Lecture 2 - Handling and Visualising Microorganism PDF

Lecture 2 - Handling and visualising microorganism Describe nutrients used by microbes Different types of growth media, and their use Liquid media: broth contains nutrients required for growth. Porous stopper allows air but not contamination. Rapid gas exchange.  Shaken to mix cells, nutrien...

Lecture 2 - Handling and visualising microorganism Describe nutrients used by microbes Different types of growth media, and their use Liquid media: broth contains nutrients required for growth. Porous stopper allows air but not contamination. Rapid gas exchange.  Shaken to mix cells, nutrients, oxygen.  Homogenous  Useful for growing large numbers Solid media: agar plates contains nutrients, solidified with agar (red seaweed extract).  Grows separate clonal colonies (arising from a single cell)  Heterogenous  Useful for observing morphological characteristics Defined Media  Ingredients are pure compounds e.g. sugars (sucrose), inorganic salts (K2HPO4) General purpose  allows growth of many microbial types so similar to Complex media e.g. nutrient broth/agar. Selective  favours growth of one specific microbial type by actively inhibiting the ability of other types to grow e.g. media containing antibiotics Differential  where different microbial types give different colour/ visual reactions e.g. media containing a pH indicator  Even GENERAL PURPOSE cannot grow all microbes. Pure cultures contain only a single species and are important because they allow us to relate microbial identity and function. It allows scientists to understand each microbes role within a complex system. Importance and limitations of pure cultures Pure cultures contain only a single species (unnatural situation but useful for research) and allows us to relate microbial identity and function. Limitations of pure cultures:  microbial types cannot be isolated in pure culture- How do we know they exist  Pure cultures are not representative of microbial diversity  Pure cultures behave differently to microbial mixtures - Microbial interactions can only be tested in mixtures Aseptic technique – what is it, why use it? Aseptic technique prevents contamination of cultures, worker, environment.  Use of sterile equipment- inoculating loop, media, pipettes, flasks  Clean work environment - disinfected surfaces, biosafety cabinet, bunsen flame  Careful handling - Be aware of ubiquity of microbes - avoid contact with hands, dust, surfaces, any non-sterile items 1 Methods of obtaining pure cultures Streak Plate: (Isolation of Pure cultures) separate cells by streaking the sample onto solid media, mechanically diluting the mixture until single cells are obtained. 1. Make a patch of sample using sterile loop 2. Flame loop to sterilize, streak out from patch 3. Flame loop, streak out from 1st set of lines 4. Flame loop, streak out from 2nd set of lines Dilution Plate: sample is mixed 1:10 with diluent to give a dilution series Some common microscopy and staining techniques Microscopy  Microbial populations (colonies on agar) are visible to the naked eye, but study of individual microbial cells needs microscopy  Need to magnify the object AND resolve fine details  Total magnification = mag. (objective) x mag. (ocular)  Resolution: the smallest distance between two points that can be seen as separate NOTE: magnification quiz q often. Objective lenses for different microbes  10x : For finding cells on slide before switching to higher magnification. Resolution = 1.1 µm  40x : For examination of larger microbial cells: fungi, algae, protists. Resolution = 0.4 µm  100x: For examination of smaller microbial cells: bacteria. Resolution = 0.2 µm  Examples: Yeast cell (fungi ~10 µm), Bacterial ~1 µm and Virus ~0.1 µm Fixing and staining microbial samples  Samples first fixed to slide by heat/ chemicals –kill and immobilises (adheres) cells to help visualisation  Cells are then stained to give contrast (most bacterial cells are transparent and invisible unless stained to create contrast)  Most biological stains are basic (+ve charge) – these bind to acidic groups (–ve charge) in proteins, DNA, cell surface The Gram Stain  Most widely used stain for bacteria – differentiates them into Gram negative (G-, pink) or Gram positive (G+, purple) cell types  The G+ bacterial cell wall is thicker than the G- cell wall, crystal violet dye is retained by G+ after an alcohol rinse whereas G-’s are decolourised and counterstained with safranin for contrast  Separation into G+ or G- is biologically meaningful, correlates with natural evolutionary groupings of bacteria Other types of microscopy Phase-Contrast Microscopy Electron Microscopy SEM and TEM  Allows visualisation of live, unfixed, unstained  Resolution of the light microscope is limited by the samples. wavelength of light – eg. viruses (~100 nm) are smaller o Determination of motility than visible light wavelengths (~400-700 nm) o Can see natural morphology  Solution: use electrons (wavelength ~0.1 nm) instead of o Can see internal structures without light to view samples à electron microscope. staining.  Scanning electron microscope (SEM) bounces electrons  Phase rings in microscope convert off sample surface; transmission electron microscope differences in refractive index into (TEM) sends electrons through a thin section. differences in contrast  Sample preparation for EM is intensive: fix, dehydrate, coat with heavy metal atoms to increase contrast. 2 Lecture 3 – Major Infectious Diseases Four Major Infectious Disease HIC/AIDS, Malaria, Tuberculosis and Influenza  First 3 named big 3, 6 mil. Deaths in 2004/ 3.5mil in 2013.  Influenza for potential pandemics. HIV/AIDS Acquired Immune Deficiency Syndrome (AIDS) later shown to be cause by a virus named Human Immunodeficiency Virus (HIV).  Genome: Single strand RNA virus (Retrovirus)  Enzyme, Reverse transcriptase (RNA-dependent DNA polymerase)  Target: Human’s Immune cells (CD4 cells (or T-cells)  Transmission: virus is present in blood, saliva, semen and vaginal secretions Single stranded RNA recognises specific cell type – T-cells (protein in membrane allows it to bind to CD4+ cells). Next viral ssRNA synthesised into dsDNA by reverse transcriptase. Viral genome gets transformed to double stranded DNA which incorporates itself thus affecting the cell. Cells reproduce, spreading viral particles. Longer undiagnosed, prognosis worsens – replication of viral cells is beyond eradication. Origins and relationship between HIV and SIV  Occurs in 2 forms, HIV-1 and HIV-2, both related to SIV, Simian Immunodeficiency Virus.  Does not cause symptoms in original monkey hosts.  SIV crossed the species barrier at least twice: o From Chimpanzee to give HIC-1 around 1930 o From the Sooty Mangabey to give HIV-2 around 1940 Global Health Observatory (GHO) data Since the beginning of epidemic, almost 78 million people have been infected and ~39 million people have died.  Globally, 36.7m were living with HIV at the end of 2016.  1m people died from AIDS-related causes worldwide in 2016, compared to 2.4 mil. in 2016  Estimated 0.8% of adults 15-49 living with HIV worldwide, large scale burden Current understanding of HIV HIV infection requires virus to into the bloodstream. Its spread via unprotected sex, sharing needles and by blood transfusion. Now detectable by DNA or antibody techniques and has been essentially eliminated from blood transfusion. Symptoms usually appear years after infection/ Proven difficult to contain, particularly countries that don’t publicise “safe sex” and lack facilitated diagnostics. Malaria  Caused by the parasite Plasmodium, a single-cell eukaryote microorganism- a protist.  Vector-borne infectious disease transmitted by mosquitoes. The bite introduces parasites from saliva into the bloodstream. Cycles as within a human and mosquito. Curable with appropriate drugs. WHO estimates that in 2016, there were 216 million clinical episodes, and 445, 000 deaths, down from 1 mil in 2000. 3.2 bil. People live in areas at risk of malaria. It is geographically isolated. Several species of Plasmodium that can infect humans; severity can vary. Well- recognised: P. vivax, P. malariae, P. ovale and P. falciparum Plasmodium life cycle Plasmodium vivax has to alternate hosts. In human it infects liver and blood cells. Complex interaction with immune system as Plasmodium is at very low levels between bouts of fever. Control of Malaria  Reduction by 65% globally since 2000 3   Can infect people many times. Immune response complex which hindered vaccine development  Use of insecticide treated nets over beds can be very effective in reducing infection.  Recently, two key proteins the need to escape RBCs identified as promising drug targets Tuberculosis Bacterial disease cause by Myobacterium tuberculosis.  Chronic bacterial infection causing high morbidity and mortality  Airborne disease usually transmitted only after prolonged exposure  1.8 million deaths in 2015  Usually infects lungs, but other organs at later stages Myobacterium tuberculosis Complex relatively impermeable cell wall and is very slow growing. Very persistent but can be cured and eliminated in many countries – but difficult to detect and has latent stage. Progression of tuberculosis if not treated Global tuberculosis status 2016  In 2016, 10.4 million people fell ill with TB, and 1.7 million died from the disease.  Over 95% of TB deaths occur in low- and middle-income countries.  TB is a leading killer of people with HIV, in 2016 40% of HIV deaths were due to TB.  Multidrug-resistant TB (MDR-TB) remains a public health crisis and a health security threat.  WHO estimates that there were 600 000 new cases with resistance to rifampicin  Estimated 53 million lives were saved through TB diagnosis and treatment between 2000 and 2016.  Ending the TB epidemic by 2030 is among the health targets of the Sustainable Development Goals.  