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MICROBIOLOGY LECTURE NOTES Left Click to go directly to a Topic. 1. History of Microbiology 2. Chemistry of Life 3. Structure of Bacteria 4. Enzymes 5. Glycolysis 6. Microbial Classification 7. The Viruses 8. Host-Parasite interactions 9. Host-Parasite interactions II

History of Microbiology:
Best to think in terms of recurring themes: 1. Cause and cure of diseases 2. Nature of Putrefaction/Fermentation 3. Controversy over Spontaneous Generation.
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Ancients felt the world filled with invisible spirits which would explain things we couldn't understand. a. Death and Disease, Disability (there has to be a reason) WE STILL STRUGGLE WITH THESE THINGS IDEAS TODAY. Greeks had anthropomorphic gods who interacted with them and could cause disease. Later Greeks lost faith in their gods and formulated other ideas. They were noted thinkers. Example: Hippocrates--disease comes from an imbalance of intrinsic factors (nutrition) and extrinsic factors--air, exercise, etc. Four elements of importance to balance: blood, phlegm, yellow bile, black bile. When these get out of balance problems occur. Bleeding to intervene. Today we infuse (add) blood with different ends in mind. Hebrews and Egyptians believed in God and an afterlife. Some biblical accounts indicate that there was a vague notion of contagion developing --"Don't sleep in the House of a Leper". But also could get leprosy by angering the Lord. Angry Jewish God changed in Christianity. Jewish God brought plagues famines and disease to Egyptians, for example. Aristotle and others believed in abiogenesis. Life came from inanimate material. Myths among many ancient people talk of the origin of man, even, from decaying corn. Shakespeare wrote about crockadiles coming from the mud of the Nile. Van Helmont wrote recipe for mice---dirty underwear, corn, in a vessel---mice come out fully formed. Solves another inexplicable problem: The origin of life. Until this is properly understood we could never understand contagion. 1546 Fracastorius of Verona wrote of contagium vivum--immersed in a syphilis epidemic at the time. Other terms "Seminaria morbi". Described transmission through inanimate objects--fomites-Through air--"ad distans" and through direct contact. Work is ignored, no evidence for any of this. It just made sense to him. 1609 Janssen and Galileo grind lenses to produce low resolution microscopes---microorganisms below the resolution achieved. But improvements were to come. Hooke--Discovers cells with an improved microscope. Schleiden and Schwann discover all plants and animals are composed of cells. 1650's Leewenhoek--Delft Holland in the textile industry and part time lens grinder. Got good resolution to allow about 3 or 4 hundred X useful magnification. He put hay and pepper into water and then looked at it through his microscope. He saw microbes in an infusion as seen below. Leewenhoek saw bacteria, protozoa, yeasts and described all the microbial forms we now know, except for viruses. Although he is not mentioned in the science literature as observing his animalcules divide, nevertheless he believed spontaneous generation was untrue and in his original papers called the idea a "bad joke" as related by Dr. Moll at the University of Amsterdam.
Left Image depicts a hay infusion. Hay is placed in pure water for a few days and protozoa, bacteria, fungi, and algae develop. The larger cells on the left are ciliate protozoa. The small rod shaped organisms in single and pairs are bacteria. The image was made black and white at Northwestern to save space. This is likely very close to what Leewenhoek saw in the 17th Centrury.

Francesco Redi, in the 1700's did a simple experiment to show flies needed parents. Used cheesecloth screening and meat. Spallanzani, a monk, in the first part of the 18th Century boiled and sealed broths. When he was careful no microbes developed. His work was criticized by Needham, a
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Welchman of the Royal Society in that other factors, excluded by Spallanzani, were needed for Sp. Gen. Notably air. Needham performed similar experiments, sloppily executed, in which microbes grew from contamination. Spallanzani died before he could clearly disprove Needham. Louis Pasteur, a noted chemist, took up the challance and utilized broths allowing air but disallowing microbes. Grew broths at different altitudes and in a dusty cellar. Used broths with cotton to show the germs accumulated on cotton. Did the Swan neck tube experiment. Ignaz Semmelweis, an eastern european physician working in a Vienna Hospital, noticed the wards where delivery occurred by midwives had 10X less Puerperal Fever and deaths than those tended to by doctors. He showed he could dramatically decrease "Childbed Fever" by having doctors wash hands in chlorine water after dissection of cadavers and between patients. Was fired for blaming deaths on doctors who didn't wash hands. Oliver Wendel Holmes wrote on the Contagiousness of Puerperal Fever". Author, Physician, and Anatomy Professor. Late 19th Century. Lister used antiseptics on wounds and during surgery. He showed they healed much faster with the antiseptic treatment. Was also the first to isolate a pure culture by serial dilution: Bacterium lactis. Pasteur wanted to isolate a bacterium in pure culture that caused disease. Began working with Anthrax. Robert Koch in Germany did too. He worked with various preparations to provide solid culture media. A woman working in his lab tried agar, recommended by a cook who used it to solidify puddings because it stayed solid at warm temps. Koch was using sterile potato slices for media. Koch isolated anthrax and formulated his Postulates. Pasteur went to work on chicken cholera and discovered one could attenuate cultures and produce artifical vaccines. Pasteur solved the riddle of rancid wines in France's vinyards. Recommended sterile technique and Pasteurization (applied to milk and became a central method for controling TB, Diptheria, and other diseases). Weinogradski and others showed soil bacteria recycle nutrients. Chamberland developed a bacterial filter. Resulted in: a. discovery of viruses b. discovery of toxins Pasteur produced attenuated rabies virus and rabies vaccination procedure. Tried it on Joseph Meister. It worked. During the 20th Century we have: 1. Development of viral culture techniques and attenuation 2. Development of the electron microscope.
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3. Discovery of antibiotics (Fleming and Dubos) 4. Discovery of Prions (Pruissner) 5. Bioengineering--removal and replication of genes--incorporating them into microbes and switching them on. 6. DNA vaccines 7. Antiviral Compounds--ribivirin, protease inhibitors. 8. Translation of the entire genome of some microorganisms e.g. yeast. All not rosy---reemergence of infectious diseases a constant problem. St Louis Encephalitis, West Nile Virus, Lyme disease, AIDS, Hanta virus Sin Nombre, Ebola outbreaks, E. coli, Antibiotic resistant bacterial strains and so on.

