Micro1_all_slides.pptx

1. Introduction and History of Microbiology Dr Roger Draheim [email protected] x 2133 SM (old) 4.19 1 “Why do I care about micro?” 2 Paper in Lancet Infectious Diseases 3 Now detected in the UK! 4 2050 projections (too modest!) 5 Intended Learning Outcomes (ILOs) To describe the differences between prokaryotes and eukaryotes To understand the evolutionary relationships of microorganisms To understand the ways in which microorganisms are classified and named To describe the impact of microorganisms on human life (medical and industrial) 6 History of microbiology Prokaryotes and eukaryotes have been in existence for billions of years Have had a significantly longer time to evolve and diversify into different forms Not only did they have the world to themselves … they had to adapt to the many changes Ice ages Heavy volcanic activity Oceans without oxygen Collisions with meteors 7 History of microbiology Not only did microorganisms survive these conditions they also transformed the earth and its atmosphere making it habitable for plants and animals that arrived much later on Bacteria appeared 4 billion years ago – Soon after the earths surface had cooled enough allow liquid water to form – Some of them are still capable of living in extreme conditions Eukaryotes: protozoa appeared probably 2 billion years ago Fungi in the last several hundred million years (may have coevolved with plants) 8 Historical roots of microbiology Discovery of microorganisms linked to the invention of the microscope Description of fruiting bodies of moulds 1664 drawings – Robert Hooke 1684 Antoni van Leeuwenhoek used simple microscope and described ‘wee animalcules’ 19th century microbiology advanced as microscopes improved 9 Historical roots of microbiology Real advances came in the late 19th century when other basic techniques were devised Major contributions from: – Robert Koch – Louis Pasteur Pasteur: – disproved the ‘spontaneous regeneration’ theory and went on to demonstrate heat sterilisation, and also developed vaccines 10 Historical roots of microbiology Robert Koch: – Demonstrated that microorganisms cause disease and developed a set of postulates – Also developed culture media for growing bacteria Major advances in the 20th century with the development of molecular biology 11 Size of microorganisms Invisible by naked eye – a few classes of worms are an exception Bacteria, and protozoa visible using light microscope – Greater than 0.3μm Viruses only visualised by electron microscopy – 0.01 – 0.30 μm 12 Size of microorganisms 13 Prokaryotes and eukaryotes Pro – early – primitive Eu – developed – true Karyon – nut/kernel (nucleus) One of the major differences between them is the presence of membrane – bound organelles, including the nucleus in eukaryotes 14 Prokaryotes Bacteria – (sometimes referred to as eubacteria) contain all known disease causing bacteria and most of the bacteria found in soil, water, animals and other environments Archaea – Are mostly anaerobes and thrive in extreme environments hot springs, freezing water, highly salty, acidic and alkaline environments 15 Eukaryotes Eukaryotic organisms include: – Algae – Fungi – Protozoa Cells of macroorganisms - animals and plants Bacteria and archaea - no evolution beyond microbial stage Eukaryotes evolved and gained: – Mitochondria – Chloroplasts – Internal membranes Bound nucleus by endosymbiosis (endosymbiotic hypothesis) 16 17 Prokaryotes vs. Eukaryotes Prokaryotes DNA not enclosed in membrane, circular and supercoiled DNA not associated with histones and low protein content No organelles Divide by binary fission Form biofilms Complex cell wall 70S ribosomes Eukaryotes Discrete nucleus with nuclear membrane Linear genetic material DNA has high histone content and non-histone proteins Organelles Divide by mitosis and meiosis Simple cell wall if present 80S ribosomes 18 Microbial diversity Mitochondria – Aerobic bacteria in cytoplasm of primitive eukaryotes – energy in exchange for safety Phototrophic bacteria incorporated into cells led to photosynthesis Within both prokaryotes and eukaryotes we see considerable microbial diversity of: – Structure – Function – Behaviour – Adaptation 19 Phylogenetic relationships Gene sequencing of 16S or 18S ribosomal RNA enables phylogenetic relationships to be calculated Amplify gene of the ribosomal RNA (rRNA) Computer compares sequences and counts every position where there is a difference ED (evolutionary distance) Construction of tree where length of line proportional to ED 20 21 Viruses Not present on the phylogenetic tree because do not contain ribosomes and therefore cannot be analysed in the same way Have very small genomes and are very diverse No highly conserved molecular ID for them If infect humans, then could not have evolved before them If infect bacteria, then probably appeared as bacteria evolving 22 Classification of Microorganisms Taxonomy and classification based originally on phenotypic and metabolic behaviour G-C content has also been used Clarified now by genotyping Microorganisms named using the binomial nomenclature, after Linnaeus 23 Systematic nomenclature Binomial system used for bacteria, fungi, protozoa and viruses Genus first, always with a capital letter, e.g. Escherichia Species follows with no capital, e.g. coli Short form E. coli or E. coli Names always written in italics or underlined 24 Microbial populations Groups of related cells live in a habitat or niche Associate with other populations in microbial communities – Form ecosystems Interact with each other in beneficial or harmful ways – Competition for nutrients and niches Antagonism – production of toxins and antibiotics 25 Microbial populations Cooperation, one group of microorganisms provide nutrients for others: – e.g. oral & gut flora Humans have 1013 cells and 1016 microorganisms associated with them Live in water, soil, plants, animals and form the microflora of their habitat Relationship with their host (if they have one) may be mutualistic, saprophytic or parasitic Multiply rapidly, ideal pathogens in or on human cells 26 2. Impact of Microorganisms on Humans Dr Roger Draheim [email protected] x 2133 SM (old) 4.19 27 Impact of microorganisms on humans The study of microbiology aims to understand how microorganisms work Knowledge of them has vastly increased the benefits to man and minimised the damage they cause Relatively few cause disease to man 1900, infection was major cause of death: – Now statistics are different 28 29 Impact of microorganisms on humans During the 20th century advances were made in public health measures: – Antibiotic discovery – Use and improved knowledge of causation of disease Complacency, over use of antibiotics, drug resistance and increased numbers of immunocompromised patients now cause problems In developing countries the picture is different and the problems persist: – Malaria – TB – Infant diarrhoea – Cholera 30 Beneficial Uses of Microorganisms Agriculture – Legumes and nodules on roots – Bacteria living in the nodules fix nitrogen, forming part of the nitrogen cycle – Reduces the need for fertilisers – Symbiotic relationship Digestive process in ruminants (sheep and cattle) – Bacteria - ruminococci, digest cellulose – Symbiosis 31 Beneficial Uses of Microorganisms Saprophytic bacteria: – Present in the soil convert complex molecules into forms accessible to plants – Take part in the carbon, nitrogen and sulphur cycles – Also source of animal and plant disease Silage making: – Hay stored anaerobically is fermented by lactobacillus sp. – Metabolise plant sugars 32 Beneficial Uses of Microorganisms Food Industry: Dairy Products - lactose in milk – Metabolised by bacteria to lactic acid Butter: – lactic acid starter culture added to cream – e.g. Lactococcus cremoris or L. lactis Cheese: – Coagulation and fermentation of milk. – Addition of other bacteria, Swiss cheese, Propionibacteria – Blue cheese, Penicillium sp. 33 Beneficial Uses of Microorganisms Yoghurt: – Low fat milk, pasteurised & inoculated with Lactobacillus bulgaricus, Streptococcus bulgaricus Probiotic yoghurts and drinks Bacteria also used in coffee and cocoa preparation Monosodium glutamate is produced from L- glutamic acid from Corynebacteria glutamicum grown aerobically on molasses 34 Beneficial Uses of Microorganisms – Vinegar: ethyl alcohol to acetic acid by genera Acetobacter or Gluconobacter – Acetobacter oxidising sorbitol produces sorbose used in the manufacture of ascorbic acid – Citric acid: fermentation using Aspergillus niger – Edible fungi, alcoholic drinks and baked goods yeasts 35 Pharmaceutical and chemical industry Antibiotics are produced by large scale fermentation processes: – From filamentous fungi and bacteria of the Actinomycete group Biological washing powders contain enzymes called subtilisins produced by: – Bacillus licheniformis, Subtilisin carlsberg Hydrolyses most types of protein bonds, pH stable and non-Ca2+ dependant 36 Pharmaceutical and chemical industry Production of cortisone and hydrocortisone – Fungus Rhizopus nigricans carries out key stereospecific hydroxylation of a cortisone precursor – Other fungi also used Vitamins and amino acids – e.g. Propiobacterium – High yields of vitamin B12 Acetone and butanol can be produced in fermentation processes: – With Clostridium acetobutylicum 37 Environmental Methane is produced from methanogenic bacteria – Development of biofuels Microbial leaching of ores – Many metals, e.g. copper, form insoluble sulphides Acidophilic bacteria are able to oxidise copper ores to soluble copper sulphate – Thiobacillus ferrooxidans 38 Environmental Sewage and waste water treatment – Anaerobic and aerobic methods Petroleum biodegradation – Can be detrimental if bacteria degrade hydrocarbons in tanks if water and air present Positive use in bioremediation of oil spillages 39 Biotechnology Genetic engineering enables gene manipulation and artificial gene products such as insulin Human insulin gene is engineered into a microorganism – Recombinant vaccines Thermostable DNA polymerase of Thermus aquaticus known as Taq polymerase, enabled more effective PCR 40 Synopsis 1. Why you should care about microbiology 2. Evolution of microbes 3. Advance of technology changed microbiology 4. History of microbiology 5. Size of microbes 6. Prokaryotes vs. eukaryotes 7. Phylogenetic relationships (viruses) 8. Systematic nomenclature 9. Microbes as “populations” 10.Impact of microbes