Bacterial_Metabolism_and_Growth_5116_M2P_2024_Benson.V12.pptx

Bacterial Metabolism and Growth Course 5116 – Molecules to Murray, Patrick R. Medical M...

Bacterial Metabolism and Growth Course 5116 – Molecules to Murray, Patrick R. Medical Microbiology, 13, 127-141.e1 © 2021, Elsevier People July 31, 2024 Inc. All rights reserved 8 AM - 9 AM (NM) 10 AM - 11 AM (FL) CDC 3906 Marc Benson, Ph.D. Reading Material Office: 317 (NM) Murray et al. Medical Microbiology, 9th ed. E-mail: [email protected] Chapter 4: In Vitro Culture Chapter 13: Bacterial Metabolism Phone: (575) 674-2317 Chapter 14: Mechanisms of Bacterial Pathogenesis Office Hours: By appointment or Link: http://ezproxy.ad.bcomnm.org/login?url=https://www.clinicalkey.com/dura/ stop by office browse/bookChapter/3-s2.0-C20180000924  Copyright Notice All reproduction or use of copyrighted materials shall comply with provisions of applicable law. Individuals are responsible for maintaining copyright compliance in good faith and with each intended use. Please consult BCOM Policy B5041 and the copyright guidelines located on https://bcomnm.org/copyright- guidelines/ for detailed information BCOM prohibits duplication, distribution, or use of copyrighted materials by students, faculty and staff unless a fair use or other exemption applies, or permission has been obtained from the work's rights holder (which may or may not be the author/creator) Infringement of copyright law may be considered a violation of the College’s Code of Professional Conduct. Anyone found liable for civil and/or criminal copyright infringement may be responsible for any monetary damages suffered by the College due to such violation(s) of this policy or related law or regulation 2  Lecture Outline 1. Pre-Lecture Clicker Questions 2. Objectives 3. Bacterial Growth 4. Oxygen Tolerance 5. Summary of Metabolic Processes 6. Biochemical Tests 7. Growth Medium 8. Medium 9. Pathogenesis 10. Post-Lecture Clicker Questions 11. Sample Questions 3 Pre-Lecture Clicker Questions 4  Lecture Outline 1. Pre-Lecture Clicker Questions 2. Objectives 3. Bacterial Growth 4. Oxygen Tolerance 5. Summary of Metabolic Processes 6. Biochemical Tests 7. Growth Medium 8. Pathogenesis 9. Post-Lecture Clicker Questions 10. Sample Questions 5  Objectives Objective Define the types of bacteria that are grouped based on their response to environmental 1: oxygen and list at least two bacterial genera for each. Objective Describe the phases of bacterial growth, including lag phase, exponential phase, stationary 2: growth phase and death phase and be able to draw the bacterial growth curve graphically. Describe the mechanisms (glycolysis, fermentation, aerobic respiration, anaerobic Objective respiration) bacteria may use to convert organic energy storage forms to forms that can be 3: used to do work in the cell (ATP, NADH, FADH). Objective Describe the basis for the following biochemical tests used to identify bacterial species: 4: catalase test, oxidase test, urease test, coagulase test. Objective Explain the basis of biochemical test used for bacterial identification and/selective media 5: used for bacteria identification discussed in class. Objective Explain the basis for the enzymatic tests for bacterial identification discussed in class. 6: 6  Lecture Outline 1. Pre-Lecture Clicker Questions 2. Objectives 3. Bacterial Growth 4. Oxygen Tolerance 5. Summary of Metabolic Processes 6. Biochemical Tests 7. Growth Medium 8. Pathogenesis 9. Post-Lecture Clicker Questions 10. Sample Questions 7  Bacterial Growth Curve Lag phase Exponential phase Stationary phase Decline Murray, Patrick R. Medical Microbiology, 13, 127-141.e1 © 2021, Elsevier Inc. All rights reserved 8  Bacterial Growth Curve Lag phase : Bacteria acclimate to environment (gene regulation, production of metabolites) No significant replication Lag phase Exponential phase Stationary phase Decline Murray, Patrick R. Medical Microbiology, 13, 127-141.e1 © 2021, Elsevier Inc. All rights reserved 9  Bacterial Growth Curve Lag phase: Bacteria acclimate to environment (gene regulation, production of metabolites) No significant replication Exponential phase (log phase): Bacterial growth and division at same rate Doubling time differs amongst bacteria Bacterium Minutes Escherichia coli 20 Staphylococcus aureus 30 Mycobacterium