CHEMISTRY OF LIFE: Introduction: 150 years ago there were natural occurances which were mysteries. Disease, Fermetation, Putrefaction--- A pile of farm silage steaming in the cool autumn--what caused these things? All were mysteries either accepted or attributed to God. Then when an invisible world came into focus with Leewenhoek we began to understand.
Search cache:N6ITmmDHARYJ:web.clark.edu/tkibota/240/Lect/Ch1_Time/Timeline.pdf history of microbiology

In this section we look at the nature of matter itself with an eye toward living matter to try to understand why chemicals behave as they do and how they interact to give us microbial life. Concepts: 1. Pure Substances or Mixtures. Elements and Compounds. Atoms and molecules and ions. Atomic mass, Atomic number, stable configurations of electrons. (Bohr Atom) Explain isotopes, Small molecules eg. Water (show visual) serves as a solute for complex array of molecules in a living cell. Passes back and forth across membranes by osmosis. Molecules tend to stick together (Hydrogen Bonding). Show: Bonds and Bonding Models. www.angelfire.com/mi/nccc/lec2.html Amounts of Things: 1. What is the atomic weight of hydrogen, H2, HCl. 2. Define a mole 3. Concentration: A mole per liter! 4. Discuss pH--- 10-4 M H+ = pH 4 IONIC AND COVALENT, POLAR COVALENT, HYDROGEN BONDS. 1. Ions and dissociation (one way things are acids)

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2. Demonstrate a change in H+ concentration over time in a bacterial culture. 3. Effects of pH e.g. basic dyes won't stain at low pH. 4. Acids decrease pH by releasing H+ ions. BASIC CHEMICAL REACTIONS: A. DECOMPOSITION: 2CuO ---> 2Cu + O2 B. SYNTHESIS: H2 + O2 ----> H2O C. DISPLACEMENT: Zn + HCl ---> ZnCl2 + H2 ORGANIC VS. INORGANIC MOLECULES: CH4 = SMALL ORGANIC MOLECULE ; NaCl or H2O = inorganic Carbon found in living organisms so compounds uniquely found in living things termed organic. They were first thought special = could not be found or created outside of life. Wohler synthesized urea in the early 19th century. Now we are making DNA and proteins, the most complex of the organic molecules. We can even make viruses.

IMPORTANT BIOLOGICAL MOLECULES:

1. Carbohydrates -- draw glucose, explain hydroxyl group and significance of them (hydrogen bonding). Show straight line and ring structure. Explain monosaccharide, disaccharide, polysaccharide. Hexose, Pentose, Trios. We will work with Glucose, Sucrose, and Lactose in the Lab. Polysaccharides structural---wood--mucopolysaccharides---slime and mucous-- and as energy sources in certain bacteria (fermentative bacteria). Also chitin.

Monomers = Single units examples, glucose and fructose= hexoses Another monomer = ribose, deoxyribose = monomers but pentoses

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ERYTHRITOL

Synthesis (dehydration synthesis or condensation reaction) Gram negative bacteria broken into two groups based on fermentation ability. Further identified by fermentation profile. Cell wall contains both glycan (sugar like molecules) and small peptides. Fermentation of sugars results in small organic acids pH goes down.

2. Fats: Two molecules, fatty acids and glycerol. Fatty acids can be saturated, unsaturated, monounsaturated, trans fatty acids. Isomers concept. Triglycerides, Phospholipids, protomembranes, nature of the double lipid membrane. Size = < 10 nm. Waxes formed from alcohols and fatty acids. Transparancies: Fatty acids are COOH acids; Fat= Glycerol by condensation with a fatty acid. Saturated Fat Unsaturated Fat Monounsaturated Fat Trans Fatty Acid Phospholipids Some bacteria store fat and can be stained with Sudan Black. Useful to store energy in fat bonds because they do not dissolve in the cell. Most organic carbon stored in the form of ringlike fats similar to cholesterol (hopanoids) in oil deposits. These are derived from bacteria.

