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MCB2010 Lect Notes Unit One: History, Overview (Ch 1) and Classification (Ch 10) Microbes in Our Lives Microorganisms are organisms that are too small to be seen with the unaided eye. Germ - refers to a rapidly growing cell. Microorganisms: Decompose organic waste Are producers in the ecosystem by photosynthesis Produce industrial chemicals such as ethyl alcohol and acetone Produce fermented foods such as vinegar, cheese, and bread Microorganisms: Produce products used in manufacturing (e.g., cellulase) and treatment (e.g., insulin) A few are pathogenic, disease-causing Knowledge of microorganisms: Allows humans to Prevent food spoilage Prevent disease occurrence Led to aseptic techniques to prevent contamination in medicine and in microbiology laboratories. Naming and Classifying Microorganisms Linnaeus established the system of scientific nomenclature. Each organism has two names: the genus and specific epithet. Scientific names Are italicized or underlined. The genus is capitalized and the specific epithet is lower case. Are Iatinized?and used worldwide. May be descriptive or honor a scientist. Scientific names Staphylococcus aureus Describes the clustered arrangement of the cells (staphylo-) and the golden color of the colonies. Escherichia coli Honors the discoverer, Theodor Eshcerich, and describes the bacterium's habitat, the large intestine or colon. A Brief History of Microbiology Ancestors of bacteria were the first life on Earth. The first microbes were observed in 1673. The First Observations 1673-1723, Antoni van Leeuwenhoek described live microorganisms that he observed in teeth scrapings, rain water, and peppercorn infusions. Origin of micro-organisms The Debate Over Spontaneous Generation The hypothesis that living organisms arise from nonliving matter is called spontaneous generation. According to spontaneous generation, a vital force forms life. The Alternative hypothesis, that the living organisms arise from preexisting life, is called biogenesis. The Theory of Biogenesis – definitive proof. Pasteur's S-shaped flask kept microbes out but let air in. Fermentation and Pasteurization Pasteur demonstrated that these spoilage bacteria could be killed by Heat that was not hot enough to evaporate the alcohol in wine. This application of a high heat for a short time is called pasteurization. The Germ Theory of Disease Early indications 1835: Agostino Bassi showed a silkworm disease was caused by a fungus. 1865: Pasteur believed that another silkworm disease was caused by a protozoan. 1840s: Ignaz Semmelwise advocated handwashing to prevent transmission of puerperal fever from one OB patient to another. 1860s: Joseph Lister used a chemical disinfectant to prevent surgical wound infections after looking at Pasteur's work showing microbes are in the air, can spoil food, and cause animal diseases. 1876: Robert Koch provided proof that a bacterium causes anthrax and provided the experimental steps, Koch's postulates, used to prove that a specific microbe causes a specific disease. Koch's Postulates 1. Organism isolation from infected animal 2. Inoculation to healthy animal 3. Observation of similar disease 4. Isolation and comparison of causative agent with original organism Vaccination 1796: Edward Jenner inoculated a person with cowpox virus. The person was then protected from smallpox. The protection is called immunity Pasteur discovered attenuation of micro-organism to produce vaccines Attenuation = the process of making an organism non pathogenic but still immunogenic (able to induce immunity) Classification Taxonomy The study of degree of similarity Phylogeny The study of the evolutionary history of organisms Ch 10 Pp 276 - 285 Classification of Biological organisms Three domains Bacteria Archaea Eukarya Protists Fungi Plants Animals Taxonomic Hierarchy - Complexity of organisms arranged in decreasing order Species Definition Eukaryotic species: A group of closely related organisms that breed among themselves Prokaryotic species: A population of cells with similar characteristics Clone: Population of cells derived from a single cell Strain: Genetically different cells within a clone Viral species: Population of viruses with similar characteristics that occupies a particular ecological niche Domain Eukarya Animalia: Multicellular; no cell walls; chemoheterotrophic Plantae: Multicellular; cellulose cell walls; usually photoautotrophic Fungi: Chemoheterotrophic; unicellular or multicellular; cell walls Of chitin; develop from spores or hyphal fragments Protista: A catchall for eukaryotic organisms that do not fit other kingdoms Identification Methods Morphological characteristics: Useful for identifying eukaryotes Differential staining: Gram staining, acid-fast staining Biochemical tests: Determines presence of bacterial enzymes Serological: Combine known antiserum + unknown bacterium Slide agglutination ELISA Western blot Dichotomous Key - a flow chart type of identification key based on only two answers or criteria to each question (branch point). Bacteria Prokaryotes, single cell Peptidoglycan cell walls Binary fission Different shapes, common ones such as bacillus, coccus, spiral For energy, use organic chemicals, inorganic chemicals, or photosynthesis Archaea: Prokaryotic Lack peptidoglycan Live in extreme environments Fungi Eukaryotes Chitin cell walls Use organic chemicals for energy Molds and mushrooms are multicellular Yeasts are unicellular Protozoa Eukaryotes, unicellular Some parasitic May be motile via pseudopods, cilia, or flagella Algae Eukaryotes Cellulose cell walls Use photosynthesis for energy Produce molecular oxygen and organic compounds Viruses Acellular Consist of DNA or RNA core Viruses are replicated only when they are in a living host cell, obligate parasites Microbes and Human Welfare Microbial Ecology 1. Balance of biological and chemical environment. Waste breakdown - recycle of organic and inorganic substances Synthesis - photosynthesis; chemical synthesis 2. Sewage treatment - removal of undesirable materials and harmful microorganisms. 3. Bioremediation - cleaning up of pollutants and toxic wastes produced by human. 4. Bio-control of insects and pests. 5. Biotechnology - recombinant DNA technology; production of drugs, vaccines, gene therapy, crop improvement. 6. Cause diseases in plants and animals - only a minority. Micro Organisms (Ch. 12) Eukaryotes The Fungi Eukaryotic Aerobic or facultatively anaerobic Chemoheterotrophic Most are decomposers, some pathogenic Mycology is the study of fungi grow in acidic and high osmotic environment Fungus Characteristics 2 main types: filamentous (mold) and non-filamentous (yeast) colonies. Molds The fungal thallus consists of hyphae; a mass of hyphae is a mycelium. Yeasts Unicellular fungi, oval, facultative anaerobes Fission yeasts divide symmetrically Budding yeasts divide asymmetrically Dimorphism Pathogenic dimorphic fungi are yeastlike at 37 degrees C and moldlike at 25 degree C Filamentous fungus Structurally, 2 types: septa hyphae and coenocytic hyphae. Functionally, 2 types: vegetative hyphae and reproductive hyphae. Vegetative hyphae: serve to absorb nutrients, reproduce by fragmentation and grow by terminal elongation. Reproductive hyphae: produce spores for reproduction, 2 types: sexual and asexual spores. Fungal Life Cycle Asexual spores Sporangiosphore Conidiospore Arthrospore Blastoconidium Conidiospores Sexual reproduction Plasmogamy Haploid donor cell nucleus (+) penetrates cytoplasm of recipient cell (-) Karyogamy + and - nuclei fuse Meiosis Diploid nucleus produces haploid nuclei (sexual spores) Sexual spores Zygospore Fusion of haploid cells produces one zygospore Fungal Diseases (mycoses) Systemic mycoses - Deep within body Subcutaneous mycoses - Beneath the skin Cutaneous mycoses - Affect hair, skin, nails Superficial mycoses - Localized, e.g., hair shafts Opportunistic mycoses - Caused by normal microbiota or fungi that are normally harmless Economic uses Penicillium - production of antibiotics Food production - Saccharomyces, Taxomyces, Mushrooms, and other food spoilage species. The Algae Eukaryotic Unicellular, filamentous, or multicellular (thallic) Most are photoautotrophs Algae Phaeophyta Brown algae (kelp) Cellulose + alginic acid cell walls Multicellular Chlorophyll a and c, xanthophylls Store carbohydrates Harvested for algin Rhodophyta Red algae Cellulose cell walls Most multicellular Chlorophyll a and d, phycobiliproteins Store glucose polymer Harvested for agar and carrageenan Chlorophyta Green algae Cellulose cell walls Unicellular or multicellular Chlorophyll a and b Store glucose polymer Gave rise to plants Bacillariophyta Diatoms Pectin and silica cell walls Unicellular Chlorophyll a and c, carotene, xanthophylls Store oil Fossilized diatoms formed oil Produce domoic acid Dinoflagellata Dinoflagellates Cellulose in plasma membrane Unicellular Chlorophyll a and c, carotene, xanthins Store starch Some are symbionts in marine animals Neurotoxins cause paralytic shellfish poisoning Oomycota Water molds Cellulose cell walls Multicellular Chemoheterotrophic Produce zoospores Decomposers and plant parasites Phytophthora infestans responsible for Irish potato blight P. cinnamomi infects Eucalyptus P. ramorum causes sudden oak death Protozoa Eukaryotic Unicellular Chemoheterotrophs Vegetative form is a trophozoite Asexual reproduction by fission, budding, or schizogony Sexual reproduction by conjugation Trophozoite and/or cyst Protozoa primary consumers, some groups are photosynthetic, some obligate parasites. 7 Phylums, medically important ones are: Amoebas Flagellates Ciliophora Sporozoite Euglenozoa Archaezoa (flagellates) No mitochondria Multiple flagella Giardia lamblia Trichomonas vaginalis (no cyst stage) Rhizopoda (amoebas) Move by pseudopods Entamoeba Acanthamoeba Apicomplexa (sporozoite) Nonmotile Intracellular parasites Complex life cycles Plasmodium Babesia Cryptosporidium Cyclospora Plasmodium Ciliophora (ciliates) Move by cilia Complex cells Balantidium coli is a human parasite, causes dysentery Euglenozoa Move by flagella Photoautotrophs Euglenoids Chemoheterotrophs Trypanosoma Undulating membrane, transmitted by vectors Euglenozoa Eukaryote Microorganisms Arthropods as Vectors Kingdom: Animalia Phylum: Arthropoda (exoskeleton, jointed legs) Class: Insecta (6 legs) Lice, fleas, mosquitoes Class: Arachnida (8 legs) Mites and ticks May transmit diseases (vectors) Chapter 11 The Prokaryotes: Domains Bacteria and Archaea One circular chromosome, not in a membrane No histones No organelles Peptidoglycan cell walls Binary fission Domain Bacteria Proteobacteria presumed to have arisen from a common photosynthetic ancestor. Most are gm - chemoheterotrophs. 