tuberculosis 800 Treponema pallidum 1980 Lag phase Exponential phase Stationary phase Decline Murray, Patrick R. Medical Microbiology, 13, 127-141.e1 © 2021, Elsevier Inc. All rights reserved 10  Bacterial Growth Curve Lag phase: Bacteria acclimate to environment (gene regulation, production of metabolites) No significant replication Exponential phase (log phase): Bacterial growth and division at same rate Doubling time differs amongst bacteria Lag phase Exponential phase Stationary phase Decline Stationary phase: Murray, Patrick R. Medical Microbiology, 13, 127-141.e1 © 2021, Elsevier Inc. All rights reserved Nutrients depleted; metabolic toxins high Cell division equals cell death 11  Bacterial Growth Curve Lag phase: Bacteria acclimate to environment (gene regulation, production of metabolites) No significant replication Exponential phase (log phase): Bacterial growth and division at same rate Doubling time differs amongst bacteria Lag phase Exponential phase Stationary phase Decline Stationary phase: Murray, Patrick R. Medical Microbiology, 13, 127-141.e1 © 2021, Elsevier Inc. All rights reserved Nutrients depleted; metabolic toxins high Cell division equals cell death Decline phase (death phase): No bacterial growth; cells dying 12  Bacterial Growth Curve Lag phase : Bacteria acclimate to environment (gene regulation, production of metabolites) No significant replication Exponential phase (log phase): Bacterial growth and division at same rate Doubling time differs amongst bacteria Bacterium Minutes Escherichia coli 20 Staphylococcus aureus 30 Mycobacterium tuberculosis 800 Treponema pallidum 1980 Stationary phase: Murray, Patrick R. Medical Microbiology, 13, 127-141.e1 © 2021, Elsevier Inc. All rights reserved Nutrients depleted; metabolic toxins high Cell division equals cell death Decline phase (death phase): No bacterial growth; cells dying 13  Lecture Outline 1. Pre-Lecture Clicker Questions 2. Objectives 3. Bacterial Growth 4. Oxygen Tolerance 5. Summary of Metabolic Processes 6. Biochemical Tests 7. Growth Medium 8. Pathogenesis 9. Post-Lecture Clicker Questions 10. Sample Questions 14  Oxygen Tolerance Bacteria tolerate certain concentrations of O2 Tool to grow, differentiate, and identify bacteria 21% O2 Bacteria have a preference for anatomical sites 0.04% CO2 based on O2 preferences Obligate Facultative Obligate anaerobe Micro- aerophile Aerotolerant anaerobe 15% O2 aerobe anaerobe 5% CO2 Bacterial Growth in Reducing Medium (Medium with a gradient of O2) Top has more oxygen Bottom has none Oxygen is toxic Prefers Requires 1% O2 O2 Requirements Oxygen Required oxygen but can grow - certain percentage of Oxygen does not matter 5-29% CO2 oxygen without - ↓ SOD (superoxide + + Clostridium + dismutase) botulinum + + ↓ - Catalase + = present Campylobacter - = absent Bordetella Escherichia jejuni Streptococcus Example pertussis coli pyogenes ↓ = present at low levels 15  Other Classes of Bacteria Requirements Type/Class Example High CO2 Capnophile Campylobacter spp. High Salt Halophile Vibrio/Staph. aureus* Optimal growth at ~ 20-45°C Mesophile Most pathogens Replicate inside host cells Obligate intracellular Chlamydia trachomatis Prefer to replicate inside host cells but can replicate outside host cells Facultative intracellular Listeria monocytogenes Replicate outside host cells Extracellular Vibrio cholerae 16 * Not true halophiles but moderately halophilic  Lecture Outline 1. Pre-Lecture Clicker Questions 2. Objectives 3. Bacterial Growth 4. Oxygen Tolerance 5. Summary of Metabolic Processes 6. Biochemical Tests 7. Growth Medium 8. Pathogenesis 9. Post-Lecture Clicker Questions 10. Sample Questions 17  Bacterial Metabolism Overview Requirements for growth: a source of carbon and nitrogen, an energy source, water Energy is derived from the controlled breakdown of various organic substrates (carbohydrates, lipids, and proteins) catabolism Cellular respiration (aerobic and anaerobic) Glycolysis, pentose phosphate, Entner-Doudoroff Tricarboxylic acid (TCA) cycle Electron transport chain (ETC) Fermentation Energy produced may then be used in the synthesis of cellular constituents (cell walls, proteins, fatty acids, nucleic acids) anabolism Bacteria utilize different carbon sources and produce different end products, enabling for the differentiation and