3. Amino acids and proteins. Primary, secondary, tertiary, quartenary structures. Pleated sheets, loops, alpha helices, soluble insoluble, structutal (keratin), and enzymes. Relationship to nucleic acid. Inhibiting one enzyme from working can eliminate an infection. For example antivirals like AZT and Protease Inhibitors can knock down viral titer in an HIV patient below measurement. Show protein structures. DNA Components. Nitrogen Bases, A,C,G,T (PURINES AND PYRIMIDINES)--Capable of bonding to one another by nitrogen bonds. Deoxyribose--capable of bonding to
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bases and to Phosphate PO4. Sequence of bases determines sequence of amino acids in protein. REVIEW OF FUNCTIONAL GROUPS AND THEIR PROPERTIES: OH, COOH, C=0, SH, PO4, NH2, C-O-C, C-SS-C, Disulfide bridge or bond. Visit PDB databases, look for Protease Inhibitors, AZT etc, http://info.bio.cmu.edu/Courses/BiochemMols/BuildBlocks/Molecules.html PRESENTATION STRATEGY: (TEST NEXT WEEK) II. Emphasize the power of stearic and electrostatic interaction---also hydrogen bonding. But the engine of all these is steric interaction. Shape is of extreme importance in understanding microbiology, disease, drug efficacy and so on. a. Review shape and overview of carbohydrates ( use transparancies) b. Review and focus in on the properties of lipids and their importance in formation of cell membranes. 1. Hydrophobic and Hydrophilic 2. Tendency to form double layers eg. Agitation. 3. Let water pass through unopposed. Let fats dissolve into them. Lets fat solubles dissolve into them. So steroids eg. Hopanoids are present in cell wall---where did all the oil come from that exist in large lakes under Saudi Arabia? Answer= Cell membranes. (alternate theory geologic hydrocarbon processed by Archaebacteria deep beneath the earth). 4. Bacterial Cells which have more lipid in the cell wall are more susceptible to lipid destroying agents-like soap. 5. In a water environment the perfect barrier is lipid. (Why?) 6. Also, in a water environment lipid energy easy to store. (Why?) II. Nucleic Acid: A. Nucleic acid components: Ribose or deoxy, Phosphate, bases with Nitrogen: Adenine, Guanine, Cytosine, Thymine, Uracil (you don't want to ingest chemicals that have the same shapes as these (base analogs--Why?) B. Components arranged into nucleotides, Hydrogen bonds hold two strands together. C. Sequence of bases is a code for construction of enzymes which have a shape which makes them effective. Stearic fidelity of enzymes is maintained by the fidelity of the base sequence. REVIEW SOME pdb FILE OF DNA ON THE COMPUTER--FIRST VIEW TRANSPARANCIES OF DNA STRUCTURE. UNDERSTAND THE "CENTRAL DOGMA".
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3. PROTEIN STRUCTURE: a. Review the structure of amino acids. Note that some are ionic, some hydrophilic, some hydrophobic. Note the condensation between carboxyl and amino groups. Dipeptides, oligopeptides, polypeptides. Describe Primary, Secondary, and Tertiary Structures. Use transparancies and terms: helix, pleated sheet, loop, etcetera. b. Go to Cabrillo on the Net and review insulin (a small protein) and analyze a "Protease Inhibitor." Questions for review???? Structure of Bacteria:

Chemicals have properties. Biochemicals have both properties and ways of interacting with other chemicals eg. Fats vs. Carbohydrates. Fats form monolayers in water. Cell parts and Cells are the basic unit of life. They have attributes: Reproduction, metabolism, storage, transport, movement. We will look at a Prokaryotic cell from the outside in with an eye toward relating cell part properties to the biochemicals which compose them. 1. External Structures: a. Glycocalyx or Capsule (computer slides) 1. Slime formation prevents dehydration. 2. allows for colony stability under adverse circumstances. Can be a big problem with biofilm development in catheters or industrial pipes. Dangerous source of infection in hospitals which is not readily removed by disinfection. 3. Non pathogenic organisms also have a capsule. Alcaligenes viscolactis is one of them. 4. Capsule can interfere with immune defenses of the host. It can interfere with phagocytosis. It swells during an infection, in many cases (Quellung reaction base of serological testing). 5. Streptococcus mutans sticks to teeth due to a capsule. Plaque becomes tartar if not removed. 6. Alcaligenes Viscolactis in milk.
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b. Flagella--protein = flagellin. Anchored through the cell wall to a series of rings which rotate. Flagella is rigid and works more like an outboard motor than a whip. Must rotate thousands of times a minute in order to push cells with all that surface through water. 4mm to 6 mm per minute in E.coli. Flagella can rotate at 2500 rpm. All spirilla have flagella (axial filaments or endoflagella) about 1/2 of the bacilli and a few cocci. DEFINITIONS: 4. Polar, lophotrichous, amphitrichous, peritrichous. 5. Runs and tumbles. Taxis, chemo, photo, thermo, magneto. When a stimulus is present tumbles are inhibited and the flagella turns counterclockwise constantly toward the stimulus. This is positive taxis. When a substance initiates tumbles the cell's flagella go clockwise and the cell tumbles, resuming its run in another direction.

c. cell walls (exceptions--Mycobacterium, mycoplasma, archaebacteria, L-forms, spheroplasts, protoplasts. 1. Surrounding, encasing molecule is peptidoglycan. Visual. NAM-NAG-NAM-NAG-NAM-NAG-NAM-NAG This is the Glycan portion of peptidoglycan. Held together by beta linkages. Each Glycan strand held together by chain of amino acids to another strand. "Chain Link Fence" analogous. 2. Examine differences between Gram + and Gram - cell walls.

To the left is a gram stain of Staphylococcus epidermis done in our lab. The spherical cocci are tiny and are gram positive because they retain the primary stain crystal violet.

Below is a gram stain of a mixed culture of Micrococcus luteus (box like arrangements of cocci) with Escherishia coli . Micrococcus is gram positive while E.coli is gram negative. E. coli consists of very small delicate rods seen pink because they lose the primary stain as they are gram negative.
The gram negative cell wall of E. coli is much thinner than the wall of Micrococcus. Coli's cell wall also contains less peptidoglycan and more lipopolysaccharide.

