BIO1342 Microbiology - All Lecture notes PDF

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BIO1342 MICROBIOLOGY LECTURE 1 Brock Biol of Microorganisms, chapter 1. History of Microbiology  Compound microscopes (more than one lens) – invented around 1595 Antonie van Leeuwenhoek (1632-1723) – Dutch cloth merchant, one of the earliest uses of a simple microscope for examining minute details ...

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BIO1342 MICROBIOLOGY LECTURE 1 Brock Biol of Microorganisms, chapter 1. History of Microbiology  Compound microscopes (more than one lens) – invented around 1595 Antonie van Leeuwenhoek (1632-1723) – Dutch cloth merchant, one of the earliest uses of a simple microscope for examining minute details of living things. One of the first scientists to discover protozoa in water. Also identified minute bloodcarrying capillaries. -

Simple microscope (200x magnification) September 17, 1683 wrote to the Royal Society about his observations on the plaque between his own teeth. These were among the first observations on living bacteria ever recorded. Called them ‘Animalcules’ (~1 m diameter)

Spontaneous generation or Abiogenesis – Aristotle (384-322 BC) – he taught that living things can be generated without the presence of life (e.g. some animals spring from putrid matter, aphids from dew, mice from dirty hay etc). First challenged by Francesco Redi in 1668. Redi believed that maggots developed on meat from eggs laid by flies. He set out his experiment: meat in a variety of flasks, some open to the air, some sealed completely, and some covered with gauze. Maggots appeared only in the open flasks. One of the first experiments in which controls are used. Aristotle’s teaching held for long though and even Redi believed it. John Needham (1713-1781) argued for the case of life force and vital atoms Lazzaro Spallanzani (1729-1799) – refuted spontaneous generation in the microscopic world Bonner suggested using the term ‘germs’

CONCLUSIVELY DISPUTED BY PASTEUR (1822-1895) Pasteur’s experiment – two swan-necked flasks with sterile nutrient broth. One got in touch with microbes and dust from air, the other did not. In the one in touch with microbes and dust microorganism grew, in the other they didn’t – this is how he disproved the hypothesis of spontaneous generation.

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ROBERT KOCH (1843-1910) – Germ theory of disease Koch’s postulates: 1. The suspected pathogenic organism must always be present in animals suffering from the disease and should not be present in healthy individuals 2. The organism must be cultivated in pure culture away from the animal body 3. Cells from a pure culture of the suspected organism should cause disease in a healthy animal 4. The organism should be reisolated and shown to be the same as the original Koch also discovered Mycobacterium tuberculosis as the causative agent of TB.

Koch’s postulated today: - Remain gold standard in medical microbiology, but it’s not always possible to satisfy all postulates for every infectious disease - Animal models are not always available (e.g. cholera, rickettsias, chlamydias) Martinus Beijerinck (1851-1931) – Enrichment -

For an enrichment culture, a medium and a set of incubation conditions are established to select for the desired organism from an inoculum

- Famously isolated the aerobic N2- fixing bacterium Azotobacter Alexander Fleming (1881-1955) – Discovery of antibiotics -

Discovered that Penicillium notatum prevented the growth of Staphylococcus aureus Awarded Nobel Prize in 1945 with Chain and Florey

Paul Ehrlich first used Salvarsan i.v. for Syphillis. It was derived from arsalinic acid which was initially used to treat sleeping sickness (reported by H.W. Thomas and A. Breinl – British physicians) but the high dosage caused toxic side effects (e.g. blindness). The structure of arsalinic acid was revised by Ehrlich and organich chemist Alfred Bertheim => Salvarsan or Arsphenamine or 606 was discovered in 1909 and it was the first modern chemotherapeutic agent. Edward Jenner (1749-1823) – Vaccination -

In 1788 an epidemic of smallpox hit Gloucestershire Jenner noted that milkmaids with cowpox did not become infected Took material from pustules of milkmaids and inoculated into a child

1798 – an inquiry into the Causes and Effects of the Variolae Vaccinae.

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Modern advances in Microbiology - Deadly diseases now shifted from microbial to non-microbial - 1995 – first genome sequences - 2004 – first large-scale mentagenomics project by Craig Venter Classification of microorganisms - Tree of life currently generated by comparing nucleic acid sequences - In particular, the nucleid acid of a molecule that is present in all living things – rRNA (ribosomal RNA) Carl Woese (1928-2012) in 1977 defined Archae (a new domain or kingdom of life) by phylogenetic taxonomy of 16S rRNA – a technique which is now standard practice. He is also the originator of the RNA world hypothesis in 1967.