5 Groups. Alpha to Epsilon (alpha) Proteobacteria - Includes many agriculturally important species. Medically important ones are: Human pathogens: Rickettsia -transmitted through insect bites, cause spotted fevers Brucella -Brucellosis, obligate parasites of mammals (beta) Proteobacteria Neisseria N. meningitidis N. gonorrhoeae Bordetella (petusis) (gamma) Proteobacteria Pseudomonas - Opportunistic pathogens Azotobacter and Azomonas. - Nitrogen fixing Legionella - Found in streams, warm-water pipes, cooling towers L. pneumophilia –causes pneumonia in human Vibrio cholerae causes cholera Pasteurella - Cause pneumonia and septicemia Haemophilus (delta) Proteobacteria (epsilon) Proteobacteria Helicobacter - causes Peptic ulcers, Stomach cancer The Nonproteobacteria Gram-Negative Bacteria Cyanobacteria - Oxygenic photosynthesis, fix nitrogen Purple and green sulfur bacteria - Anoxygenic photosynthesis Chlamydiae - medically important ones: C. trachomatis - Trachoma, STD, urethritis Spirochaetes -with axial filaments Treponema pallidum, causes Syphilis Firmicutes Gram-positive Clostridiales Clostridium, Endospore-producing, Obligate anaerobes. Epulopiscium Bacillales - Bacillus, Endospore-producing rods Staphylococcus - Cocci Lactobacillales - Generally aerotolerant anaerobes, lack an electron-transport chain Lactobacillus Streptococcus Enterococcus Listeria Mycoplasmatales - Wall-less, pleomorphic M. pneumoniae causes pneumonia in human Actinobacteria - Gram-positive Actinomyces Corynebacterium diphtheriae causes Diphtheria in human Mycobacterium, TB and Leprosy bacteria belongs to this group Streptomyces Domain Archaea Methods of study (Ch 3) Units of Measurement 1 m = 10-6 m = 10-3 mm 1 nm = 10-9 m = 10-6 mm 1000 nm = 1 um 0.001 um = 1 nm Microscopy: The Instruments A simple microscope has only one lens. In a compound microscope the image from the objective lens is magnified again by the ocular lens. Total magnification = objective lens x ocular lens Resolution is the ability of the lenses to distinguish two points. A microscope with a resolving power of 0.4 nm can distinguish between two points 0.4 nm apart. The maximum resolution of a light microscope is 0.25 uM. Shorter wavelengths of light provide greater resolution. Refractive index is the light-bending ability of a medium. The light may bend in air so much that most of them miss the small area of the high-magnification lens. Immersion oil is used to keep light from bending. Electron Microscopy Uses electrons instead of light. The shorter wavelength of electrons gives greater resolution. Uses electromagnetic lens for focusing Transmission Electron Microscopy (TEM) 10,000-100,000 resolution 2.5 nm Stains - benzene derivatives called aniline or synthetic dyes. Each stain consist of 2 chemical groups Chromophore group - imparts color, contains unsaturated chemical bonds that absorb specific wavelengths of light. Auxochrome group - ionising radical, increase solubility of the dye and react with substrates. Stain Classification: based on chromophore group In a basic dye, the chromophore is a cation. e.g. Methylene blue, crystal violet, widely used to stain bacterial cells In an acidic dye, the chromophore is an anion. e.g. Eosin, nigrosine - staining the background instead of the cell and is called negative staining. Simple Stains Use of a single basic dye is called a simple stain. A mordant may be used in conjunction with stain to hold the stain or coat the specimen to enlarge it. Differential Stains Differentiate between different bacteria. Examples: Gram stain and Acid-fast stain. The Gram stain classifies bacteria into gram-positive and gramnegative. Acid-fast stain differentiate bacteria into acid fast and nonacid fast organisms. Differential Stains: Gram Stain Primary stain = Crystal Violet Mordant = iodine Decolorizer = 95% alcohol with or without acetone Counter stain = Safranin Acid fast stain Acid-fast stain – used for staining Mycobacterium sp. Acid-fast stain - carbolfuchsin driven into Cell by heat. Decolorizing agent - acid-alcohol, Carbolfuchsin is more soluble in waxy layer of cell wall of acid-fast organisms. Non-acid fast organisms do not have wax in cell wall to retain stain. Counter stain - methylene blue. Cannot stain acid fast organism but can stain the now empty non-acid fast organism. Differential Stains: Acid-Fast Stain Cells that retain a basic stain in the presence of acid-alcohol are called acid-fast. Non-Acid-fast cells lose the basic stain when rinsed with acid-alcohol, and are usually counterstained (with a different color basic stain) to see them. Special stains stain special structures such as Endospore and flagella stains. Negative stain: for capsule and negative staining Colloidal stain to stain background, simple stain to stain cell, Capsule remains unstained. Endospore stain: Heat is required to drive a stain into endospores. Flagella staining requires a mordant to make the flagella wide enough to see. Endospore stain Heat is required to drive a stain into endospores. Negative stain - staining background only Flagella stain - increase thickness of flagella Chapter 4 Prokaryotes and Eukaryotes Prokaryotic Cells Comparing Prokaryotic and Eukaryotic Cells Prokaryote comes from the Greek words for prenucleus. Eukaryote comes from the Greek words for true nucleus. Prokaryote One circular chromosome, not in a membrane No histones No organelles Peptidoglycan cell walls Binary fission Average size: 0.2 -1.0 um to 2 - 8 um Basic shapes: coccus, bacillus, spiral (vibrios, sprilla, spirochetes. Unusual shapes Star-shaped Square Arrangements Pairs: diplococci, diplobacilli Clusters: staphylococci Chains: streptococci, streptobacilli Glycocalyx Outside cell wall Usually sticky A capsule is neatly organized A slime layer is unorganized & loose Extracellular polysaccharide allows cell to attach Capsules prevent phagocytosis Flagella Outside cell wall Made of chains of flagellin Attached to a protein hook Anchored to the wall and membrane by the basal body Flagella Rotate flagella to run or tumble Flagella proteins are H antigens forms different serological groups (types of the same strain). (e.g., E. coli O157:H7) Motile Cells Axial Filaments Endoflagella In spirochetes Anchored at one end of a cell Rotation causes cell to move Other appendages Fimbriae allow attachment Pili-shorter than flagella, one or two per cell. Pili are used to transfer DNA from one cell to another Cell Wall Prevents osmotic lysis Made of peptidoglycan (in bacteria) Peptidoglycan Polymer of disaccharide N-acetylglucosamine (NAG) & N-acetylmuramic acid (NAM) linked by polypeptides Gram-positive cell walls Thick peptidoglycan Teichoic acids In acid-fast cells, contains mycolic acid Gram-Positive cell walls Teichoic acids: Lipoteichoic acid links to plasma membrane Wall teichoic acid links to peptidoglycan Polysaccharides provide antigenic variation Gram-Negative Outer Membrane Lipopolysaccharides, lipoproteins, phospholipids. Forms the periplasm between the outer membrane and the plasma membrane. Protection from phagocytes, complement, antibiotics. O polysaccharide antigen, e.g., E. coli O157:H7. Lipid A is an endotoxin. Porins (proteins) form channels through membrane Gram-Negative Outer Membrane - extra layer of LPS Gram Stain Mechanism Crystal violet-iodine crystals form in cell Gram-positive Alcohol dehydrates peptidoglycan CV-I crystals do not leave Gram-negative Alcohol dissolves outer membrane and leaves holes in peptidoglycan CV-I washes out Damage to Cell Walls Lysozyme digests disaccharide in peptidoglycan. Penicillin inhibits peptide bridges in peptidoglycan. Protoplast is a wall-less Gram-positive cell. Spheroplast is a Gram-negative cell with partially damaged cell wall. Protoplasts and spheroplasts are susceptible to osmotic lysis. Plasma Membrane Phospholipid bilayer Peripheral proteins-embedded protein on either side of Membrane, easily removable. Integral proteins-may be transmembrane, removable only by detergent disruption of the bi-layer. Transmembrane proteins Fluid Mosaic Model Membrane is as viscous as olive oil. Proteins move to function Phospholipids rotate and move laterally Plasma Membrane Selective permeability allows passage of some molecules such as small Molecules, lipid soluble molecules, gases. Contain enzymes for nutrient breakdown or energy production. Movement Across Membranes Passive processes Simple diffusion: Movement of a solute from an area of high concentration to an area of low concentration. Facilitative diffusion: Solute combines with a transporter protein in the membrane and being carried across the membrane. Osmosis-movement of water across membrane Osmosis Movement of water across a selectively permeable membrane from an area of high water concentration to an area of lower water. Osmotic pressure The pressure needed to stop the movement of water across the membrane. Active processes-transport of molecules against concentration gradient Active transport requires a transporter protein and ATP. Transporters are specific, there is no alternation to the transport material Group translocation-exclusively in Prokaryotes, transported material is chemically altered. e.g. glucose is transported as glucose phosphate in bacteria. Cytoplasm Cytoplasm is the substance inside the plasma membrane Nuclear Area Nuclear area (nucleoid)-contains a single, circular, double-Stranded DNA bacterial chromosome. Ribosomes - made up of 2 subunits, a larger one and a small one Large subunit - 50 S Small subunit - 30 S Together, they form a 70S ribosome Inclusions Metachromatic granules (volutin) Polysaccharide granules Lipid inclusions Sulfur granules Carboxysomes Gas vacuoles Magnetosomes Endospores Resting cells Resistant to desiccation, heat, freezing, drying and chemicals Bacillus, Clostridium Sporulation: Endospore formation Germination: Return to vegetative state Require special stain to reveal-Schaeffer-Fulton endospore stain. Eukaryotic Cells Comparing Prokaryotic and Eukaryotic Cells Prokaryote comes from the Greek words for prenucleus. Eukaryote comes