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Lecture 1

BIOL 4140 Lecture 1: Microbiology

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BIOL 4140

Microbiology: Introduction 1/18/2017 4:02:00 PM Intro- How microbes effect us and why we should care about them What are microbes? • Organisms so small that they are not easily visible without a microscope • They are diverse- bacteria, archaea, eukaryotes (fungi, protists, animal parasites), viruses, prions Pathogens were the first to grab our attention • = microbes that cause diseases How they effect us…. • Microbes can spoil or preserve foods • Tooth Decay • Needed for vitamin C production—deficiency was an issue for sailors/ pirates • C. Difficile infection- fatal infection; resident gut microbiota (without antibiotic perturbation) often blocks infection • Chronic wound infection 1/20- history of microbiology History of Microbiology- to understand how things change and develop Biology started with the discovery of the microscope discovery of microorgnaisms • Antoine Van Leeuwenhoek • Following Hooke’s 1665 use of microscopes to observe plant cells • Established that there were forms of life that were not visible to the naked eye; originally referred to them as animalcules (tiny animals) • Observed bacteria from his teeth plaque Sterilization and development of germ theory • Lazzaro Spallanzani- evidence against “spontaneous generation” of living creatures o Boiling broth would sterilize it and kill microorganisms in it o New microorganisms could only settle in broth if it was exposed to air • John Snow- tracing the source of cholera; One of the pioneers of epidemiology Broad Street Cholera outbreak- major outbreak in London; many died • Dominant theory was miasma theory- diseases such as cholera and plague spread by “bad air” from rotten organic matter • John snow investigates.. skeptical of miasma theory; interviewed locals and mapped area o Found that sections where the closest source of water was the Broad street pump had the most cases of cholera public water pump was the source of the outbreak • Anomaly: there were no cases in the Broad Street Brewery—got beer from boiled water Vaccination- Edward jenner- Smallpox vaccine= world’s first vaccine “The father of immunology” • Observed that milk maids were immune to smallpox Jenner postulated that contact with cow pox blisters protects them from small pox o It was known that infection with cowpox could prevent against infection with smallpox o Investigators tested cowpox vaccine with success • Jenner established immunity • In 1840, the vaccine was offered to the public by the British government • “Saved more lives than the work of any other human” What are the challenges against vaccination now? Development of Germ Theory- Louis Pasteur • Founder of medical microbiology • Germ Theory 1861= growth and reproduction of microbes within their host can cause diseases • Suggested forms of sterilization: filtration, exposure to heat, or exposure to solution/ chemical solutions • Developed a method for attenuating a virulent pathogen for immunization (inspired by Jenner’s success with smallpox vaccination) Classification of bacteria- Ferdinand Julius Cohn • His classification of bacteria was based on shapes- rod, spherical, spiral, thread Microbes as causal agents of disease • Robert Koch- he found that the blood of cattle who were infected with anthrax always had large numbers of Bacillus anthracis • He could transmit anthrax by injecting a small sample of blood from an infected animal into a healthy animal • Founded the scientific method of microbiology Koch’s postulates; to identify disease-causing agents • The microorganism or other pathogen must be present in all cases of the disease • The pathogen can be isolated from the diseased host and grown in pure culture • The pathogen from the pure culture must cause the disease when inoculated into a healthy, susceptible laboratory animal • The pathogen must be re- isolated from the new host and shown to be the same as the originally inoculated pathogen Discovery of viruses, environmental microbiology • Martinus Beijerinck- discovery of viruses; development of enrichment culture techniques • Sergei Winogradsky- developing the concept of chemoautotrophy; Nitrogen cycle; Winogradsky columns Discovery of Archaea- Carl Woese • Based on differences in Ribosomal RNA • Wasn’t accepted without resistance at first Notable discoveries st • 1878 Lister- developed the 1 method to isolate a pure bacterial culture (lactic fermentation of milk) • 1880 Pasteur- developed a method for attenuating a virulent pathogen for immunization (inspired by Jenner’s success