on health 11.Industrial (agriculture, pharma, environmental, biotech) impact of microbes 41 3. Systems of Microbial Classification Dr Roger Draheim [email protected] x 2133 SM (old) 4.19 42 Intended Learning Outcomes (ILOs) – To describe some of the ways in which bacteria may be classified: size, shape and cell wall structure – To describe the components and structure of Grampositive, Gram-negative and acid-fast cell walls – To relate cell wall structure to the function and characteristics of bacteria 43 Bacterial classification Size: measured in microns (1mm = 0.001mm) – Bacteria range in size from 0.2mm (Chlamydiae) up to 10mm – Smallest are at the limit of resolution of the light microscope Shape (mophology): – Cocci – Bacilli – Ovoid – Tapered – Curved – Spiral – Hyphae-like structures May vary within species 44 Bacterial classification Cellular arrangements: – Clusters – Chains – Diploid – Tetrads – Palisades Motility Presence or absence of spores Growth characteristics: – Speed – Morphology on selective and non-selective media – Atmosphere – Temperature 45 Bacterial classification Biochemical profile Serological tests Metabolic end products as detected by HPLC, GLC Genetic analysis by rRNA typing, etc. – G-C content Staining characteristics: Gram, acid-fast 46 Bacterial morphology 47 cocci (sing. coccus) 48 Bacterial arrangement 49 bacilli (sing. bacillus) / rods 50 Curved bacteria 51 4. Overview of Cell Wall Structure Dr Roger Draheim [email protected] x 2133 SM (old) 4.19 52 Cell envelopes Provides – Protection from osmotic lysis – Protection from host defences – Shape and rigidity – Semi-permeable barrier May form up to 1/3 of the dry weight of the bacterium 53 Cell envelopes The prokaryotic cell envelope can be defined as: – The complex of membranes and associated macromolecules which together form the boundary between the inside and the outside of the cell They are the interface between bacteria and the diverse spectrum of environments that they inhabit 54 Types of bacterial cell walls Gram-positive: – High internal osmolarity – Need thicker cell wall Gram-negative: – Thinner but more complex cell wall Acid-fast: – Walls contain complex lipids 55 Gram-positive vs. Gram-negative 56 Overview of G+ and G- 57 Model of G+ and G- cell walls 58 Gram staining 59 Differentiation by Gram staining Differences because of the physical nature of the bacterial cell walls Peptidoglycan acts as a permeability barrier preventing loss of the initial stain: – crystal violet (CV) Iodine is the second solution to be added – Mordant (coordination complex) – CV-I complex (larger than CV or I alone) 60 Gram staining Decolourisation step: – Thought to shrink the pores of the thick peptidoglycan and CV-I complex retained Gram-negative bacteria have a thin layer of peptidoglycan, with less cross-linking and larger pores CV-I complex removed more easily through G- cell wall 61 Gram staining 62 Removal of cell walls Cell walls can be removed in osmotically stabilising solutions Without walls, Gram-positive organisms become spherical protoplasts Gram-negatives become spheroplasts Survival depends on keeping them in hypertonic solution of sucrose L forms are variants that have lost the ability to synthesise a cell wall 63 5. Detailed Cell Wall Structure Dr Roger Draheim [email protected] x 2133 SM (old) 4.19 64 Overview of G+ and G- 65 Cytoplasmic membrane A cytoplasmic membrane contains: – Ion pumps to maintain membrane potential and synthetic enzymes – A coiled portion of the membrane, the mesosome – The mesosome acts as an anchor to bind and pull apart daughter chromosomes during cell division 66 Peptidoglycan (murein) Common to all types of bacterial cell wall Composed of basic structure of 2 sugar derivatives connected in b1-4 linkages: – N-acetyl glucosamine (NAG) – N-acetyl muramic acid (NAM) Form a repeating structure called glycan tetrapeptide Glycan chains connected by peptide cross links of 4 amino acids to give rigidity 67 Peptidoglycan 68 Peptidoglycan Cross linking of NAM with a tetrapeptide Characteristic for each specific bacterium In Gram-positive bacteria, cross linking involves a peptide interbridge In S. aureus each interbridge peptide consists of 5 molecules of the amino acid glycine connected by peptide bonds (peptapeptide) 69 Peptidoglycan 70 Peptidoglycan specifics Forms mesh like exoskeleton but is porous enough to allow diffusion of metabolites Essential for survival in hostile conditions Can interfere with phagocytosis, is mitogenic for lymphocytes and has pyrogenic activity b1-4 bond can be degraded by lysozyme 71 Peptidoglycan: diversity Tetrapeptide contains both D and L amino acids – (D a.a. not normally used in nature) First 2 amino acids may vary for different organisms 3rd amino acid essential for cross linking D-amino amino acids, include lysine and diaminopimelic acid (DAP) Links to D-alanine in position 4 of another chain 72 Peptidoglycan Precursor form of the peptide has an extra D-ala, which is released during the cross linking step Prefabricated subunits are assembled on the inside of the cell Brought to the surface by a large phospholipid called bactoprenol and attached to the existing structure 73 Additional components In Gram-positive bacteria, PG forms multiple layers and often cross linked in 3 dimensions to give strong rigid cell wall Teichoic acid is also present in the wall: – Acidic polysaccharide Contains glycerophosphate or ribitol phosphate residues Negatively charged and contribute to overall negative charge of cell surface 74 Teichoic acid and lipoteichoic acid Glycerol containing acids are bound to membrane lipids of Gram-positives: – Called lipoteichoic acids Mediate attachment to host cells Sugars, choline or D-alanine may be attached to the hydroxyls of the ribose or glycerol: – Providing antigenic determinants May determine serotype of bacteria 75 Proteins Form part of the cell wall M protein of Group A Streptococcus: – Present as fine processes called fimbriae – Protects from phagocytosis Protein A of S. aureus: – Binds Fc region of IgG – Competes with neutrophil receptors for the Fc region of opsonising antibodies 76 Gram-negative cell walls Thinner, but more complex Cytoplasmic membrane and thin layer of peptidoglycan (5-10% only of weight) in the periplasmic space between the CM and the inner surface of the outer membrane Periplasmic space contains a variety of hydrolytic enzymes necessary to break down large macromolecules for metabolism 77 Periplasmic space Enzymes include proteases, phosphatases, lipases, nucleases and carbohydrate degrading enzymes Sugar transport systems and binding proteins Components of chemotaxis system, sensing environment outside of the cell In pathogenic bacteria, there will be virulence factors such as collagenases, hyaluronidases, proteases and beta-lactamase 78 Outer membrane Unique to Gram-negative prokaryotes Maintains structure and forms permeability barrier to large molecules: – e.g. lysosyme Provides protection from adverse environmental conditions such as the human digestive system Outer edge of outer membrane is different from any other biological membrane 79 Outer membrane Amphipathic molecule known as lipopolysaccharide (LPS) LPS is also known as endotoxin Powerful stimulator of the immune response – – Activates B cells – Macrophages – Stimulated to release IL1 and 6, tumour necrosis factor Leads to fever, and if large amounts present, septic shock and multi organ failure 80 LPS 3 structural sections: – Lipid A – Core polysaccharide – O polysaccharide Lipid A anchored in the membrane: – Is responsible for the endotoxic activity – Very toxic to host – Released in large quantities when cells die – Consists of 6 fatty acid chains and 2 glucosamine residues 81 LPS Core polysaccharide: – Branched and contains 9-12 sugars – Contains unusual sugar 2 keto-3-deoxy-octanate (KDO) – Core region constant for many species of bacteria O antigen: – A long linear polysaccharide of 50-100 repeating saccharide units of 4-7 sugars per unit – Distinguishes serotypes of bacterial species 82 Structure of LPS 83 Membrane pores Porin protein forms channels in the membrane: – Roughly 100 on cell surface – 10 angstrom (Å) diameter Act as transport proteins for larger metabolites including: – Maltose – Oligosaccharides – Vitamin B12 – Nucleosides Permeable to hydrophilic molecules, less so to hydrophobic molecules Less sensitive to antibiotics 84 Gram-positive vs. Gram-negative 85 Overview of G+ and G- 86 Gram Staining 87 Acid-fast bacteria Mycobacterium: – Have a peptidoglycan layer attached to an arabinogalactan polymer – Surrounded by a wax-like lipid coat of mycolic acid, cord factor and wax D Acid-fast staining Interferes with phagocytosis 88 Acid-fast cell wall 89 Cell wall structure/function Gives bacteria: – Characteristic shape – Protection – Transport mechanisms – Aids pathogenicity Advantages to man: – Classification – Provides selective toxicity for anti-microbials 90 Synopsis 1. Different methods of bacterial classification (size, shape, motility, sporulation, biochemical characteristics, metabolism, genetic analysis, etc…) 2. Bacterial morphology 3. Cellular envelopes 4. Classes of bacetrial cell walls 5. Differences between G+ and G6. Gram staining and how differentiation occurs 7. Cell wall removal 8. Individual components (CM, PG, TA, LTA, proteins, PP space, OM, LPS, porins) 9. Acid-fast bacteria and associated cell wall structure 91 6. Flagella and Motility Dr Roger Draheim [email protected] x 2133 SM (old) 4.19 92 Intended Learning Outcomes (ILOs) – To describe the structure and function of bacterial flagellae, pili, fimbriae, capsules, ribosomes and storage granules – To describe the formation of endospores and their subsequent germination 93 Bacterial structure 94 Flagella (sing. flagellum) Long thin appendages that originate from the cytoplasmic membrane Extend through the cell wall into the surrounding medium Enable bacteria to be mobile Very thin (20nm) Not visible by light microscope unless stained 95 Flagella Observed mostly in: – Gram-negative rod-shaped bacteria. – Gram-positive rods Vary in number/arrangement on bacterial surface Bacterial characteristic used during classification Composed of flagellin (protein) – 30-40 kDa 96 Flagellar arrangement 97 Flagellar structure / arrangement 98 Flagellar structure / arrangement 99 Flagella Fundamental structure: – Not straight but helical – Constant distance between 2 adjacent curves known as wavelength – Is constant for a given organism Composed of protein subunits of flagellin 100 Flagellar components Three main components of a flagellum: – The motor known as the basal body – The hook Consisting of a single class of proteins at the base of the flagellum connecting the filament to the motor – The filament 101 Flagellar structure 102 Flagellar basal body Basal body is anchored in the cytoplasmic membrane and cell wall Small rod passing through a system of rings Multiple rings are present in Gram-negative bacteria: – An outer ring in OM/LPS – One in peptidoglycan – One in the cytoplasmic membrane – One in the cytoplasm In Gram-positive bacteria, one fewer inner ring present (why?) 103 Flagella basal body 2 Mot (motor) proteins are anchored either side of the MS ring These drive the flagellar motor: – Causing a torque that rotates the filament The Fli proteins act as a motor switch Energy supplied by the passage of protons from outside the cell into the cytoplasm via the basal body mot complex – nearly 1000 protons for a single rotation 104 Flagellar basal body 105 Flagella genetic control In E. coli and S. typhimurium, over 40 genes necessary for motility. – Major groups known as fla, fli and flg Several functions: – Structural proteins – Exporting of flagellar components through the cell wall – Biochemistry of synthesis Flagella grow from the tip, flagellin moves up the hollow core 106 40 proteins, really? 