Introduction to Microorganisms PDF

Introduction to Microorganisms Sean D. Reid, Ph.D. Microbiology and Immunology [email protected] Learning Objectives • Demonstrate knowledge of vocabulary relevant to microbiology. • Demonstrate an understanding of the medically important microbial pathogens. o Bacteria, viruses, fungi, parasites...

Introduction to Microorganisms Sean D. Reid, Ph.D. Microbiology and Immunology [email protected] Learning Objectives • Demonstrate knowledge of vocabulary relevant to microbiology. • Demonstrate an understanding of the medically important microbial pathogens. o Bacteria, viruses, fungi, parasites, prions • For the medically important microbial pathogens, demonstrate knowledge of: o Morphology, structure, basic genetics, and growth characteristics as discussed • Demonstrate an understanding of host-pathogen interactions including: o Commensalism, colonization, infection and disease • Demonstrate knowledge of the basics of microbial pathogenesis. o Differentiate between strict and opportunistic pathogens. o The infectious cycle Why is the study of microbial pathogens important? Beyond the immune system, what makes the difference? SANITATION (INFRASTRUCTURE) • Prevents exposure • Biggest impact • Prevents illnesses transmitted by WATER, food, direct contact VACCINATION • Prevents infection in exposed • Significant impact • Limited by R&D, biology ANTIBIOTICS • Cure infections • Significant impact • Losing effectiveness? Size of Microorganisms E. coli: ~1µm diameter, 2µm length 5 Bacteria (Prokaryotes) • Single cell organisms with both DNA and RNA, but no defined nucleus o Double stranded DNA organized into a circular chromosome – nucleoid. o Some carry plasmids – small DNA molecule that is separate from chromosomal DNA. • Most possess a cell wall which contains peptidoglycan. • Size is from 0.1–10 μm. • Possess 70S ribosomes. o Comprised of 50S and 30S subunits. o Are frequent drug targets. Classification Schemes for Bacteria Taxonomic Ranks Formal Rank Example Kingdom Division Class Order Family Genus Species Subspecies Prokaryotae Gracilicutes Scotobacteria Eubacteriales Enterobacteriaceae Escherichia coli O157H7 Only the rank of family, genus, species, and subspecies and are commonly used and usually just the genus and species. Anotherdeadmicrobiologistella shamelesslylookedoverii Upper case Lower case Source: Bergey’s Manual of Determinative Bacteriology, 9th ed. Criteria Used for Classification (Partial List) Phenotype (observable trait) Morphology- cell shape and size Staining reactions- especially Gram staining reaction Metabolism- biochemical reactions Growth characteristics including colony size, shape, and color Serologic reactions- antigen-antibody reactions Antibiotic sensitivity or resistance Phage typing- using bacteriophages to identify bacterial strains Spore forming Analytical Protein types- determined by gel electrophoresis Lipid and cell wall fatty acid content Genotype (molecular) DNA hybridization- to determine closeness of relationship between various bacteria DNA sequence and ribotyping PCR based assays Plasmid analysis Bacterial Classification Based on Morphology (Shape) Cocci Rods Filamentous Curved Spiral The cell shapes that occur among unicellular true bacteria. Coccus, rod, curved, spiral, and filamentous. • Often shapes derived from Greek. • Streptos: twisted, as in a twisted chain • Staphyle or staphulē: bunch of grapes Bacterial colony morphology varies widely. Bacterial normal flora can provide protection against or be a source of infection. Viruses • Viruses contain either DNA or RNA, and are incapable of propagation outside a living cell. o Obligate intracellular parasites. • Virion– complete virus particle • Phage– virus that infects bacteria • Capsid– outer protein coat that protects the viral nucleic acid • Single-stranded or doublestranded DNA or RNA in capsid o May take a variety of forms (e.g., circular, linear). • Size ranges from 20-300nm. Fungi • Eukaryotic cells that possess DNA and RNA, a defined nucleus and a cell wall (different from bacteria) o Unicellular- yeasts o Filamentous- molds o Dimorphic- both Yeast Filamentous mold Dimorphic Parasites • An organism that lives on and at the expense of another organism o Protozoa: complex unicellular organisms with a defined nucleus and other organelles o Helminths (worms): multicellular organisms o Members of both groups have complex lifestyles. Toxoplasma gondii A pair of adult schistosomes (worms) http://www.oculist.net/downaton502/prof/ebook/duanes/pages/v4/v4c046.html http://www.cab.unimelb.edu.au/cab_schisto.htm Prions • Prions (PrPC or PrPSC) are noncellular, infectious proteins. • Human cells make a normal protein (PrPC) encoded by the PRNP gene. • Mutations in PRNP result in an abnormally shaped protein, PrPSC. • PrPSC convert PrPC into PrPSC. • The abnormal protein accumulates and forms clumps that damage/destroy