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LECTURE 2 Evolution – The tree of life Brock Biology of Microorganisms (Chapter 13) – Microbial Evolution and Systematics. Carolus Linnaeus – Swedish botanist – started grouping. Traditionally, life has been organised based on similarities. Linnaen classification - Based exclusively on morphological similarity - Hierarchical system independent of evolutionary theory, no assumptions on ‘relatedness’ are made Phylogenetic relationships are more meaningful: Phylogeny: study of the evolutionary history of organisms (both living and extinct) – started by Häckel in 1866. Tree of life – monophyletic tree of organisms – one origin MONERA – at the bottom. Group related organisms together and exclude non-related organisms – based on phenotype and genotype Morphology/Phenotype often very informative of relationships Need homologous characters when comparing! (homology is an absolute, not a spectrum) -

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Homologs: a gene/organ etc related to a second gene/organ by descent Orthologs: genes in different species that evolved from a common ancestral gene by speciation and kept original function. OR: Orthologs are homologous genes that are the result of a speciation event (e.g. Gene A present in one organism, this organism evolves into two separate organisms which also carry gene A => Orthologs) Paralogs: genes related by duplication within a genome and evolved new functions OR: Paralogs are homologous genes that are the result of a duplication event

Comparing non-homologs results in incorrect relationship assumptions! Monophyly – a monophyletic group is a group of organisms that consists of a species and all its descendants.

Non-monophyly:

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Paraphyly: e.g. birds and reptiles Polyphyly: Same species that have different evolutionary ancestry (e.g. in different parts of the world) Other morphological issues: Parallel evolution: independent evolution of same feature from same ancestral condition (e.g. sabre tooths in Europe and South America – one (SA) is related to marsupials, the other isn’t) Convergent evolution: independent evolution of same feature from different ancestral condition (e.g. hedgehog and porcupine; hedgehog is a mammal while porcupine is a rodent) Secondary loss: reversion/conversion back to an ancestral state/condition (e.g. loss of legs in snakes)

Morphology fraught with danger (genotype better?) – a lot of microbes, for example, don’t exhibit phenotypic characters. Advantages of molecular data – many genes are present in all organisms; data set is as large as genome size (allows for many comparisons to be made)

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Before the 1970s life was thought to consist of two domains: prokaryotes (bacteria) and eukaryotes Carl Woese identified a third domain – the Archaebacteria in 1977.

Archaebacteria – the third domain of life: - Many are extremophiles: can grow at extreme temperatures, salinity or pH (e.g. hot springs in Yellowstone – up to 100C, lakes in Africa – pH 11 or 12 and high salinity) EUKARYA, BACTERIA, ARCHAE – THE THREE DOMAINS OF LIFE e.g. human, wombat, capybara => human & capybara – non-marsupials; wombat – marsupial – NOT ENOUGH INFORMATION TO DRAW A CONCLUSION ABOUT THE ORIGIN (could base it on location, type of mammal, structure etc) We need more information: an outgroup - Take jellyfish for example – use as an outgroup – how related are the three organisms to it? – helps identify However, no outgroup known – we don’t know of any other form of life outside the three domains

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- Can use gene duplications as Outgroup. e.g Gene A becomes gene A and gene A’. Compare alignments of both genes in all three domains and you get a tree, but it has no direction. Use alignments of both A and A’ together and see similarities and probabilities – this tree now has direction. The rooted universal tree: - Currently there are a few known gene duplications - All show the same topology for the universal tree - Eukaryotes and Archaebacteria are sister groups (one within the other) Where did life evolve from? -

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Not Black smokers – temp at them is over 350C, ATP instantly degrades at 150C. The most an organism can endure (a litotroph) is around 120C White smoker – much cooler – 40-90C, methane- and hydrogen-rich, alkaline and trace metals; abiogenic hydrocarbon production. Twelve years ago they were biochemically categorised – more likely to support life. They’re ancient, can ‘live’ over 10k years, up to 60m tall, fossilised versions found (360 MYA sample). Hollow and full of tiny chambers with walls of pyrite (ironsulfide) which is also found in our electron-transport chains – core bioenergetics driver Early reaction vessels? – chemical evolution driven by a pH, temp and redox gradient. Difference in gradients harvested by ATPases etc. In biological systems, energy is conserved solely by the use of gradients. NO GRADIENTS – NO LIFE. Then Prokaryote diversity starts to form – after early reaction vessels, when lipids are formed, they are deposited around the walls. The lipids in Archaebacteria are very different from any other ones we know.