107 Flagella motility Enables movement at rate of up to 60 cell lengths per second Movement different in polar and peritrichous flagella Peritrichous: – In straight line – counter(anti)clockwise – Tumbling – clockwise smooth swimming Polar: – More rapid spinning 108 Chemotaxis driven by flagella rotation Motility and chemotaxis Normal movement is randomly composed of: – Runs and tumbles Chemotaxis: – Response to different chemical gradients Compare the chemical state of environment to that sensed seconds before and if attracted move up the gradient By altering the CW:CCW ratio can move up/down gradient Movement away if the chemical is repellent 110 Flagella antigens and phase variation Salmonella spp. Typed by their O (LPS) and H (flagellar) antigens >2000 serotypes (different O and H antigens) Have the ability to differentially express the protein antigen as either: – Phase 1 or phase 2 - phase variation 111 7. Endospores Dr Roger Draheim [email protected] x 2133 SM (old) 4.19 112 Endospores Two genera of medical importance produce highly resistant endospores These enable them to survive under adverse environmental conditions for long periods of time – Bacillus and Clostridia Spherical or oval structures, dormant or resting phase Survive in soil for up to 30 years – B. anthracis – C. tetanus – C. perfringens 113 Endospores Resistant to: – Drying – Heat – Pressure – Many chemical disinfectants Killed by heating to 120oC for 15-20 mins Size, shape and location of spores within stationary phase of vegetative cells are helpful for identification Appear as non-staining refractile areas within the cell 114 Endospores 115 Endospores 116 Endospores 117 Structure of endospores 118 Endospores Formation of spores stimulated by environmental conditions: – Lack of nutrients – Change of temperature – Redox potential 3 stages: – Activation – Germination – Outgrowth 119 Germination of endospores Activation: – In vitro accomplished by heating at sub-lethal but elevated temperature Germinate when placed in nutrients – Loss of resistance to heat and chemicals – Loss of calcium dipicolinate and cortex components – Acid-soluble spore proteins degraded Germination: converts back to vegetative cell relatively rapidly 120 Outgrowth of endospores Visible swelling as a result of – Water uptake – Synthesis of new RNA, proteins and DNA. Cell emerges from broken spore and begins to divide 121 8. Other subcellular components Dr Roger Draheim [email protected] x 2133 SM (old) 4.19 122 Pili and fimbriae Fine hair like filaments on the surface of many Gram negative bacteria – Not involved in motility Fimbriae composed from 20 kDa protein Pili generally longer Both involved in attachment of bacteria to host cells – Bind to lectins on cell surfaces, e.g sugar binding proteins – Gives tissue specificity 123 Pili and fimbriae Bacteria within a human host are constantly losing and reforming fimbriae – fragile structures Aid in evasion of the immune system Bacteria, e.g. N. gonorrhoea can change the antigenic structure of their pili / fimbriae. 124 Sex pili and conjugation Used by conjugative plasmids to transfer copies of itself to a new host – F+ and F- (F indicates fertility) Sometimes other genetic material is mobilised during conjugation After conjugation the previously negative cell has a copy of the plasmid and is able to pass it on to another cell Efficient manner for transfer of antimicrobial resistance 125 Sex pili and conjugation 126 Sex pili and conjugation The F plasmid of E. coli can also mobilise the chromosome to be transferred through cell to cell contact Episome: integrates into the chromosome and leads to transfer of large regions of the host genetic material and extensive genetic recombination. – Hfr strain (high frequency recombination) Cells already containing a plasmid are poor recipients for the same or similar plasmid 127 Hfr-specific recombination 128 Bacterial conjugation 129 Capsules and slime layers Consist of polysaccharide or sometimes protein. – General term is glycocalyx Capsule – Thick viscous gel outside and attached to cell wall Slime – Capsule loosely attached to cell wall and easily washed off Both are hydrophilic – Appear as halos around cells in a Gram stain 130 Capsules 131 Capsules and slime layers Variable thickness: – Klebsiella spp. and Streptococcus pneumoniae up to 10 m thick Microcapsule of E. coli and Salmonella spp: – Too thin to be seen on light microscope – Detected by antisera Capsule not always expressed under lab conditions 132 Composition of capsules Mainly acidic polysaccharide – The acidic groups being glucuronic acid or phosphate. Most are immunogenic and stimulate host antibody response Exceptions: – Group B meningococci - capsule contains Nacetylneuraminic acid which is found on host cells – Hyaluronic acid of S. pyogenes capsule can lead to host cell damage because found on host cells 133 Advantage to bacteria Protect cells from desiccation and toxic material Attachment to host cells Soluble material released into solution blocks opsonising antibodies Resist complement alternative pathway Resist phagocytosis by neutrophils 134 Bacterial chromosomes Single circular chromosome of double stranded DNA, called a nucleoid 300 to 400  m in length and supercoiled like a rubber band Genes arranged linearly along the chromosome Size range from 600 to 9500kb (9.5Mb) Composed of nucleotides: – Adenine – Guanine – Cytosine – Thymine (uracil in RNA) 135 Prokaryotic protein synthesis Takes place on ribosomes, 70S (30S+50S) in the cytoplasm – 30S contains 16S RNA (small subunit) – 50S contains 23S RNA (large subunit) Up to 10,000 ribosomes per cell – Up to 35% of dry weight is RNA in growing cells Bacterial mRNA is polycistronic (multiple genes on one mRNA) – Can be translated by several ribosomes simultaneously. 136 Prokaryotic protein synthesis Protein translation via: – tRNA and mRNA 30S subunit initiation complex + 50S subunit Location of protein encoding genes determined by looking for Open Reading Frames (ORF): – Start codon – Codon (triple sequences) – Stop codon 137 Pro vs. Euk Txn & Tln 138 Cytoplasm – carbon storage polymers Amorphous gel containing: – Enzymes and ions and a variety of granules – Accumulation of food reserves (polysaccharides, lipids or polyphosphates) One of the most common inclusion bodies consists of poly- -hydroxybutyric acid (PHB) Used as storage for carbon and energy storage polymers 139 Also cytoplasmic... Glycogen also used as storage product – Starch-like polymer of glucose subunits Smaller than PHB granules Genetic elements: – Plasmids: circular and autonomously replicating – Transposons: “jumping” genetic cassettes 140 Synopsis 1. Flagellar structure and arrangement 2. Flagellar components 3. Flagellar basal body structure + function (rotation) 4. Bacterial motility and how it works 5. Phase variation of genes 6. Pili and fimbriae 7. Bacterial conjugation (F+/F- and Hfr) 8. Capsules and slime layers 9. Bacterial chromosomes 10.Prokaryotic transcription / translation 11.Cytoplasmic entities (storage, plasmids, transposons) 12.Endosopres (formation and germination) 141 9. Introduction to Bacterial Growth and Nutrition Dr Roger Draheim [email protected] x 2133 SM (old) 4.19 142 Intended Learning Outcomes (ILOs) To understand the nutrient and growth requirements of bacteria To understand the various phases of the bacterial growth curve and how they are measured To understand the influence of environmental factors on bacterial growth (including laboratory conditions) To understand the fundamental bacterial metabolic pathways and how their usage in classification 143 Bacterial nutrition and growth In order to grow, bacteria require (for classification): – A source of energy (energy) – The raw materials to build new cell components (carbon source) All cells have the ability to direct chemical reactions and organise molecules into specific structures Chemical substances from outside the cell are transported inside and transformed 144 Anabolism and catabolism Anabolism (anabolic reactions) – The processes by which these substances are transformed into new cellular components – Biosynthesis Catabolism (catabolic reactions) – Biosynthesis requires energy, which is also required for motility and transport. Most obtained from the oxidation of chemical compounds which are broken down into simpler forms – The released energy is conserved 145 Catabolic and anabolic reactions Catabolism results in energy + nutrients + waste products Anabolism - synthesis of cell components from nutrients Different bacterial cells have different nutritional requirements and this provides a method for classification 146 Nutritional requirements Most bacteria require a source of carbon, hydrogen, oxygen and nitrogen, an energy source, water and various ions 95% of microbial cells’ dry weight is composed of: carbon, oxygen, hydrogen, nitrogen, sulphur, phosphorus, potassium, calcium, magnesium and iron Called macroelements / macronutrients because required in large amounts. The first 6 are