An estimated 53 million lives were saved through TB diagnosis and treatment between 2000 and 2016. Recent history of tuberculosis in developed countries Discovery of anti-tubercular drugs in 1940s & 1950s led to; 1. effective tuberculosis cure 2. dismantling of public health control systems 1980 developed countries saw a re-emergence of TB due to development of drug resistance in the bacteria and the increase in the number of immunocompromised people with HIV/AIDS  Peak of 27,000 cases of TB in the US alone in 1993, mostly people living in poor housing conditions and with access to limited medical care. The Australian situation (2017) One of the lowest TB incidence rates. Approx. 1300 new cases each year with majority of people who were born overseas. MDR- TB diagnosed is low, approx. 1-2% cases classified. Influenza Viral disease: Genome – ssRNA (single strand RNA)  Airborne route and inhalation. Infection largely confined to respiratory tract.  Generally mild pain with inconvenience, range from asymptomatic to life threatening.  Flu virus spreads around in winter seasons of two hemispheres.  Reported 2014 ~3-5 million cases of severe illness per year resulting in ~375000 deaths Model of Influenza Virus Haemagglutin (HA) and Neuraminidase (NA) cover surface of the virus. Both are immunogenic. Influenza Proteins 1 Antibodies against HA and NA is protective. Generates strong selection for these proteins to change to escape the immune response. Thus, new mutations accumulate = enormous variation.  antigenic drif. Occasionally re-assortment of RNA segments between strain of virus giving much greater antigenic change  antigenic shif. 4 Known Influenza Pandemics  1918 pandemic unusual as >50% fatalities were adults betw. 20-40 yrs.  Estimates 5% killed by 1918  25 mill. Killed in first 6 months compared to HIV/AIDS which killed 25 mill in first 25 years. Influenza Vaccines  Major disease in winter causing significant number of people to take time off work. Also, can be lethal  Vaccine produced each year – major current vaccine in embryonated hen’s eggs and takes months to accumulate.  H/e, cell culture grown vaccines are now becoming available.  No cure Lecture 4 - General Features of Prokaryotes Know the general features of microbes and understand the definitions of the Domains Bacteria, Archaea, and Eukarya and their key distinguishing features Strongly influence or dominate all environments: Mass: Bacteria and Archaea largest component of biosphere Number of Individuals Genetic/ Biochemical diversity Pathogens/parasites are commensals, co-operators or mutualists:  Inform regulatory pathways: Enteric NS and Endocrine sys  Requires for postnatal development  Support nutrition Microbes are a part of us; strong influence on our health, food, environment:  Managing infectious diseases must consider the impact of those management practices on other microbes on and around us.  Food export/import and Travel both increases spread of microbes.  Disinfectants and antibiotics change the selective pressures on microbes in our immediate environment.  Our diet changes the selective pressures on microbes in our digestive tract. Understanding distinctive aspects of structure and function are critical. Reliably discriminating different types of microbes i.e. structurally unique molecules that make something distinctive such as peptidoglycan or evolutionarily divergent molecules (e.g. RNA polymerases of bacteria share similarity to Archaea than Eukarya). Features that are specific to Bacteria and widely present are relatively safe broad- spectrum antibacterial targets. Ancient bacterial features homologous molecules present in most modern bacteria and are absent from humans, animals and plants. Features that vary between different bacteria are useful in bacterial diagnostics and classification and are potentially useful as specific antibacterial targets. Recognize the implications of cell features that are conserved within the domain Bacteria for applications in microbiology Generic Properties that differentiate microbes from macro-organisms Microbes are microscopic biological entities that can autonomously replicate (free-living cells) or can subvert another organism to their own replication (viruses and intracellular parasites) Small size means:  Few resources required to complete a generation  High surface area to volume ratio 5 Much faster and more efficient replication than microbes  They can outcompete for nutrients unless controlled. Apart from size, there are no general properties that distinguish all microbes from us. Different types of microbes have different properties, and we need to look at this molecular scale. Eukaryotes: A structural organization term – cells with membrane separation of transcription processes  The Domain Eukarya are exclusively Eukaryotes.  Predominantly microbial, also include the multicellular macro-organisms (animals & plants): o Fungi: distinct clade that are typically heterotrophic, non-motile, and saprophytic (extracellular digestion) e.g. Aspergillus o Algae: an ‘umbrella’ term for photosynthetic microbial Eukarya e.g. Volvox o Protozoa: an ‘umbrella’ term for heterotrophic, motile, ingestive cells e.g Amoeba Recognize the implications of structural features that are variable in the domain Bacteria (both between different species and in one species) Bacterial Morphology: Clinical Relevance  Morphology is useful for identification and classification  Both cocci, but differ in arrangement: clusters vs. chains  Spirochetes are rare pathogens. Cell shape very useful for diagnosis e.g. spirochetes observed in genital sores = syphilis  Gastric ulcers + spirilla = Helicobacter pylori  Respiratory distress + irregular rods in palisades = Diphtheria Summary Three domains: Archaea, Bacteria and Eukarya. The three domains were originally distinguished by phylogenetic analysis of rRNA – many other biochemical and evolutionary analyses support this and now overwhelming support for this view of the tree of life. Archaea – Distinct membranes. No known pathogens. All microscopic and have prokaryote structure. Bacteria – Highly biochemically diverse and include many pathogens. Vast majority are microscopic and have prokaryote structure (but some visible to naked eye and some do have a membrane-bound nucleus). All known cellular pathogens of plants and animals are either Bacteria or Eukarya (no Archaea). Eukarya – Highly morphologically diverse. Microscopic and Macroscopic forms. Include many pathogens. Complex multicellularity common.  The morphology of Bacteria can change with environment or life history stage.  It is important to recognize what features of bacterial cells are unchanging, constant features suitable for detection and identification – this is the basis of a stable and useful classification system for reliable diagnostics. 6 Be able to give examples of physiologically variable properties in Bacteria (environmentally influenced expression) Overview of Bacterial cell structure Typical structure and function of a Prokaryote All cells have homologous machinery for replication, transcription and translation – but evolutionary divergence means some differences.  Bacteria differ from Eukarya (and Archaea) in details of Replication, Transcription and Translation  Eukarya, including fungi and protozoans, very similar in basic processes Be able to describe possibilities and constraints on targeting processes of replication, transcription and translation for antimicrobial drug design The nucleoid: Where the chromosome is  Bacteria usually have a single circular chromosome  Chromosome (d.s. DNA) + structural proteins (histones) together comprise the nucleoid (tightly packed)  Bacterial nucleoid is NOT membrane bound (not a true ‘nucleus’ e.g. eukaryotes) The nucleoid: not the only DNA in Bacteria Bacteria often contain accessory DNA elements that can replicate independently of the chromosome: Plasmids -  made of circular or linear DNA  no protein coat Bacteriophage -  Made of linear double strand or single strand DNA or RNA  And they have a protective protein coat Replication and Transcription: essential functions and significance as drug targets  Nucleoid includes the core genome of the cell (Plasmids and phage: accessory functions of parasites)  Information flows from nucleoid to messenger RNA (via RNA polymerase) to protein (via ribosomes)  Antibiotics can target the nucleoid: ciprofloxacin inhibits DNA synthesis, while rifampin inhibits RNA synthesis. Ribosomes: Protein synthesis factories  Small (~20 nm) and very numerous (~20,000/cell)  Complex structure, made of many different subunits: both ribosomal RNA (rRNA) and ribosomal proteins  The 16S rRNA sequence is strongly conserved in all bacteria  can use PCR to diagnose disease  Small + large subunits: o Small – 30s o Large – 50s Ribosome: Function and significance  Functions: Translation of mRNA into a protein  Proteins (enzymes) central to all aspects of life  translation is a critical cellular function  Many different antibiotics inhibit ribosome function e.g. streptomycin, tetracycline, chloramphenicol Although many antibiotics target replication, transcription and translation machinery; they are typically only safe/useful for Bacteria 7 Examples of Broad-Spectrum antibacterial antibiotics that target processes also present in eukaryote cells  Effectiveness derives from the essential nature of these processes.  Specificity to Bacteria (safety) derives from evolutionary divergence.  Broad-spectrum against bacteria derives from conserved nature of these processes. In clonal population or single organism, all cells have identical DNA but may express it differently Bacterial cells can be morphologically and biochemically distinct via response to changing environments. E.g.  