from the Greek words for true nucleus. Flagella and Cilia Microtubules Tubulin 9 pairs + 2 arrangements Cell wall Plants, algae, fungi Carbohydrates Cellulose, chitin, glucan, mannan Glycocalyx Carbohydrates extending from animal plasma membrane Bonded to proteins and lipids in membrane Plasma Membrane Phospholipid bilayer Peripheral proteins Integral proteins Transmembrane proteins Sterols Glycocalyx carbohydrates Plasma Membrane Selective permeability allows passage of some molecules Simple diffusion Facilitative diffusion Osmosis Active transport Endocytosis Phagocytosis: Pseudopods extend and engulf particles Pinocytosis: Membrane folds inward bringing in fluid and dissolved substances Eukaryotic Cell Cytoplasm - Substance inside plasma membrane and outside nucleus Cytosol-Fluid portion of cytoplasm Cytoskeleton-Microfilaments, intermediate filaments, microtubules Cytoplasmic streaming-Movement of cytoplasm throughout cells Organelles Membrane-bound: Nucleus Contains chromosomes ER Transport network Golgi complex Membrane formation and secretion Lysosome Digestive enzymes Vacuole Brings food into cells and provides support Mitochondrion Cellular respiration Chloroplast Photosynthesis Peroxisome Oxidation of fatty acids; destroys H2O2 Eukaryotic Cell Not membrane-bound: Ribosome Protein synthesis Centrosome Consists of protein fibers and centrioles Centriole Mitotic spindle formation Nucleus Endoplasmic Reticulum Ribosomes 80S Membrane-bound Attached to ER Free In cytoplasm 70S In chloroplasts and mitochondria Golgi Complex Lysosomes Vacuoles Mitochondrion Chloroplast Endosymbiotic Theory Chapter 13 Viruses virus (Latin for poison) Contagium vivum fluidum (a living infectious fluid) A non-cellular entity which consists mainly of protein and nucleic acid (DNA or RNA), can replicate only after entry into specific type(s) of living cells. Virus 1. No metabolism and, few or no enzymes of their own. 2. No intrinsic motility. 3. Cannot grow on artificial lab media. 4. Do not respond to physical stimuli in their environment. Viruses Viruses contain DNA or RNA And a protein coat Some are enclosed by an envelope Some viruses have spikes Most viruses infect only specific types of cells in one host Host range is determined by specific host attachment sites(receptors) and cellular factors Types of Virus Class 1: Animal viruses Class 2: Plant viruses Class 3: Bacterial viruses (bacteriophages or phages) Viral Structure An infectious viral particle, a virion, is composed of nucleic acid (DNA or RNA) surrounded by a protein coat (Capsid). Nucleic Acid - A virus can have either DNA or RNA but never both. DNA may be: A. Single stranded, circular B. Single stranded, linear C. Double stranded, linear Viral Structure RNA may be: A. Single stranded, linear B. Double stranded, linear Viral Structure Capsid and Envelope The capsid (nucleocapsid) is composed of protein subunits called Capsomeres. Some capsidsare made-up of only one type of protein, in other viruses several types of protein may be found. Many viruses have an envelope, consists of combination of lipids, proteins, and CHO, an outer membranous layer surrounding the nucleoapsid. A virus without envelope is called naked virus or nonenveloped virus. In some viruses envelope may be covered by spikes. Viral Structure Spikes: 1. Are CHO and protein complex 2. 3. 4. Can be used as means of identification. Help viruses attach to host. Cause clumping of RBC called Hemagglutination. Helical Viruses Polyhedral Viruses Complex Viruses Growing Viruses Viruses must be grown in living cells. Bacteriophages form plaques on a lawn of bacteria. Animal viruses may be grown in living animals or in embryonated eggs. Animal and plants viruses may be grown in cell culture. Continuous cell lines may be maintained indefinitely. Virus Identification Cytopathic effects Serological tests Detect antibodies against viruses in a patient Use antibodies to identify viruses in neutralization tests, viral hemagglutination, and Western blot Nucleic acids RFLPs PCR Multiplication of Bacteriophages (Lytic Cycle) Attachment Phage attaches by tail fibers to host cell Penetration Phage lysozyme opens cell wall, tail sheath contracts to force tail core and DNA into cell Biosynthesis Production of phage DNA and proteins Maturation Assembly of phage particles Release Phage lysozyme breaks cell wall Lytic cycle Phage causes lysis and death of host cell Lysogenic cycle Prophage DNA incorporated in host DNA The lysogenic cells are immune to reinfection by the same type of phage. The lysogenic cells may exhibit new properties, such as resistance to antibiotics, production of toxins, and specialized transduction. The Lysogenic Cycle Specialized Transduction Multiplication of Animal viruses Attachment Viruses attaches to cell membrane Penetration By endocytosis or fusion Uncoating By viral or host enzymes Biosynthesis Maturation Release Production of nucleic acid and proteins Nucleic acid and capsid proteins assemble By budding (enveloped viruses) or rupture Attachment, Penetration, and Uncoating Release of an enveloped virus by budding Multiplication of DNA Virus Pathways of Multiplication for RNA-Containing Viruses Multiplication of a Retrovirus Cancer Activated oncogenes transform normal cells into cancerous cells. Transformed cells have increased growth, loss of contact inhibition, tumor specific transplant and T antigens. The genetic material of oncogenic viruses becomes integrated into the host cell's DNA. Oncogenic Viruses Oncogenic DNA Viruses Adenoviridae Heresviridae Poxviridae Papovaviridae Hepadnaviridae Latent Viral Infections Virus remains in asymptomatic host cell for long periods Cold sores, shingles Persistent Viral Infections Disease processes occurs over a long period, generally fatal Subacute sclerosing panencephalitis (measles virus) Prions Infectious proteins Inherited and transmissible by ingestion, transplant, & surgical instruments Spongiform encephalopathies: Sheep scrapie, Creutzfeldt-Jakob disease, Gerstmann-Stressler-Scheinker syndrome, fatal familial insomnia, mad cow disease PrPC, normal cellular prion protein, on cell surface PrPSc, scrapie protein, accumulate in brain cells forming plaques Chapter 5 Microbial Metabolism Metabolism is the sum of the chemical reactions in an organism. Catabolism¡V breakdown of complex substance, usually hydrolytic reactions, exogonic. Anabolism ¡V synthesis of complex substance from simple ones, usually endogonic. Microbial Metabolism Catabolism provides the building blocks and energy for anabolism. Almost all such biochemical reactions involve enzymes. Chemical reactions in a cell can be grouped into many pathways. A metabolic pathway is a sequence of enzymatically catalyzed chemical reactions in a cell. Metabolic pathways are determined by enzymes. Enzymes are encoded by genes. Chemical reactions The collision theory states that chemical reactions can occur when atoms, ions, and molecules collide. Activation energy is needed to disrupt electronic configurations. Reaction rate is the frequency of collisions with enough energy to bring about a reaction. Reaction rate can be increased by enzymes or by increasing temperature or pressure. Enzymes Biological catalysts Specific for a chemical reaction; not used up in that reaction Apoenzyme: protein Cofactor: Nonprotein component Coenzyme: Organic cofactor Holoenzyme: Apoenzyme + cofactor Important Coenzymes Important coenzymes in the biological systems. NAD+ NADP+ FAD Coenzyme Some metal ions are important cofactors. Enzymes Efficiency of enzymes are measured by turnover number = max number of substrate converted to product / sec The turnover number is generally 1-10,000 molecules per second. Enzyme Classification Enzymes are named ¡Vase, grouped into six classes Oxidoreductase Transferase Hydrolase Lyase Isomerase Ligase Oxidation-reduction reactions Transfer functional groups Hydrolysis Removal of atoms without hydrolysis Rearrangement of atoms Joining of molecules, uses ATP Factors Influencing Enzyme Activity Enzymes can be denatured by temperature and pH Factors Influencing Enzyme Activity Temperature - optimal temperature = 37C above the optimal, enzymes undergo denaturation at lower temperature reaction, rate decrease. Factors Influencing Enzyme Activity pH ¡V optimal pH Activities decrease above and below the optimal pH Factors Influencing Enzyme Activity Substrate concentration - within limits, enzymatic activity increases as substrate concentration increases until saturation Factors Influencing Enzyme Activity Inhibitors ¡V poisons, such as cyanide, arsenic and mercury Competitive inhibition - Competitive inhibitors ¡V a substrate look alike, compete with the normal substrate for the active site of the enzyme Reversible ¡V in and out of the active site, can be overcome by increasing substrate Irreversible ¡V irreversible binding of active site. Factors Influencing Enzyme Activity Factors Influencing Enzyme Activity oncompetitive inhibition Inhibitors act on other parts of the apoenzyme or on the cofactor (allosteric site) and decrease the enzyme¡¦s ability to combine with the normal substrate. Metabolic Pathway Metabolic pathway can involve multiple enzymes, each acting in successive step in a chain The product(s) from a step in the chain can serve as a substrate for the next reaction in the chain Factors Influencing Enzyme Activity Feedback inhibition occurs when the end product of a pathway inhibits an enzyme¡¦s activity in the pathway. Other factors Enzyme Repression (Genetic Control) - End product binds with DNA and stops its production. Enzyme Induction - Enzyme is synthesized only if the inducer (substrate is present). Ribozymes Ribozymes ¡V enzymatic RNA molecules RNA that cuts and splices RNA in eukaryotic cells. Oxidation-Reduction Oxidation - reaction where there is a removal of electrons from an atom or molecule, often produce energy. Reduction - reaction where there is a gain of electrons an atom or molecule. Redox reaction is an oxidation reaction paired with a reduction reaction, most common in biological systems Oxidation-Reduction In biological systems, the electrons are often associated with hydrogen atoms. Biological oxidations are often dehydrogenations. Dehydrogenation ¡V oxidation reaction involving the removal of a protons and an electrons at the same time. NAD+ an electron acceptor, is reduced to NADH, the stored energy of NADH can be