the components of carbohydrates, lipids, proteins and nucleic acids 147 Macroelements / macronutrients Potassium, calcium, magnesium and iron exist as cations and have a variety of roles: Potassium – required for activity of enzymes including those involved in protein synthesis Calcium – Heat resistance of spores Magnesium – enzyme co-factor, complexes with ATP, stabilizes ribosomes and cell membranes Iron (Fe²⁺ and Fe³⁺) – in cytochromes and a cofactor for enzymes and electron carrying proteins 148 Micronutrients / trace elements Trace elements needed by most cells: – Manganese – Zinc – Cobalt – Molybdenum – Nickel – Copper Part of enzymes and cofactors, aid in the catalysis of reactions and maintenance of protein structure 149 Classification based on metabolism Carbon (along with H/O) often found together in same nutrient source … heterotrophs (require “other” carbon source) Exceptions are autotrophs (CO2 automatically from air) – Which use carbon dioxide as sole or principal carbon source – Many are photosynthetic Microorganisms use wide range of carbon sources – Burkholderia cepacia can utilise over 100 different carbon sources – others are fastidious and use very few carbon sources – vary among different species (classification) 150 Sources of energy for anabolic reactions All organisms require energy 2 sources of energy available to microorganisms: – – Light energy trapped during photosynthesis: phototrophs Energy derived from oxidising organic or inorganic molecules: chemotrophs 151 Source of electrons 2 sources of electrons: – Lithotrophs – Rock eaters’ use reduced inorganic substances as electron source Organotrophs extract electrons from organic compounds 152 Summary of metabolic classification 153 Classification of microorganisms Most non-photosynthetic bacteria, including most pathogens, fungi and protozoa are chemoorganoheterotrophs Use: – Organic chemical energy source (not photo) – Organic electron donor (not litho) – Organic carbon source (not auto) 154 Medically relevant bacteria Pathogenic bacteria derive their energy from metabolising sugars, fats and proteins Growth requirements vary within this group from: – Those who can survive on inorganic nutrients + a simple carbon source such as glucose (E. coli) – To complex growth requirements (Treponema pallidum). 155 Another criteria for differentiation If use same nutrients as most naturally occurring members of the species – Known as a prototroph (prototrophic) May mutate so that they cannot synthesise a molecule necessary for growth and reproduction, – e.g., a particular amino acid (leucine) – known as an auxotroph (leucine auxotroph) 156 N / P / S metabolism Microorganisms need to incorporate large quantities of these to grow May be available from same sources as carbon etc, but often use inorganic source as well Nitrogen needed for synthesis of: – – – – – – – Amino acids Purines Pyrimidines Carbohydrates Lipids Enzyme co-factors Other substances Many use the nitrogen in amino acids. Also reduce nitrate to ammonia and incorporate by assimilatory nitrate reduction 157 N / P / S metabolism Phosphorous present in nucleic acids, phospholipids, ATP, cofactors, proteins and other cell components. Use inorganic phosphate sources and incorporate directly Sulphur needed for synthesis of amino acids, cysteine and methionine, some carbohydrates, biotin and thiamine 158 Growth factors Growth factors are organic compounds required because they are essential cell components or precursors and cannot be synthesised by the microorganism – – – – Amino acids Purines and pyrimidines Vitamins Examples: Enterococcus faecalis needs 8 different vitamins for growth. Haem and niacin are necessary for H. influenzae to grow 159 10. Nutrient Uptake by Bacteria Dr Roger Draheim [email protected] x 2133 SM (old) 4.19 160 Nutrient uptake Need to be able to transport necessary substances into the cell Have to pass through selectively permeable plasma membrane Many different nutrient molecules needed, complex task using several different transport systems Facilitated diffusion, active transport and group translocation 161 Facilitated diffusion Rate of diffusion increased by use of carrier proteins, permeases, embedded in membrane Selective for particular solute Driven by concentration gradient across membrane, so is reversible Allows lipid insoluble molecules to enter the cell e.g. glycerol transport in E. coli, S. typhimurium, Pseudomonas, Bacillus and many others. 162 Facilitated diffusion 163 Active transport Allows uptake against a concentration gradient and therefore allows concentration of solutes inside the cell Substrate-binding proteins in the periplasmic space (PBPs) of Gram-negative bacteria bind the molecule to be transported and interact with membrane transport proteins to move the solute inside the cell Energy source is ATP E. coli transports a variety of sugars (arabinose, maltose, galactose, ribose) via PBPs 164 Active transport (w/PBP) 165 Active Transport Proton motive force (PMF) can also be used to drive active transport. The following examples function as a single protein. Lactose permease of E. coli is a good example: – Single protein transports lactose inward as proton enters cell at same time, symport. – E. coli also uses proton symport to take up amino acids and organic acids Antiport when, e.g. E. coli pumps sodium outward and protons inward. Sodium gradient drives uptake of sugars and amino acids. 166 Active transport (syn/antiporters) 167 Group translocation (PTS) Molecule transported into cell while being chemically altered, e.g. by phosphorylation Called phosphoenolpyruvate: sugar phosphotransferase system (PTS) Aerobic bacteria lack these systems, but found in facultative anaerobes and some obligate anaerobes E. coli uses PTS to take up glucose, fructose, mannitol and sucrose. 168 Group translocation (PTS) 169 11. Bacterial Growth Dr Roger Draheim [email protected] x 2133 SM (old) 4.19 170 Growth / binary fission Bacterial replication is a coordinated process resulting in the production of 2 equivalent daughter cells – Binary fission Requires sufficient metabolites and cascade of regulatory events Cell cycle involves replication of genetic material and cell division, but not always at the same rate although coordinated 171 E. coli cell cycle During the cell cycle the cell will double in length and then divide into two cells by transverse fission Cell division usually takes place 20 minutes after replication has finished Newly formed DNA is attached to adjacent sites on the plasma membrane and initiates a cross wall or septum growth and division of the two cells 172 Binary fission As the membrane grows the daughter chromosomes are pulled apart Initiates process of cell division, formation of septum The time required for a complete growth cycle dependent on nutritional and genetic factors 173 Binary fission (E. coli) 174 Bacterial Growth Growth rate is change in cell number or cell mass per unit time Interval for the formation of two cells from one called a generation (doubling time) Pattern of population increase, where the number of cells doubles in each time period is referred to as exponential growth 175 1. Lag phase When a microbial population is inoculated into a fresh medium, growth does not usually begin immediately - lag phase - during which they may need to: – resynthesise essential constituents – cells need time to recover from damage – transfer from rich to poor medium requires synthesis of new enzymes Length of lag phase varies considerably with conditions of bacteria and nature of the medium 176 2. Exponential (Log) phase Exponential phase (log phase): Influenced by temperature, culture medium and genetic characteristics Microorganisms are growing and dividing at the maximal rate possible for their genetic potential, culture medium and conditions. – Constant rate of growth Used in biochemical and physiological studies 177 3. Stationary phase Stationary phase - essential nutrient used up, waste products accumulate to unacceptable levels NO net increase or decrease in cell number – Usually at a population level of 10⁹ /ml – Dependent on level of nutrient availability, physical conditions: oxygen availability if aerobic, build up of toxic waste products – e.g. Streptococci producing lactic and other organic acids from sugar fermentation Several genes necessary for survival sur genes 178 4. Death phase Death phase Cell death slower than exponential growth. Also logarithmic Growth curve characteristics reflect events in a population of cells Bacterial growth can be demonstrated on solid culture media with the formation of colonies or in liquid samples via cell counts Viable cell count better with solid media 179 Bacterial growth curve 180 Growth Kinetics Exponential phase can be used to study bacterial physiology and also necessary for industrial culture Time for population to double is the generation time (or doubling time) 181 Measurement of growth Cell counts using counting chambers Larger cells such as fungi and protozoa can be counted on a coulter counter Spectrophotometry Spread plate and pour plate techniques Membrane filtration systems Flow cytometry 182 12. Environmental Factors Governing Bacterial Growth Dr Roger Draheim [email protected] x 2133 SM (old) 4.19 183 Environmental factors affecting bacterial growth Source of nitrogen, carbon, essential ions and nutrients Temperature Water, salt levels pH Atmosphere (O2 levels) 184 Essential elements Carbon, oxygen, nitrogen, hydrogen, sulphur, phosphorous, potassium, magnesium, calcium, iron, sodium and chloride Many bacteria have the ability to synthesise molecules called siderophores to sequester iron from host iron stores 185 Water – 80% or more of the cell mass is water – During growth, nutrients and waste products enter and leave in solution and so free available water (Aw) is a necessity – Mostly the cytoplasm of the cell has higher solute concentration and so water tends to diffuse into the cell – Positive water balance 186 Salt (osmolarity) Some bacteria have adapted to higher salt levels (seawater has 3% salt) – Halophiles Halophiles require salt levels of 2.8M to saturation of 6.2M Modify structure of proteins and membranes Extreme halophiles accumulate levels of potassium to remain hypertonic, 4-7M Halotolerant bacteria can tolerate some reduction in Aw 187 Effect of pH Most environments have a pH of between 5 and 8-9 and most organisms are able to grow within these parameters Acidophiles grow optimally between pH 0 and 5.5 Neutrophiles between 5.5 and 8 Alkalophiles 8.5-11 – With extreme alkalophiles at 10 or above Most bacteria and protozoa are neutrophiles Most fungi require prefer slightly acid conditions, pH4-6. Algae also favour acidity. 