identification of specific bacteria 18  Do not memorize Bacterial Metabolism Glycolysis, pentose phosphate, Entner-Doudoroff Summary Pentose phosphate Glycolysis Entner-Doudoroff (Hexose monophosphate shunt) (Embden-Meyerhof) Prokaryotes only 2 NADPH 2 NADH 1 NADPH Nucleic acid precursors 2 ATP production 1 ATP production Aromatic amino acid precursors Eg. Pseudomonas spp. 2 steps: Oxidative and nonoxidative Neisseria spp. (Gram + rare Enterococcus faecalis) 2-keto-3-deoxy-6- Ribulose 5- phosphogluconate Glucose phosphate (KDPG) Nucleotide Ribose 5- Xylulose-5- Synthesis phosphate Phosphate Glyceraldehyde- Fructose-6- Pyruvic Acid 3-phosphate Phosphate (GP) Pyruvic Acid 19  Bacterial Metabolism Fermentation A process where organic molecule acts as an electron donor and one or more of its organic products act as a final election acceptor, resulting in energy production - anaerobic Major tool utilized to differentiate and identify bacteria Salmonella spp. Sugar (not pink) E. coli (pink) Pyruvic Acid (Lactose fermentation) MacConkey Agar Gasses Tests for the ability to ferment lactose Lactic acid Butyric acid Propionic acid Ethanol (CO2, H2) 20  Do not memorize Bacterial Metabolism H+ H+ Cytochrome H+ Intermembrane Electron Transport Chains Space C Complex Complex Complex Complex The ETC is localized to the plasma I II III IV ATP e- membrane (plasma membrane) of Matrix NADH NAD+ Succinate Fumarate O2 H2O Synthase ATP bacterial cells Mitochondrial H+ ADP Often differ from mitochondrial ETC Periplasmic H+ High Oxygen H+ Low Oxygen H+ Electrons may enter ETC at Space multiple points and exit through e- multiple terminal oxidases Cytoplasm ATP Synthase Cytochrome bo NADH NAD + Cytochrome bd complex complex ATP Shorter, so less energy production H2O O2 O2 H2 O ADP H+ E. coli More complex, with aerobic and e- H+ anaerobic pathways Periplasmic H+ H+ Cytochrome C H+ NO3 - NO 2 - NO N2O N2 Space Tool utilized to differentiate and Complex I Complex III Complex IV Denitrification Enzymes identify bacteria Cytoplasm e- ATP Synthase Oxidase test NADH NAD + O2 H2O aerobic pathway anaerobic pathway ATP Nitrate Reduction test Pseudomonas aeruginosa H+ ADP 2  Lecture Outline 1. Pre-Lecture Clicker Questions 2. Objectives 3. Bacterial Growth 4. Oxygen Tolerance 5. Summary of Metabolic Processes 6. Biochemical Tests 7. Growth Medium 8. Pathogenesis 9. Post-Lecture Clicker Questions 10. Sample Questions 22  Biochemical Tests Overview Purpose: Differentiation of bacteria based on varying biochemical pathways Most biochemical tests will be discussed in future lectures Examples used today: Catalase test Oxidase test Coagulase test Urease test  Biochemical Properties The catalase enzyme is used by bacteria and Biochemical Tests Catalase Test eukaryotes to eliminate oxidative damage when H2O2 is produced from molecular O2 H2O2 and other reactive oxygen species kill bacteria Immune Phagolysosome cell Oxidative Burst 2O2 Oxygen ↓ 2O2- Superoxide radical ↓ Hydrogen H2O2 Peroxide Nucle ↓ Hypochlorous us HOCl acid  Biochemical Properties The catalase enzyme is used by bacteria and Biochemical Tests Catalase Test eukaryotes to eliminate oxidative damage when H2O2 is produced from molecular O2 Catalase H2O2 and other reactive oxygen species kill bacteria H2O2(aq) → 2H2O(aq) and O2(g) Reiner K. 2010. Catalase Test. American Society for Microbiology, Washington, DC. www.asm.org. Accessed 22 July 2024. The catalase enzyme is used by bacteria to Bubbles inactivate one pathway of the antioxidant defense response of the immune cell. Used to differentiate bacteria Immune Phagolysosome cell Oxidative Burst 2O2 Oxygen ↓ 2O2- Superoxide radical ↓ Procedure Catalase H2O2 X Hydrogen Peroxide 1. Hydrogen peroxide added to each slide 2. Inoculum added to hydrogen peroxide ↓ Nucle us 2H2O + O2 X HOCl X Hypochlorous acid 3. If gas bubbles are present, bacteria is catalase positive  Biochemical Tests Oxidase Test Biochemical Properties Cytochrome oxidase Some bacteria use oxygen as a final electron acceptor in their electron transport chain - reduce oxygen to form water tetramethyl-ρ-phenylenediamine Periplasmic dihydrochloride space (no color change -) Cytochrome Plasma oxidase Membrane complex indophenol Cytoplasm production Bacteria O2 H H2+O (purple +)  Biochemical Tests Oxidase Test Biochemical Properties Cytochrome