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3. Explain LPS and its role as an endotoxin and fever producer. 4. Also mention Spheroplasts and Protoplasts, L forms. Chlamydia and Rickettsia. d. pili and fimbriae: Structures that allow sticking to surfaces---important in pathogenesis eg. UTI'"s, biofilms, teeth, glass, rocks (Pellicle formation in culture). Some Pili specialized. Sex pilus. 1946 experiment with minimal media showed genes transfered requiring contact. Define: auxotroph, prototroph, mutation rate. Minimal media (Glucose salts media). Mating types. F+ and F- (movie)

e. other structures: 1. Chromatin Body--Long cirucular piece of DNA. Couple of thousand genes. It is haploid and relatively easy to manipulate eg...stick other genes into using bioengineering techniques. 2. Plasmids---small cirucular pieces of DNA (F factor is on a plasmid, Antibiotic resistance carried on plasmids). They are easily transferred from one cell to another. During sexual transfer recipient becomes F+. 3. Ribosomes---70s----50 + 30. Two parts. e. Mesosomes, endospores, fat globules, polyphosphate, Sulphur granules, magnetosomes, photosynthetic structures. BIOCHEMICAL REACTIONS AND ENZYMES I. Structure supports function. Function is physiology and metabolism. Metabolism is the sum of the biochemical reactions inside a cell. Lets take a closer look at how biochemical reactions or just ordinary chemical reactions occur. A. Collision theory: This makes sense as an explanation of how two molecules may interact to form bonds. One bangs into another. If the collision is energetic enough--we see this as the reaction mixture being hot enough---or as Activation Energy---and if the vulnerable parts of the molecules are the ones that get hit, we get bonds breaking and new bonds forming.
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H2O2 + I- ----> IO- + H2O IO- + H2O2 ----> O2 + H2O + ITwo points should be made about the above reaction. It takes two steps to happen. I- is not used up in the reaction. The reaction occurs due to contact with I- where bonds are broken and new bonds formed. What bond is broken and what new bond forms? Also, I- is an inorganic small ion. Small inorganic species that speed reactions are called catalysts. ENERGY DIAGRAM WITH AND WITHOUT A CATALYST. Define: Activiation Energy, catalyst (two examples), exergonic reaction, endergonic reaction. Show hydrogen peroxide and Iodide catalyst and catalase reaction. Explain how catalase identifies organisms Staph and Strep.

Enzyme attributes: 1. Large protein molecules at approx 40,000 amu, Glucose substrate about 180 amu. ;2. Denatured by heat--cooked potato vs. raw potato. Catalyst versus enzyme. 3. Lowers Activation energy by substrate binding at special place on the enzyme called the "Active Site"! There are other important sites on the enzyme but the active site is most important for catalysis.
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4. Cofactors which bind and release from the Active site are often required (not always)...Vitamins and minerals act as cofactors. B vitamins often needed for energy reactions. 5 Efficient and able to process thousands of substrates per second. So only a small amount of enzyme needed to do reactions. 6. Can be interfered with. Example: Penicillin combines with an enzyme which is needed to put peptide cross links in cell wall material (Why is penicillin more active against gram positives?) 7. Delicate in that environmental conditions for use are narrow---pH, Temperature, Osmotic pressure, concentrations of substrates must be withing proper limits. Extremes can denature and kill cells. FACTORS THE AFFECT THE ACTIVITY OF ENZYMES: 1. TEMPERATURE 2. PH 3. CONCENTRATION OF SUBSTRATE 4. CONCENTRATION OF ENZYME GRAPH THE AFOREMENTIONED VARIABLES AGAINST REACTION RATE.. ENZYME INHIBITION: 1. Competitive Inhibition. Show succinic to fumaric acid and malonic acid inhibitor.

Succinic Acid Fumaric Acid

Malonic Acid Inhibits the above reaction.

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2. Show structure of folic acid and PABA:

All inhibitors act at the active site. The inhibition is reversible by adding more substrate. New drugs are made which retain the properties of the inhibitor but which have new properties, like remaining in the urine longer etcetera. When an inhibitor "looks like" the substrate of an enzyme it can inhibit it either reversibly or irreversibly. In irreversible inhibition the contact usually occurs in a place by covalent bonding and not just by stearic reactions.

Feedback Inhibition, Allosteric Inhibition, Endproduct Inhibition. Precursor Activation, Energy link control. Positive and Negative Feedback. All involve ligands and receptor. INHIBITION AT THE LEVEL OF THE GENE. FIRST DISCOVERED WAS THE LACTOSE ARRAY OF GENES THAT E.COLI HAS. CALLED THE LAC OPERON--JACOB AND MONOD WORKED OUT THE MODEL: Wavy line made at R is repressor protein from R = repressor gene. I is the Incucer gene and a, b, c, and d are structural genes (make enzymes for lactose fermentation. Inducer can bind with repressor protein and "shut off". I----a------------b--------------c--------------d-----------R------------> /\/\/\/\/\/\/\/ Structural genes for lactose
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metabolism & repressor (protein always on)(constitutive enzyme) Another graphic with a promoter gene, which must be activated to turn system on:

LITHOTROPHS PHOTOTROPHS ORGANOTROPHS BREAK TO: 1. HETEROTROPHS (PARASITES, SAPROPHITES, Holotrophs) 2. Also Mutualistic bacteria and Commensals. Metabolism is all reactions, break to: A. Catabolic Reactions
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B. Anabolic Reactions. Example of catabolism is Glycolysis:

MICROBIAL CLASSIFICATION: Includes the following:


