CSF-6250 Integrative Pathophysiology & Biomechanics in Health Care 2 PDF

CSF-6250 Integrative Pathophysiology & Biomechanics in Health Care 2 Module: Microbes in Human Health and Disease Unit 03 – Bacteria Verena Van Fleet, PhD, MS, MEd Professor, Department of B...

CSF-6250 Integrative Pathophysiology & Biomechanics in Health Care 2 Module: Microbes in Human Health and Disease Unit 03 – Bacteria Verena Van Fleet, PhD, MS, MEd Professor, Department of Basic Sciences Unit 3 Objectives - Differentiate prokaryotic and eukaryotic cells. - Recognize structural and functional features of bacterial species useful for diagnostic purposes. - Recognize methods to measure microbial growth. - List factors that affect microbial growth. - Name the three processes of horizontal gene transfer in bacteria. - Recognize the purpose and applications of PCR and CRISPR NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 2 Lesson 1: Prokaryotic Cells Salmonella typhimurium invading cultured cells https://www.wikidoc.org/index.php/File:SalmonellaNIAID.jpg#filehistory NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 3 The Prokaryotic Cell Found in all bacterial cells 4 NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu Prokaryotic and Eukaryotic Cells Prokaryotic Cell Eukaryotic Cell Small, simple CM Large, complex (0.1-5 mm) Cytoplasm (10-100) mm) Unicellular DNA and Uni- or multicellular No nucleus RNA Nucleus present Circular DNA Ribosomes Linear DNA No membrane- Membrane-bound bound organelles organelles present 5 NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu Bacterial Cell Shapes and Arrangements Shape and arrangement are species-specific and useful in diagnosing bacterial infections 6 (2Yteplasmic Membrane) CM and Cytoplasm of Prokaryotic Cells Lase Numbers CM: higher in test – Lipid bilayer: No cholesterol tubes – Proteins: Higher protein content Low Lover intest takes Ribosomes: – 70S (vs 80S in eukaryotic cells) – Target of certain antibiotics Eukaryotes: small subunit: 40S large subunit: 60S complete ribosome: 80S Inclusions: – Storage of certain resources in polymerized form – Clinical Example: Volutin (poly-phosphorus) in Corynebacterium diphtheriae Volutin granules, made visible with specific volutin stain NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 7 External Structures of Prokaryotic Cells: Cell Wall - Major component: - Peptidoglycan - Protection from osmotic pressure - Several types of antibiotics inhibit peptidoglycan synthesis - E.g., penicillin ↳ Prevent bacteria from growing NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 8 External Structures of Prokaryotic Cells: Gram-positive and Gram-negative Cell Wall - Two common structural types anchor ↓ Thickerinitive Not in ↓ ther Lipopolysaccharide (LPS) Lipid A = Endotoxin NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 9 External Structures of Prokaryotic Cells: Gram-positive and Gram-negative Stain Differentiation of Gram-positive and Gram-negative cells NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 10 External Structures of Prokaryotic Cells: Clinical Significance of Cell Wall Type Gram-positive Gram-negative Susceptibility to penicillin high low Susceptibility to lysozyme high low Susceptibility to lysis by complement low high Sensitivity to heat and disinfectants low high Endotoxin no yes Exotoxin some species a few species NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 11 External Structures of Prokaryotic Cells: Acid Fast Cell Wall - These Gram-positive bacteria are covered with a wax-like substance called mycolic acid - Stain pink with the acid-fast stain - Clinically important acid-fast bacteria: Mycobacterium tuberculosis and Mycobacterium leprae NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 12 External Structures of Prokaryotic Cells: Capsule – Not found in all species – Outside of cell wall – Cannot be stained; made visible with ‘negative stain’ – Glycocalyx = ‘sugar coat’ (sticky coat) – Capsule: Something – Highly organized and defined – Consisting of polysaccharides or proteins aputa patheter – Can be a virulence factor (disguising cells from the immune system and resisting phagocytosis) – Slime layer: – Less organized, not tightly bound to the cell – Consisting of polysaccharides, glycoproteins, or glycolipids – Function: – Retention of water – Biofilm formation NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 13 External Structures of Prokaryotic Cells: Flagella, Fimbriae - Flagella - Visibility through ‘flagella stain’ - Presence and arrangement is species-specific - Fimbriae - Sticky, bristle-like appendages (made of protein) Salmonella with - Usually many peritrichous flagella - Purpose: biofilm formation and fimbriae From: bio.libretext.org NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 14 External Structures of Prokaryotic Cells: Pilus/Pili Pili (singular: pilus) - Hollow tubes (made of protein) - Longer than fimbriae, shorter than flagella Pilus - Make a connection between two cells - Only 1-2 per cell - Purpose: Transfer of DNA between two cells ( ‘conjugation’) Fimbriae E. coli bacteria with fimbriae and a pilus connecting the cells NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 15 Endospores – Found in some gram-positive genera of bacteria – Clinically important: certain species of Bacillus and Clostridium – Dormant stage (low nutrients, temperature, pH, …) – Resistant to commonly used methods of sterilization – Not to be confused with reproductive spores of fungi! Vegetative Cells Endospores – Species-specific endospore morphology: Sensitive to extreme Resistant to extreme temperatures and radiation temperatures and radiation Gram-positive Do not absorb Gram stain; need special endospore stains (1,4) central endospore, (2,3,5) Physiological water content and Dehydrated; no metabolic terminal endospore, (6) lateral enzymatic activity activity endospore Capable of growth and Dormant; no growth or metabolism metabolic activity NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 16  Could be Commonly Used Stains Oh boards know purpse of the stain T- Also called: ‘Negative stain’ NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 17 Lesson 2: Bacterial Metabolism NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 18 Metabolism - Metabolism = catabolism + anabolism - Ultimate function: growth and reproduction NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 19 Carbohydrate Catabolism AEROBIC ANAEROBIC +O2 Glucose -O2 2 NAD+ Glycolysis 2 ATP 2 NADH 2 Pyruvate Fermentation Pathways NADH CO2 2 NAD+ 2 Acetyl-CoA Organic Acids Ketones Alcohols CO2 TCA Cycle NADH CO2 Oxidative FADH2 ATP Phosphorylation Lots of ATP 20 Fermentation Pyruvate Glucose ATP - Reasons for not completely oxidizing glucose and/or other nutrients (to CO2): - Lack of O2 - Lack of genes for some enzymes of the cellular respiration process - The final electron acceptor is an organic metabolite NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 21 Carbohydrate Catabolism Aerobic vs Anaerobic Aerobic Respiration Fermentation O2 required Yes No Mostly oxidative Substrate-level Phosphorylation phosphorylation phosphorylation Organic metabolite Final e-Acceptor O2 (pyruvate,…) ATP/Glucose Lots (30-32) Few (2) NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 22  Fermentation Products End Products Pathway Example Microbes Commercial Products Acetone, butanol, Acetone-butanol- Clostridium acetobutylicum Commercial solvents, gasoline ethanol, CO2 ethanol alternative Ethanol, CO2 Alcohol Candida, Saccharomyces Beer, bread Formic and lactic acid, Butanediol Klebsiella, Enterobacter Chardonnay wine ethanol, acetoin, 2,3 buanediol; CO2; H2 Butyric acid, CO2, H2 Butyric acid Clostridium butyricum Butter Lactic acid Lactic acid Streptococcus, Sauerkraut, yogurt, cheese Lactobacillus Acetic, formic, lactic, Mixed acid Excherichia, Shigella Vinegar, cosmetics, and succinic acids; pharmaceuticals ethanol, CO2, H2 Acetic acid, propionic Propionic acid Propionibacterium, Swiss cheese acid, CO2 Bifidobacterium CO2 and organic acids → pH drop NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 23 Use of Microbial Metabolism in Medicine - Growing bacteria to study them - Use of unique metabolic capabilities to identify bacteria (diagnosis) - Tests are supposed to be - Simple - Cheap - Fast - Clearly differentiating between positive and negative results NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 24 Use of Microbial Metabolism in Medicine Enzyme 1 Enzyme 2 Enzyme 3 Substrate Product Some substrates Some enzymes Some products can only be are only found in are only found by catabolized by certain bacterial certain bacterial certain bacterial species/strains species/strains species/strains NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 25 Lesson 3: Bacterial Growth and Nutrition NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 26 Microbial Growth Microbial ‘growth’: – Does not refer to size of cells, but to size of the bacterial culture or the number of cells – Under good circumstances, growth is exponential with a generational time as short as 20 minutes NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 27 Microbial Growth and Nutrition Optimal conditions for growth – Chemical: Macro- and micronutrients, oxygen, water – Physical: Temperature, pH, osmolarity NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 28 Microbial Growth and Nutrition: Chemical Requirements Need for nutrients that contain the all elements of life – Nitrogen (N): Needed in higher proportion compared to humans due to the high nitrogen content in peptidoglycan (cell wall) – Oxygen (O): Depends on species Bethood Oxygen I by high concentration Oxygen High butNeed Oxygen (Strept throat) Low NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 29 Microbial Growth and Nutrition : Chemical Requirements Need for nutrients that contain the elements of life – Macronutrients – Micronutrients – Trace elements – Growth factors (vitamins, vitamin-like compounds) ↳ species specific NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 30 Microbial Growth and Nutrition: Physical Factors-pH Growth rate highest at optimum pH of growth than Pathogens Less & 1) protozoa (in cytoplasm) NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 41 What are Plasmids? - Small circular DNA molecules - Replicate independently - Do not contain essential genes for metabolism - Some plasmids are called ‘factors’ - R factor = ‘resistance factor’; encodes for a capability to survive antibiotics and/or heavy metals - F factor = ‘fertility factor’ (see process of conjugation) - ‘Virulence factor’; encodes for a capability of a strain or species to cause disease (e.g., capsule formation, see process of transformation) - ‘Bacteriocin factor’; kill bacterial cells that are competitors NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 42  The Central Dogma of Molecular Biology DNA Replication All cellular organisms have proof-reading functions: → detect and fix mutations and Gene Expression Transcription errors when DNA is replicated Please watch the short animation linked at the end of this unit (from Bauman, Microbiology). Processes in essence the same Translation in prokaryotes and eukaryotes NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 43 Genetic Recombination and Transfer - Genetic recombination: Exchange of segments, typically genes, between two DNA molecules - Recombinants: Cells with DNA molecules that contain one or more new nucleotide sequence - Vertical gene transfer: An organism replicates its genome and provides copies to descendants, including permanent mutations; transfer from one generation to the next generation - Horizontal gene transfer: Donor cell contributes part of its genome to a recipient cell; transfer within generation → important for genetic variability in species that have no sexual reproduction NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 44 Horizontal Gene Transfer in Prokaryotes uptake Of Requires a free DNA bacteriophage infection Please watch the three short animations on these three processes linked at the end of this unit (from Bauman, Microbiology). NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 45 Gene Technology: PCR - PCR = polymerase chain reaction - Groundbreaking technology to amplify DNA (or RNA) in vitro - Areas of application: - Sensitive diagnostic tests (in the early stage of infectious diseases) - Forensic science - Biomedical research - … - Repetitive process consisting of three steps: 1. Denaturing 2. Priming 3. Extension Please watch the three short animations on PCR, linked at the end of this unit (from Bauman, Microbiology). NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 46 Gene Technology: CRISPR - CRISPR = clustered, regularly interspaced, short palindromic repeats - CRISPR-Cas = CRISPR-associated enzymes - CRISPR-Cas9 applied in gene editing (adding or removing of nucleotides) - Areas of application: - Medicine: Treatment of genetic disorders (first successful application in a patient with sickle cell anemia in 2023); treatment of viral infections (e.g., HIV) - Biotechnology: Creation of medicines - Agriculture: Development of disease resistant crops - Epidemiology: Disease of RNA virus disease outbreaks (Ebola, Zika,…) NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 47 Summary – Comparison of pro- and eukaryotic cells – Features of bacterial cells useful for identification and diagnosis – Bacterial metabolism – Bacterial growth and nutrition – Bacterial genetics – Principles of two important gene technology methods (derived from bacterial genetics) NORTHWESTERN HEALTH SCIENCES UNIVERSITY | nwhealth.edu 48