188 Effect of pH When obligate acidophiles are placed in neutral pH: – cytoplasmic membrane melts (dissolves) – cells lyse Obviously need high levels of hydrogen ions for stability Need for buffers when bacteria grown in artificial media to maintain optimum pH 189 Temperature Bacteria and archaea survive over a wide temperature range from -22 to >1000C Extremophiles can tolerate extreme cold or heat – Related to structure and enzymes Because they are unicellular, temp surrounding the microorganism has a direct effect upon it and may influence enzyme catalysed reactions High temperatures denaturing enzymes, affect the lipid bilayer etc. 190 Temperature Cardinal temperatures: – minimum, optimum and maximum – optimum usually closer to maximum than minimum Psychrophiles: grow well at 00C and optimum 150C or lower, max around 200C – Cell membranes have high levels of unsaturated fatty acids in membrane. – Cell membrane disrupted above 200C 191 Temperature Psychrotrophs – Psychrotolerant organisms can grow at lower temperatures with optimum of 20-300C – Can grow at 00C and max 350C – Found in soil water, milk and dairy products. – e.g. Listeria monocytogenes Most bacteria associated with human infection are mesophiles growing optimally between 9 and 40 degrees 192 Temperature Thermophiles grow at 550C or higher – Found in composts, hay stacks, hot water lines and hot springs More heat stable enzymes and protein synthesis systems operating at higher temps and more saturated lipids in membranes Hyperthermophiles, a few grow at 900C or 1000C and do not grow well at less than 550C – Found in hot areas of the sea floor 193 Oxygen requirements Bacteria vary in their requirement for oxygen Aerobes are capable of growth in full oxygen tension (21% in atmosphere) Oxygen (O2) serves as terminal receptor for electron transport chain in aerobic respiration 194 Classification by O2 Many bacteria are facultative anaerobes: – With appropriate nutrient and culture conditions they can grow under aerobic or anaerobic conditions, but grow better in the presence of oxygen Aerotolerant – Anaerobes can tolerate oxygen and grow in its presence but do not use it Microaerophiles – Are aerobes that can only use oxygen if it is present at levels below that of the atmosphere. – Limited capacity to respire or have oxygen labile enzyme 195 Classification by O2 Anaerobic organisms lack a respiratory system that can use oxygen as a terminal electron receptor Obligate (strict) anaerobes are killed by oxygen. – Lack the enzymes to decompose the toxic products of oxygen. (Hydrogen peroxide, superoxide and hydroxyl radicals) Strict and aerotolerant anaerobes cannot generate energy through respiration and use fermentation and anaerobic pathways – Contain flavin enzymes which react with oxygen to yield toxic products 196 Classification by O2 Strict anaerobes are killed by oxygen but can be found in otherwise aerobic environments. – e.g. Mouth where aerobes use up the oxygen and enable them to survive Products of oxygen are extremely toxic and so many bacteria have enzymes that protect them (aerotolerant): – Superoxide dismutase and catalase 197 Bacterial culture media Understanding growth requirements can help in the design of culture media for a given genera and enable selectivity to be developed Knowledge of metabolic pathways helps to design identification systems / media 198 Synopsis 1. Anabolic / catabolic reactions 2. Microelements/micronutrients and macroelements/trace elements 3. Classification based on metabolism (sources of carbon, energy, electrons) 4. Prototroph vs. auxotroph 5. Nutrient uptake (facilitated diffusion, active transport: PBPs and syn/antiporters, group translocation/PTS) 6. Binary fission (E. coli cell cycle) 7. Four phases of bacterial growth (diagram/measuring) 8. Factors affecting growth (elements, water, osmolarity, pH, temp, oxygen) and their use in classification 9. Bacterial culture medium 199 13. Introduction to Bacterial Metabolism Dr Roger Draheim [email protected] x 2133 SM (old) 4.19 200 Intended Learning Outcomes (ILOs) To understand: – the different classes of electron acceptors – the three “stages” of metabolism – how electron acceptors correlate with energy liberated (production of reducing equivalents) – biosynthesis from metabolic (by-)products – bacterial classification based upon metabolic abilities 201 Bacterial metabolism overview Chemoorganotrophs oxidise organic molecules to liberate energy They vary in their energy sources and use of electron acceptors Proteins, polysaccharides and lipids are broken down into simpler molecules and energy is released Nutrients are channelled into a few common pathways so enzymes are used efficiently 202 Bacterial metabolism overview Most of the energy released during the process of catabolism is generated by: – The movement of electrons through electron transport chains – More negative reduction potentials to ones with more positive reduction potentials This means that aerobic respiration is more efficient (O2 vs. NO2-, NO3- or SO4-) 203 It’s all about the electron acceptors! Variety used, three main processes in bacterial metabolism, depending on the genus Fermentation – an organic electron acceptor – Gives electrons to an endogenous acceptor – Usually an intermediate derived from the catabolism of the original nutrient – May be pyruvate that acts as electron acceptor 204 It’s all about the electron acceptors Aerobic respiration: electrons donated to – an exogenous acceptor, oxygen Anaerobic respiration: electrons donated to – an exogenous acceptor other than oxygen e.g nitrate or sulphate 205 Overview 206 Three stages of catabolism First stage – Proteins, polysaccharides and lipids – first stage catabolism – Proteins to amino acids – Lipids to glycerol – Polysaccharides to monosaccharides 207 Feeding pathways ex. monosaccharides 208 14. Second- and Third-stage metabolism Dr Roger Draheim [email protected] x 2133 SM (old) 4.19 209 Glucose to pyruvate Second stage: – Further breakdown to pyruvate or acetyl-CoA – Glycolytic pathway – Embden-Meyerhof-Parnas (EMP pathway) – Pentose phosphate pathway – Entner-Doudoroff pathway Most common is the glycolytic pathway Takes place in cytoplasm of bacteria 210 Glycolysis / Embden-Meyerhof-Parnas (EMP) pathway 211 Pentose phosphate pathway 212 Entner-Doudoroff pathway 213 Glucose to pyruvate Pentose phospshate pathway may be used at the same time as the glycolytic and Entner-Doudoroff Can operate either aerobically or anaerobically and is also important in biosynthesis Entner-Doudoroff pathway: – Only used in a few bacteria, e.g. Pseudomonas spp. and very few Gram-positives (exception is Enterococcus faecalis) EMP / PPP / ED all have pyruvate as end product 214 Maximising energetic return Third stage – Aerobic, anaerobic respiration and fermentation Pyruvate can be oxidised by pyruvate dehydrogenase to carbon dioxide and acetyl-CoA – Which can enter the TCA cycle Fermentation can take place in the absence of aerobic or anaerobic respiration 215 TCA cycle (Krebs / Citric acid cycle) 216 Aerobic respiration Most energy in form of ATP released when pyruvate degraded aerobically to carbon dioxide Acetyl-CoA enters TCA cycle Complete cycle functional in: – many aerobic bacteria – free living protozoa – most algae and fungi E. coli does not use the full TCA cycle under anaerobic conditions or when glucose concentration is high TCA cycle provides carbon skeletons for biosynthesis 217 Aerobic respiration Products are carbon dioxide + ATP + NADH + FADH Most ATP is generated when NADH and FADH are oxidised in the electron transport chain Oxidative phosphorylation with oxygen as final acceptor Net energy gain for glycolysis TCA cycle and electron transport is 38 ATP / glucose 218 Anaerobic respiration Many bacteria have electron transport chains that can operate with exogenous electron acceptors other than oxygen Oxidative phosphorylation with nitrate, sulphate or carbon dioxide as the terminal acceptors Not as efficient as aerobic respiration in production of ATP because have less positive reduction potentials 219 Anaerobic respiration Facultative anaerobes such as enteric bacteria can use nitrate as terminal acceptor in absence of oxygen Obligate anaerobes cannot use oxygen at all Those using carbon dioxide or carbonate are called methanogens Because reduce carbon dioxide to methane 220 Bacterial respiration Less H+ with non-O2 acceptor Less ATP generated with non-O2 acceptor 221 Replenishment of NAD+ / FAD+ Without replenishment glycolysis will stop 222 15. Fermentation and Classification Dr Roger Draheim [email protected] x 2133 SM (old) 4.19 223 Fermentation In the absence of respiration NADH is not oxidised by the electron transport chain – no electron acceptor is available If NAD not regenerated then glycolysis will stop Many organisms solve this by: – Slowing or stopping pyruvate dehydrogenase activity – Using pyruvate as an electron and hydrogen acceptor for the reoxidation of NADH in a fermentation process – May lead to production of ATP 224 Fermentation Different classes of fermentation which are often characteristic of particular groups: – Lactate (Streptococcus, Lactobacillus) – Ethanol (Yeast) – Propionate (Propionibacterium) – 2,3-Butanediol (Enterobacter, Serratia) – Also acetate, butanol, acetate, isopropanol and butyrate by mixed acid fermentation and butanediol fermentation 225 Fermentation examples ethanol fermentation lactic acid fermentation mixed acid fermentation 226 Energy usage and biosynthesis Energy derived from respiration used in proton motive force (PMF) for: – Flagella rotation – Active transport Products used in biosynthesis of cellular constituents – Peptidoglycan – Lipopolysaccharide (LPS) – Proteins – Nucleic acids 227 Bacterial classification Knowledge of the form of respiration and fermentation allows classification of bacteria and also provides a series of tests to help with their identification Examples – Fermentation of a variety of sugars – Growth requirements aerobic or anaerobic 228 Bacterial classification Possession of metabolic enzymes Biochemical reactions including: – Products of fermentation – e.g. VP (Voges-Proskauer) reaction where pyruvate is fermented to acetoin and reduced to 2,3 butanediol – Acetoin detected by addition of alpha naphthol – Positive in Klebsiella, Enterobacter, Serratia and Hafnia spp. 229 2,3-butanediol fermentation 230 Metabolism for classification Forms the basis of many ID systems for example: – API systems (strips) – Clostridia spp. ferment butyric acid – Anaerobic bacteria can be identified by using GLC Peaks produced by different products are analysed 231 Classification of bacteria 232 Tying it together … it’s all about the protons! 