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1. Nomenclature 2. Taxonomy (which relates to Nomenclature) 3. Identification There has been an evolution, over the past 50 years, in the classification schemes used for microorganisms. Early on we had: Fauna and Flora (or Plants and Animals) Later---1960's we have the introduction of 3 Kingdoms: Plants, Animals, and Protists. Then Whittaker's 5 Kingdom System: Plant, Animal, Fungi, Protists, and Prokaryotes (Monera). Currently, with the advent of molecular genetics techniques we have three Domains serving as higher Taxa over Kingdom. Each domain has its Kingdoms. Three domains = Eucarya, Archaea, and Eubacteria. The domains arose because work with ribosomal RNA ca. 1500 base long units, showed clearly the natural evolutionary history of the cell types. Ribosomal RNA evolves more slowly than DNA because of a slower mutation rate. So examination of base sequences more clearly shows natural relationships (evolutionary relationships) between cells. Classification: 1. Binomial system of nomenclature. 2. Latin or Greek or latinized names. Bergey's Manual is the authority. It is constantly changing. Sarcina lutea to Micrococcus luteus and Streptococcus faecalis into Enterobacter faecalis. Zillions of more examples. Taxonomic Heirarchy: = Artificial mechanism to classify living things. But it can be used on any group of related types. For example:

Taxonomy of a Car Taxonomy and of Escherischia coli

DOMAIN
KINGDOM DIVISION CLASS ORDER FAMILY GENUS SPECIES STRAIN AUTOMOBILES AMERICAN MADE MINIVAN GENERAL MOTORS PLYMOUTH VOYAGER LE

MECHANICAL DEVICES INTERNAL COMBUSTION PROKARYOTES GRAM NEGATIVE SCOTOBACTERIA EUBACTERIALIS

EUBACTERIA

ENTEROBACTERIACEAE ESCHERISCHIA COLI O157:H7

Definition of a species is different in bacteria:


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1. Not based on sex---many do not reproduce sexually 2. Some have "out of species" sexual transfer. We are evolving in our definition of species from highly similar in characteristics to a genetic definition. Also, strain is a subgroup of species. Characteristics of bacteria: 1. Morphological---have definite limitations for classification. Hundreds of species have the same morphology. 2. Chemical Characteristics: Cell Wall type currently defines Division. Another example are the sulphur bacteria with S present internally. Differential Staining can help with this. 3. Metabolic Characteristics. Fermenters, Citrate Utilizers, Catalase possessers, Urease possesers, Acetyl-methyl carbinol producers are all examples of metabolic characteristics we use in biochemical tests in the lab. 4. Genetic Testing. We can now, with the host of enzymes we possess, magnify and sequence DNA by a variety of methods. If we know the genome we will have an "exact" definition of species. Viruses: I. Taxonomy--We use the same scheme eg. Class, Order, Family, Genus, Species. We don't need to go higher than Family for all common viruses. A. Examples of families: Picornaviridae, Poxviridae, Retroviridae. B. Genera have the suffix "virus" after them. For Picornaviridae Enterovirus (alimentary tract) species e.g. poliovirus 1, 2, 3 cardiovirus (neurotropic) species e.g. mengovirus ;rhinovirus (nasopharyngeal region) species e.g. Rhinovirus 1a apthovirus (cloven footed animals ) species e.g. FMDV-C hepatovirus (liver) species e.g. Hepatitits A virus C. Species is most difficult to assign. For example if we look at the immunodeficiency viruses we have: Retroviridae, lentiviruses, then HIV 1 HIV 2, SIV, BIV, Visna virus, FIV and so on. But there are many transitional genotypes in between, for example HIV 1 and HIV 2. Where do we put them. Strains? II. Virus types based on nucleic acid type and covering. We have: Double stranded DNA type example is T bacteriophage.
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We have Single stranded DNA type. Strand can be sense strand or a negative sense strand. We have RNA Viruses: 1. Positive sense strand RNA = mRNA 2. We have negative sense strand RNA, which must make a + strand, or do reverse transcription = Retroviridae = HIV and cancer causing viruses. 3. RNA viruses mutate like crazy. They don't have the repair enzymes available to them for replication. Also, virus is so small that RNA would have to be significantly larger to make an enzyme. One 100 amu amino acid needs 1000 triplet to code for it. What restriction does that place on the virus? This is why protein coat is usually made of 2 or 3 proteins that are made by the genome over and over and then assemble into simple geometric shapes like a 3-D Jigsaw puzzle. Virus can use the same gene over and over instead of having more nucleic acid, which could not pack into a multiprotein coat.

