3. The pathogen from the pure culture must cause the disease when it is inoculated into a healthy, susceptible laboratory animal. 4. The pathogen must be isolated from the inoculated animal and must be shown to be the original organism. Exceptions to Koch’s postulates Some pathogens can cause several disease conditions. Some pathogens cause disease only in humans. - Ethical considerations – cannot intentionally inoculate someone with an infectious agent. Some pathogens cannot be cultures. Disease symptoms can be linked to several pathogens. Classifying infectious diseases Symptom A subjective change in a body function that is felt Syndrome by a patient as a A specific group of result of a disease. signs and symptoms that Sign accompany a An objective disease. change due to a disease that a person can observe and measure. Diseases classified in terms of how they behave within host and within given population. Communicable disease - Any disease that can spread from one host to another. - Disease in which infected person transmits infectious agent. - Directly or indirectly. Contagious disease - A disease that is easily spread from one person to another. - Very communicable.  - Spread easily and rapidly from one person to another. Noncommunicable disease - A disease that is not transmitted from one person to another. - Caused by o Microorganisms that normally inhabit the body and occasionally produce disease. o Microorganisms reside outside body and produce disease only when introduced into the body. Occurrence of a disease Understand disease – understand occurrence. Incidence (frekwensie) - Fraction of a population that contracts a disease during a specific time. - Measure of the number of new cases if a characteristic that develop in a population in a specified time period. - Indicator of the spread of the disease. Prevalence (Voorkoms) - Fraction of a population having a specific disease at a given time, regardless of when it first appeared. - Proportion of a population who have a specific characteristic in a given time period, regardless of when they first developed the characteristic. - Takes into account old and new cases. - Indicator of how serious and how long a disease affects the population. E.g. - Incidence of AIDS in 2007 in the USA was 56 000 but the prevalence in 2007 was 1 185 000. Allows scientists to estimate the range of disease’s occurrence and tendency to affect some groups of people more than others. Frequency of occurrence Sporadic disease - A disease that occurs occasionally in a population. Endemic disease. - A disease that is constantly present in a certain population. - E.g. Common cold. Epidemic. - A disease acquired by many hosts in a given area in a short time. - E.g. Influenza. Pandemic. - An epidemic that occurs worldwide. Severity or duration of a disease Acute disease - Symptoms develop rapidly but only last for a short time. - E.g. Influenza. Chronic disease - Disease develops slowly but lasts a long time. - Body’s reaction may be less severe. - E.g. Tuberculosis. Subacute disease - Symptoms between acute and chronic. Latent disease  - Disease with a period of no symptoms when the causative agent (pathogen) is inactive but then becomes active to produce symptoms of the disease. Immunity of the population Rate disease/epidemic spread – determined by immunity of population. Vaccinations - Long-lasting or lifelong protection. - Protects enough individuals population prevent disease’s rapid spread to those in population not vaccinated – herd immunity. o The presence of immunity in most of a population. o Outbreaks limited to sporadic cases. o Not enough susceptible individuals support spread of disease to epidemic proportions. People immune – carry pathogen but not have disease. - Reducing occurrence disease. Immune individuals are barrier for the spread infectious agent. Extent of host involvement Local infection - Pathogens are limited to a small area of the body. Systemic (generalized) infection - An infection throughout the body. - Spread by blood or lymph. Focal infection - Systemic infection that began as a local infection. Sepsis - The presence of a toxin or pathogenic organism in blood and tissue. Septicemia (blood poisoning) - The proliferation of pathogens in the blood, accompanied by fever, sometimes causes organ damage. - Common example of sepsis. Bacteremia - A condition in which there are bacteria in the blood. Toxemia - The presence of toxins in the blood. - E.g. tetanus. Viremia - The presence of viruses in the blood. The state of the host resistance also determines the extent of infections Primary infection - An acute infection that causes the initial illness. Secondary infection - An infection caused by an opportunistic microbe after the primary infection has weakened the host’s defenses. Epidemiology The study of where and when diseases occur and how they are transmitted in populations. Epidemiologists - Determine the etiology of a disease.  - Identify other important factors concerning the spread of disease. - Assemble data and graphs to outline incidence of disease. The Centers for Disease Control and Prevention (CDC) - Collects and analyzes epidemiological information in the United States. Study Unit 9 Microbial mechanisms of pathogenicity Pierce, Chapter 15 (p 449 -470) Microbes don’t try to cause disease: - Microbial cells are getting food and defending themselves. When the balance between host and microbe is tipped in favour of the microbe, an infection or disease results. Learning these mechanisms of microbial pathogenicity is fundamental to understanding how pathogens are able to overcome the host’s defenses. Number of Portals of entry invading Penetration or Damage to host Portals of exit Mucous membranes microbes evasion of host cells Generally the same as Respiratory tract. Siderophores. the portals of entry for a Gastrointestinal defences given microbe: Capsules. Direct damage. tract. Toxins. Mucous Cell wall components. Genitourinary Endotoxins. membranes. Enzymes. tract. Exotoxins. Skin. Antigenic variation. Conjunctiva. Lysogenic conversions. Parental route. Adherence Invasins. Skin. Cytopathic effects. Intracellular growth. Parental route. How microorganisms enter a host Pathogenicity - The ability of a microorganism to cause disease by overcoming the defenses of a host. Virulence - The degree of pathogenicity of a microorganism. Portals of entry - The avenue by which a pathogen gains access the body. Portals of entry Mucous membranes - Respiratory. - Gastrointestinal. - Genital. - Conjunctiva. Skin - Hair follicles. - Sweat glands. Parental route (direct deposition beneath skin or membrane). Mucous membranes Access by penetrating mucous membrane lining respiratory tract, gastrointestinal tract, genitourinary tract and conjunctiva. Respiratory tract Gastrointestinal tract Genitourinary tract Easiest. In food and water via Sexually. Most frequently used. contaminated fingers. Microbes cause sexually Microbes inhaled into Most destroyed by HCl + transmitted infections nose or mouth in drops of enzymes in stomach or by (STIs): bile in small intestine.  moisture and dust Survive – cause disease. - Penetrate unbroken particles. Hepatitis A, typhoid fever, mucous membrane. Common cold, etc. - Others require cut or pneumonia etc. Pathogens eliminated by abrasion. faeces. HIV infections, genital Transmitted to other warts etc. hosts by contaminated food, water or fingers. Skin Important defense against disease. Unbroken – impenetrable. Access through openings skin (sweat gland ducts, hair follicles etc.) Conjunctiva - Delicate mucous membrane lines eyelids and covers white eyeballs. - Effective barrier. - Some diseases: o E.g. Conjunctivitis, trachoma, etc. Parental route Parental route - A portal of entry for pathogens by depositing directly into the tissues beneath the skin and mucous membranes. Punctures, injections, bites, cuts, etc. Enter body – don’t necessarily cause disease: - Occurrence disease depend several factors. E.g. HIV, hepatitis virus, bacteria causing tetanus etc. Preferred portal of entry Many pathogens have a preferred portal of entry. - Prerequisite to being able to cause disease. - Gain access through another portal – disease might not occur. Number of invading microbes Few microbes enter body – overcome by host’s defenses. Many microbes – disease. Likelihood disease increase as number of pathogens increase. 𝐈𝐃𝟓𝟎 - Infectious dose for 50% of a sampled population. - The number of microorganisms required to produce a demonstratable infection in 50% of the test host population. - Virulence of a microbe. - E.g. Bacillus anthracis Portal of entry 𝐈𝐃𝟓𝟎 Skin 10 – 50 endospores. Inhalation 10 000 – 20 000 endospores. Ingestion 250 000 – 1 000 000 endospores. 𝐋𝐃𝟓𝟎 - Lethal dose of 50% of the sampled population. - The lethal dose for 50% of the inoculated hosts within a give period.  - Potency of a toxin. - E.g. Toxins Toxin 𝐋𝐃𝟓𝟎 Botulinum 0.03 ng/kg. Shiga toxin 250 ng/kg. Staphylococcal enterotoxin 1 350 ng/kg. Adherence Almost all pathogens have some means of attaching themselves to host tissue at portal of entry. Adherence - Attachment of a microbe or phagocyte to another’s plasma membrane or other surface. Attachment pathogen and host is accomplished by means of a surface molecule on pathogen – adhesin or ligand – that binds to complementary surface – receptors – on host cells. - Glycocalyx: Streptococcus mutans. ▪ Attach to surface of teeth by glycocalyx. - Fimbriae: Escherichia coli. Majority if adhesins are glycoproteins or lipoproteins. Receptors on the host cell are typically sugars (mannose). Adhesins different strains same species can vary in structure. Microbes have the ability to come together in masses, cling to surfaces etc. - forming biofilms - E.g. o Dental plague on teeth. o Algae on wall of swimming pool. o Scum accumulated in shower door. - Forms where microbes adhere to particular surface that is moist and contains organic matter. - Adhere, secrete glycocalyx that further attaches bacteria to each other and surface. - Resist disinfectants and antibiotics. Penetration or evasion of host defenses How do bacteria overcome host defenses? Capsules Some bacteria make glycocalyx material that forms capsules around cell walls. - Increase virulence. Resists host’s defenses by preventing phagocytosis (process by which cells of body engulf and destroy microbes). Capsule prevent phagocytic cell from adhering to bacterium. Host can overcome this by producing antibodies against the capsule. - Encapsulated bacteria easily destroyed. E.g. Streptococcus pneumoniae. Cell wall components Cell wall bacteria contain chemical substances contribute to virulence. M protein resists phagocytosis. - A heat- and acid-resistant protein of streptococcal cell walls and fibrils.  - Streptococcus pyogenes. - Found cell walls and fimbriae. - Attachment bacterium to epithelial cells host and helps bacterium resist phagocytosis by white blood cells. - Increases virulence. Opa protein inhibits T helper cells. - A bacteria outer membrane protein; cells wilt Opa form opaque colonies. - Neisseria gonorrhoeae. Mycolic acid (waxy lipid) resists digestion by phagocytes. - Mycobacterium tuberculosis. Enzymes Virulence some bacteria aided by production extracellular enzymes and related substances. Chemicals digest materials between cells and form or digest blood clots etc. Coagulase - Bacterial enzyme that causes blood plasma to clot. - Coagulates fibrinogen in blood. - Fibrinogen converted by coagulase into fibrin (threads form blood clot). o Clot protect bacterium from phagocytosis and isolate other defences host. Kinases - An enzyme that removes a P from ATP and attaches it to another molecule. - A bacterial enzyme that breaks down fibrin (blood clots). - Digest fibrin clots. Hyaluronidase - An enzyme secreted by certain bacteria that hydrolyzes hyaluronic acid and helps spread microorganisms from their initial state of infection. - Hydrolyzes hyaluronic acid – polysaccharide holds together certain cells. - Tissue blackening infected wounds. Collagenase - An enzyme that hydrolyzes collagen. - Facsilitate spread of gas gangrene. - Break down protein collagen – connective tissue muscles etc. IgA proteases - Defence against adherence of pathogens to mucosal surfaces – IgA antibodies. - Pathogen ability to produce enzymes, IgA proteases, destroy antibodies. - Destroys IgA antibodies. Antigenic variation Adaptive immunity - Specific defensive response body to infection or antigen. - Presence antigens – body produce antibodies. o Bind to antigen and inactivate or target for phagocytosis. Antigenic variation - Changes in surface antigens that occur in a microbial population. - Body immune response against pathogen: o Pathogen already altered antigens and unaffected by antibodies. Penetration into the host cell cytoskeleton Microbes attach to host by adhesins. Interaction triggers signals in host cell that activate factors result entrance some bacteria. Mechanism provided by host cell cytoskeleton.  Invasins: - Surface protein produced by Salmonella, Typhimurium and Escherichia coli that rearranges nearby actin filaments in the cytoskeleton of a host cell. o Membrane ruffling – disruption of cytoskeleton of host cell. - Salmonella alters host actin to enter host cell. - Use actin to move from one cell to the next. o Listeria. Damage to host cells How do pathogens damage the host cells? Pathogen overcomes host’s defenses, can damage host cells in 4 basic ways: 1. Using host’s nutrients. 2. Cause direct damage in the immediate vicinity of the invasion. 3. Producing toxins, transported by blood and lymph, that damages sites far removed from the original site of invasion. 4. Introducing hypersensitivity reactions. 1. Using host’s nutrients: Siderophores. Iron needed to growth pathogenic bacteria. Concentration free iron in human body low. Pathogens secrete protein – Siderophore - Bacterial iron-binding proteins. - Take away iron from iron-transport proteins. - Bind to iron even more tightly. Alternative: - Pathogens that bind directly to iron-transport proteins and hemoglobin. - Toxins to kill cells to release iron. 2. Direct damage Direct damage as pat