neurons creating microscopic sponge-like holes. • Outcome is neurologic disease. o Spongiform encephalopathies Interventional strategies against prion diseases Adriano Aguzzi, Markus Glatzel, Fabio Montrasio, Marco Prinz & Frank L. Heppner Nature Reviews Neuroscience 2, 745-749 (October 2001) Comparison of Medically Important Microbial Pathogens Characteristic Viruses Bacteria Fungi Parasites (Protozoa and Helminths) Cells No Yes Yes Yes Approximate diameter (μm) 0.002-0.2 1-5 3-10 (yeasts) 15-25 Nucleic acid DNA or RNA DNA and RNA DNA and RNA DNA and RNA Nucleus None Prokaryotic Eukaryotic Eukaryotic Ribosomes Absent 70S 80S 80S Mitochondria Absent Absent Present Present Outer surface Protein capsid Rigid wall w/ and lipoprotein peptidoglycan envelope Rigid wall w/ chitin Flexible membrane Motility None Some None Most Replication Not binary fission Binary fission Budding or mitosis Mitosis Host-Pathogen Interactions Vocabulary changing (Infect Immun. 68:6511-6518) • Pathogen: A microorganism capable of producing pathology (disease) • Pathogenicity: The ability to cause disease • Infection: Invasion of the host by microorganisms which may or may not lead to disease • Virulence: A quantitative measure of pathogenesis o ID50 infectious dose: the number of organisms (or amount of a toxin) required to produce an infection in 50% of the test animals o LD50 lethal dose: the number of microorganisms (or amount of a toxin) required to kill 50% of the test animals • Virulence factor: Microbial product that permits a pathogen to cause disease Vocabulary: Classifications of bacterial infections Medical Microbiology. 4th edition. Baron S, editor. University of Texas Medical Branch at Galveston; 1996. Key Concept Disease occurs from a combination of microbial and host response. Disease occurs from a combination of microbial and host responses. The two primary determinants of infectious disease outcome are the status of the host immunity and the virulence properties of the pathogen. host microbe microbe host The degree of host damage is a better way to classify infections (current thinking). A. Acquisition followed by elimination (physical defenses or immunity). B. Acquisition may result in damage and disease in certain hosts. C. Commensal microbes can cause disease if commensalism is disturbed by immune impairment or alterations in host microbial flora. D. Colonization may be terminated by an immune response. E. Colonization may lead to disease if sufficient damage ensues. Source: Infection and Immunity, 68:6511-6518. F. Colonization may lead to a state of persistence (immune response not able to eradicate infection). G. An immune response or therapy may eradicate the infection, but the damage may be irreversible. H. If sufficient damage is incurred death may result. I. Persistent infections may reactivate and cause overt disease. Steps in the Infectious Cycle of Most Pathogens Step Requirement Entry/Attachment Enter the host, move to a surface, attach to a surface. Local or general spread Evade immediate local defenses. Replication/Colonization Increase bacterial numbers. Evasion of host defenses Evade immune and other defenses long enough for the full cycle in the host to be completed. Damage (pathology/disease) Not strictly necessary but often occurs. Shedding from body (transmission) Leave body at a site and on a scale that ensures spread to fresh hosts. Most Common Portals of Entry for Pathogenic Microorganisms a. Respiratory tract (Streptococcus pneumoniae, Bordetella pertussis) b. Gastrointestinal tract (Salmonella, E. coli, Vibrio cholerae) c. Skin and mucous membranes (Staphylococcus aureus, Streptococci) d. Genitourinary tract (Neisseria gonorrhoeae, Chlamydia) e. Direct inoculation o o o o Insect borne (vector), e.g. Rickettsia Opportunistic - e.g. surgery or indwelling device Cuts and scrapes Fomites Bacteria exhibit specificity (tropism) during attachment. Bacterium Tissue Corynebacterium diphtheriae Pharynx Neisseria gonorrhoeae Urogenital epithelium Streptococcus mutans Tooth surfaces Streptococcus salivarius Tongue surfaces Vibrio cholerae & Escherichia coli Small intestine epithelium Staphylococcus aureus Nasal membranes Staphylococcus epidermidis Skin • Molecular adhesins on microbes attach to surface receptors on host cells. • Adhesin-receptor interaction determines tissue specificity (tropism). • Most pathogens have multiple attachment mechanisms. • Often very visible to the immune system! Local or General Spread (Dissemination) of Bacteria • Host factors that contribute to dissemination o Movement of fluids (secretions, blood, lymph etc.) o Cellular trafficking o Localized inflammation that causes damage • Bacterial factors that contribute to dissemination o Motility • Swimming, swarming, twitching o Localized production of enzymes (toxins, proteases, DNases) Flagella are surface components that can mediate motility towards