Look at the viruses on the above, in particular polio virus. It has the icosohedral shape with 20 triangles. And each triange is composed of 3 proteins(trangulation number = 3). This is very efficient allowing the entire coat to be composed of 3 proteins, saving space for polio's RNA. Helical viruses appear as if they were a tube but really they are helixal. A circle of proteins 1-7 make a ring, then similar rings are stacked up. Once again this economizes in the amount of genetic material the virus has to lug around. Encapsulated viruses have a lipoprotein envelope that surrounds the capsid. The capsid can be icosohedral (as in HIV) or another shape. Much of the capsid material is derived from the membrane of the cell the virus infects and is picked up on the way out. It has peplomers (spikes) derived from virus genetic material and on the surface are proteins which allow for attachment of the virus to the host cell. Viral Life Cycles: There are several steps in the life cycle of a virus: Attachment: The virus must contact a homologous protein on the surface of the cell. Attachment involves recognition of virus surface proteins with cell surface proteins. The virus requires a receptor. Often the lack of a receptor can explain why viruses are specific to a cell type or species.
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Penetration: The virus enters the cell. This can occur by different mechanisms. For example, the virus can be taken in by a process like phagocytosis. Or the virus can inject its DNA into the cell while leaving the capsid outside. Or the membrane (enveloped virus) can fuse with the membrane of the cell and slip in. Uncoating: Once inside the virus sheds its outer protein coat to release its genetic material and enzymes. This can happen by phagocytosis or by a breakup of the capsid. Nucleic acid then can begin to suppress normal cell processes to begin its reproductive activity. DNA viruses may enter the nucleus for replication. RNA viruses generally stay in the cytoplasm. Some DNA viruses and Retroviruses combine with host cell DNA and become Latent. DNA or retroviruses may have longer effects on the infected cell than a Lytic virus. Although we are seeing some persistent infectious effects with what were previously thought of as Lytic viruses. Protein Synthesis, Replication: The virus genetic material replicates to many copies of itself and synthesizes the proteins of its capsid and also any virus derived enzymes it carries. These components "Self Assemble" as a result of their conformation and charge distribution. Release: The cell bursts in lytic viruses---T phage and polio---or simply sheds virus as in HIV. The cell generally dies regardless of the process of release. To view a series of animations of the life cycles of phage and animal viruses go here. Click the link below!
http://www.cat.cc.md.us/courses/bio141/lecguide/unit2/adlyt.html

Prions and Viroids: Despite the conventional wisdom that all infectious agents are composed of DNA or RNA it appears that Prions are a class of protein, derived from normal brain protein,
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where a mutation gave rise to a particle that interacts with the normal protein changing its shape to the infectious Prion. This bypasses the nucleic acid replication needed in all other known infectious agents. It appears, at this point, that these infectious particles produce a disease that results in slow neurodegeneration. In Great Britain hundreds of thousands of cattle were slaughtered because they showed signs of Mad Cow Disease, caused by a prion which it is believed, was transmitted to cattle by feeding them renderings of sheep, including brain material. Sheep have been known to suffer a prion disease called Scrapie. The worry now is that these prion diseases seem to cross species lines with more ease than conventional pathogens. Evidence suggests that a human neurodegenerative disease "Creutzfeld Jacob Syndrome" may be caused by the new varient prion that is associated with Mad Cow. So far these problems in the human population have been extremely rare. Viroids: Viroids are naked bits of Rna that seem to have the ability to transmit themselves without associated proteins to cause infectious disease in some plants. It is believed that these particles are derived from transposons, or DNA that jumps from place to place inside normal cells. This is suggestive of a possible mode of origin of viruses.

Summary: Virues introduce their genetic material into cells. The virus genome either interacts with the host genome (it can incorporate and become part of
the genome or not) Incorporation and latency is termed lysogeny. If the virus actively reproduces and bursts the cell releasing new virus (lytic phase) the cell is immediately destroyed. Even if the virus is lytic--as in polio (RNA virus) the majority of new virus produced during a life cycle is mutated to the point of being unable to effectively infect new cells. The consequences of these events is unknown but may be the cause of disease in human, animal, and plant populations.

Host Parasite interactions in Disease:


Some definitions are in order: Disease: a deviation from the "normal" healthy state. Mostly thought of in the context of infectious disease but causes can be other than microbial. In this respect Koch was overgeneralizing to say "All disease is caused by microorganisms". There are also: 1. Genetic Diseases: Tay-Sachs, Sickle Cell Disease, Manic-Depressive illness, Cystic Fibrosis. 2. Developmental Diseases: Birth defects 3. Degenerative Diseases: Arthritis, Necrosis and calcification of injuries, Cirrhosis of the Liver etcetera. However, we will concern ourselves here with infectious diseases caused by microorganisms. Bacteria, viruses, protozoa, yeasts, and fungi all cause disease in man, animals and plants. Pathology: The study of disease. The prefix path relates in some way to disease: Pathogenicity: This is the ability of an agent to cause a disease. Pathogenesis: Is the manner in which the disease develops Etiology : Is the cause of the disease Infection: Root of this word implies to "dip into" or to "stain" it implies pathogens growing in the body.
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Question: Does an infection always result in disease? Are we all infected now? What is a subclinical infection? What is the "Carrier State"? Focal infections are infections where the etiology is an organism growing and damaging tissue in an area of the body where it is not normally present. Example is E. coli in UTI's