used to generate ATP. The Generation of ATP Nutrients are usually highly reduced compounds(energy rich), stepwise oxidation of nutrients release stored energy. Energy released can be stored in ATP. ATP is generated by the phosphorylation of ADP. The Generation of ATP 3 mechanisms of phosphorylation Substrate level phosphorylation ¡V direct transfer of P from substrate to ADP Photo-phosphorylation - only in photosynthetic cells Oxidative Phosphorylation ¡V electrons are Transferred through an electron transport chain Substrate-level phosphorylation is the transfer of a high-energy PO4- to ADP. The Generation of ATP Oxidative Phosphorylation Energy released from the transfer of electrons (oxidation) of one compound to another (reduction) is used to generate ATP by chemiosmosis. Photo-phosphorylation Only in photosynthetic cells Light causes chlorophyll to give up electrons. Energy released from the transfer of electrons (oxidation) of chlorophyll through a system of carrier molecules is used to generate ATP. Energy from ATP and NADPH is used for the synthesis of sugar (carbohydrate) Metabolic Pathways of Energy Production Aerobic Respiration of CHO A process in which carbohydrates are completely oxidized into H2O and energy (ATP) involves three major steps: 1. Glycolysis 2. Kreb¡¦s cycle 3. electron transport chain Final electron acceptor is almost always an inorganic molecule, most commonly oxygen. Anaerobic Respiration of CHO: fermentation(Patial Oxidation) A metabolic processes that release energy from a sugar or other organic molecule Does not require oxygen or an electron transport chain, and use organic molecules as the final electron acceptor Metabolic Pathways Glycolysis The oxidation of glucose to pyruvic acid, produces ATP and NADH. Preparatory Stage 2 ATPs are used Glucose is split to form 2 Glucose-3-phosphate Energy-Conserving Stage 2 Glucose-3-phosphate oxidized to 2 Pyruvic acid 4 ATP produced 2 NADH produced Glucose + 2 ATP + 2 ADP + 2 PO4¡V + 2 NAD+ ? 2 pyruvic acid + 4 ATP + 2 NADH + 2H+ Alternatives to Glycolysis Pentose phosphate pathway: Uses pentoses and NADPH Operates with glycolysis Entner-Doudoroff pathway: Produces NADPH and ATP Does not involve glycolysis Pseudomonas, Rhizobium, Agrobacterium Cellular Respiration ?Oxidation of molecules liberates electrons for an electron transport chain ?ATP generated by oxidative phosphorylation Intermediate Step ?Pyruvic acid (from glycolysis) is oxidized and decarboyxlated ?1 NADH is generated from each pyruvic acid Krebs Cycle ?Oxidation of acetyl CoA produces NADH and FADH2 The Electron Transport Chain ?ETC accepts hydrogen ions released during previous two steps ?A series of carrier molecules that are, in turn, oxidized and reduced as electrons are passed down the chain. ?Energy released can be used to produce ATP by chemiosmosis. ?End products include: Thirty- four 34 ATP and water Chemiosmosis ¡V Generation of ATP is coupled to the transfer of protons across a membrane Respiration ?Aerobic respiration: The final electron acceptor in the electron transport chain is molecular oxygen (O2). ?Anaerobic respiration: The final electron acceptor in the electron transport chain is not O2. Yields less energy than aerobic respiration because only part of the Krebs cycles operations under anaerobic conditions. ?Energy produced from complete oxidation of 1 glucose using aerobic respiration ?ATP produced from complete oxidation of 1 glucose using aerobic respiration ?36 ATPs are produced in eukaryotes. Fermentation Releases energy from oxidation of organic molecules Does not require oxygen Does not use the Krebs cycle or ETC Uses an organic molecule as the final electron acceptor Alcohol fermentation. Produces ethyl alcohol + CO2 Glucose ----------> Ethylalcohol+CO2 Example: Sccharomyces Cerevisiae Lactic acid fermentation. Produces lactic acid. Glucose ----------> Lactic Acid Examples: Lactobacillus Bulgaricus Homolactic fermentation. Produces lactic acid only. Heterolactic fermentation. Produces lactic acid and other compounds. produce both organic acids (lactic, acetic, succinic) and alcohol from glucose. Example: E. coli Photosynthesis Photo: Conversion of light energy into chemical energy (ATP) Light-dependent (light) reactions Synthesis: Fixing carbon into organic molecules Light-independent (dark) reaction, Calvin-Benson cycle Oxygenic: 6 CO2 + 12 H2O + Light energy = C6H12O6 + 6 O2 + 6 H2O Anoxygenic: CO2 + 2 H2S + Light energy = [CH2O] + 2 A + H2O Cyclic Photophosphorylation (anaerobic organism) End Product: ATP only Noncyclic Photophosphorylation (aerobic organisms) End Products: Oxygen, ATP, and NADPH Dark reaction(Calvin Benson cycle) Reaction takes place in three stages. Carboxylation Phase Carbon (from carbon dioxide) is fixed/attached to RuBP(Ribulose 1,5 Biphosphate) to produce PGA (3-Phosphoglyceric Acid). Reduction Phase Utilizes ATP and NADPH (from light reactions) to produce PGAL (Glyceraldehyde3-Phosphate). The Regeneration phase PGAL is converted into glucose and RuBP. Reaction Summary 6CO2 + 12H2O + Light ----- C6H12O6+6O2+6H2O Photosynthesis Chapter 6 End Products of Microbial Growth Microbial growth = increase in number of cells, not cell size The Requirements for Growth: Physical Requirements Temperature Minimum growth temperature Optimum growth temperature Maximum growth temperature Growth Temperature Range Min Psychrophiles Mesophile Thermophiles 0 25 40 Temperature 0C) Opt 15 37 56 Max 20 40 85 The Requirements for Growth: Physical Requirements pH Neutrophiles Acidophiles Alkalophiles pH range 6.5 ¡V 7.5 E. coli, S. aureus grow in acidic environments Helicobacter pylori (bacteria), most fungi and some algae. pH range 8 ¡V 11 Alcaligenes faecalis Buffer is used to prevent extreme pH changes Osmotic Pressure Osmophiles: organisms that can tolerate high solute concentration in their environment,which normally cause plasmolysis Saccharophiles Halophiles Extreme or obligate halophiles require high osmotic pressure Facultative halophiles tolerate high osmotic pressure. Example: S. sureus Chemical Requirements Carbon Structural organic molecules, energy source Chemoheterotrophs use organic carbon sources Autotrophs use CO2 Nitrogen In amino acids, proteins. Most bacteria decompose proteins Some bacteria use NH4+ or NO3. A few bacteria use N2 in nitrogen fixation Sulfur In amino acids, thiamine, biotin. Most bacteria decompose proteins Some bacteria use SO42ƒ{ or H2S Phosphorus In DNA, RNA, ATP, and membranes. PO4 is a source of phosphorus Trace Elements Inorganic elements required in small amounts, usually as enzyme cofactors Oxygen Aerobes capable of growing in the presence of atmospheric oxygen. Synthesize enzymes like catalase, superoxide dismutase, or peroxidase. These enzymes help eliminate toxic forms of oxygen such as hydrogen peroxide, superoxide free radicals etc. Example: Mycobacteria legionella Anaerobes Obligate Anaerobes: Grow only in the absence of oxygen. Some may require 5-10% CO2 for growth. Example: Clostridium perfringens caused gas gangrene) Facultative Anaerobes: Prefer to grow in the presence of oxygen but can also grow in its absence. Examples: E. coli, S. aureus Aerotolerant Anaerobes. Prefer to grow in the absence of oxygen but can also grow in its presence. Example: Lactobacillus plantarum Microaerophiles Require low concentration (2-10%) of oxygen for growth. Enzymes are either absent or ineffective. Pathogens use host's enzyme to eliminate toxic oxygen. Examples: T. pallidum, Campylobacter jejuni Toxic Forms of Oxygen is generated as a consequence of oxygen Survival. Singlet oxygen: O2 boosted to a higher-energy state Superoxide free radicals Peroxide anion (H2O+ O-) Hydroxyl radical (OH) Organic Growth Factors Organic compounds obtained from the environment, Vitamins, amino acids, purines, pyrimidines Growing micro-organisms in the laboratory: Culture = Microbes growing in/on culture medium Culture Medium: Nutrients prepared for microbial growth Requirements: Sterile-No living microbes Inoculum = Introduction of microbes into medium Two main forms of culture media: Liquid: Broth containing nutrients Solid: Broth supplemented with a solid growth support. Solid support = Agar - Complex polysaccharide, used as solidifying agent for culture media in Petri plates, slants, and deeps. Generally not metabolized by microbes. Liquefies at 1000C, solidifies ~400C Categories of culture media according to composition: Chemically Defined Media (simple/synthetic media) - Exact chemical composition is known. Example: Glucose salt broth. Complex Media: Contain one or more compounds with undefined chemical Substances - Extracts and digests of yeasts, meat, or Plants. Example: Nutrient broth, Nutrient agar Special purpose culture media Anaerobic Culture Methods Reducing media - Contain chemicals (thioglycollate or oxyrase) that combine O2. Medium was heated first to drive off dissolved O2. Anaerobic (Gaspak) jar Sodium Bicarbonate + Sodium Borohydride + Water Candle jar Selective Medium Allows the growth of certain type(s) of microbes while inhibiting the growth of other. Examples: Sabouraud¡¦s Agar (pH 5.6, for the isolation of fungi) Suppress unwanted microbes and encourage desired microbes. Differential Medium Distinguishes between different types of micro-organisms through the physiological reactions unique to those microbes. Example: Blood Agar differientiates bacteria on the basis of hemolysis. Beta hemolysis (clear zone) Alpha Hemolysis (greenish zone) Gamma Hemolysis (no lysis) Selective and Differential Media Selective and differential Example: Mannitol Salt Agar differentiates between different types of staphylococci on the basis of mannitol fermentation. S. Epidermidis is negative for fermentation S. aureus is positive for mannitol fermentation. EMB Agar (contains crystal violet, eosin and lactose. select for gram negative bacteria. Lactose differentiates between those that ferment lactose ( red to pink colonies) from those that do not (colorless) Enrichment Media Encourages growth of desired microbe Assume a soil sample contains a few phenol-degrading bacteria and thousands of other bacteria. Inoculate phenol-containing culture medium with the soil and incubate Transfer 1 ml to another flask of the phenol medium and incubate Further transfer 1 ml to another flask of the phenol medium and incubate Only phenol-metabolizing bacteria will be growing A pure culture contains only