233 Synopsis 1. 2. 3. 4. 5. 6. 7. Overview of central concepts of bacterial metabolism Three processes with different electron acceptors Three stages of catabolism First: feeding reactions Second: EMP/PPP/ED to pyruvate Third: TCA cycle (Krebs or citric acid cycle) Respiration / fermentation to replenish reducing equivalents 8. Biosynthesis from biochemical intermediates 9. Metabolism classification of bacteria 234 17. Introduction to Fungi Dr Roger Draheim [email protected] x 2133 SM (old) 4.19 235 Intended Learning Outcomes (ILOs) To describe the structure of various fungi To outline the differences between yeast and filamentous fungi To describe how fungi are employed by mankind To describe the health hazards posed by fungi 236 Overview of fungi Contain: – Nucleus – Vacuoles – Mitochondria Spore-bearing Generally reproduce sexually or asexually Cell walls of polysaccharides, cellulose and/or chitin Saprophytic More than 100,000 species of fungi known – Only a few are of clinical importance 237 Classification of fungi May be divided into two groups: – Filamentous fungi (moulds) Hyphae extend as a result of transverse divisions forming a mycelium – Yeast Characteristic form is a single cell Reproduction by division or budding Bud attached to pseudohyphae 238 Filamentous fungi Filamentous fungi examples: – Aspergillus – Penicillium – Trichophyton Consists of – surface mycelium – aerial hyphae Cells of hyphae often contain many nuclei Conidia (asexual spores) are found at the end of the hyphal branches Spores are numerous and spread easily through air They are common contaminants in laboratories and a source of allergies 239 Life cycle of filamentous fungi 240 Filamentous fungi On agar plates colonies have a dusty appearance – Colonies are often brightly coloured Some species produce sexual spores – These are resistant to drying, heating and freezing – Not as resistant to heat as bacterial spores Some species produce fruiting bodies and mushrooms Mushrooms produce sexual spores (basidiospores) – Can be dispersed through the air 241 Aspergillus spp. 242 Yeasts Yeasts – Candida – Saccharomyces – Cryptococcus Unicellular Occur as spheres, ovals or cylinders Grow by – Budding (S. cerevisiae) – Fission (Schizosaccharomyces pombe) 243 Saccharomyces cerevisiae (bakers / budding yeast) 244 Dimorphic fungi Some fungi show both mycelial and yeast forms and are known as dimorphic fungi Dimorphic fungi: – Form hyphae at environmental temperatures – Occur as yeast cells in the body – Switch being temperature-induced Some yeasts form a filamentous phase (opposite) – Candida albicans – This phase is involved in pathogenicity 245 Importance of fungi BENEFITS Food Decay as part of nutrient cycles Antibiotic production Extensively used in other commercially important fermentation processes Potential for pest control Molecular biology HAZARDS Infections Production of toxins (Mycotoxins) Food spoilage Rot and decay Plant pathogens 246 Food Mushrooms – Some filamentous fungi produce fruiting bodies Quorn – A fungal protein used as vegetarian meat substitute Yeast tablets – Dietary supplement, rich in protein and riboflavin (vitamin B2) – Yeast used in brewing and baking Moulds used for ripening cheeses 247 Food The yeast Saccharomyces cerevisiae is used in the brewing of alcoholic beverages and in the leavening of bread Continental lagers use bottom-fermenting yeast such as S. carlsbergensis Filamentous fungi are involved in the mould ripening of cheeses, e.g. Penicillium roqueforti and P. camemberti. 248 Antibiotic Production Most antibiotics are produced by – Streptomyces spp. – filamentous fungi Especially species of Aspergillus and Penicillium Other antibiotics from fungi: – Fumigillin (anti-amoebal) – Griseofulvin (antifungal) – Cephalosporins 249 Penicillin 1929: – Fleming discovered that staphylococcal growth on a petri dish was inhibited by a contaminating fungus – Penicillium notatum 1940: first isolated by Florey and Chain 1941: first clinical application Modern processes use Penicillium chrysogenum 250 Penicillin notatum 251 Fungi in biotechnology Fungi are extensively used in commercial processes to produce: – Chemicals for food industry – Chemical and pharmaceutical industries Products include: – Citric acid – Kojic acid – Itaconic acid – Steroids (transformations) – Glycerol – Ethanol – Enzymes e.g. amylases, penicillin acylase 252 Hazards involving fungi Infections: by filamentous moulds and yeasts, superficial and deep mycoses Production of toxins (mycotoxins) which can cause disease Food spoilage Rot and decay Plant pathogens 253 17. Fungal Infections Dr Roger Draheim [email protected] x 2133 SM (old) 4.19 254 Fungal infections Fungal infections are called mycoses They can be divided according to the site of infection into: – Superficial mycoses – Cutaneous mycoses – Subcutaneous mycoses – Systemic (deep) mycoses – Infections of the immunocompromised host are called opportunistic mycoses 255 Cutaneous mycoses Caused by fungi that invade only superficial keratinised tissue: – Skin – Hair – Nails Most important groups are the dermatophytes: – Epidermophyton – Microsporum – Trichophyton 256 Morphology and identification Colonies can be cultured on: – Sabouraud agar (pH 5.6) – Agar containing cyclohexamide (inhibits saprophytes) Non-dermatophytes do not grow in the presence of cyclohexamide Conidia may be observed on slide cultures and aid in the identification Sources of dermatophytes are anthrophilic (human over other animals), zoophilic (animals over humans) and geophilic (prefer soil) 257 Identification of dermatophytes All have characteristic cultural and microscopic appearance used for identification Cause characteristic skin lesions called ‘ringworm’ (rash is circular with a ring-link appearance) or tinea Description of symptoms often includes part of the body – Tinea pedis (foot) – Tinea corporis (body), etc… – Also cause brittle or thick nails 258 Trichophyton rubrum 259 Microsporum canis 260 Epidermophyton floccosum 261 Infections caused by dermatophytes Tinea pedia (athlete’s foot) Most prevalent of all dermatophytes Toe webs infected with Trichophyton spp. or E. floccosum Initially causes itching between toes and formation of vesicles which rupture and discharge fluid Skin becomes macerated and peels 262 T. mentagrophytes (Athlete’s foot) 263 Athlete’s foot Cracks prone to secondary bacterial infection Nail infection may follow Individual may become hypersensitive and develop dermatophytids (vesicles) elsewhere on the body: – Especially the hands Athlete's foot is only found in people wearing shoes Infection is spread by communal showers and changing rooms Prevention by hygiene, especially keeping toes dry Treat with antifungal creams or powders – e.g. Daktarin 264 Ringworm (Tinea corprois, Tinea cruris) Dermatophytosis of non-hairy skin gives rise to annular lesions of ringworm Varying degrees of inflammation may be found, the most common Isolates are – E. floccosum – T. rubrum – T. mentagrophytes Treat with antifungal cream 265 M. canis ringworm (from feline) 266 Tinea capitis (ringworm of the scalp) Microsporum in childhood Trichophyton in adults Infection begins on skin of scalp and then down wall of hair follicle May appear as alopecia with scaling, black dot ringworm Trichophyton may also infect beard hair Treatment: – Remove hairs – Treat with Griseofulvin or shampoo with Miconazole 267 tinea capitis 268 Non-dermatophyte superficial infections Non-dermatophytes are often faster growing and grow on Sabouraud agar only (pH altered and without cyclohexamide) Cause problems for immunocompromised patients who have limited cellular immunity 269 Aspergillus Aspergillosis describes a group of mycoses caused by spp. of the filamentous fungus Aspergillus Infection comes from exogenous source Pulmonary aspergillosis may occur in distinct forms – One, aspergilloma, is a fungus ball growing in a preexisting cavity (e.g. tuberculosis) – Often asymptomatic or patient has a cough 270 Aspergillus Invasive aspergillosis is caused by A. fumigatus in immunocompromised patients Widespread destruction of tissue as fungus grows Treat with Amphotericin B Allergic aspergillosis occurs in patients with elevated IgE levels – 10-20% of asthmatics react to A. fumigatus Treatment is with corticosteroids Endocarditis may occur in immunosuppressed patients or those who have undergone open heart surgery Therapy depends on antifungals and surgical removal of infected tissue 271 Aspergillus fumigatus 272 Systemic mycoses Coccidioides immitis Grows as mould in soil Coccidioidomycosis is a lung infection caught after inhaling arthrospores Found in semi-arid regions, mainly southwest USA and Northern Mexico. Also called San Joaquin valley fever Usually asymptomatic or self-limiting lung infection, – Mild cough – Chest pains – Headache 273 Coccidioides immitis Dissemination occurs in less than 0.5% of cases – Often immunocompromised patients Chronic cases involve localised cavities in lungs filled with spherules (cylindrical bodies) of C. immitis Other tissues involved – Bones – Liver – Meninges – Brain – Skin – Heart Can be treated with Amphotericin B High death rate in disseminated cases 274 Coccidioides immitus 275 Histoplasma capsulatum Grows as mould in soil and in culture, and as a mould or yeast in animal tissues Intracellular parasite found in soil rich with droppings of birds and bats – Occurs in USA, endemic in Ohio and Mississippi River valleys Histoplasmosis is usually asymptomatic or flulike symptoms with fever and cough – 250,000 new cases each year in USA 276 Histoplasma capsulatum Microconidia inhaled Symptoms of coughing, fever and joint pain Infections are mostly self-limiting but patient is often left with discrete calcified lesions in the lung Chronic form can develop in adults; large cavities develop in lung from new infection or by reactivation of old infection Sometimes dissemination occurs, most