The Host
The animal, plant, bacterium, or human infected is termed the host. Infection is an interaction of host and pathogen. Both have factors that ward off the other. Here we will discuss some host factors. NON-SPECIFIC HOST FACTORS: Normal microbiota (normal flora or indiginous flora): These are organisms which normally reside in and on the body. Staphylococcus epidermis, Lactobaccilli in the female urogenital system, Propionibacteria and Corynebacteria on the skin, Streptococcus species in the mouth and so on. These organisms have an inhibitory effect on other organisms by: 1. Competition for nutrients 2. Production of chemicals like bacteriocins which directly inhibit other organisms. Escherischia coli and a number of anaerobes (belonging to Clostridium and other genera) occupy the colon. 3. By tying up receptors that pathogens need to colonize a host. Normal microbiota are added at birth and come from the birth canal. Later, when a baby begins nursing and eating food more organisms are added. The development of the digestive system would be abnormal without organisms present. If the normal microbiota gets out of balance due to antibiotic therapy a normal inhabitant may selectively survive and cause problems. Digestive problems after antibiotic therapy are almost always due to Clostridium difficile. Its spores probably resist the antibiotics then germinate after therapy is over. Result = diarrhea and other complaints. Urogenital and Oral Yeast infections also can result from imbalances in host microbiota. The white patches of yeast shown cannot be scraped off because the Candida yeast produces hypae like a mold which invades and anchors the organism to the tissue. Transients are organisms that cause no harm but remain for a short time on the host. OTHER NON-SPECIFIC FACTORS: 1. Physical Barriers: Skin and mucous membranes, mucous, ciliary ladder, coughing , sneezing, diarrhea, peristalsis, Bony encasements eg. skull, ear wax and other secretions. 2. Chemical Barriers: pH of the skin, urogenital tract, in males length of the urethra prevents UTI's, females not as lucky. pH of earwax about 4, or female urogenital system about 4---secretion of glycogen in mucous with hydrolysis and fermentation by lactobacilli keep pH down. Disruption may result in yeast infections. Transferrin binds iron at the first sign of an infection. For many organisms the result is too little iron for growth. In semen there is spermine, in tears lysozyme,
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often clots form due to infection to wall off the invader. There are also some white blood cells and macrophages which eat anything (non-specific) others are specific and only eat things tagged with antibody.

Kinds of Relationships between Host-Microbe:


1. Mutualism: Both benefit---E. coli in the intestine produces vitamin K and other nutrients, Lactobaccilli maintain urogenital pH around 4 in vagina. 2. Commensalism: Most of the normal flora benefit from the host but confer no advantage back, except, in their numbers maintain diversity and stave off infection. 3. Parasitism: The microbe benefits at the expense of the host. Parasites can be macroscopic (worms) or microbes. Infectious disease is a form of parasitism as is infestation. 4. Opportunism: Opportunists are organisms that are a part of the normal flora but, because of circumstance, become pathogens. Examples include: appendicitis, punture wounds (tetanus)--and the many organisms that infect immunocompromised individuals like those with HIV and who are undergoing chemotherapy, or have their immune systems compromised for another reason. Click here: Patient before death of Opportunistic Pseudomonas aeruginosa from HIV immune suppression. Synergy is when two organisms cause worse problems than either of them alone. Examples can be found in mixed infections like gangrene and HIV. Click to see X-RAY and clinical images of: SYNERGISTIC GAS GANGRENE Communicable and Non-Communicable Diseases: Classify these: TB, Influenza, Measles, Tetanus, Appendicitis, botulism. Communicable diseases: can be sporadic, endemic, or epidemic. Epidemiologists study communicable disease incidence and prevalence. Prevalence is the proportion of people in a population with a disease. Incidence is the amount of people with the disease. Epidemiology studies why and how people within populations get a disease and how the disease progresses through the population. They study ways to stem a disease so that it affects fewer people and try to stop it. The Communicable Disease Center in Atlanta studies epidemiology and reports every week on the state of health and the progress of various diseases across the United states. Would you like to read the weely issue? Click here for a copy of this week's. Free subscriptions too at: MORBIDITY AND MORTALITY WEEKLY REPORT. EMERGING AND RE-EMERGING INFECTIOUS DISEASES:
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Over the last twenty years we have had outbreaks of several new diseases and reemergences of some older ones. Examples include: Toxic shock syndrome (Staphylococcus aureus) Necrotizing Fascitis (Flesh eating Disease) Lyme Disease HIV Disease Hanta virus Sin Nombre Pulmonary Syndrome E. coli 0157:H7 Legionaires Disease ( a chlamydia like organism attacking macrophages in the human lung) Ebola Virus Influenza strains (Hong Kong virulent strain) West Nile Fever (Encephalitis in New York City) All of the above are the result of new ways of living and modern transportation. The number of new emerging diseases seems to be accellerating and is a constant challenge to epidemiologists. Some terminology: Nosocomial Infections: Those aquired in the hospital---it is estimated that from 20,000 to 80,000 infections are aquired in the hospital. Many resistant to antibiotics. The hospital is a sea of organisms in a reservois of immuno-compromised people full of a variety of antibiotics. Zoonotic infections (Zoonoses): Infections passed from animals to man. Some of these are extremely virulent: Hanta virus pulmonary syndrome, Lyme disease, Influenza etc. In many cases the organism is much less toxic to the animal eg Hanta. Incubation Period: The length of time between penetration of the pathogen and first symptoms or signs. For example, polio virus has an incubation period of from 5 days to two weeks. Prodrome: After infection but before full blown illness we have vague feelings of "something coming on". Symproms are mild and may include achiness, stuffiness, mild headache and so on. Period of Illness: The host experiences all the symptoms and signs of the disease. This is a point where either death or recovery follows. Period of Decline: Symptoms begin to ease. Fever gets lower, aches and pains recede. Convalesence: Period of weakness and recovery following illness. Convalescent serum contains a multitude of antibodies against the pathogen and used to be used to treat people in the Illness stage. CLASSIFICATION OF DISEASES BY INCIDENCE:
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Sporadic Diseases --- tetanus---pops up in a population from time to time. Endemic Disease: Endemic typus - rat flea is the vector so it is geographically isolated to areas where there are infected rat fleas. Epidemic Typhus: Human Body Louse is the vector so it can spread through an entire population of individuals infected with lice. Also, airborne illnesses like influenza become epidemic. Acute: Severe symptoms appear and person is in an infectious crisis. Chronic: Symptoms are milder but last a long time. Many illnesses that are chronic are difficult to manage or eradicate. Latent: Symptoms are followed by periods of wellness where symptoms again recur later. A good example is the natural history of a Syphilis infection.