Different size and shape in starved or fed cells  Different flagellation levels in cells of one population Transcription influences expression because the amount of mRNA regulates the amount of gene product (protein) produced Imposes some constraints on how we use morphology to differentiate  Low gluc, high Lac: Lactose binds to repressor protein which removes it. Low glucose sends signals of lack or glucose which signals CRP repressor which turns transcription on - even without or with little glucose.  High gluc, low lac: Blocked by repressor proteins that bound to the DNA stop transcription. Bacteria trade off fine detail control for make fast large morphological pathways. Hence, they have multiple genes that work together expressed together. Global expression patters enable flexible response to environmental change and give rise to within-population diversity. Lag, stationary and death phase  gene expression different in each phase. Sigma factors  RNA molecules recruited by certain promoters (we don’t have).  Different sigma factors recognize different promoters  Different promoters are associated with different genes  Turning off one sigma factors means large suites of genes sharing the cognate promoter are turned off  Turning on a new sigma factor means a new set of genes sharing the cognate promoter are turned on Antibiotics can safely target the divergent replication, transcription and translation machinery of Bacteria Summary:  All three share basic processes of replication, transcription and translation. Bacteria are highly divergent from Eukarya. 8   DNA replication machinery is essential and can be targeted by antibiotics (Gyrase/Ciprofloxacin).  Transcription machinery is essential and bacterial versions are different from Eukarya (RNA pol has sigma factor) and can be targeted by antibiotics. (RNA pol/Rifampicin).  Translation machinery is essential and the bacterial version has some structural differences to eukaryal one and can be targeted by antibiotics. (Ribosome/streptomycin).  Bacterial genomes are more flexible than eukaryote ones – they can acquire new genes from plasmids or bacteriophage that are readily expressed. Traits such as possession of toxins or antibiotic resistance are capable of being ‘suddenly’ acquired.  How Bacteria manage their DNA has some differences to Eukaryotes. The key enzymes of replication and division are so divergent that they represent unique drug targets.  The small genome size of Bacteria means they can regulate global changes in gene expression relatively simply. We know they do this by looking at expression profiles over time (large sets of genes all turn on or off together). We know they do this in co-ordinated fashion because they can turn on or off in response to specific signals (lac operon example).  This genetic flexibility has consequences for: o targeting variably expressed structures o targeting metabolic pathways o classification and diagnostics of Bacteria. Lecture 5 - Special Features of Prokaryotes The nature of Prokaryote genome organization and gene expression means they are extremely flexible in terms of both gene content and expression pattern: For Bacteria phenotypic variation must be used very carefully in classification and considered in management strategies. Describe the typical structure of Gram-negative and Gram-positive cell envelopes Primary components are the cell wall and cell membrane(s). Function: Protective barrier against environment, separates cytoplasm from exterior. Prokaryote structure – significance Different morphologies have different surface area/ volume ratios. Morphology Coccus Rod Filament SA/V ratio Low Medium High Good for Survival Compromise Nutrient uptake Gram negatives have a different rigid cell envelops in comparison to Gram positives. Gram-positive peptidoglycan wall is connected to plasma membrane -thicker. Gram-negative has thin peptidoglycan. Diderm this outer membrane and plasma membrane. Comparison of cell envelope in gram + and gram – bacteria Peptidoglycan is on the outside of the plasma membrane of gram positive and there is another membrane on the outside of gram-negative bacteria. The Gram stain differentiates Gram-negative (red e.g. Escherichia (E.coli) and Gram-positive bactera (purple e.g. Streptococcus). Overall Plasma Membrane Structure 9  Function of the plasma membrane  Selective permeability: influx of nutrients, efflux of waste selectivity critical: can’t let cytoplasm our or toxins back in. The membrane is active.  Metabolic processes of the membrane e.g. electron transport, respiration, and lipid biosynthesis all occur in the membrane.  Site of environmental signal transduction  membrane receptors switch on regulatory protein  activates transcription  They can grow above 100 degrees as long as the pressure is enough to keep the water lipid. Explain the function of peptidoglycan in the Bacterial cell wall. Explain why it can be safely targeted by antibiotics with examples. Plasma membrane: Clinical and other significance  Organisms have a minimum temperature below which they cannot grow. This is due to the plasma membrane; if you get below, they go rigid and go solid (e.g. fridge). When the membrane gets rigid, the cell can’t work because there are proteins in the membrane.  Enzymatic processes go faster as temperature increases. Not a symmetrical rate. Collapses as proteins become less stable and lose structure. Also membranes will lyse and become disordered. Plasma membrane: structural differences Ether bond that links hydrocarbon to the head group. Archaea sometimes are monolayer and very rigid structures that allow thermophiles to survive. Eukaryal membranes commonly have sterols. Aromatic rings compounds that give additional flexibility. Ergosterol in fungi is essential, distinctive  can exploit for antifungals. Plasma membrane: target for microbial control Disrupts membrane structure  leakage of cytoplasm  cell death occurs. Examples are:  Synthetic: detergents (e.g. SDS), antiseptics (e.g. Benzalkonium)  Antibiotics: polymyxin, lantibiotics (e.g. Nisin targets plasma membrane gate)  Innate Immune system: Defensins, Cathelicidins Block lipid synthesis  dysfunctional membrane and cell death occurs. Example:  Antifungals: Block ergosterol e.g. Azoles (Canestan) 10 Plasma membrane: All domains, some variation in lipid composition Explain the function and distinctive aspects of the plasma membranes in Bacteria, Fungi and protists. Explain how it can safely be targeted by antibacterial and antifungal drugs. The Bacterial Cell Wall contains Peptidoglycan Cell wall depends on type of bacteria:  Gram + : peptidoglycan + teichoic acids  Gram - : peptidoglycan + outer membrane Peptidoglycan (PG)  Structural polymer made of sugars and amino acids unique to bacteria. Made of repeating di-saccharide unit; N-acetylglucosamine (NAG) + N-acetylmuramic acid (NAM). PG cross-linked by peptide bridges. Significance Function is osmotic protection. Cytoplasm very salty  higher pressure; membrane alone can’t resist. Often essential as enzymes of innate immunity target PG (lysozymes)  lysis and death of cell. Many antibiotic target PG biosynthesis e.g. penicillin, cephalosporins, vancomycin. Many Eukaryote patter recognition receptors target PG and initiate signalling pathways inform organism of Bacteria presence (Vibrio-Squid mutualism; Tracheal cytotoxin in whooping cough pathogenesis). Peptidoglycan monomer structure The conserved features of peptidoglycan make it a target for:  Lysozyme – bactericidal enzyme of innate immunity degrades peptidoglycan  Antibiotics – penicillin, cephalosporin. Blocks synthesis of peptidoglycan 11 Explain the structure and function of the outer membrane of Gram-negative Bacteria. Describe the significance of the Lipopolysaccharide Lipid A and O-antigen components. Outer membrane structure (Gram-negative only) Outer membrane: Function and Significance Border control: protective barrier against toxins e.g. antibiotics, bile and is also an entry port Presence of many sugars in LPS defines bacterial surface properties: hydrophilic, negative charge O-antigen is highly variable, allow Gram negative bacteria to evolve changes in surface  evades immune system (perhaps better thought of as ‘cross-talk’) Lipid A component is target for patter recognition receptors usually triggers inflammatory response so known as endotoxin  systemic toxic effects in gram negative infections. Cell surface polysaccharides most cells have glycosylated surface molecules (Bacteria, Archaea and Eukarya) But the type of polysaccharide and what it is attached to varies. Recognize the significance and applications of surface adornments such as capsule, pili, fimbriae and flagella Glycocalyx, capsule and slime layer  Glycocalyx is a layer of polysaccharides that is secreted by bacteria, and present on the outside of cell envelope  Capsule is a well-organised glycocalyx that is difficult to remove from the surface of cells  Slime layer is a more diffuse glycocalyx that can be removed by washing the cells  Capsule stain: India ink stains cells and background back but not capsule which appears clear Glycocalyx: Functions and Significance  Used in attachment – esp. the formation of biofilms  Protection against desiccation and other stresses  Help bacteria to evade immune system – particularly makes it difficult for phagocytes to recognise bacteria  Not that useful for targeting because some bacteria can turn them on and off.  Example: Klebsiella pneumoniae, colonies are mucoid, contributes to virulence Outer membrane structures ofen show variable expression porins and glycocalyx  Fimbriae and pili: attachment and sex  Thin protein strands found on surface of gram-negative bacteria  Fimbriae are very thin (~5nm diameter), very numerous (up to 1000 per cell), used for attachment to surfaces. 12   Pili are thicker (~10nm diameter), less numerous so only a few per cell may exist. They are used for transferring DNA i.e. for sex.  