often in old age or infancy or with immunosupression Treatment with intravenous Amphotericin 277 Histoplasma capsulatum 278 Yeast infections Candida appears as Gram-positive, oval budding yeast, 2-3 mm x 46 mm Forms pseudohyphae in culture and tissues On Sabouraud agar it produces soft, cream coloured colonies with characteristic yeasty smell The submerged growth consists of pseudomycelia 279 Candida albicans 280 Infections Mouth – Oral thrush – White patches inside mouth – Most common in infants and AIDS patients Growth of Candida in the mouth is enhanced by: – Corticosteroids – Antibiotics – High levels of glucose – Immunodeficiency 281 Candidiasis 282 Infections Female genitalia – Vaginal thrush or vulvovaginitis – Irritation, discharge, intense itching – Acid pH is normally maintained by bacteria, suppressing Candida – Diabetes, pregnancy, progesterone and antibiotics predispose 283 Infections Skin – Occurs in warm parts of body – Often follows immersion in hot water Nails Lungs and other organs – May be secondary infection of lungs, kidney and other organs under predisposing conditions (e.g. tuberculosis or cancer) Chronic mucocutaneous candidosis – Sign of immunodeficiency in children 284 Pityriasis (Tinea) versicolor Superficial skin infection caused by yeast – Malassezia furfur Characterised by pale or dark patches of skin Part of normal skin flora in majority of adults where it causes no problems Requires fatty acids for growth and therefore media has to contain oil 285 Pityriasis (Tinea) Versicolor 286 Treatment of fungal infections The range of antifungals is poor compared to antibacterials Suggest a reason for this? There is interest in developing new drugs 287 ANTIFUNGAL DRUGS DRUG MECHANISM USES Amphotericin B Binds to ergesterol in cell membrane Broad spectrum, systemic Flucytosine Accumulated by permease Converted to fluorouracil Synergistic with amphotericin B Azoles (e.g. Fluconazole, Ketoconazole, Itraconozale) Inhibit ergesterol synthesis Systemic Griseofulvin Accumulates in keratinised tissue, interacts with fungal microtubules Dermatophytes Nystatin As amphotericin B Topical 288 Synopsis 1. 2. 3. 4. 5. 6. 7. Properties / classification of fungi (filamentous fungi and yeast) Life cycle / properties of filamentous fungi Life cycle / properties of yeasts Dimorphic fungi and transitions Benefits of fungi (food / antibiotics / penicillin / biotechnology) Hazards involving fungi (infections / toxins / food spoilage / rot + decay) Infections / mycoses (superficial / cutaneous / subcutaneous / systemic (deep) / opportunistic) 8. Identification of dermatophytes 9. Tinea pedis / tinea corprois / tinea cruris / tinea capitis 10.Aspergillosis 11.Systemic mycoses (Coccidiodes immitis / Histoplasma capulatum) 12.Yeast infections (Candida / Pityriasis) 13.Antifungals 289 18. Introduction to Viruses Dr Roger Draheim [email protected] x 2133 SM (old) 4.19 290 Intended Learning Outcomes (ILOs) To understand – the history and background of virology and viruses – viral classification, nomenclature and replication – the classification of protozoa – the detection, prevention, diagnosis and treatment of the various protozoan classes 291 History of virology Viral infection has played a significant role in medical history – The nature of viruses has only fairly recently been understood Roman Empire weakened by measles and smallpox epidemics, rabies was common Spanish conquest of Mexico aided by the fact that the Aztecs killed by smallpox brought by the conquerors 292 History of virology Early control of viral infection came in the eighteenth century – discovered that in Turkey some protection from smallpox was gained by inoculating children with dried material from healed scabs This had originated in China in the 10th century 1796 Jenner first used cowpox to protect against smallpox 293 History of virology With the invention of porcelain filters that would filter out bacteria it was discovered that leaf extracts from plants could still produce disease after filtration Observed that tobacco mosaic virus only multiplied in living plants but could survive in a dried state Then discovered that hoof and mouth disease and yellow fever were not bacterial Discovery by Peyton Rous in 1911 – A virus caused cancer in chickens 294 History of viruses Tobacco mosaic virus crystallised in 1935 By the late 1930’s researchers concluded that viruses were complexes of nucleic acid and proteins only able to reproduce in living cells Viruses cultivated in 6-8 day old fertilised chicken eggs – Cultivated in chorioallantoic membrane and allantoic cavity Now grown in cell culture and form plaques / destroyed cells or have a cytopathic effect on the cells 295 Terminology Virus – A genetic element containing either RNA or DNA that replicates in cells but is characterised by having an extracellular state Virion – The complete virus particle, the nucleic acid surrounded by a protein coat and sometimes other material Retrovirus – A virus whose RNA genome has a DNA intermediate as part of its replication cycle 296 Viruses Responsible for many important acute diseases e.g. childhood illnesses: – Mumps – Measles – Chickenpox Chronic diseases – HIV Dangerous diseases: – Yellow fever – Lassa fever – Ebola – Smallpox Tumour formation - warts and cancers 297 Distinguishing features of viruses Extremely small size – 20-300nm (1nm = 10-3  m) Pass through bacteria-retaining filters Invisible by light microscope Impossible to grow on culture media Simple structure: nucleic acid + protein + some have lipid outer membrane 298 Distinguishing features of viruses Lack intracellular structures, e.g. ribosomes Some surrounded by envelope but no outer wall (peptidoglycan, etc…) Genetic information carried in nucleic acid Classified on the basis of the hosts they infect – Animal viruses – Plant viruses – Bacterial viruses (bacteriophages) Also classified on structural characteristics 299 Distinguishing features of viruses Each type of virus has one type of nucleic acid – Either RNA or DNA and provides a basis for classification RNA viruses are the only organisms to have their genome in this form – sometimes the RNA genome consists of several segments 300 Distinguishing features of viruses Replication: – Lack of protein synthesis, apparatus and energy producing systems makes them obligate intracellular parasites Incapable of binary fission – Do not grow and divide as bacteria do Intracellular release of nucleic acid which directs viral protein production and its own replication 301 Distinguishing features of viruses Proteins and nucleic acid synthesised independently and ultimately assembled into identical mature virus particles which are then released from the cell Often aggregate and can be seen in infected cells as inclusion bodies Not affected by antibiotics and other chemotherapeutic drugs Inhibited by interferon 302 303 Viral structure Mature virus called a virion Genetic material may be in the form of RNA or DNA (never both) either single stranded ss or double stranded ds dsDNA and ssRNA are most common Single continuous piece of nucleic acid – but there are exceptions 304 Viral structure Exceptions: – Reoviruses 10 fragments of dsRNA (rotavirus), Bunyaviruses 3 of ssRNA Orthomyxoviruses 8 ssRNA(influenza) – Retroviruses Rous sarcoma virus, HIV have two complete copies of RNA genome Capsid: protein covering Nucleocapsid: nucleic acid with capsule 305 Viral structure Capsid composed of subunits – Called capsomeres Icosohedral viruses – Rigid 20 faced box enclosing nucleic acid Helical viruses – Cylinder around nucleic acid Envelope – Loose membrane consisting of lipid bilayer derived from host cell membrane into which viral (not host cell) proteins are inserted. – Spikes project from the envelope - organised aggregates of viral glycoprotein 306 Viral structure: general principles With the exception of poxviruses, all DNA viruses are icosahedral All helical viruses are RNA viruses RNA can be icosahedral or helical All helical viruses are enveloped All RNA viruses except Picornavirus, Calciviridae, and Reovirus are enveloped 307 308 309 Icosahedral viruses Appear to be spherical but closer on EM reveals shape examination 20 faces which are equilateral triangles – Composed of hexons 12 vertices or corners composed of pentons All composed of protein subunits 310 Helical viruses Filamentous capsomeres arranged in helical symmetry to a hollow cylinder along the which the nucleic acid is wound form inside of Each capsomere is a single protein subunit In their envelopes, viruses appear roughly spherical – Nucleocapsid is flexible enough to coil up within the loose fitting lipid membrane 311 Unusual viruses Helical nucleocapsid supercoiled into the form of a hollow sphere surrounded by an icosahedral shell and further enclosed by an envelope – HIV Unusual viruses: pox viruses, e.g. Orf virus, particle ovoid or brick shaped ‘complex’ structure – Neither icosahedral nor helical 312 313 Enveloped or non-enveloped? Non-enveloped viruses are able to survive better in the environment than enveloped – Are often bile resistant Therefore can infect via intestine, e.g. – – – – Rotavirus Norovirus Hepatitis A Poliovirus Spread easily – common cold Enveloped viruses less hardy in the environment, spread by droplets – (Influenza) saliva, – (EBV) – Sexual contact (HIV) 314 Enveloped viruses 315 Classification of viruses Classified on the basis of a small number of characteristics Structure – – Icosahedral, helical or complex Enveloped or non-enveloped Nature of the nucleic acid – Whether DNA, RNA, ss or ds – Whether ss is + strand (coding) or – strand (non-coding) 316 Nomenclature Family with viridae as suffix – Herpesviridae, – Poxviridae Genera name with suffix virus – Herpes Simplex virus Species, often named after specific disease or numbered – HSV1 – Herpes zoster – Measles 317 19. Introduction to Protozoa I Dr Roger Draheim [email protected] x 2133 SM (old) 4.19 318 Introduction to protozoa Classification and importance of protozoa Description of the major protozoal infections in the UK and worldwide Control of these infections: – Treatment – Prevention 319 Classification Protozoa Apicomplexa Sporozoea Plasmodium Toxoplasma Cryptosporidium Pneumocystis?? Sarcomastigophora Mastigophora (flagellates) Trypanasoma Leishmania Giardia, Trichomonas Sarcodina (amoebae) Entamoeba Naegleria Acanthamoeba 320 Importance Infections are generally not common in the UK – Diseases such as malaria are endemic in the tropics Many protozoan diseases are more prevalent in the Third World as a result of poverty and survival of the vectors that transmit them Some infections have become more prevalent in the UK associated with immunosuppression e.g. HIV/AIDS – Toxoplasma – Pneumocystis – Cryptosporidium 321 Protozoal infections in the UK Trichomonas vaginalis Toxoplasma gondii Giardia lamblia Cryptosporidium parvum Pneumocystis carinii Plasmodium (malaria) – Encountered in patients who have visited endemic areas 322 CLASSIFICATION Protozoa Apicomplexa Sporozoea Plasmodium Toxoplasma Cryptosporidium Pneumocystis?? 