one species or strain A colony is a population of cells arising from a single cell or spore or from a group of attached cells A colony is formed from a colony-forming unit (CFU = one single live organism) Nutritional Patterns Photoautotrophs Organisms that obtain energy from light and use carbon dioxide as their major carbonsource. Examples: Cyanobacteria, Algae,and Plants. Chemoautotrophs Organisms that use inorganic compounds such as hydrogen sulfide (H2S), ammonia (NH3), elemental sulfur (S) etc.as their energy source and they use CO2 as their sole source of carbon. Examples: Thiobacillus thiooxidans Thiobacillus ferrooxidans Photoheterotrophs: Organnisms that use light as their energy source but cannot utilize CO2 as carbon source, instead they use organic compounds such alcohols and sugars. Chemoheterotrophs: Organisms that use organic compounds as both energy and carbon source. Most are saprobes but some are parasites. Examples: Most bacteria, protozoans, all fungi, and animals. Growth takes place through binary fission. A. The parent cell enlarges. B. Chromosome (DNA) duplicates. C. Central transverse septum forms that divides the cell into two daughter cells. Some microorganism grow by budding or by fragmentation. Generation Time: The time required for the cell population to double. Bacterial Growth Curve Lag phase: Intensive metabolic activities, synthesis of DNA and enzymes. No increase of cell number, preparative phase for cell division.? Log growth phase (exponential growth phase) Active cell reproduction, shortest generation time, a sensitive phase (sensitive to adverse condition). Stationary phase: Cell # reproduced = cell # dead due to depletion of nutrients plus excretion of metabolic waste in the medium; unavailability of oxygen or carbon dioxide; increase in density of cells? Death Phase: Number of dead bacteria is greater than the number of new bacteria produced. Time for death phase varies from bacteria to bacteria. Direct Measurements of Microbial Growth Plate Counts: Perform serial dilutions of a sample Inoculate Petri plates from serial dilutions. Advantages: Only viable cells are counted, allows isolation of colonies. Disadvantages: Too expensive, requires overnight incubation. After incubation, count colonies on plates that have 30-300 colonies (CFUs) Direct Measurements of Microbial Growth Filtration Direct Microscopic Count Estimating Bacterial Numbers by Indirect Methods Turbidity Measurements Advantage: Quick Disadvantages: Count includes both living and non-living microbes. Cannot discriminate between microbes and other particles. Expensive Metabolic activity Dry weight Chapter 8 Terminology Genetics Study of heredity, what genes are, how they carry information, how information is expressed, and how genes are replicated Gene Segment of DNA that encodes a functional product, usually a protein Codon: a sequences of three bases in mRNA that specifies a particular amino acid in the translation process. Genome All of the genetic material in a cell or organism Genomics Molecular study of genomes Chromosome: structures that contain the DNA of organism. Genotype Genes of an organism Phenotype be Expression of the genes, characteristics that can observed DNA and Chromosome Bacteria contain a single circular chromosome made of DNA. A typical bacteria chromosome has about 4 million base pairs and is about 1mm long (1000 times larger than the bacterial cell) Structure of DNA (Deoxyribose Nucleic Acid) Flow of Genetic Information DNA Double stranded helical polymer of nucleotides associated with proteins. Each nucleotide is composed of: 1. A "Backbone" of deoxyribosesugar(pentose) and a phosphate group 2. One of the following four nitrogen bases: adenine(Purine), guanine(Purine) thymine(Pyrimidine),cytosine(Pyrimidine) Thymine is always paired with adenine, and guanine is always paired with cytosine(complementary base pairing). Strands held together by hydrogen bonds between AT and CG Strands are antiparallel DNA DNA DNA DNA is copied by DNA polymerase In the 5„S „_ 3„S direction Initiated by an RNA primer Leading strand synthesized continuously Lagging strand synthesized discontinuously Okazaki fragments RNA primers are removed and Okazaki fragments joined by a DNA polymerase and DNA ligase DNA DNA DNA replication is semiconservative The Flow of Genetic Information DNA mRNA amino acids proteins 1. Proteins are polymer of amino acids and are essential for the survival of all living cells. 2. The genetic code (codon) in a mRNA molecule gives the amino acid sequence for a protein. 3. There are 64 possible codons (61 sense and 3 nonsense (UAA, UAG, UGA) hat code for 20 amino acids. 4. The genetic code is redundant; that is, there is more than one codon for each amino acid, except for tryptophane (UGG) and methionine 5. for (AUG)which have only one code word each. The genetic code that initiates the message is AUG, which codes methionine. Transcription DNA is transcribed to make RNA (mRNA, tRNA, and rRNA) Transcription begins when RNA polymerase binds to the promotor sequence Transcription proceeds in the 5„S „_ 3„S direction Transcription stops when it reaches the terminator sequence Translation mRNA is translated in codons (3 nucleotides) Translation of mRNA begins at the start codon: AUG Translation ends at a STOP codon: UAA, UAG, UGA Translation Conversion of mRNA information into amino acid sequence (protein) at the ribosome. The 30s subunit of the ribosomes have two specific sites on their surface A-Site: is the entry site for tRNA whose anticodon recognizes the codon on the mRNA. P-Site: is the exit site for naked tRNA, it also carries the growing polypeptide chain. Regulation of Bacterial Gene Expression Constitutive enzymes are expressed at a fixed rate Other enzymes are expressed only as needed Repressible enzymes Inducible enzymes Repression Operon Regulation of Gene Expression Mutation Change in the genetic material Mutations may be neutral, beneficial, or harmful Mutagen: Agent that causes mutations Spontaneous mutations: Occur in the absence of a mutagen Mutation Base substitution (point mutation) Missense mutation Nonsense mutation Frameshift mutation Mutation Ionizing radiation (X rays and gamma rays) causes the formation of ions that can react with nucleotides and the deoxyribose-phosphate backbone. Nucleotide excision repairs mutations Mutation UV radiation causes thymine dimers Light-repair separates thymine dimers The Frequency of Mutation Spontaneous mutation rate = 1 in 109 replicated base pairs or 1 in 106 replicated genes Mutagens increase to 10? or 10? per replicated gene Selection Positive (direct) selection detects mutant cells because they grow or appear different. Negative (indirect) selection detects mutant cells because they do not grow. Genetic Transfer and Recombination Vertical gene transfer Horizontal gene transfer Transformation Recombination Conjugation Genetic Recombination Exchange of genes between two DNA molecules Crossing over occurs when two chromosomes break and rejoin Transduction Plasmids Conjugative plasmid Carries genes for sex pili and transfer of the plasmid Dissimilation plasmids Encode enzymes for catabolism of unusual compounds R factors Encode antibiotic resistance Plasmids Transposons Segments of DNA that can move from one region of DNA to another Contain insertion sequences for cutting and resealing DNA (transposase) Complex transposons carry other genes Chapter 7 The Control of Microbial Growth Antimicrobial Agent: substances that kill microbes or prevent their growth. (antibacterial, antifungal, Microbicidal: antimicrobial agents that kill microorganisms. (bactericidal, fungicidal, virucidal). ? Microbistatic antimicrobial agent that inhibit the growth of microbes. Sterilization: the process of destroying or removing all forms of microbial life. Asepsis is the absence of significant contamination. Aseptic surgery techniques prevent microbial contamination of wounds. Terminology Disinfection: a process (physical or chemical) that kills the vegetative forms of microbes, but does not necessarily kill their spores. A disinfectant (germicidal) is a substance used on inanimate objects. Antisepsis: a process which applies antiseptic to the surface of the body (skin and mucous membrane) to kill or inhibit the growth of microbes. Sanitization: a process which reduces microbial population to levels that are considered safe by public health guidelines. Bacterial populations die at a constant logarithmic rate. Effectiveness of antimicrobial treatment depends on: Number of microbes Nature of microbe Temperature, pH Agent concentration physiological state Presence of interfering (extraneous) organic matter Actions of Microbial Control Agents Damage to cell wall Examples: lysozyme, lysostaphin, penicillins. Alternation of membrane permeability Examples: polymixin-b, cepacol Damage to proteins and nucleic acids Examples: radiations, alcohols, acids, and chloramphenicol. Physical Methods of Microbial Control Heat Thermal death point (TDP): Lowest temperature at which all cells in a culture are killed in 10 min. Thermal death time (TDT): Time to kill all cells in a culture Decimal reduction time (DRT): Minutes to kill 90% of a population at a given temperature Methods of Physical Control High Temperature ¡V moist heat, dry heat Low Temperature Desiccation Radiation Filtration Moist Heat One of the most effective and widely utilized means of killing microbes. It causes denaturation of vital proteins such as enzymes. Endospores of B. Anthracis are destroyed by moist heat within 2-15 minutes at 100 C, while dry heat takes 180 minutes at 140 C. Moist Heat-Autoclave Moist heat denatures proteins Autoclave: Steam under pressure operated at15 psi at 121 C for 15 min Boiling water - Water brought to boiling will kill vegetative microbes only. Endospores can survive boiling for several hours. Sub boiling temperature Pasteurization reduces spoilage organisms and pathogens Equivalent treatments 63¢XC for 30 min High-temperature short-time 72¢XC for 15 sec Ultra-high-temperature: 140¢XC for High-temperature short-time 72°C for 15 sec Ultra-high-temperature: 140°C for 1 sec Thermoduric organisms survive Dry Heat Sterilization kills by oxidation Flaming Incineration Hot-air sterilization ?used to sterilize substances impermeable to or damaged by moisture. e.g. oil, powders Low Temperature (microbistatic) Low temperature inhibits microbial growth Refrigeration Deep freezing - may not kill the microorganisms and may in fact preserve them along with the material being frozen. Frozen cultures can be stored indefinitely at ?70 C or in tanks of liquid nitrogen at 196 C. Physical control of microbial growth Desiccation (microbistatic) - Drying vegetative cells of microbes inhibits their metabolic activities. The length of time microbes survive after desiccation depends on the following factors:? 