Pili are often encoded by conjugative plasmids which are self-transmissable between bacterial cells. Flagella Bacteria can be motile or non-motile. Most motile bacteria swim using flagella – threadlike protein structures (~ 20 nm x 20 µm) extend out from cell wall. The presence or absence of flagella and their distribution pattern around the cell is helpful for bacterial identification. Structure of Flagellum  Thousands of identical subunits of flagellin protein self-assemble into the filament  Filament is hollow and allows transports of flagellin units from the cytoplasm to growing up.  Thousands of flagella which are coiled into the membrane. Works like a boat propeller.  Polar which is one and then peritrichous flagella where there are numerous flagella.  Eukaryotes flagella wiggle because of a motor within the flagella whereas as bacterial flagella rotate like a corkscrew because of a motor stuck in the cell envelope. Function and Significance of Flagella  Flagellum filament is helical, rotates like a boat propeller to push bacterium forward  Source of energy for the flagellum motor is the membrane proton gradient  Advantage of flagella: allow chemotaxis = movement toward nutrients, or away from toxins  Disadvantages of flagella: - need protein to build, - costs energy to run, highly immunogenic (H antigen) and target of pattern recognition receptors (TLR5) Summary  The fundamental difference in the cell envelope between gram + (one membrane/thick peptidoglycan) and gram – (two membranes, thin peptidoglycan).  Much of the diversity seen in bacterial surface structures is aimed at three things: o Changing how other organisms perceive bacterial presence (immune evasion) o Changing how the Bacterial cell interfaces with the external environment (motility, permeability) o Sticking to surfaces (Fimbriae) Be able to describe the significance of endospores for sterilization and diagnostics Endospores Some Gram-positive bacteria (e.g. Bacillus, Clostridium) make endospores to survive under stressful conditions. Endospores are:  Metabolically inactive, but can germinate to yield new vegetative cells if conditions are favourable  Extremely tough - survive harsh conditions – e.g. heat, desiccation, radiation, disinfectants  Three major layers: Coat (protein), cortex (peptidoglycan) and core (DNA, ribosome, etc). Clinical and other significance  Spore-forming bacteria are difficult to kill by standard methods  require autoclaving (steam under pressure); we can’t say that we’ve sterilised something until we’ve killed the endospores.  Many clinically-important bacteria are spore-formers: e.g. Bacillus anthracis  anthrax Cell envelope summary: Structures/Functions/Significance Cell envelope of Bacteria very different to all Eukarya.  Bacterial cell wall has Peptidoglycan / targeted by penicillin and innate immune components such as lysozyme  Two broad bacterial cell wall organisations - Gram negative and Gram positive Cell membrane (plasma membrane) common to all domains. 1. Fungal membrane different from animal, has ergosterol targeted by azoles. 2. Bacterial plasma membrane has distinct molecules. Lipid II targeted by antibiotics. Other cell envelope features on Bacteria contribute additional functions 13   Gram negatives also have outer membrane – distinctive feature is LPS. A proinflammatory molecule and used in serotyping.  Glycocalyx/capsule – protection. Used for serotyping.  Flagella – motility. Used for serotyping and may be vaccine target.  Fimbriae and pili – protein structures for attachment (fimbriae/pili) and export/transfer of molecules including DNA transfer (sex pili). Used for serotyping and may be vaccine targets. Lecture 6 – Bacterial Identification and Classification The need for a hierarchical, monothetic taxonomy - Different problems require identification at different scales - Sheer numbers of species require organization (Classification) Purpose of monothetic taxonomy: simplify education by substituting one name for many facts. Through placing organisms into groups allowing:  Organization of huge amounts of information  Predictions and/or hypotheses about organisms and their properties  Essential for accurate identification of organisms  Different end-users to use the same database for different applications Morphological characteristics are useful in determinative schemes but limited for systematics. Be able to show applications with stains and limits with flagella. Rapid methods for identification: specific stains Gram stain reveals cell wall type. Thick walls retain crystal violet in alcohol wash. Spore stain reveals endospores in bacteria belonging to the genera Bacillus and Clostridium. Endospores retain malachite green dye after water rinse. Bacterial cells are counterstained with safranin. Why are stains useful? Cell wall type reliably discriminates into two kinds (Gram+ve or –ve). Nature of endospores discriminates further within the Gram +ves (eg. anthrax, tetanus, botulism). Can be used in a hierarchy of tests to guide identification. Flagellate and non-flagellate species are found in all three domains of life: archaea, eukarya and bacteria. Presence or absence of flagella is VERY useful for identification. However, the flagella are NOT homologous – they just look similar (convergence). Taxonomically, it would NOT be useful to classify all flagellate organisms together on this feature Physiological characteristics are useful in determinative schemes but limited for systematics. Be able to show inability to make nested exclusive physiological groups Differences in these characteristics reflect differences in the genes of the cell AND differences in the expression patterns of those genes These reflect what a cell actually does - useful to discriminate differences that are of practical significance. 14 15   Different properties are relevant to different fields. NOTE: Nitrogen-fixation ONLY Bacteria and Archae. Methanotrophy ONLY Bacteria. Methanogenesis ONLY Archaea. Nitrification ONLY Bacteria and Archaea Microbial classification presents unique challenges Simple morphology under light microscopy  limited set of easily observable features Convergent evolution e.g. many diverse species have developed spherical cells (cocci) as the optimum shape Horizontal gene transfer: features of one microbe may be acquired from multiple different sources (homologous genes are not necessarily orthologous) Facultative metabolism: “variable phenotype” one species may have several different possible ‘physiologies’ depending on how it is cultured e.g. genus Rhodobacter Prokaryotes Taxonomy Traditional morphological/ phenetic route of biological classification gave species descriptions BUT did not yield hierarchy of exclusively shared traits  unreliable predictive capacity. Taxonomy must be based on measurable aspects of microbes. The applications of classification and identification have distinct requirements - Determinative taxonomy is focussed on identification - Systematic taxonomy is focussed on classification Until recently microbiology had a determinative classification rather than a systematic classification. In determinative schemes identification is based on a vast library of biochemical and morphological test data that is used in numerical taxonomy schemes. Still widely used in pathology labs for commonly encountered clinical microbes. E.g. API strip: Analysis of carbon source utilization and enzyme activities based on colour production from indicator dyes – not used for pathogens but still reproducible way to distinguish organisms. Molecular sequence variation provides an evolutionary history that is not otherwise obtainable for microbes. Describe use of 16S rRNA to make trees 16 Diversity at the molecular scale All cells have a ribosome with the same basic structure. The sequence of the ribosome components varies. This sequence variation reflects the amount of evolutionary separation. Sequence variation in homologous macromolecules approximates the general biological difference in the organism. DNA, RNA, and protein sequences Sequencing nucleic acids and proteins has had a great impact on microbial identification and classification Huge amount of information. E.g. one DNA sequencing machine can read ~ 20,000,000,000 bases per day Availability of universally-present marker genes and proteins to allow comparisons across ALL species Don’t need to culture microbes to get sequence data Polyphasic taxonomy – describe integrated use of phylogenetic, genomic and morpho-physiological data for classification Modern microbiology employs “polyphasic taxonomy” where classification is underpinned by nucleic acid sequence data and physiological data. Phylogeny: The extent of divergence between homologous features is measured. Information about an organisms evolutionary relationship to all other organisms is incorporated into classification schemes Phenotype: Morphotype and physiology Information about an organisms structure and function - what it actually does – is incorporated into classification schemes Historically – it was recognized most members of the same species show >70% genome similarity in DNA:DNA hybridization. Core genes present in all strains:  Transcription/Translation/DNA processing  Central metabolic pathways  Basic cell envelope components (PG, membrane) Accessory’ genes, not present in all strains:  Alternate Carbon sources  Antibiotic resistance  Surface molecules (Fimbriae, capsule polysaccharides)  Phage genes Presence/absence of metabolic genes often useful to define species attributes. Higher taxa more usefully defined by genes encoding conserved features: structural (e.g. cell envelope) or essential processes (replication/transcription/ translation) Phylogenetic analyses of rRNA sequences led to the first useful taxonomic hierarchy for Microorganisms. 