323 Plasmodium Causes malaria in areas of the world where the mosquito vectors can breed Tropics between 60 degrees north and 40 degrees south, mainly in: – – – – Africa India Far East South America Four species of plasmodium – – – – P. falciparum P. vivax P. malariae P. ovale Complex life cycle involving liver and blood cells and several different stages of parasites 324 Plasmodium life cycle 325 Plasmodium (malarial parasite) 326 Toxoplasmosis Caused by infection with common parasite, Toxoplasma gondii – Can infect all mammal and bird species and found worldwide Only passed on if enter the food chain Up to one billion of world population is infected with T. gondii. 327 Toxoplasma gondii A definitive host is the one in which sexual reproduction takes place Any other host is called a reservoir – – – – – – Definitive host is the domestic cat Mammals can act as reservoir 75% of feline population has antibodies 25% of human population has antibodies Between 7-34% of people in the UK have been infected Majority asymptomatic or flu like symptoms A latent infection can be reactivated later in life if predisposing conditions arise Two main at-risk groups – Immunosuppressed – Pregnant women 328 Acquired toxoplasmosis Can be lethal to the immunosuppressed Low numbers of infection in England and Wales – 2007 - 107 – 2008 - 65 reported to HPA Symptoms include – fever, headaches, fatigue – swelling of lymph glands – infection of brain, lung, heart, liver 329 Congenital toxoplasmosis If contracted during pregnancy the infection can cross the placenta and infect the foetus Severity of disease depends on stage of pregnancy – Rare in early pregnancy but can lead to miscarriage, stillbirth and birth defects Infection in last 28 weeks very rarely leads to problems at birth but symptoms may develop much later in 20’s and 30’s Pregnant women can be tested by looking for a rising titre of antibodies Education about the risks is important, encouraging prevention of infection 330 Treatment All congenital cases should be treated Pyrimethane plus sulphadiazine is the recommended treatment Pyrimethane is teratogenic and should not be used in the first trimester Spiramycin, an inhibitor of bacterial protein synthesis, is effective but is not licensed in the UK for this use If treatment fails, termination may be considered 331 332 Cryptosporidium parvum Causes cryptosporidosis In healthy adults the disease causes mild gastrointestinal upset and is self-limiting Potentially lethal to the immunosuppressed No effective chemotherapy About 6,000 cases pa, mainly associated with drinking water 333 Cryptosporidium parvum Can be spread in swimming pools Very low infectious dose One cyst is believed to be sufficient to cause infection Outbreaks have been reported due to drinking water containing between 1 an 9 cysts per litre Ozone is effective in killing cysts but is not routinely used in UK 334 Contamination of drinking water Giardia and cryptosporidium cause major problems for the water industry – Cysts enter water sources from human or animal faeces or run-off from a slaughterhouse – Water is first treated by filtration This should remove all pathogens. Many outbreaks arise from failure of this stage – Water is then treated by chlorination This does not kill the cysts – Cysts are present in small numbers and are difficult to detect – Large volumes of water must be tested (1m3) 335 Cryptosporidium parvum 336 Pneumocystis carinii Uncertain taxonomy Morphology suggests protozoa rRNA sequence suggests fungus Major cause of death in AIDS patients Treatment strategies for HIV have lowered numbers of patients developing full blown AIDS 337 Diseases Atypical interstitial cell pneumonia (pneumocystosis) –Pneumonia which does not respond to antibiotics Spread by droplet infection and close contact Common in infants, especially in crowded institutions such as hospitals and orphanages in central Europe It is estimated that 85% of AIDS patients will become infected Low level of antibodies in healthy adults suggests that subclinical infection is common and disease may result from reactivation rather than new infection 338 Diagnosis / treatment Diagnosis – Cannot be cultured from sputum – Requires invasive techniques: broncho-alveolar lavage or open lung biopsy – In such samples the organism can be detected using silver or immunofluorescent stains Treatment – Cotrimoxazole (sulphamethoxazole plus trimethoprim) is drug of choice – Pentamidine can be used as an alternative 339 20. Introduction to Protozoa II Dr Roger Draheim [email protected] x 2133 SM (old) 4.19 340 CLASSIFICATION Protozoa Sarcomastigophora Mastigophora (flagellates) Trypanasoma Leishmania Giardia, Trichomonas 341 Trypanosomes Three species of flagellated protozoan Trypanosomes cause disease: – T. brucei gambiense – T. brucei rhodiense cause African trypanosomiasis or sleeping sickness – T. cruzi causes South American trypanosomiasis, Chagas disease 342 Sleeping sickness Spread by Tsetse fly and restricted to equatorial Africa T. brucei remains extracellular – First in the tissues near the insect bite and then in the blood, where it divides rapidly and continuously Swollen chancre appears near bite, also swollen lymph nodes in neck Parasite spreads rapidly in blood – fever and splenomegaly, and heart involvement Headache and psychological changes and then coma 343 Sleeping sickness Patients often left with neurological and mental disability Survival in the blood as a result of antigenic variation of glycoprotein coat Diagnosed by LN biopsy or lumbar puncture and raised serum IgM levels Treatment with tryparsamide and melarsoprol Difficult to control tsetse fly 344 Chagas disease T. cruzi spread by reduviid bug (kissing) bug found in the walls of housing in poor areas All species of mammal act as reservoirs of infection and parasite can live in host macrophages and cardiac muscle cells Causes chronic disease and involves heart and intestinal tract Death often as a result of myocarditis 345 Chagas disease Diagnosed by parasites in blood film or in later stages by serology Very difficult to treat – Prevention is better Improved housing conditions and living standards Vectors difficult to control 346 Leishmania Leishmania cause disease in South and central America – New World leishmaniasis in India, Middle East Africa and Mediterranean coast – Old world leishmaniasis. Old world, dogs act as reservoir of infection Two types of disease, visceral (liver and spleen) and cutaneous. Caused by different species Visceral causes chronic disease, kala-azar with eventual liver failure 347 Leishmania Cutaneous disease characterised by large ulcer developing from small papule on the skin Heals with scarring and patient is relatively immune to further infection Called Baghdad boil and Delhi sore In immunodeficient patients, cutaneous becomes like leprosy and visceral is a major complication of HIV in the tropics 348 Trichomonas vaginalis Normally non-pathogenic flora of vagina May cause trichomonas vaginitis associated with: – itching and burning – foul-smelling discharge Men may be asymptomatic carriers inflammation of the urethra may occur 349 T. vaginalis Reproduction appears to be exclusively by binary fission Morphology – Ovoid – 4 free anterior flagella – 5th flagellum is recurved and attached to the body to form an undulating membrane – Single nucleus – No mitochondria - energy is generated by hydrogenosomes 350 Diagnosis, prevention and treatment Diagnosis – Microscopy of vaginal swab Prevention – Toilet hygiene – Safe sex – Do not share clothes or toilet articles Treatment – Metronidazole for both partners – Incidents of resistance have been reported 351 Giardia lamblia (duodenalis) Formerly known as G. intestinalis Trophozoites have 2 nuclei, – Giving characteristic owl’s face Causes giardasis (beaver fever) Inhabits lumen of duodenum or upper ileum Carriage can range from asymptomatic to malabsorption syndrome Onset is associated with watery, but not bloody, diarrhoea 352 Route of infection Cysts are shed in human and animal faeces Infection arises by ingesting cysts, which excyst in the host’s gut Spread can be person-to-person in unsanitary conditions The most common route is by drinking improperly treated water There are about 6,000 cases pa in England and Wales 353 Detection and Treatment Detection: – Diagnosis is by detection of cysts in the faeces – Shedding of cysts is irregular, so several samples must be taken at different times Treatment – Infection is usually self-limiting, but if necessary may be treated with metranidazole 354 CLASSIFICATION Protozoa Sarcomastigophora Sarcodina (amoebae) Entamoeba Naegleria Acanthamoeba 355 Entamoeba histolytica Intestinal protozoan infection occurs worldwide, but most often in subtropical and tropical countries, where prevalence may exceed 50% Trophozoite stages live in large intestine and reproduce by binary fission Resistant encysted forms produced and passed from body and act as infective agents in the environment Contaminate food and drink 356 Synopsis of protozoan infections Site of Infection Organism Route of Entry GI Tract Cryptosporidium Giardia Entamoeba Ingestion Blood Plasmodium Trypanasoma Insect bite Lung Pneumocystis Inhalation Vagina Trichomonas Contact All Tissues Toxoplasma Ingestion 357 Synopsis 1. 2. 3. 4. 5. 6. 7. 8. History of virology / viruses Terminology of virology Distinguishing features of viruses Replication of viruses Structures of viruses Classification of viruses Introduction / Classification of protozoa Plasmodium / Toxoplasmosis /Cryptosporidium / Pneumocystis (diagnosis and treatment) 9. Trypanosomes (sleeping sickness / Chagas disease / Leishmania) 10.Diagnosis / prevention / treatment 11.Giardia (detection / treatment) 12.Summary of protozoan infections 358

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