An unfortunate commentary about our current culture is that diseases pass through borders (witness HIV) unhampered, but cures do not. This contributes to the problems of infectious disease. Reservois = Source of the disease (the poor)---yet we do not agressively treat the poor.

PORTALS OF ENTRY: Skin Mucous membranes (Respiratory, GI, STD's) We mentioned that pathogens must: 1. Enter the host 2. Attach to host tissues 3 Damage the host 4. Grow and multiply withing the host 5. (In the case of a communicable disease---escape the host) Lets look at the above in a bit more detail: A. Skin: parenteral route (inoculation), rusty nail, burns, all provide entry. Sex and inhalation or ingestion provide mucous membranes to a pathogen evolved to take advantage of these sites. Bacterial Pathogens have proteins called "adhesins" or ligands which promote attachment. M protein of streptococcus aids attachement and evasion of host
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defenses. Bacterial Enzymes that help it infect: 1. Leucocidins: Kills phagocytes, WBC's, 2. Hemolysins: Lyse RBC's also some WBC's to disrupt area of infection. 3. Coagulase: Staphylococcus aureus coagulates blood (this same enzyme is what activates the Staph Latex Test in lab). Clots can wall off growing organism from blood and phagocytes, although it limits the spread of the organism. 4. Kinases: Streptokinase a good example. These break up clots to allow for spread of the organism. Streptokinase used medically to dissolve coronary clots in a coronary attack. 5. Hyaluronidase: Dissoves cellular cement-- responsible for blackening of wounds as in gangrene infections. 6. Collagenase: Breaks down collagen in connective tissue. Gelatin extracted from collagen. 7. Sideophores: scavage iron out of body fluids. 8. Invasins: These enzymes can act on actin protein to rearrange the cellular skeleton so that the pathogen can penetrate cells (Salmonella can do this).

DAMAGE TO HOST
The damage to a host could be: 1. Direct Damage by enzymes or toxins at the infection site. 2. Systemic damage due to toxins 3. Induction of the immune system toward hypersensitivity reactions. TOXINS: Exotoxins: Are produced inside a cell and released to the outside. Some of the most poisonous substances know to man are exotoxins. For Example: 1 nanogram (10-9)g of botulism toxin will kill a guinea pig. Tetanus toxin produced in very small amounts in a puncture wound will kill an adult. Organisms that produce potent exotoxins need not be invasive to produce
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illness. The illness comes from small amounts of exotoxin toxin. Exotoxins are generally produced by gram positive organisms. Endotoxins: Endotoxins are less toxic than exotoxins, generally, and are released during lysis and death of gram negative organisms. They can produce fever, shock, and blood clots.

Exotoxin
Gram positive organisms Some Cytotoxins Some Neurotoxins Some Enterotoxins Extremely Toxic Antitoxin can be produced Proteins

Endotoxin
Gram negative organisms Pyrogenic (Fever Producing) Enterotoxins Clot Blood and Cause Fever Not as Toxic but may cause lethal shock No antitoxins Lipid from LPS of Gram Neg Cell Wall

SPECIFIC HOST DEFENSE:


When we refer to a specific host defense we are talking about immunity. Some cellular phagocytosis of pathogens is "non-specific" but some is the result of specific immunity. Terms: Antigen: A substance which stimulates the immune system. It generally must be large and is usually protein. However, sometimes small molecules (penicillin) can combine with larger body molecules and become antigenic, and stimulate the immune system. Antibody: A protein molecule produced by a class of lymphocyte call B lymphocyte, which is soluble and can combine with the antigen that stimulate it and produce a variety of responses to deactivate the antigen. Immunity is aquired through contact with antigens, which allows for a rapid production of antibody and a higher titer of antibody in the blood than before contact. The way this occurs is modeled in the "CLONAL SELECTION THEORY".

CLASSIFICATION OF AQUIRED IMMUNITY


Naturally Aquired
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Artificially Aquired
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Active Antigen enters body naturally and the body produces antibody and specialized lymphocytes

Passive

Active Antibodies are produced in response to vaccines body produces them and special lymphocytes

Passive Preformed antibodies are given in immune serum produced by another host (convalescent serum or horse serum). Introduced into the body through injection

Antibodies pass from mother to fetus through the placenta or to infant through breast feeding her milk.

CLONAL SELECTION THEORY AND ACTIVATION OF ANTIBODIES During fetal development specialized cells of the developing immune system move about the body and desensitize to self proteins. In other words, the fetal immune system recognizes self from antigen and this lasts throughout life. Any self antigens which will stimulate an immune response are somehow destroyed during this period. There is an area of the DNA in precursor cells of the lymphocytes which produce antibody shapes. The DNA mutates to the point of producing a large number of B lymphocytes (one of two categories of lymphocytes, the other being T) which then can produce a large number of different types of antibodies. Many of these antibodies are expressed on the surface of the B cell (They poke out). If a B cell combines with a particular antigen for which its antibody fits it will be stimulated to reproduce a clone of itself which will produce a large number of this antibody. Hence we account for large numbers of specific antibodies. The role of T cells is to stimulate the B cells and activate them by producing chemicals (chemokines) which act as cellular hormones. The T cells get stimulated by Macrophage which digest foreign cells and present antigen to T cells by direct contact. After this process some B cells of the specific antigen producing type remain as memory cells. Other B cells were stimulated by antibody and T cells to become plasma cells which produce antibody. DIAGRAM OF CLONAL SELECTION AND IMMUNE ACTIVATION

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