at smallpox vaccination) • 1881 Koch- developed solid media for cultivating cells on the surface of the plate (simplified the process of isolating microbes) • 1884 Koch- identified tubercle bacillus as the agent that caused Tuberculosis and formulated the Koch postulates Noble prize in 1905 • 1889 Beijernick- developed enrichment cultures for isolating a pure culture if Rhizobium • 1891 Ehrlich- proposed that antibodies are responsible for immunity against diseases Noble prize in 1908 • 1893 Smith and Kilbourne- established that ticks carry Babesia microti (which causes babesiosis in animals and humans), providing a foundation for animal host studies and insect vectors • 1915/1917 Twort & d=Herrelle- discovery of bacteriophage (virus that infect bacteria) • 1928 Griffith- discovered transformtion in bacteria, establishing the field of molecular gentics • 1929 Felming- discovered penicillin and its effect on bacteria, starting the “Antibiotic Era” Nobel prize in 1945 • 1935 Stanley- crystallized tabacco mosaic virus and showed in remained infectious Nobel prize in 1946 • 1941 Beadle and Tatum- used fungus Neurospora crassa to establish the link between enzymes and corresponding genes (one gene- one enzyme) Nobel prixe in 1958 • 1943 Luria and Delbruck- shows that inheritance in bacteria follows Darwinian principles Nobel prize in 1969 • 1946 Lederberg and Tatum- discovered conjugation in bacteria • 1949 Enders, Weller, and Robbins- developed technique to grow viruses in culture Nobel prize in 1954 • 1952 Lederberg and Zinder- transduction: transfer of genetic information by viruses o a phage of salmonella can carry DNA from one bacterium to another • 1961 Brenner, Jacob and Meselson- used phage-infected bacteria to show that ribosome’s are the site of protein synthesis • 1982 Prusiner- discovery of prions Nobel prize in 1997 • 1983 Mullis- used heat-stable enzyme from Thermus aquaticus to establich PCR Nobel Prize in 1993 • 1993-2013 Mojica, Bolotin, Siksnys, Doudna, Church and Zhang- Discovery of CRISPR-Cas9 bacterial adaptive immunity machinery, which enabled flexible gene editing Nobel prize to come soon Transition to Genomic era • Genomics has revolutionized every aspect of microbiology, from tools to conceptual frameworks • Whole genome sequencing has become a common tool to reveal the genetic potential of organisms • Application not limited to analysis of genes • Sequencing to us is what microscope was to van Leeuwenhoek Tools for modern microbiology • Genomics: Study of the DNA/ genomes of organisms • Transcriptomics: Studying RNA molecules in one cell or a potulation of cells • Metabolomics: Studying molecules involved in cell metabolism • Proteomics: Studying the profile, structure, and abundance of protiens Form single species studies to communities- many microbial functions happen within assemblies of interacting species called communities, not as acts of isolated species • What microbes do depends on the environment theyre in, and that environment depends on other organisms • Requires a shift in studies towards examining microbial activities in communities o Examining monocultures might not be enough 1/ 23 Prokaryotes and Eukaryotes 1/18/2017 4:02:00 PM Bacteria- unicellular organisms • prokaryotic because they lack a nucleus • around for more than 3 billion years; evolved into complexity with a lot of capabilities • 4 categories by shape= rod, spherical, spiral, curved • divide asexually; divide by binary fission; may have flagella for motility • Gram staining tells differences in structure • Example= E. Coli; rod shaped bacteria o Mostly harmless- commonly found in intestines o Beneficially by producing vitamins (Vit. K) and prevent colonization by pathogens o Also pathogenic strains—can cause food poisoning like symptoms o Popular- can be grown and cultured easily- most widely studied prokaryotic organism • Example= H. pylori- famous because of role in causing ulcers o Relatively small o Can remain in our stomach without causing any problem- more than ½ people have it in upper GI tract and over 80% of individuals infected will never show symptoms o To avoid stomach acid, they use flagella to embed themselves in stomach lining mucus ▪ In some people can cause inflammation ulcers can become cancerous Archaea- differ from bacteria in cell wall structure; also prokaryotes with no clear nucleus • Avidity to extreme environmental conditions • Can also produce asexually by binary fission (use flagella) • Not as well studied- harder to culture/ reproduce and some are anaerobic • Example- Korarchaeum cryptofilum- found at high temperatures but found in low abundances • Example- Methanococcus