1. The type of the microorganism 2. The material in which the organisms are dried. 3. The completeness of the drying process. ? 4. The physical conditions involved, such as temperature and humidity. Physical Methods of Microbial Control Radiation damages DNA Ionizing radiation (X rays, gamma rays, electron beams) Nonionizing radiation (UV) (Microwaves kill by heat; not especially antimicrobial) Chemical Methods of Microbial Control Principles of effective disinfection Concentration of disinfectant Organic matter pH Time Chemical Methods of Microbial Control Evaluating a disinfectant Use-dilution test 1. Metal rings dipped in test bacteria are dried 2. Dried cultures placed in disinfectant for 10 min at 20°C 3. Rings transferred to culture media to determine whether bacteria survived treatment Types of Disinfectants Biguanides. Chlorhexidine Disrupt plasma membranes Halogens. Iodine, Chlorine Oxidizing agents Bleach is hypochlorous acid (HOCl) Heavy Metals. Ag, Hg, Cu Oligodynamic action Denature proteins Surface-Active Agents or Surfactants Chemical Food Preservatives Organic Acids Inhibit metabolism Sorbic acid, benzoic acid, calcium propionate Control molds and bacteria in foods and cosmetics Nitrite prevents endospore germination Antibiotics. Nisin and natamycin prevent spoilage of cheese Aldehydes Inactivate proteins by cross-linking with functional groups (–NH2, –OH, –COOH, —SH) Glutaraldehyde, formaldehyde Gaseous Sterilants Denature proteins Ethylene oxide Peroxygens Oxidizing agents O3, H2O2, peracetic acid Chapter 20 Antimicrobial Drugs Chemotherapy - The use of drugs to treat a disease Antimicrobial drugs - Interfere with the growth of microbes within a host Antibiotic - Substance produced by a microbe that, in small amounts, inhibits another microbe Selective toxicity - A drug that kills harmful microbes without damaging the host 1928 Fleming discovered penicillin, produced by Penicillium. 1940 V Howard Florey and Ernst Chain performed first clinical trials of penicillin. The Action of Antimicrobial Drugs Antibacterial Antibiotics Inhibitors of Cell Wall Synthesis Penicillin Natural penicillins Semisynthetic penicillins Penicilinase-resistant penicillins Extended-spectrum penicillins = Penicillins + Beta-lactamase inhibitors Carbapenems Monobactam Cephalosporins 2nd, 3rd, and 4th generations more effective against gramnegatives Polypeptide antibiotics Bacitracin - Topical application against gram-positives Vancomycin - Glycopeptide important "last line" against antibiotic resistant S. aureus Antimycobacterium antibiotics Isoniazid (INH) - Inhibits mycolic acid synthesis Ethambutol - Inhibits incorporation of mycolic acid Antibacterial Antibiotics Inhibitors of Protein Synthesis Chloramphenicol - Broad spectrum, binds 50S subunit, inhibits peptide bond formation Aminoglycosides Streptomycin, neomycin, gentamycin - Broad spectrum changes shape of 30S subunit Tetracyclines - broad spectrum, interferes with tRNA attachment Macrolides - Gram-positives, binds 50S, prevents translocation Erythromycin - Gram-positives, binds 50S, prevents translocation Streptogramins - gram-positives, binds 50S subunit, inhibits translation Synercid - Gram-positives, binds 50S subunit, inhibits translation Oxazolidinones, Linezolid - Gram-positives, binds 50S subunit, prevents formation of 70S ribosome Antibacterial Antibiotics Injury to the Plasma Membrane Polymyxin B - Topical, combined with bacitracin and neomycin in over-the-counter preparation Antibacterial Antibiotics Inhibitors of Nucleic Acid Synthesis Rifamycin - Inhibits RNA synthesis, antituberculosis Quinolones Fluoroquinolones Ciprofloxacin Inhibits DNA polymerase - Urinary tract infections Antibacterial Antibiotics Competitive Inhibitors Sulfonamides (Sulfa drugs) - Inhibit folic acid synthesis, Broad spectrum Antifungal Drugs Inhibition of Ergosterol Polyenes - Amphotericin B, Azoles Miconazole - Triazoles Allylamines Inhibition of cell wall synthesis Echinocandins Inhibition of nucleic acids Flucytocin - Cytosine analog Inhibition of mitotubules (mitosis) Griseofulvin - for superficial mycoses Tolnaftate - for athlete's foot; action unknown Anti-viral drugs Nucleoside and nucleotide analogs Acyclovir, deoxyguanosine Protease inhibitors Enzyme inhibitors Inhibit attachment Inhibit uncoating Interferons prevent spread of viruses to new cells Enzyme Inhibitors for HIV Protease inhibitors Indinavir Inhibit attachment Zanamivir for Influenza Inhibit uncoating Amantadine Interferons - prevent spread of viruses to new cells Antiprotozoan Drugs Antihelminthic Drugs Disk-Diffusion Test – qualitative test for effectiveness of drug Broth Dilution Test – qualitative and quantitative test Antibiotic Resistance A variety of mutations can lead to antibiotic resistance. Mechanisms of antibiotic resistance 1. Enzymatic destruction of drug 2. Prevention of penetration of drug 3. Alteration of drug's target site 4. Rapid ejection of the drug Resistance genes are often on plasmids or transposons that can be transferred between bacteria. Antibiotic Resistance – Main causes Misuse of antibiotics selects for resistance mutants. Misuse includes: Using outdated, weakened antibiotics Using antibiotics for the common cold and other inappropriate conditions Use of antibiotics in animal feed Failure to complete the prescribed regimen Using someone else's leftover prescription Effects of Combinations of Drugs Synergism occurs when the effect of two drugs together is greater than the effect of either alone. Antagonism occurs when the effect of two drugs together is less than the effect of either alone. The Future of Chemotherapeutic Agents Antimicrobial peptides Broad spectrum antibiotics from plants and animals, e.g. Squalamine (sharks) Protegrin (pigs) Magainin (frogs) Antisense agents - Complementary DNA or peptide nucleic acids that binds to a pathogen's virulence gene(s) and prevents transcription Chapter 14 Principles of Disease and Epidemiology Pathology - Study of disease Etiology - Study of the cause of a disease Pathogenesis - Development of disease Infection - Colonization of the body by pathogens Disease - An abnormal state in which the body is not functionally normally Normal Microbiota and the Host Transient microbiota may be present temporarily Normal microbiota permanently colonize the host since birth, acquired from environment. Symbiosis is the relationship between normal microbiota and the host. Normal Microbiota and the Host: In commensalism, one organism is benefited and the other is unaffected. e. g. corynebacteria on eye surface saprophytic mycobacteria in ear. In mutualism - a type of symbiosis where both partners are benefited. e. g. E. coli in intestine synthesis vitamin K and obtain nutrients from the intestine. In parasitism - one organism is benefited at the expense of the other. * Some normal microbiota are opportunistic pathogens-microbes which ordinarily do not cause disease in their normal habitat may become harmful at a different site or when host resistance is weakened. e. g. E. coli in urinary tract or bladder Pneumocystis carinii in AIDS patients Microbial antagonism is competition between microbes. Normal microbiota protect the host by: occupying niches that pathogens might occupy e. g. Inhibition of yeast by normal flora E. coli in intestine produces bacteriocin to inhibit the growth of pathogens. produces acids to inhibit growth of other bacteria. Probiotics are live microbes applied to or ingested into the body, intended to exert a beneficial effect. Koch’s Postulates Koch's Postulates are used to prove the cause of an infectious disease. Exceptions to Koch’s postulates 1. Fastidious pathogens – Treponema pallidum 2. Different pathogens with similar symptoms 3. Multiple diseases cause by same organism e.g. Mycobacterium tuberculosis in lung and elsewhere Strep. Pyrogenes in throat and in heart valve Classifying Infectious Diseases Symptom - A change in body function that is felt by a patient as a result of disease Sign - A change in a body that can be measured or observed as a result of disease. Syndrome - A specific group of signs and symptoms that accompany a disease. Classifying Infectious Diseases Communicable disease - A disease that is easily spread from one host to another. e. g. measles, chickenpox, TB Contagious disease - A disease that is easily spread from one host to another. Noncommunicable disease - A disease that is not transmitted from one Host to another. tetanus, E. coli Occurrence of Disease Incidence - Fraction of a population that contracts a disease during a specific time. Prevalence - Fraction of a population having specific disease at a given time Sporadic disease - Disease that occurs occasionally in a population e. g. Typhoid fever Endemic disease - Disease constantly present in a population e. g. common cold Epidemic disease - Disease acquired by many hosts in a given area in a short time. Pandemic disease - Worldwide epidemic. Severity or Duration of a Disease Acute disease - Symptoms develop rapidly Chronic disease - Disease develops slowly Subacute disease - Symptoms between acute and chronic Latent disease - Disease with a period of no symptoms when the patient is inactive e. g. shingles(Varicellovirus) Extent of Host Involvement Local infection - Pathogens limited to a small area of the body e. g. Boils and abscesses Systemic infection - An infection throughout the body. e.g. Measles Focal infection - Systemic infection that began as a local infection Bacteremia - Bacteria in the blood Septicemia - Growth of bacteria in the blood Toxemia - Toxins in the blood Viremia - Viruses in the blood Primary infection - Acute infection that causes the initial illness Secondary infection - Opportunistic infection after a primary (predisposing) infection Subclinical disease - No noticeable signs or symptoms (inapparent infection) Predisposing Factors Make the body more susceptible to disease - gender, genes, environment, age, etc. Short urethra in females Inherited traits such as the sickle-cell gene Climate and weather Fatigue Age Lifestyle Chemotherapy Stages of A Disease 1. Incubation Period – depends on factors like virulence, number of infectious agent and host resistance. 