17  Characteristics that are non-essential or readily subject to horizontal gene transfer are unpredictable within this hierarchy. Many accessory genes (see Venn diagrams) are strain-specific and useful as epidemiological markers or for targeted vaccines. In the real world two further key points: 1. Polyphasic taxonomy ABSOLUTELY REQUIRES that you isolate and characterize the organism in the laboratory but using the scheme to identify organisms doesn’t. 2. The majority of naturally occurring microbes are not yet cultured, but their classification can be predicted by sequence information. Lecture 7 – Introduction to Medical Microbiology Current problems and emerging threats posed by microorganisms Infectious diseases: Leading causes of death worldwide Main causes of death in low-income countries Leading infectious killers worldwide Infectious diseases are one of the biggest causes of death worldwide, accounting for around 25% of all deaths – WHO 2004. Leading cause in low-income countries, most preventable or curable but can’t due to economic/ political reasons. Infectious diseases remain among the leading causes of death worldwide for three reasons: 1. Emergence of new infectious diseases i.e. discovery of new human pathogens e.g. Ebola hemorrhagic fever; New infectious diseases continue to evolve and ‘emerge’. Changes in human demographics, behavior, land use, etc are contribute by changing transmission dynamics. May involve exposure to animal or arthropod carriers of disease. 2. Persistence of intractable infectious diseases; Increased/ imprudent use of antimicrobial drugs and pesticides led to development of resistant pathogens, allowing many diseases to make a comeback (e.g. TB, malaria). Recently, decreased compliance with vaccination policy has also led to re- emergence of diseases such as measles and pertussis, which were previously under control. E.g. Malaria - many drugs are now quite useless against the malaria pathogen Staphylococcal resistance to methicillin in hospitals TB – increasing resistance to the drugs used to treat it 3. Re-emergence of old infectious diseases; Natural genetic variations, recombination’s, and adaptations allow new strains of known pathogens to appear to which the immune system has not been previously exposed and is therefore not primed to recognize (e.g. influenza). HIV – recognised in 1981 Cholera – usually associated with poor hygiene SARS – 2002/3 Infections secondary to HIV and Tuberculosis H1N1 influenza (Swine flu) “Old diseases” making a comeback because of H5N1 influenza (Bird flu) resistance etc. West Nile Virus (WNV) Term to describe symbioses Factors - host and microbe - determine where the host-microbe relationship fits in the symbiosis spectrum Normal Flora (NF): resident microbes associated with healthy individuals. Benefits: Aid digestion Stimulate immune system Supply essential growth requirements Aid resistance to infection Their interaction with the host is generally called ‘commensalism’, meaning they derive benefit from the association without harming their host. 18 Symbiosis: Living together of organisms. Spectrum of host/microbe association: - Commensalism o One partner benefits o Other partner not affected o ‘Commensals’ o Normal Flora - Mutualism o Both partners benefit o Often obligatory: eg ruminants, GIT flora o ‘Mutualists/Symbionts’ - Parasitism o One partner benefits o Other partner harmed o ‘Parasitic organisms’ e.g. B. anthracis - anthrax o Pathogen = parasitic organism that causes specific disease However, once again the distinction between parasitism and commensalism is not always absolute - the boundary between the 2 depends on how we define ‘harm’. E.g. For viruses, reproduction depends on taking over and usually destroying the host cell - but frequently this harm is not noticed as it is at too low a level to be detected by the host The Symbiosis Spectrum Factors governing symbiosis: 1. Number of organisms: ↑ numbers = shift to parasitism e.g. poor hygiene - buildup of normally harmless organisms until they cause harm 2. Virulence of organism: talk more about this in next slide 3. Host defense/resistance: healthy hosts have a high degree of resistance to most microorganisms. Virulence: degree of pathogenicity. ↑ virulence → likely to cause harm Characteristics of parasitism - advantages and disadvantages to the parasite Goals for parasitic Organisms Live and reproduce efficiently NOT harm host; may trigger immune response Symptoms can aid reproduction: – Transmission: cough, sneeze, pustule – Host damage aids spread Advantages of parasitism Food & shelter – Food & energy – Host enzymes: break down macromolecules Example: Tapeworm – taenia spp. o Eat contaminated/inadequately cooked meat o Attach to human intestine with hooks and suckers on head o Absorb intestinal fluids 19  Reproduction/replication – Main goal – Co-ordinate development with availability of suitable hosts Example: Influenza virus - viruses can only replicate in living cells. If these form part of a tissue, then released virus particles can quickly infect neighboring cells. Disadvantages of parasitism Hostile environment – Constantly under threat – Special survival mechanisms eg spores, cysts, rapid reproduction Reliance on host for growth and reproduction – Host damaged/dies → parasite dies – Balance growth, replication Dynamics of the host-parasite relationship Lecture 8 -Transmission of Epidemiology of Disease Be familiar with terms used in epidemiology Epidemiology: scientific discipline that evaluates the occurrence, determinants. Distribution and control of health and disease in a human population. Can be caused be:  Environment, genetic, infection, unknown, interaction, lifestyle Koch’s Postulates 1. The same pathogen must: be present in every case of the disease 2. The pathogen must: be isolated from the diseased host and grown in pure culture 3. The pathogen from the pure culture must: cause the disease when inoculated into a healthy, susceptible laboratory animal 4. The pathogen must: again be isolated from the inoculated animal and must be shown to be the original organism Know how to recognise an epidemic Four main types: Sporadic diseases are those that occur occasionally and at irregular intervals e.g. typhoid caused by Salmonella, which occurs from time to time due usually to contaminated water/food. An outbreak is the sudden, unexpected occurrence of a disease in a limited segment of the population, e.g. Ebola outbreak in Zaire. An endemic disease is one which maintains a steady low-level frequency at moderately repetitive intervals e.g. the common cold, increasing in the winter months due to changes in our behaviour and susceptibility and malaria which is also endemic in some parts of the world. 20 An epidemic is a sudden increase in the level of disease above the expected levels, e.g. influenza; usually have some incidence of influenza each year, occasionally when a very large segment of the population contracts the disease. A pandemic is an increase in disease in a large population over a very wide geographic area e.g. some influenza epidemics have developed into pandemics; global. Understand the relationship between epidemiology and the infectious disease cycle Infectious disease caused by an infectious agent or its products. Goals of epidemiologist is to: control speed of spread AND eliminate from population. 2 major types of epidemics that are recognised in infectious disease epidemiology. Common source epidemic:  Usually results from a single contaminated source e.g. contaminated food (e.g. Salmonella poisoning) or water (e.g. Legionnaries’ disease)  Recognise source through factor the sufferers all had in common  Characterised by a sharp rise in the number of people infected and a relatively fast decline (assuming the cause of the infectious is established and eliminated) Propagated epidemic:  Usually results from introduction of a single infected individual into a susceptible population  A significant proportion of the population will have been infected and will be either immune – or dead (Note: in general, pathogens don’t all kill their hosts)  Eventually the disease will decline through lack of hosts  this kind of epidemic has a much flatter bell-shaped curve Herd Immunity Need number of susceptibles below a threshold level whereby the pathogen can’t propagate effectively. Larger the proportion of a population is immune, smaller the probability of effective contact, hence, group resistance. So susceptibles can be effectively immune through immunity of the herd. Can lead to complacency e.g. immunization programs. Some opting out because of low incidence e.g. whooping cough but as a result of immunized population – if level drops below threshold, herd immunity will break down. Know the links in the infectious disease cycle Understand how Influenza illustrates the infectious disease cycle Cycle contains 5 parts: Pathogen- Source (reservoir) of pathogen- Transmission to host- Susceptibility of host- Exit from host HA trimer and NA tetramer of Influenza virus important in attacking… 4 main groups of Influenza: A, B, C and a 4th. A is the most prevalent and distributed, infects animals and birds. Antigenic variation within Group A  16HA types and 9 NA types  Use these variants to classify strains  All strains infect birds Antigenic drift: small antigenic changes due to accumulation of mutations  altered HA and NA protein. Small changes not effectively recognised by immune system. Antigenic shift: large antigenic change due to reassortment of the genomes  large scale protein alteration  not recognised at all, can produce epidemics or pandemics. The Source/Reservoir of the pathogen: the location in which it is normally found and from which it is transmitted to the host. Sources/reservoirs can be:  Animate e.g. another human or animal 21   Inanimate e.g. water or food Frequently, the source is another human, and this person is known as a carrier. Carriers can:  Be ill with the disease, or  Appear healthy, they may o be recovering