maripaludis- found in salt marsh sediments; obligate anaerobes; produces methane Fungi- mushrooms, molds and yeasts; eukaryotic cells with a true nucleus • Molds= multi-cellular organisms; form filamentous tubes called hyphae that help absorb material • Yeasts- unicellular; may produce sexually or asexually • Obtain nutrients by absorbing organic material from their environment; can spread and reproduce by releasing spores • Example- saccharomyces cerevisiae- oval-shaped yeast; harmless and beneficial o Only pathogenic in blood stream and immunosuppressed patients o Can be grown and cultured easily for lab; most widely studied eukaryotic microbe; used to produce alcohols from sugars Protozoa- unicellular aerobic eukaryotes (nucleus); complex organelles and obtain nourishment by absorption and ingestion • Divide based on mode of locomotion o 1. Flagellates produce their own food and their whip-like structure to propel forward o 2. Ciliates- tiny hair to beat to produce movement o 3. Amoeboid- false feet/ pseudopodia used for feeding and locomotion o 4. Sporozoans- non-mobile • essential in marine environment • Example= amoeba proteus- eukaryote with phospholipids bilayers membrane similar to other eukaryotic organisms Algae- diverse group of photosynthetic organisms which aren’t closely related • Poorly defined • Live in water, soil, rocks and produce oxygen • Believed that they are the origins of green land plants • Example= cyanobacteria- photosynthetic; nitrogen fixing bacteria that live mostly in soil and water o Chloroplasts found in plants have evolved from cyanobacteria endosymbiosis o Harmful- concern for drinking water supplies; can clog filters and aren’t safe for consumption o Application in biotechnology because so versatile- agriculture, generation of renewable energy Helminths- multi-cellular animal parasites; visible to the eye; live and feed on host, receiving nourishment by disrupting hosts’ nutrient absorption weakness and disease • May not all be bad; modulation of immune system may prevent allergies and autoimmune diseases o  co-evolution of parasites with host aren’t as harmful • Example= A. lumbricoides- round worm; affect 1 billion worldwide; eggs live in soil • Once swallowed, they go in small intestines and become adults • Female worms produce as many as 200,000 eggs per day for a year Viruses- non-cellular entities consist of nucleic acid core; different from everything else; microorganisms, yet not living organisms • Cannot reproduce or feed themselves/ metabolize without a host • Infest prokaryotic & eukaryotic cells and exploit the cellular machinery of the host to replicate • Example= lambda phage- infects E. coli; typical structure of outer protein capsid enclosing genetic material; can enter into lytic phase which it kills and lyses cell to produce offspring o Used as model organism for studying bacteria-phage interaction and used as molecular genetics tool for cloning Prions- infectious agent composed entirely of protein; disease due to abnormal folding of regular proteins • Folding can be used as a guide to mis-fold other proteins in the cell reason they can be pathogenic • Animal prion diseases- example= mad cow • Human prion disease are often in NS- o CJD- can lead to death Microbes are diverse- Bacteria are more diverse! (picture on graph—top shows bacteria diversity, bottom is Archaea) • 1/25 Taxonomy 1/18/2017 4:02:00 PM Taxonomy- the classification, description, identification and naming of living organisms • Carlos Linnaeus- proposed Linnaean taxonomy, a system of categorizing and naming organisms using a standard format o Divided natural world into 3 kingdoms: Animals, Plants and Minerals • Developed into a hierarchy, based on kingdom, class, order, family, genus and species Phylogeny (based on evolutionary relations)= evolutionary history of organisms • Arranging organisms by how closely related they are phylogenic tree (tree of life) • Initially based on visual classifications • Linnaeus- developed a new way to categorize animals and plants Haeckel- proposed the four kingdoms Whittaker- added fungi to the tree of life Tree of life- based on similarity to humans Endosymbiotic theory- history of how mitochondria came about • an organism originally trapped inside an organism based on similarity between organelles and bacterial cells- proposed by Margulis Classification hierarchy • Life domain kingdom phylum class order family genus species Classification by appearance- most intuitive way of characterizing; helpful for identification Classification by traits • Temperature Response o Psychrophiles- low temperature; below/ around freezing; can cause