2. Prodromal period – mild symptoms 3. Illness – sever symptoms, immune response from patient 4. Decline – diminished illness and symptoms 5. Convalescence – return of body to pre-disease state, can be contagious and spread disease. Reservoirs of Infection Reservoirs of infection are continual sources of infection. 1. Human —people with disease; latent and non- symptomatic Carriers. e.g. AIDS; diphtheria, typhoid fever, hepatitis AIDS, gonorrhea 2. Animal — Rabies, Lyme disease Some zoonoses may be transmitted to humans Nonliving zoonoses — Botulism, tetanus 3. Non-living reservoirs – soil and water Clostridium botulinum; C. tetani; contaminated water. Transmission of Disease Contact Direct - Requires close association between infected and susceptible host e.g. viral diseases such as flu, AIDS, Hepatitis Bacterial disease such as syphilis. Indirect - transmission by means of a non-living Object, fomites e. g. towels, bedding, drinking cups. Droplet - Transmission via airborne droplets, coughing, sneezing. Vehicle - Transmission by an inanimate reservoir such as water, air, food waterborne - cholera, shigella foodborne - tapeworms and food poisoning. airborne - droplets; dust, e.g. TB Vectors Arthropods, especially fleas, ticks, and mosquitoes Mechanical -Arthropod carries pathogen on feet Biological -Pathogen reproduces in vector Transmission of Disease Nosocomial (Hospital-Acquired) Infections Are acquired as a result of a hospital stay. 5-15% of all hospital patients acquire nosocomial infections Factors affecting nosocomial infections 1. Microorganims in hospital – hospital is a major reservoir of pathogens, most are opportunistic pathogens. e. g. Antibiotic resistant Staphlococcus aureus; E. coli and Pseudomonas. 2. Host condition – compromised host such as burn patients or patients with weakened immune system are common subjects. 3. Chain of transmission – direct contact transmission between staff and patient, or, between patient and patient. 4. Indirect contact transmission through fomites and air-bourne transmission. Emerging Infectious Diseases Diseases that are new, increasing in incidence, or showing a potential to increase in the near future. Contributing factors: Evolution of new strains e.g. V. cholerae O139 Inappropriate use of antibiotics and pesticides Antibiotic resistant strains Changes in weather patterns Hantavirus Evolution of existing microbes Modern transportation West Nile virus Ecological disaster, war, expanding human settlement Animal control measures Lyme disease Widespread use of antibiotics and pesticides Global warming Public Health failure Diphtheria Epidemiology The study of where and when diseases occur Centers for Disease Control and Prevention (CDC) Collects and analyzes epidemiological information in the U.S. Publishes Morbidity and Mortality Weekly Report (MMWR) www.cdc.gov Morbidity: incidence of a specific notifiable disease Mortality: deaths from notifiable diseases Morbidity rate = number of people affected/total population in a given time period Mortality rate - number of deaths from a disease/total population in a given time Chapter 15 Microbial Mechanisms of Pathogenicity Pathogenicity The ability to cause disease Virulence The extent of pathogenicity Portals of Entry Mucous membranes - respiratory tract, gastrointestinal tract, Urogenital tract and conjunctiva. Skin - impenetrable barrier if not broken, micro-organisms can grow in openings, fungus can grow on keratin surface. Parenteral route - direct deposit of microorganism beneath skin or onto mucous membrane. Numbers of Invading Microbes ID50: Infectious dose LD50: Lethal dose (of ID50 and LD50 changes e.g. Vibrio cholera for 50% of the test population a toxin) for 50% of the test population according to other factors. ID50 lower if stomach acid is neutralised Factors affecting pathogenicity 1. Capsules – resist phagocytosis. e.g. in Strep. pneumoniae; B. anthracis, protective effect neutralised by antibodies. 2. Cell wall components – Waxes of Mycobacterium resist digestion. 3. Enzymes – exoenzymes destroy WBCs. Hemolysins – lyse RBC, produce by Staph and Strep; Mechanisms of Microbial Pathogenesis 1. Direct damage Entrance and exit from host cell damage host. 2. Toxins Exotoxins- proteins, mostly are enzymes used by Microbes. Mostly produced in gm +, Exotoxin genes carried in plasmids. e.g. Diphtheria toxin – tox gene carried by lysogenic plasmid. Neurotoxins – interferes nerve impulse transmission Enterotoxins – affects GI tract linings Endotoxins - Find in the outer cell wall of gm – bacteria = lipid A of LPS Toxemia - Presence of toxin the host's blood Toxoid - Inactivated toxin used in a vaccine Toxin structure Exotoxin - mostly a 2 polypeptide chain structure, A chain = active chain B chain = binding chain e.g. Botulinum toxin – neurotoxin, blocks neurotransmitte release to muscle, resulting in paralysis. Tetanus toxin – neurotoxin, causes spasm. Vibrio enterotoxin – 2 chain toxin, affect fluid balance of GI epithelium. Antitoxin - Antibodies against a specific toxin Endotoxin – LPS of Gram negative bacteria Pathogenic effect of Virus – damage to host cells Cytopathic Effects of virus a) Stop cellular synthesis b) Release of lysosomal lysozyme, resulting in cell lysis c) Production of inclusion bodies d) Cause synctium formation e) Change cellular function f) Induce antigenic changes and target cells to be destroyed by host immune system g) Induce chromosomal damage h) Lost of contact inhibition Chapter 16 Nonspecific Defenses of the Host Susceptibility - Lack of resistance to a disease Resistance - Ability to ward off disease Nonspecific resistance - Defenses against any pathogen Specific resistance - Immunity, resistance to a specific pathogen Host Defenses Mechanical Factors Skin Epidermis consists of tightly packed cells with Keratin, a protective protein Mucous membranes Ciliary escalator: Microbes trapped in mucus are transported away from the lungs Lacrimal apparatus: Washes eye Saliva: Washes microbes off Urine: Flows out Vaginal secretions: Flow out Chemical Factors Fungistatic fatty acid in sebum Low pH (3-5) of skin Lysozyme in perspiration, tears, saliva, and tissue fluids Low pH (1.2-3.0) of gastric juice Transferrins in blood find iron NO (Nitrous Oxide) inhibits ATP production Normal Microbiota Microbial antagonism/competitive exclusion: Normal microbiota competes with pathogens. Non-specific Cellular defenses – mostly in blood Formed Elements In Blood White Blood Cells Neutrophils: Phagocytic Basophils: Produce histamine Eosinophils: Toxic to parasites, some phagocytosis Monocytes: Phagocytic as mature macrophages Fixed macrophages in lungs, liver, bronchi Wandering macrophages roam tissues Lymphocytes: Involved in specific immunity Inflammation Phases in inflammation: Redness Pain Heat Swelling (edema) Acute-phase proteins activated (complement, cytokine, kinins) Vasodilation (caused by histamine, kinins, prostaglandins, leukotrienes) Margination and emigration of WBCs Tissue repair Effect of Inflammation Fever: Abnormally High Body Temperature Hypothalamus normally set at 37°C Gram-negative endotoxin cause phagocytes to release interleukin 1 Hypothalamus releases prostaglandins that reset the hypothalamus to a high temperature Body increases rate of metabolism and shivering to raise temperature When IL-1 is eliminated, body temperature falls. (Crisis) The Complement System Serum proteins activated in a cascade. Effects of Complement Activation Opsonization or immune adherence: enhanced phagocytosis Membrane attack complex: cytolysis Attract phagocytes Some bacteria evade complement Capsules prevent C activation Surface lipid-carbohydrates prevent MAC formation Enzymatic digestion of C5a Interferons (IFNs) Alpha IFN & Beta IFN: Cause cells to produce antiviral proteins that inhibit viral replication Gamma IFN: Causes neutrophils and macrophages to phagocytize bacteria Chapter 17 Specific Defenses of the Host: The Immune Response • Innate (nonspecific) - Defenses against any pathogen • Immunity - Specific antibody and lymphocyte response to an antigen • Antigen (Ag) - A substances that causes the body to produce specific antibodies or sensitized T cells • Antibody (Ab) - Proteins made in response to an antigen Terminology • Serology - Study of reactions between antibodies and antigens • Antiserum - Generic term for serum because it contains Ab • Globulins - Serum proteins • Gamma globulin - Serum fraction containing Ab Serum Proteins The Immune Response • Acquired immunity - Developed during an individual's lifetime • Humoral immunity - Active at the body fluid level (blood, lymph, mucus, and other body secretions) Only B-cells are directly involved, which produce antibodies. Effective mostly against bacteria, bacterial toxins, and viruses. • The cellular Immunity - Active at cellular and blood level. T-cells are involved, they produce lymphokines(a group of proteins that regulate the activities of other cells). Antibodies are not involved. Effective mostly against intracellular bacteria, virus and fungi. • Naturally acquired active immunity resulting from infection • Naturally acquired passive immunity Transplacental or via colostrum • Artificially acquired active immunity Injection of Ag (vaccination) • Artificially acquired passive immunity Injection of Ab Antibody Structure IgG antibodies • Monomer • 80% of serum antibodies • Fix complement • In blood, lymph, intestine • Cross placenta • Enhance phagocytosis; neutralize toxins & viruses; protects fetus & newborn • Half-life = 23 days IgM antibodies • Pentamer • 5-10% of serum antibodies • • • • IgA • • • • • IgD • • • • • IgE • • • • • Fix complement In blood, lymph, on B cells Agglutinates microbes; first Ab produced in response to infection Half-life = 5 days antibodies Dimer 10-15% of serum antibodies In secretions Mucosal protection Half-life = 6 days antibodies Monomer 0.2% of serum antibodies In blood, lymph, on B cells On B cells, initiate immune response Half-life = 3 days antibodies Monomer 0.002% of serum antibodies On mast cells and basophils, in blood Allergic reactions; lysis of parasitic worms Half-life = 2 days Antibody titer: • Is the amount