from the disease o be yet to become ill i.e. infected but not yet showing symptoms but still harbor the pathogen. Diseases that are passed from animals to humans are known as zoonoses. Frequently the animals are domestic or are rats, both of which live in close proximity to humans. Know how the links can be broken to control an epidemic Understand the relationship between virulence and transmission in the host-pathogen interaction Avian Influenza A H5N1 Bird Flu  Asia, Europe, Africa  Can be passed from birds to humans, not human-to-human  has resulted in around 150 human deaths and mass cullings of birds  wild waterfowl have become a reservoir for this strain and have spread it around the world  BUT fear is that if this strain were to undergo re-assortment with a human Influenza virus, could start a pandemic. Genetic Reassortment (Recombination) In the case of human Influenza, generally the pathogen is passed from human to human. But antigenic shift can sometimes occur in animals. Human Influenza virus can also be transmitted to animals, especially pigs  co-infection may lead to recombination or genome segments SARS – Associated Coronavirus (SARS-CoV) Acute Respiratory Syndrome. Note that severe (intensity) and acute (temporal) do not mean the same thing in medical terminology. Coronaviruses (solar corona-like appearance) are large enveloped viruses with +ve strand RNA. Large club-shaped envelope spikes which aid attachment and entry to host cells, as in this electron micrograph. Like Influenza A virus, Coronaviruses infect a variety of mammals and birds. Until recently Coronavirus infection in humans produced only mild upper respiratory tract infection. They are the 2 nd most prevalent cause of the common cold. In China in 2002 a new type of pneumonia with sudden and severe onset was noted. Resulted in ~ 8,000 cases worldwide with a ~ 10% death rate. New  SARS-associated-Co-V. Transmission can be: Direct  Air-borne transmission – usually on respiratory secretions or dust through coughing, sneezing or talking.  Contact transmission – person-to-person or through an intermediary (e.g. utensil). Indirect  Vehicle transmission – single inanimate vehicle transmits pathogen to multiple hosts but does not support reproduction e.g. food and water.  Air-borne transmission – virus can remain viable after 24 hours on an object. Control of Epidemics Control depends on identifying the components of an infectious disease that are responsible for an epidemic i.e. part of the cycle that is most susceptible to control. Control is generally at 1 of 3 points: 1. Reservoir:  Reduce or eliminate the source of infection e.g. infected food/water  Quarantine carriers and diseases individuals  Destroy animal carriers e.g. eliminate rodents, birds carrying bird flu 2. Transmission:  Stop infection going from 1 host to another – disease stops  Change human behaviour – wash food, don’t drink contaminated waste  Destroy insect vectors with insecticides AND Control animal vectors – rodent control programs, mosquitoes, fleas 3. Susceptibility  Best way to make host less susceptible is to boost resistance 22   Administer better nutrition  Vaccination/immunisation programmes. This is the point where Influenza is controlled. Vaccines are available but are costly due to having to come up with new ones because of antigenic drift and shift over time (usually just advised for those most at risk eg elderly) Virulence and Mode of Transmission Virulence: degree is pathogenicity. Strongly influenced by mode of transmission, host health and ability to live outside of host. Lecture 9: Pathogenicity and Virulence Factors Pathogenicity: The ability to cause disease Virulence: The extent or degree of pathogenicity Microbial strategies and/or products that allow them to colonise, invade, evade and harm their host Examples of microbial virulence factors What are virulence factors? A microbial strategy or product that contributes to virulence or pathogenicity i.e. traits and products (e.g. toxins). Other essential factors: ability of the organism to derive food and energy from host (‘housekeeping’ functions) 1. Virulence Factors  Aid colonisation of host tissues  Allow the microbe to penetrate host tissues and grow  Prevent or reduce the host response  Cause direct damage to host tissues through toxicity Bacterial Virulence Mechanisms  Adherence  Cytotoxic proteins  Evasion of phagocytic and  Invasion  Endotoxin immune clearance  By-products of growth such  Superantigen  Capsule as gas, acids  Induction of excess  Resistance to antibiotics  Toxins inflammation  Intracellular growth  Degradative enzymes 2. Colonisation  Most infections are initiated by the attachment of the microbe to host tissues.  Attachment can be relatively non-specific and mediated by capsules or slime e.g. bacteria attaching to teeth using dental plaque  Or attachment can require the interaction between special structures on the microbe and on host cells. Colonisation – Adherence Mechanisms Adhesins/Ligands: bind to receptors on host cells Glycocalyx Streptococcus Mutans Dental Plaque Fimbriae Escherichia Coli GIT Neisseria Gonorrhoeae GUT Tapered end Treponema Pallidum Syphilis Fimbriae: Bacteria adhere to host cells using either pili (or fimbriae), rod shaped proteins extending from the bacterial surface. or a fimbrial adhesins. Both mediate binding to host cell. Colonisation – Influenza Virus Many viruses attach to receptors on cell surfaces e.g. proteins with transport functions. Influenza is a bit different. Remember that HA = haemagglutinin, 3 copies make up the HA coat spike; NA = neuraminidase (4 copies make up the NA coat spike). The HA molecules bind to sialic acid on the surface of human epithelial cells. 3. Host Cell Penetration & Growth 23 Most microbrial pathogen follow colonisation by cell invasion and growth. Some microbes are taken up by membrane fusion. E.g. Measles virus bids to receptor then releases virus contents inside. Other microbes are taken up by endocytotis. E.g. Influenza follows binding to the host cell surface by inducing the cell to endocytose it. The virus contents are then released into the cell from the endosome. Some bacteria can trick normally non-phagocytic cells into engulfing them. They do this by inducing changes in the actin component of the cell cytoskeleton – this results in the cell engulfing the bacterium. Bacterial surface proteins that provoke phagocytic ingestion are called invasins or invasion factors – it is not yet clear how these cause the actin rearrangements that result in phagocytosis. Host cell Penetration and Growth – Histoplasmosis Other microbes invade naturally phagocytic cells - usually part of the host immune system and are used to engulf and eliminate foreign objects (using the body’s defence system for invasion) e.g. Histoplasma capsulatum: soil-borne fungus. Spores inhaled, in immunocompromised host, hides and grows inside alveolar macrophages, transport pathogen to other parts of the body. Endemic in parts of the USA around the Ohio river. A fungus. The pathogen enters lung -> budding yeast -> pneumonia and systemic illness. Host Cell Penetration and Growth – Tuberculosis Mycobacterium tuberculosis: initiates disease by hiding inside alveolar macrophages and replicating, triggers inflammatory response, leads to tubercle formation Host Cell Penetration and Growth – Enzymes  Coagulase  Coagulates blood  Kinases  Digests fibrin clots  Hyaluronidase  Hydrolyse Hyaluronic Acid  Collagenase  Hydrolase Collagen  IgA proteases  Destroy IgA antibodies  Siderophores  Iron binding Proteins  Antigenic Variation  Alter Surface Proteins Microbes also use a number of degredative enzymes to help them spread through the tissue e.g. hyaluronidase (degrades hyaluronic acid in connective tissue) also nucleases, elastases etc. Siderophores take iron from host. 4. Evading the Immune Response Variety of different approaches:  Encapsulation  Inhibition of chemotaxis  Antigenic mimicry  Inhibition of phagocytosis  Antigenic masking  Inhibition of phagolysosome fusion  Antigenic shift  Resistance of lysosomal enzymes  Anti-immunoglobulin proteases  Intracellular replication  Destruction of phagocyte Also, the use of capsules to evade the immune response. Hydrophilic capsule: Encapsulation common means of inhibiting phagocytosis e.g. by Cryptococcus neoformans. 24   Mimic host surface antigens e.g. Streptococcus pyogenes capsule consists of hyaluronic acid, present in human connective tissue.  Mask antigens by coating themselves with host proteins e.g. coat with fibronectin, a protein found on many host cell surfaces. How cells survive inside phagocytic cells Phagocytic immune cells, such as monocytes, PMNs (polymorphonuclear neutrophils) and macrophages e.g. M. TB and Histoplasma capsulatum. By inhibiting fusion of the phagosome (in which they are contained) with the lysosome (which is a bag full of enzymes used to kill off things picked up by the phagosome).  Some can resist or inactivate lysosome enzymes. Clearly, microbes that survive phagocytosis by these cells are a big problem as they are growing and surviving within the very cells that are designed to eliminate them from the host body. The Process of Phagocytosis  Different kinds of bacterial toxins: exo- and endo-toxins How some of these toxins exert their effects on host cells and damage the human host Examples of toxin-mediated disease processes Microbial Toxins: Exotoxins and Endotoxins Disease is frequently determined by the production of microbial toxins – especially in the case of bacteria. These toxins are classified as either exotoxins or endotoxins. EXOTOXINS Exotoxins, as the name implies, are toxins that are released from bacterial cells, although some are only released when the cell lyses. These toxins are important medically in food poisoning (e.g. Staphylococcal enterotoxin) and also cause a lot of symptoms of microbial disease. Toxins can be used by the microbe to:  Destroy part of the cell  Inhibit metabolic functions i.e. aid in tissue damage and invasion Exotoxins can be encoded on chromosomal genes, though frequently they are on extrachromosomal elements called plasmids and prophage. Plasmid = self-replicating DNA circle often carrying genes for non-essential functions e.g. antibiotic resistance and toxins Prophage = integrated phage that can carry toxin genes. Microbial Exotoxins There are three types: 1. A-B toxins - The majority of exotoxins Composed of 2 portions – dubbed ‘A’ and ‘B’, which are joined together by disulphide bonds. Can be simple – only 1 A and 1 B portion or compound – where B has multiple subunits.  