contamination of refrigerated food o Mesophiles- room/ body temperature; the ones we typically deal with when we deal with pathogens o Thermophiles- high temperature (in compost) o Hyperthermophiles- very high temperature • Based on oxygen dependency o Aerobes- (strict/ obligate aerobes) ▪ Need/ use oxygen for growth ▪ Subgroup= Microaerophiles- technically aerobes; use oxygen for growth but are poisoned by high concentration of oxygen o Anaerobes- can grow in the absence of oxygen ▪ Subgroup= aerotolerants- don’t use oxygen but not harmed by it o Facultative anaerobes- have both capabilities; can survive in both aerobic and anaerobic environments • Classification by Metabolic Capabilities o Source of energy ▪ Chemotrophs- obtain energy by the oxidation of compounds  Chemoliphotrophs- inorganic compounds  Cehmoorganotrophs- get oxygen from organic compounds ▪ Phototrophs- obtain energy from light o Carbon Dependency ▪ Autotrophs- produce complex organic compounds from simple substances present in its surrounding ▪ Heterotrophs- cannot fix carbon from inorganic sources but uses organic carbon for growth o Metabolic dependency ▪ Prototrophs- capable of synthesizing all compounds needed for growth ▪ Auxotrophs- cannot synthesize a particular compounds required for growth; need to obtain it from something else • Classification by Life-style o Association ▪ Endosymbiosis: Symbiont lives within the body or cells of the host; many instances of endosymbiosis are obligate ▪ Ectosymbiosis- on the surface of the host, including internal surfaces such as the lining of the GI tract • Cell wall staining o Gram-positive or Gram-negative- depending on cell wall structure • Growth on specific resources- growth plates are specific to certain types of organisms o Can potentially differentiate closely related organisms Taxonomy in the light of genetics (by genotype) • Has become the most dominant way of classifying microbes • American biologist Carl Woese discovered what appeared to be a living record in the evolution of organisms • Ribosomal RNA gene: conserved sites (C) and hypervariables sites (V) o rRNA gene is one of only a few genes present in all cells o conserved sites are similar among many organisms o Hypervariable sites diversity as organisms evolve o Conserved areas allow universal primers for amplifying this portion of the genome o Sequencing of the hypervariable areas allows differentiation of species • Ribosomal DNA sequencing for classification o Target rDNA depends on the branch of organism ▪ Prokaryotes- use 16S subunit ▪ Eukaryotes- use 18S subunit o Database of rDNA sequences allows identification of microbes o **Isolation of microbe DNA extraction Amplify RNA gene using universal primers sequence a portion (16S or 18S) of the rRNA by PCR Compare the sequence of the Database • Tools for classifying microbes (mostly bacteria) o RDP o NCBI BLAST • Advantage of Classifying: Access to previous work o You know what you’re dealing with and certain characteristics about it o Staphylococcus Epidermis- the things we can tell about it from databases; we don’t need to start from scratch even when we have just isolated the organism The challenge of defining microbial species • Historically, a species is defined as a group of individuals that actually or potentially interbreed in nature. In this sense, a species is the biggest gene pool possible under natural conditions o BUT ▪ Many organisms form hybrids ▪ Many microbes reproduce mainly asexually o …The definition of a species isn’t defined for a microbe ▪ we need to change our definition of a species… • Species identification based on rRNA gene similarity- using 97% 16S similarity as a measure to distinguish species in general works well o Limitations still… ▪ Identification limited to those already included in the database ▪ Early categories based on 527 bp portion of 16S subunit in bacteria, but later transitioned to full 16S ▪ 97% similarity may not be the best choice (subjectively; all primates would be grouped together) ▪ based on whole genome sequencing, there are exceptions and inconsistencies Pan-genome versus Core-genome (definitions aren’t very set in stone) • Core-genome= contains gene families shared by all the organisms within a group***  • Pan-genome contains all the genes families including shared and strain specific genes • Picture: showing 4 different strains of E. Coli… out of 4,000 genes there are about 3,071 that are shared between all strands… AND there are genes only present in one strand • Current species definition is mainly based on core genome*** History of Microbial Impact 1/18/2017 4:02:00 PM Humans have been living with and using