of Ab in serum Immune system cells communicate via cytokines • Interleukin-1 Stimulates TH cells • Interleukin-2 Activates TH, B, TC, and NK cells • Interleukin-12 Differentiation of CD4 cells • -Interferon Increase activity of macrophages • Chemokines Cause leukocytes to move to an infection Cell-Mediated Immunity • Specialized lymphocytes, mostly T cells, respond to intracellular Antigens • After differentiating in the thymus, T cells migrate to lymphoid tissue • T cells differentiate into effector T cells when stimulated by an antigen(Ag) • Some effector T cells become memory cells T Cells • Helper T Cells (CD4, TH) * TH1 Activate cells related to cell-mediated immunity • TH2 Activate B cells to produce eosinophils, IgM, and IgE • Cytotoxic T Cells (CD8, TC) • Destroy target cells with perforin • Delayed Hypersensitivity T Cells (TD) • Associated with allergic reaction, transplant rejection, and tuberculin skin test • • Suppressor T cells (TS) Turn off immune response when Ag no longer present Helper T Cells Cell-mediated Cytotoxicity Nonspecific Cells • Activated macrophages: Macrophages stimulated by ingesting Ag or by cytokines • Natural killer cells: Lymphocytes that destroy virus-infected cells, tumor Ch 21 Microbial disease of skin and eye Skin •Salt inhibits microbes •Lysozyme hydrolyzes peptidoglycan •Fatty acids inhibit some pathogens Mucous Membranes •Line body cavities •Epithelial cells attached to an extracellular matrix •Cells secrete mucus •Some have cilia Normal Microbiota of the Skin •Gram-positive, salt-tolerant bacteria •Staphylococci •Micrococci •Diphtheroids Microbial Diseases of the Skin Staphylococcal Skin Infections •S. epidermidis •Gram-positive cocci, coagulase-negative •Staphylococcus aureus •Gram-positive cocci, coagulase-positive •Leukocidin •Exfoliative toxin Staphylococcal Skin Infections •Folliculitis •Infections of hair follicles •Sty •Folliculitis of an eyelash •Furuncle •Abscess; pus surrounded by inflamed tissue •Carbuncle •Inflammation of tissue under the skin •Impetigo of the newborn •Toxemia •Scalded skin syndrome •Toxic shock syndrome Streptococcal Skin Infections •Erysipelas •Impetigo Invasive Group A Streptococcal Infections •Streptokinases •Hyaluronidase •Exotoxin A, superantigen •Cellulitis •Necrotizing fasciitis •Sore throat, endocarditis, fresh eating syndromes Infections by Pseudomonads •Pseudomonas aeruginosa •Gram-negative, aerobic rod, opportunistic pathogen •Pyocyanin produces a blue-green pus •Pseudomonas dermatitis •Otitis externa •Post-burn infections Acne •Comedonal acne •Occurs when sebum channels are blocked with shed cells •Inflammatory acne •Propionibacterium acnes •Gram-positive, anaerobic rod, feeds on glycerol in sebum •Treatment: •Preventing sebum formation (isotretinoin) •Antibiotics •Benzoyl peroxide to loosen clogged follicles •Visible (blue) light (kills P. acnes) •Nodular cystic acne •Treatment: isotretinoin Warts •Papillomaviruses – spread by direct contract, genital warts spread through sexual contact. •Treatment: •Removal •Imiquimod (stimulate interferon production) •Interferon Poxviruses •Smallpox (Variola) Transmitted by respiratory route, no known animal reservoir •Smallpox virus (Orthopox virus) •Variola major has 20% mortality •Variola minor has <1% mortality •Monkeypox •Prevention by smallpox vaccination Herpesviruses •Varicella-zoster virus (Human herpes virus 3), causes chicken pox, Shingles and Reyes syndrome. Spread by respiratory route. •Transmitted by the respiratory route •Causes pus-filled vesicles •Virus may remain latent in dorsal root ganglia Shingles •Reactivation of latent HHV-3 releases viruses that move along peripheral nerves to skin. Herpes simplex 1 and Herpes simplex 2 •Human herpes virus 1 and HHV-2 •HSV1 - Cold sores or fever blisters (vesicles on lips, thru oral contact) •Herpes gladiatorum (vesicles on skin) •Herpes whitlow (vesicles on fingers) •Herpes encephalitis (HHV-2 has up to a 70% fatality rate) •HHV-1 can remain latent in trigeminal nerve ganglia •HHV-2 – transmitted thru sexual contact, can remain latent in sacral nerve ganglia •Acyclovir may lessen symptoms Measles (Rubeola) •Measles virus •Transmitted by respiratory route •Macular rash and Koplik's spots •Prevented by vaccination •Encephalitis in 1 in 1000 cases •Subacute sclerosing panencephalitis in 1 in 1,000,000 cases Rubella (German Measles) •Rubella virus •Macular rash and fever •Congenital rubella syndrome causes severe fetal damage •Prevented by vaccination •Fifth Disease Human parvovirus B19 produces milk flu-like symptoms and facial rash •Roseola •Human herpesvirus 6 causes a high fever and rash, lasting for 1-2 days Cutaneous Mycoses •Dermatomycoses: tineas or ringworm •Metabolize keratin •Trichophyton infects hair, skin, nails •Epidermophyton infects skin and nails •Microsporum infects hair and skin •Treatment •Oral griseofulvin •Topical miconazole Subcutaneous Mycoses •Sporotrichosis •Sporothrix schenckii enters puncture wound •Treated with KI Candidiasis •Candida albicans (yeast) •Candidiasis may result from suppression of competing bacteria by antibiotics •Occurs in skin; mucous membranes of genitourinary tract and mouth •Thrush is an infection of mucous membranes of mouth •Topical treatment with miconazole or nystatin Microbial Diseases of the Eye •Conjunctivitis (pinkeye) •Haemophilus influenzae •Various microbes •Associated with unsanitary contact lenses •Neonatal gonorrheal ophthalmia •Neisseria gonorrhoeae •Transmitted to newborn's eyes during passage through the birth canal •Prevented by treatment newborn's eyes with antibiotics •Chlamydia trachomatis •Inclusion conjunctivitis •Transmitted to newborn's eyes during passage through the birth canal •Spread through swimming pool water •Treated with tetracycline •Trachoma •Greatest cause of blindness worldwide •Infection causes permanent scarring; scars abrade the cornea leading to blindness •Herpetic Keratitis •Herpes simplex virus 1 (HHV-1) •Infects cornea, may cause blindness •Treated with trifluridine •Acanthamoeba keratitis •Transmitted from water •Associated with unsanitary contact lenses Ch 22 Microbial diseases of the Nervous system Microbes enter the nervous system via: •Skull or backbone fractures •Medical procedures •Along peripheral nerves •Blood or lymph Microbial Diseases of the Nervous System •Bacteria can grow in the cerebrospinal fluid in the subarachnoid space of the CNS •The blood brain barrier (capillaries) prevents passage of some materials (such as antimicrobial drugs) into the CNS •Meningitis - Inflammation of meninges •Encephalitis - Inflammation of the brain The Meninges and Cerebrospinal Fluid Bacterial Meningitis - Symptoms •Fever, headache, stiff neck •Followed by nausea and vomiting •May progress to convulsions and coma •Diagnosis by Gram stain of CSF •Treated with cephalosporins Haemophilus influenzae Meningitis •Occurs mostly in children (6 months to 4 years) •Gram-negative aerobic bacteria, normal throat microbiota •Capsule antigen type b •Prevented by Hib vaccine Neisseria Meningitis, Meningococcal Meningitis •N. meningitidis •Gram-negative aerobic cocci, capsule •10% of people are healthy nasopharyngeal carriers •Begins as throat infection, rash •Serotype B is most common in the U.S. •Vaccine against some serotypes is available Streptococcus pneumoniae Meningitis, Pneumococcal Pneumonia •Gram-positive diplococci •70% of people are healthy nasopharyngeal carriers •Most common in children (1 month to 4 years) •Mortality: 30% in children, 80% in elderly •Prevented by vaccination Tetanus •Clostridium tetani •Gram-positive, endospore-forming, obligate anaerobe •Grows in deep wounds •Tetanospasmin released from dead cells blocks relaxation pathway in muscles •Prevention by vaccination with tetanus toxoid (DTP) and booster (dT) •Treatment with tetanus immune globulin Botulism •Clostridium botulinum •Gram-positive, endospore-forming, obligate anaerobe •Intoxication due to ingesting botulinal toxin •Botulinal toxin blocks release of neurotransmitter causing flaccid paralysis •Prevention: •Proper canning •Nitrites prevent endospore germination in sausages •Treatment: supportive care and antitoxin •Infant botulism results from C. botulinum growing in intestines •Wound botulism results from growth of C. botulinum in wounds . Leprosy •Mycobacterium leprae •Acid-fast rod that grows best at 30°C •Grows in peripheral nerves and skin cells •Transmission requires prolonged contact with an infected person •Tuberculoid (neural) form: Loss of sensation in skin areas; positive lepromin test •Lepromatous (progressive) form: Disfiguring nodules over body; negative lepromin test Poliomyelitis •Poliovirus •Transmitted by ingestion •Initial symptoms: sore throat and nausea •Viremia may occur; if persistent, virus can enter the CNS; destruction of motor cells and paralysis occurs in <1% of cases •Prevention is by vaccination (enhanced-inactivated polio vaccine) Rabies virus (Rhabdovirus) •Transmitted by animal bite •Virus multiplies in skeletal muscles, then brain cells causing encephalitis •Initial symptoms may include muscle spasms of the mouth and pharynx and hydrophobia •Furious rabies: animals are restless then highly excitable •Paralytic rabies: animals seem unaware of surroundings •Preexposure prophylaxis: Infection of human diploid cells vaccine •Postexposure treatment: Vaccine + immune globulin Arboviral Encephalitis •Arboviruses are arthropod-borne viruses that belong to several families. •Prevention is by controlling mosquitoes Cryptococcus neoformans Meningitis (Cryptococcosis) •Soil fungus associated with pigeon and chicken dropping •Transmitted by the respiratory route; spreads through blood to the CNS •Mortality up to 30% •Treatment: amphotericin B and flucytosine African Trypanosomiasis •Trypanosoma brucei gambiense infection is chronic (2 to 4 years) •T. b. rhodesiense infection is more acute (few months) •Transmitted from animals to humans by tsetse fly •Prevention: elimination of the vector •Treatment: Eflornithine blocks an enzyme necessary for the parasite •Parasite evades the antibodies through antigenic variation Transmissible Spongiform Encephalopathies •Caused by prions •Sheep scrapie •Creutzfeldt-Jakob disease •Kuru •Bovine spongiform encephalopathy •Transmitted by ingestion or transplant or inherited •Chronic, fatal Transmissible Spongiform Encephalopathies

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