B component is responsible for cell binding – they bind to specific host cell surface molecules - glycoproteins, glycolipids, or sometimes proteins. Specificity determines which cells toxins attack  A component is the enzymatic part of the molecule that actively damages the cell. It is translocated into the cell cytoplasm following binding of the B portion and cleavage of the disulphide bond. Translocation can be:  Directly through the cell surface  Following endocytosis Most A portions work by ADP-ribosylating target cell proteins, rendering them inactive by altering their shape and function. 25 ADP-ribosylation: mechanism used by the A part of the toxin. It cleaves part of the active target protein and attaches an ADP- ribosyl group, inactivating the protein. E.g. Cholera: Vibrio Cholerae Cholera toxin carried by a lysogenic phage that integrates into the Vibrio cholerae genome. Lysogenic phage carries A-B toxin gene. ADP-ribosylates regulatory component of adenylate cyclase ↑ adenylate cyclase → ↑ cAMP → fluid & electrolyte efflux Severe vomiting and profuse watery diarrhoea ('rice-water stools') Other A portions can have different effects e.g. Shiga toxin of Shigella dysenteriae cleaves host cell RNA thereby damaging the ribosome and preventing protein synthesis. Some otherA-B proteins you may have heard of: Diphtheria, Tetanus and Anthrax toxin. 2. Membrane-Disrupting Toxins These toxins lyse cells by disrupting the integrity of their membranes. There are 2 kinds of these: Channel forming – these toxins insert into the host cell membrane and form channels, allowing the cytoplasmic contents to leak out and water to enter – eventually the host cell swells and bursts eg Staphylococcus aureus Phospholipase – snips off the charged head groups from membrane phospholipids – destabilising the membrane = cell lyses. These toxins are toxic for many types of cells and they don’t have specific targets. E.g. Gas Gangrene Alpha-toxin of Clostridium perfringens – has phospholipase action and can lead to gas gangrene. Sub-terminal and distending. 3. Superantigens Rare strains of Staphylococcus aureus produce TSS-toxin. Unusual bacterial toxins which inappropriately stimulate immune cells, causing overproduction of bioactive molecules like cytokines = can cause shock; circulatory system & organ systems fail. The inset also shows the characteristic skin peeling of TSS. ENDOTOXINS Endotoxin is part of lipopolysaccharide (LPS) that is a component of Gram negative cell walls. Lipopolysaccharide Lipid A is the toxic part of this molecule – it is embedded in the Outer Membrane, with the core polysaccharide and the O-antigen extending outward from the bacterial cell surface. As the lipid A is usually embedded, it exerts its effects only when the bacterial cell lyses. Particularly acts on components of the immune system, initiating complement and blood coagulation cascades, and stimulating over- production of various immune system molecules leading to fever = septic shock.  Shock = life-threatening drop in blood pressure  Septic shock = caused by bacteria Effects are dose-related – exposure to large quantities of endotoxin, such as during bloodstream infection, can lead to fever, hypotension, septic shock and death. Also be caused by bacteria colonising wound infections or indwelling catheters and getting into the blood stream. 26 Gram Positive Bacteria: Inflammatory Components On the other hand, Gram +ve bacteria don’t have this endotoxin i.e. lipid A. But they can induce an apparently identical inflammatory response, which seems to be due to their cell wall molecules, peptidoglycan and teichoic acid and LTA. Lecture 10 - Host Defence Mechanisms Understand the role of the host defense system in the prevention of establishment of disease Normal Flora (NF) : compete for space, resources, nutrients and chemicals e.g. lactic acid bacilli in female genital tract maintain low pH and inhibit colonization by pathogens. Opportunistic pathogens: controlled by environmental limitations but if removed gain access to bloodstream of tissues and disease can result. E.g. viridans group streptococci are NF of mouth, if enter bloodstream can cause endocarditis. Factors the compromise host - ↓ resistance to infection  Malnutrition, alcoholism  Immunosuppression e.g. HIV  Cancer, diabetes  E.g. Bacteroides spp. NF in gastrointestinal tract.  Trauma Through abdominal surgery can enter  Antibiotics bloodstream. Dynamic relationship betw. Host & Pathogen. Many direct factors that influence this eg  Nutrition  Physiology  Fever  Age, Either Very Young Or Very Old – Susceptibility To Infections Increases. Babies Are At Risk Once Their Maternal Immunity Declines And The Immune Systems Of The Very Old Are Also Compromised.  Genetics Also, many indirect factors that influence the host-pathogen relationship  Personal hygiene  Socioeconomic status  Living conditions Know various ways in which the human body is equipped to prevent colonisation and establishment of disease Resistance is both non-specific and specific body mechanisms. Innate defenced with which the host is born. Non-specific host defences: PHYSICAL AND MECHANICAL Skin Defences 27 Mechanical barrier of thick epidermis cells - dry and inhospitable. Glands and follicles secreting antibacterial substances. Only penetrated at breaks. E.g. infection with hepB virus or HIV using blood contaminated needles. Also  Mucous Membranes Mucosal membranes have a tightly packed epithelium covered by secreted mucus which  resists penetration  traps microbes  often antimicrobial secretions too e.g. lysozyme which cleaves peptidoglycan and is especially effective against Gram +ves Our eyes, respiratory, gastrointestinal and urogenital tracts are lined with mucosal membranes to prevent attachment, colonisation and invasion of undesirable pathogens. Under the mucosa lies the Mucosal Associated Lymphoid Tissue, acting like MALT. Respiratory System Humans inhale ~104 microbes/day. Our nose contains tiny hairs that mechanically filter the inhaled air of organisms and foreign matter. In the respiratory tract, the mucociliary blanket or ‘escalator’ acts to remove foreign bodies. Thick, sticky mucosal surface traps foreign bodies in mucus. Cilia beat upwards away from the lungs, expelling the mucus. Streptococcus pneumoniae can only invade the lungs and cause pneumonia if damaged e.g. as in chronic bronchitis Gastrointestinal Tract  Stomach – gastric juice pH 2-3 (HCl, enzymes, mucus)  Ileum – enzymes, bile, GALT, peristalsis  Colon – normal flora, lysozyme, peptides Microbes that reach the stomach are often killed by the highly acid conditions. In the small intestine, pancreatic enzymes, bile, intestinal secretions, Gut Associated Lymphoid Tissue and peristalsis In the colon, NF and their products help to prevent establishment of pathogenic microbes. Genitourinary Tract  Normally sterile – low pH, urea  Distal urethra – some m/os  Vagina – lactic acid bacteria  For males, there is a distance barrier for microbes, not present in females, leading to a higher incidence of UTIs.  Urine – low pH and urea can kill microbes.  Vagina – produces glycogen that lactic acid bacteria degrade keeping the pH low and unfavourable for many microbes. Eye The conjunctiva lines the interior surface of the eyelids and the eyeball secretes mucus. Kept moist by the flushing action of tears from the lachrymal glands. Tears also contain lysozyme Flushing Mechanisms Last of the physical barriers that limit the microbial load at a particular site: 1. Tears 2. Saliva is secreted (1L/day) providing a flushing action. 3. Urine is voided from the bladder 4-10 times per day, carrying microbes with it. 28 CHEMICAL BARRIERS Compounds produced by the body/ normal flora contained on it. Chemical Barriers can be: Enzymes: Lysosomes, tears, mucous and saliva. Breaks down the cell wall in Gram positive organisms such as staphylococci and streptococci. Bacteriocins: Many NF organisms produce toxic proteins that are lethal to related species. Complement Complement is a series of 9 proteins (C1-C9). Always present in serum (i.e not formed in response to infection) 3 pathways of complement activation – the alternative pathway is an important non-specific defence 1. Normally inactive and activated when Ag/Ab reactions occur 2. Reacts in a cascade - to aid clearance from foreign organisms by induction of inflammatory response with Ab, causing bacterial cell lysis 3. Aids phagocytosis e.g. Complement +Ab-m/o more liable to phagocytosis Fibronectin Important glycoproteins which are proteins with polysaccharide moieties include: Fibronectin which mediates non-specific clearance by coating foreign cells causing clotting, and by blocking attachment of foreign organisms to epithelial cells thus limiting colonisation. Interferon Another glycoprotein is interferon. Produced by many eukaryotic cells in response viral infection. Interferon produced by an infected cell is excreted and acts on adjacent cells where it interferes with viral multiplication. 4. Is species specific, not virus specific 5. Has been of interest as possible anti-viral drugs and also anti-cancer drugs ( basal cell carcinoma) Expensive as species specific, but with genetic engineering techniques commercial production has become possible.  