a surface and dissemination. Directional motility mediated by the flagellum is called chemotaxis. Swimming motility occurs in liquid and semisolid environments. E. coli are about 1 µm in diameter by 2 µm long and swim at about 30 µm/s. In human terms, this is 15 X ~6 ft = 90 ft/sec or ~60 miles/hr! Twitching motility is pilusmediated and allows bacteria to move along mucosal surfaces. •Twitching motility is a form of solid surface translocation. •Flagella independent •Occurs in a wide range of bacteria. o Pathogenic Neisseria o Moraxella o Pseudomonas •Twitching motility occurs via retraction and extension of the pilus (grappling hook model). Twitching motility is pilus-mediated and allows bacteria to move along mucosal surfaces. Microbiology, 1999, 145:2863 Toxin production by Streptococcus pyogenes allows dissemination. Immune Evasion Strategies of Pathogens Stealth Hide - capsules, biofilms Run - antigenic variation, modify surface “Just fit in” - molecular mimicry Frontal attack Kill - toxins Disarm - toxins, proteases, peptidase Invade Properties of Capsules •Usually high molecular weight but composed of polymerized simple sugars- glucose, mannose, galactose. •In rare cases can be protein or amino acids- e.g., Bacillus anthracis capsule is composed of the polymer D-glutamate. •Often expressed in vivo but not in vitro - may be phase variable. •Main functions: Prevent phagocytosis by PMN. Adherence and colonization. Prevent complement and Ab deposition. Prevent desiccation. Bacterial Biofilms: A Common Cause of Persistent Infections What is a biofilm? A collection of aggregated bacteria bound to a surface and embedded in a selfproduced polymeric matrix Why do bacteria form biofilms? Bacteria within biofilms are inherently resistant to antimicrobial agents: Phagocytic cells, antibiotics, ROS, chemical agents An Example of a Clinically Important Biofilm Dental caries Biofilm Sugars from diet provide nutrients for bacteria. Acid Damage to enamel. Tooth decay Inflammation Immune Evasion Strategies of Pathogens Stealth Hide - capsules, biofilms Run - antigenic variation, modify surface “Just fit in” - molecular mimicry Frontal attack Kill - toxins Disarm - toxins, proteases, peptidase Invade How Changes in DNA Sequence Lead to Antigenic Variation COOH NH2 Pilin protein of N. gonorrhoeae Conserved region Mechanism of antigenic variation for N. gonorrhoeae pilin. Antigenic variation occurs via homologous recombination between the expressed locus (pilE) and a silent locus (pilS). Variable region pilS (silent gene) N. gonorrhoeae chromosome Recombination Pilus (fimbriae) pilE (expressed gene) pilS (silent gene) N. gonorrhoeae chromosome pilE (expressed gene) New pilin protein (theoretically over a million different pilin subunits) COOH NH2 Antigenic variation of Neisseria gonorrhoeae pilin Conserved region Variable region Immune Evasion Strategies of Pathogens Stealth Hide - capsules, biofilms Run - antigenic variation, modify surface “Just fit in” - molecular mimicry Frontal attack Kill - toxins Disarm - toxins, proteases, peptidase Invade Molecular MimicryYou can’t kill what you don’t see! • Numerous bacteria either produce or decorate their cell surface with host-like proteins. o Fibronectin and other molecules of the extracellular matrix are common examples - deposited on numerous bacteria. o Capsule of Streptococcus pyogenes is made of hyaluronic acid. o Sialic acid is often found on the surface of Neisseria, Haemophilus. o Factor H binding (Lyme disease spirochetes). • Molecular mimicry can result in autoimmune disorders. Immune Evasion Strategies of Pathogens Stealth Hide - capsules, biofilms Run - antigenic variation, modify surface “Just fit in” - molecular mimicry Frontal attack Kill - toxins Disarm - toxins, proteases, peptidase Invade Immune Evasion Strategies of Pathogens •Bacterial toxins work by distinct mechanisms. •Some exported out of cell and some are directly injected. •Many target host immune cells. A. Damage membranes (pore forming toxins). B. Cleave host surface components. C. Modulate signal transduction pathways. D. Protein synthesis inhibitors. Key Concept Toxins inhibit host cells via distinct mechanisms. Some are cytotoxic (kill host cells), and some are cytotonic (alter cellular pathways but don’t kill cells). The targeted cells are often professional phagocytes. Organization of the Information - Make a Grid! Microbiology Features Name of Organism Clinical Features Vibrio cholerae Severe watery diarrhea •G- curved rod •Oxidase + •Motile •O1, O139 serotypes What disease? How does it cause disease? Which agent? Epidemiological Features Virulence/ pathogenesis Features Children/ adults in developing countries •Cholera toxin •TCP •Several other toxins Who is at risk? When are they at risk? Why does the organism cause disease? Treatment •Oral rehydration •Doxycycline •Limited vaccine effectiveness Is there a treatment?