microorganisms for much longer than they have been able to see them • Example: People were making fermented beverages • Example: Cheese, yogurt and bread making • What do we gain by looking into the details of microbial contribution? o Safety of the food product The Iceman- prehistoric humans had a limited grasp on the causes of disease; still they attempted to treat illnesses and infections • Otzi the Iceman- a 53000 year old mummy found frozen in ice of Otzel Alps on Australian-Italian border • Infected with the eggs of the parasite human whipworm, which may have caused abdominal pain and anemia • Also evidence of bacterium that causes Lyme disease • Trying to treat his infections with the woody fruit of a fungus with laxative and antibiotic properties, which was discovered tied to his belongings • Also had tattoos Early Notions of Disease and Containment • Several ancient civilizations appear to have had some misunderstanding that disease could be transmitted by things they couldn’t see o Quarantining leprosy patients o Miasma theory= transmission of disease via air of rotten material o Sewage in Rome- built a channel to carry sewage out from the city into the river to avoid accumulation of waste, and prevent disease What Causes Disease? • Suspicion of possibly invisible causes for disease o  Hippocrates: Diseases have natural causes from within patients on their environments, rather than supernatural causes o Thucydides- survived the plague in Athens and recorded the observation that survivors didn’t get re-infected even when exposed ▪ immunity o Varro—“certain minute creatures” in air and water were responsible for contracting disease Bubonic Plague AKA Black Plague • AKA black death • Caused by bacteria Yersenia Pestis • Killed ¼-1/2 of people in eastern Mediterranean years • Killed 50-100 million people in the 1300’s—most popular event 1918 Flu pandemic • another pandemic caused by viruses; unusually deadly; 500 million people infected around the world and killed 5% of world population (50-100 million people) • Contributed to 12 year drop of human’s life expectancy • Most influenza outbreaks target juvenile, elderly, or already weakened patients; pandemic killed previously healthy young adults Great Irish Famine • The famine in Ireland cause potato crop to fail significant drop in food resources famine 100 million died and 100 million had to migrate • Cause is potato blight, which destroyed potato crops throughout Europe • Possibly caused by microbial eukaryote, phytophthora infestans A look back to the past • Infections and food spoilage have tainted our impression of microbes the impression that they can be harmful • BUT things have been getting better… o Life expectancy (mostly caused by infections) has increased! o Infections aren’t killing as many people as they used to • Increase in life expectancy due to vaccination and antibiotics Vaccination- active immunity to diseases can be acquired by natural or intentional exposure to disease through vaccination • Use weakened/ inactive forms of microbes o  body will produce antibodies to fight them • more modern techniques involve cloning genes to mass produce the antigens that are producing the reactions Antibiotics, the good • Produces by organisms to fight • Treating a broad range of infections increasing life span • Lowering the bacterial load prevent infection (during surgery • Higher yield of livestock in animals, it promotes growth and prevents disease Antibiotics, the bad • AAD- diarrhea • Colon inflammation (colitis) o  Malabsorption characterized by a celiac- like syndrome o  impaired absorption of medications o ^ both due to inflammation • Impaired metabolism and absorption of vitamins disrupted • Rise of antibiotic resistant organisms • Susceptibility to other infections Rise of antibiotic Resistant Strains • Evolution drives microbes toward developing resistance How can Antibiotic Resistance Spread? • We eat it when we use it in livestock resistance o When its present in our food (meat products) • Patients use antibiotics to treat things that aren’t a bacterial infection (such as a cold)/ all infections being treated the same way o Resistance rises when people who don’t need antibiotics are treated with them • ** if we develop high enough resistance, its not effective anymore The Threat Is Real • Nevada dies of bacteria that was resistant to several microbiotics What Can we DO?! • Responsible administration of antibiotics o Avoid long-term, unnecessary, sub-lethal doses • Discovery of new antibiotics o Not enough corporate support o Research on previously uncultivated organisms
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