Anti-Viral Action of Interferon 1. Virus infection 2. IFN synthesis and excretion 3. FN binds plasma membrane of another cell 4. Triggers production of enzymes rendering cell resistant to viral infection by inhibiting viral protein synthesis. BIOLOGICAL BARRIERS Normal Flora mentioned before. Inflammation – Acute or Chronic Acute Inflammation Reaction to tissue injury and infection. Cardinal signs: redness, heat, pain, swelling and altered function of tissue Begins when injured cells release chemical signals that attract neutrophils and other white blood cells to the site. 1. Increased blood flow and dilation that bring more antimicrobial factors. Capillary permeability that allows escape of fluid & cells producing swelling. WBCs attracted to the site stick to wall of capillary & squeeze through 2. Rise in temperature; fever 3. Formation of fibrin Clot so remain localised 4. Phagocytosis: phagocytose pathogen to neutralise and eliminate. WBCs undergo chemotaxis to site of infection & attack pathogen Chronic Inflammation  Slow process  Permanent Tissue Damage 29   Intracellular Pathogens e.g. M. tuberculosis, M leprae, T pallidum Stimulated by the persistence of bacteria. If bacteria can’t be killed off during inflammation, body tries to wall them off by forming granuloma. E.g. mycobacteria have cell walls with high lipid content, making them relatively insensitive to phagocytosis. The bacteria that cause leprosy and syphilis also often survive within macrophages. Phagocytosis Phagocytic cells recognise, ingest and kill many microbes. Receptors:  Non-specific = related to complement cascade for detection and phagocytic destruction of foreign microbes.  Specific: o Filamentous haemagglutinin which is found on the surface of the organism Bordetella pertussis that causes whooping cough o Mannose: which is a major component of bacterial cell walls and of some virus envelopes and immune complexes. It is found in the cell walls of many Gram negative fimbriated bacteria such as Proteus o Klebsiella capsules which also contain polysaccharides that will bind to the mannose receptor o Lipopolysaccharide in the cell wall of the protozoan Leishmania Outcomes of Phagocytosis 4 outcomes: 1. digestion and destruction by phagocyte 2. no effect because not phagocytosed 3. destroy phagocyte 4. grow within phagocyte Specific Immune Response So, when a microbe invades, there are 2 fundamentally different immune responses:  non-specific immunity, as we’ve just discussed  specific or adaptive immunity The specific immune response has 3 major functions 1. Recognise anything foreign 2. Respond to it and eliminate it or render it harmless and so prevent disease 3. Remember it 4 characteristics distinguish specific and non-specific immunity  specific to particular pathogen  remember and act quickly  enormous diversity of Abs  discriminate between self and non-self Specific immune response has 2 branchs: Humoral branch (or Ab-mediated) B-cells interact with Ag and differentiate into Ab-secreting plasma cells. Ab binds Ag and tags it for destruction Cell-mediated branch Subpopulations of T-cells are activated by Ag produce cytokines that facilitate both humoral and cell-mediated responses. Kill virus-infected cells Both B- and T-cells also differentiate into memory cells to become active in subsequent responses. The immune system distinguishes between ‘self’ and ‘non-self’ Self and non-self ANTIGENIC DETERMINANTS An antigen = a substance that elicits an immune response. Usually large, complex molecules Each antigen can have several antigenic determinant sites or epitopes. Antibodies recognize and bind to these epitopes 30 ANTIBODIES - STRUCTURE Lecture 11 – Medically Important Bacteria: Gram Negatives REVISION – How are bacteria classified? Through their phenotype:  Gram reaction and morphology  Carbon sources, energy sources  Electron acceptors (eg. aerobic/anaerobic) Through their genotype:  Ribosomal RNA sequence  Other DNA, RNA, protein sequence Classification of Bacteria  Bacteria will be organised by their phylogeny – a systematic approach  Phenotypes important for identification or pathogenicity  Systematics agrees with phenotype, eg. all endospore formers are Gram positive  In other cases, the classifications disagree, eg. anaerobic growth occurs in diverse bacteria  Both approaches are useful – it depends on the question being asked ! Name eight different medically-relevant Gram-negative bacteria, and describe: phylogeny, microscopic morphology, normal habitat , the human disease(s) caused and any distinctive/ unique features Phylum Genus Example Proteobacteria Escherichia E.coli  Facultative anaerobic,  Most strains are NF, and beneficial eg. E.coli K12 heterotrophic, Gram negative rods biosynthesizes vitamin K  Found in gut of humans and  Some strains pathogenic (eg. O157, O111); these are animals food or water-borne  Part of family pathogens ◊ diarrhoea, fever Enterobacteriaceae: contains many  Virulence factors: other pathogens: Salmonella, endotoxin (all), Klebsiella, Yersinia enterotoxin (some)  Widely used in microbiology as a Selective + differential agars: on model organism XLD lactose positive, H2S negative.  Motile by peritrichous flagella Salmonella  Facultative anaerobic, heterotrophic, G-ve rods  NF in animal gut, pathogenic to humans  S.enterica: food-borne infection, self-limiting diarrhoea 31   S.typhi: water-borne, typhoid fever, potentially fatal Virulence factors: endotoxin, enterotoxin, cytotoxin XLD agar: lactose negative, H2S positive. Salmonella enterica – flagella = motile Vibrio V. cholerae  Facultative anaerobic,  The most pathogenic Vibrio species: heterotrophic, gram negative curved o causes cholera: severe diarrhoea rods o virulence factor: cholera toxin (an exotoxin)  Motile, various flagella  curved cell, polar flagellum, bundles of pili arrangements  Transmission  Motile, various flagella usually by faecal contamination arrangements of water Habitat is primarily marine, but can cause  V.cholerae is gut infection more rarely a food-borne disease (seafood) Bundles of pili  Pseudomonas P. aeruginosa  Aerobic, heterotrophic G-ve rods,  Nosocomial infection (hospital acquired) – esp. burns. motile (polar flag.)  Virulence factors: multiple antibiotic resistance,  Ubiquitous in soil and water, haemolysin, proteases opportunistic pathogens  Large genome (6 Mb) P. aeruginosa transmitted from nurse’s finger nail to many heart Metabolically versatile surgery patients  Metabolic versatility helps P.aerug. to colonise diverse niches (eg. fingernail, heart)  P.aeruginosa infections are common, but person-person transmission is rare Neisseria N. meningitidis (Meningococcal)  Aerobic,  Cells adhering to cilia in the respiratory tract heterotrophic, G-ve  Inflammation of meninges (membrane around brain) = diplococci fever, rash, headache, confusion, death  Habitat: mammalian mucous  Serious, rapidly progressing disease – needs rapid diagnosis membranes and treatment (antibiotics)  Virulence factors:  Analysis: microscopic examination of CSF o capsule: evasion of immune response N. gonorrhoeae (Gonorrhoea) o fimbriae: adhesion to tissues  Sexually transmitted  Diagnose by microscopic examination  Safranin: epithelial cells and Neisseria appear pink  Note adherence of Neisseria cells to the epithelial cells Rickettsia R.prowazekii (Epidemic typhus)  Aerobic, heterotrophic, G-ve,  Headache, fever, rash (up to 50% mortality) – overcrowded coccobacilli conditions  transmitted by body louse  Intracellular parasites of arthropods –  Girulence factors: adhesin, phospholipase eg. fleas, lice, ticks  Cannot be grown in vitro – only in tissue culture. Bacteria are very dependent on host metabolism  Small ‘degenerate’ genome (1 Mb) = specialised lifestyle  Related to mitochondrion  Transmission to humans occurs via bites or faeces of arthropods = various fever diseases Bacteroidetes Bacteroides B. fragilis  Anaerobic, heterotrophic, G –ve rods  Opportunistic pathogen: cause infection if it escapes the  NF, but can be opportunistic gut eg. abcesses, septicemia, appendicitis pathogens  Virulence factors: capsule, antibiotic resistance  Bacteroides are most abundant cells in  Resistance genes are on conjugative transposons human body  Several beneficial species: digestion of carbohydrates, exclude pathogens by 32  competition Spirochetes Treponema T. pallidum  Anaerobic, heterotrophic, G-ve,  Causes Syphilis: a sexually transmitted disease (STD) spirochaetes.  Effects: 10: chancres, 20: rash, 30: nervous system damage  Obligate parasites, require animal cells  Can’t be grown in standard media ‘Degenerate’ small for growth genome (1 Mb)  Use axial filament for structure and corkscrew motility Lecture 12 – Medically Important Bacteria: Gram Positives Both gram positive and negative bacteria are medically important Phylum Genus Example Gram negative Chlamydiae Chlamydia C. trachomatis  Aerobic, heterotrophic, G-ve  Causes urethritis (STD) and trachoma (eye infection) cocci Prescott Hypertrophy of eyelids in trachoma Chlamydia is  Obligate intracellular serious pathogen for koalas parasites of humans &  An ‘energy parasite’ – dependent on host cells for ATP and animals other metabolites  Cause sexually-transmitted  Virulence factors: disease and eye infection o unusual cell wall allows growth inside phagocytes  Cannot be grown on agar, o has no peptidoglycan  intrinsic resistance to all small genome (1 Mb) antibiotics targeting PG. consistent with host- dependence Gram positive Firmicutes Bacillus – primarily soil organisms B. Anthracis  facultative  Facultative anaerobic,  Causes anthrax: highly infectious and deadly disease anaerobic heterotrophic, G+ve rods  Usually zoonotic: transmitted from animals (cattle, sheep) rods or cocci  Ubiquitous in environment,  some make esp. soil