LECTURE 3 Structure of Bacteria (Chapters in Brock: parts of 3, 4 and 5) Cell morphology: - Coccus (Streptococcus pnemounea) - Rod (E.coli) - Spirillium

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Spirochete (Treponema – causes syphilis) Budding bacteria Filamentous bacteria (Streptomycin – used in many antibiotics)

Cell size: -

S/V Ratio: surface area to volume ratio; the bigger the radius, the smaller the S/V ratio. Small bacteria – large S/V ratio  Faster uptake of nutrients More cells per given resource  more mutations Drives evolution

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Cytoplasmic membrane: Phospholipid bilayer – hydrophobic (fatty acids) and hydrophilic (polar groups) In bacteria usually Phosphatidylethanolamine – polar head group Strengthened by hopanoids (molecules) (the equivalent to cholesterol) Not a rigid structure Essential for mycoplasmas

Phospholipid bilayer – archaea and bacteria – Ester linkage in Bacteria (and eukaryotes) but Ether linkage in Archaea for fatty acids – fundamental difference. Archaebacteria bilayer made of repeating Isoprene unit instead of palmitic acid (e.g.

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in bacteria and eukaryotes). Diglycerol thetraether – makes the structure of archaebacteria more rigid => enables them to survive hostile environments Membrane and secreted proteins -

Signal recognition particle facilitates that certain proteins get embedded in the lipid bilayer/membrance

Cell wall - Peptidoglycan (murein) – repeating chain of carbohydrates and acid (NAcetylglucosamine and N-Acetylmuramic acid)  can be up to 90% of the cell wall in Gram +ves and is about 10% in Gram –ves Outer membrane - Second lipid bilayer in Gram –ves (on top of the peptidoglycan, but not the same as it) This is what gives Gram –ves their characteristics. Second bilayer: Lipid A, Core polysaccharide, O-specific polysaccharide

Rough appearance Smooth appearance -

Miles Misra dilution – for counting bacteria Light microscopy: R=0.5 /numerical aperture 10x ocular lens and 10x objective lens = 100x magnification if light source is 500mm and N.A for 10x is 0.25 => R=1000nm = 1 m, hence we can see bacteria

Gram-negative bacterial cell wall – exterior phospholipid bilayer is elaborated and different from gram+

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Gram-positive bacteria – they don’t have a second bilayer, just a much thicker peptidoglycan layer. The difference in the two allows for differential staining (Gram staining – used in medical microbiology all the time). Gram staining: 1. A thin film of cells is applied to a slide and allowed to air dry 2. Crystal violet dye is added for one minute. It a basic dye – positively charged and binds with high affinity to negatively charged molecules (such as nucleic acids within the cell and to structures on the cell surface). 3. Rinse with water 4. Add iodine solution which forms a complex with the crystal violet and later makes the dye hard to remove from some of the cells 5. Rinse with water 6. Add ethanol to decolorize the cells (flood for 20 seconds). Ethanol extracts the crystal violet-iodine complex from gram-negative, but not from gram-positive cells (because they have a very thick cell wall). 7. Counter stain with Safranin – the gram-negative cells pick up the dye (and stain in pink) while the gram-positive cells remain violet. 8. (The slide is rinsed with water before viewing under a microscope).

CELL SURFACE STRUCTURES Capsules (can be either carbohydrate or protein): Bacillus anthracis causes Anthrax – has a very big capsule. Poly-D-glutamic acid (not known how it’s attached, assembled etc). - Can be polysaccharide or protein or both (a lot are carbohydrates). - Play a role in pathogenesis and biofilm formation. If you get rid of/knock off Anthrax’s genes (in a particular plasmid) responsible for the synthesis of its capsule, it’s no longer toxic.

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Biofilms (multispecies collection of bacterial cells) – the predominant bacterial phenotype in nature. The may form: - On solid substrates in contact with moisture - On soft tissue surfaces in living organisms - At liquid air interfaces They produce Extracellular Polysaccharide (EPS) and form/live around it. Full of extracellular DNA. Hugely important in infection (classic example is CF). Formation: - When they’re floating around, they’re called planktonic cells - When they’re on the surface – sessile cells Fimbriae and pili: - Predominantly proteinaceous, but can be glycosylated. - Involved in pathogenesis, biofilms and conjugation. - Involved in twitching motility (e.g. pseudomonas aeruginosa)

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Flagella (multimeric protein complex transversing both inner and outer membranes) Helical in shape Uses the same principle as a proton pump The motor proteins spin and this is how bacteria move Different localisation: - Peritrichous (they’re everywhere :D) - Polar – at one end - Lophotrichous – several flagella at one end, but can also have several flagella at another one - Amphitrichous – single flagella at both ends

ENDOSPORES -

Dormant stage of a lifecycle

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Extra layers of complex material – spore coat, high calcium, dipicolinic acid. They can stay dormant for years and be triggered and germinate to produce vegetative cells. Persister cells (antibiotics don’t kill ALL bacteria and those that aren’t killed enter a state and are known as persister cells – this is probably what causes chronic infections) – not genetic mutants.

LECTURE 4 Structure of bacteria – antibiotics (Chapters in Brock: parts of 15 and 27)

CELLULAR STRUCTURES/PROCESSES AS ANTIBIOTIC TARGETS -

An antibiotic targets an essential process – a gene without which that bacteria can’t live  the product of that gene is the target of a given antibiotic

Targets: - Cell wall (synthesis) - DNA replication (DNA gyrase, Topoisomerase II etc) - RNA transcription – RNA polymerase - Ribosomes (because they’re big, have got rna and protein components) - Folic acid and metabolism (some of the oldest antibiotics – trimethoprim (interferes in the pathway), sulphonamides)

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Antibiotics for the different targets: Cell wall synthesis: - Cycloserine - Vancomycin - Bacitracin - Penicillins - Cephalosporins - Monobactams - Carbapenems DNA gyrase: - Nalidixic acid and Cirprofloxacin  Quinolones - Novobiocin RNA elongation: - Actinomycin DNA-directed RNA polymerase: - Rifampin - Streptovaricins Protein synthesis (50S inhibitors): - Erythromycin (macrolides) - Chloramphenicol - Clindamycin - Lincomycin Protein synthesis (30S inhibitors): - Tetracyclines - Spectinomycin - Streptomycin - Gentamicin - Kanamycin - Amikacin - Nitrofurans Protein synthesis (tRNA): - Mupirocin - Puromycin Lipid biosynthesis: - Platensimycin Cytoplasmic membrane structure and function: - Polymyxins - Daptomycin Folic acid metabolism (oldest antibiotics):

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Trimethroprim Sulfonamides

ANTIMICROBIAL AGENTS Naturally occurring antimicrobials: antibiotics -

Penicillin came from a fungus (discovered by Alexander Fleming) not a naturally occurring

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Streptomyces (filamentous bacteria) – producers of a lot of antibiotics. Belong to Actinomyces (A).

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EFB = endospore-forming bacteria (not all)

Modification of natural antibiotics => semi-synthetic antibiotics Naturally occurring: -

Aminoglycosides (not widely used today because of neurotoxicity and nephrotoxicity; a lot of antibiotics have high affinity for the 30S subunit which we as humans also have, hence they can be toxic) – antibiotics that contain amino sugars bonded by glycosidic linkage (e.g. kanamycin, neomycin, amikacin). Toxic because they act on protein synthesis – on the ribosomes.  Considered reserve antibiotics for when other antibiotics fail.

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Macrolides – contain lactone rings bonded to sugars (e.g. erythromycin ) Broad-spectrum antibiotic that targets the 50S subunit of ribosome

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Tetracyclines – contain four rings; widespread medical use in humans and animals; broad-spectrum inhibition of protein synthesis; target 30S ribosomal unit

(Penicillin target – broad-spectrum against gram-negative – cell wall synthesis;)

SYNTHETIC ANTIMICROBIAL DRUGS: -

Quinolones – inhibition of DNA gyrase, damage to the DNA -> can’t replicate  Bind the A subunit of DNA gyrase (A2B2) (DNA gyrase – topoisomerase 2 that makes nicks in the DNA)  Resistance mediated by decreased binding (mutations in the DNA structure that will prevent binding)

The range of activity of antimicrobials depends on what the antimicrobial targets. Range of activity:

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Penicillins – gram-positive bacteria Sulfonamides – both gram+ and gramQuinolones – a range of microorganisms (as all microorganisms have to replicate DNA) Isoniazid – more specific, mycobacteria (TB) Polymyxins (synthetic peptide, produced from gram+) – on gram-, affects the outer phospholipid bilayer WORLDWIDE PRODUCTION AND USAGE

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ß-lactam: Penicillins, cephalosporins – semi-synthetic penicillins macrolides: against protein synthesis ß-Lactams antibiotics are one of the most important groups of antibiotics of all time. They interfere with cell wall synthesis  Include: penicillins, cephalosporins, and cephamycins Penicillins - discovered by Alexander Fleming - Primarily effective against gram-positive bacteria - Some synthetic forms are effective against some gram-negative bacteria - Target cell wall synthesis

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G kills bacteria by disrupting the formation of new cell wall material during bacterial growth A gram-positive cell wall consists of long glycan chains, each with alternating units of the sugar derivatives M and G. The G-M-tetrapeptide unit is called a glycan tetrapeptide. Peptide interbridges link the chains together into a meshwork creating an enormous and strong molecule called peptidoglycan. When the cell is growing and forming new cell wall material, the glycan chains are broken by bacterial enzymes so that new glycan tetrapeptides can be insterte...