oxidase Procedure Some bacteria use oxygen as a final electron Filter paper is saturated with the oxidase reagent (tetramethyl-ρ-phenylenediamine dihydrochloride) acceptor in their electron transport chain - Bacteria added to filter paper reduce oxygen to form water Purple color before 30 seconds = oxidase positive The enzyme is not present in all bacteria and (indophenol production) thus can be used to differentiate bacteria in the clinical lab tetramethyl-ρ-phenylenediamine Periplasmic dihydrochloride space (no color change -) Cytochrome Plasma oxidase Membrane complex indophenol Cytoplasm production Bacteria O2 H H2+O (purple +)  Biochemical Tests Coagulase Coagulase Biochemical Properties - Negative The coagulase enzyme interacts Staphylococci with coagulase-reacting factor in (CoNS) serum and subsequently converts Coagulase soluble fibrinogen to insoluble fibrin, which causes clumping. + clumping Staphylococcus During infection, fibrin may aureus surround bacterium and protect from phagocytosis. Differentiates between Procedure Staphylococcus aureus and the 1. Serum added to sample tubes coagulase-negative Staphylococci 2. Each inoculated with pure bacteria sample 3. Incubate for 37°C 4. Invert tube to evaluate consistency (clumping) of serum 28 Sturm. 2010. Catalase Test. American Society for Microbiology, Washington,  Biochemical Tests Urease Test Urease Urea + 2H2O → 2NH3 + CO2 + H2O ammonia Proteus + - E. coli Biochemical Properties Urease is produced by certain bacteria to modulate pH (intracellular and extracellular) Procedure Bacteria are added to broth medium containing urea Plays a role in amino acid and nucleic acid synthesis Urease converts urea to ammonia and CO2 Used to differentiate bacteria pH indicator turns pink in basic conditions (ammonia), indicating presence of urease 29  Lecture Outline 1. Pre-Lecture Clicker Questions 2. Objectives 3. Bacterial Growth 4. Oxygen Tolerance 5. Summary of Metabolic Processes 6. Biochemical Tests 7. Growth Medium 8. Pathogenesis 9. Post-Lecture Clicker Questions 10. Sample Questions 30  Culture Medium Overview Purpose: Culture medium is used to: A) Grow microbes B) Select for the growth of specific microorganisms C) Differentiate amongst microorganisms Culture media – substance in which an organism grows; may include additional material to aid in growth, selection, and differentiation of microbes Types: Nonselective – supports growth of many organisms Selective – supports growth of specific organisms usually by limiting growth of others Differential – used to differentiate organisms in a mixture Specialized – ingredients support the growth of specific organisms  Culture Medium Overview Other definitions to be familiar with: Inoculum – introduction of a microorganism to medium Culture – growth of microorganisms Chemically-defined – every ingredient is known Enriched – addition of extra nutrients (blood, etc,) Fastidious organism – difficult to grow in nonselective culture media; requires specific ingredients Reducing – medium with low oxygen (growth of anaerobes) 32  Culture Medium Nonselective Agar Nonselective agar - supports growth of many organisms Trypticase soy broth/agar – Digested soybean meal and casein; full of nutrients Nutrient broth/agar – peptone and beef extracts; full of nutrients Blood agar – trypticase soy with sheep blood (enriched) Chocolate agar – trypticase soy with lysed sheep blood (enriched) Thioglycolate broth – Sodium thioglycolate added to promote a reducing (anaerobic) environment; used to grow anaerobic bacteria and can be used to differentiate oxygen usage Sabouraud dextrose agar – growth of fungi Trypticase soy agar Blood agar Chocolate agar Thioglycolate broth Sabouraud agar CDC Image: 12464 CDC Image: 12448 CDC Image: 11980 CDC Image: 1046  Culture Medium - Selective/Differential Agar Selective – supports growth of specific organisms usually by limiting the growth of others Differential – differentiation of organisms in a mixture  Culture Medium - Selective/Differential Agar Selective – supports growth of specific organisms usually by limiting the growth of others Differential – differentiation of organisms in a mixture MacConkey agar – selective for Gram-negative bacteria (bile salts and crystal violet prevent Gram positive bacterial growth; differential for lactose-fermentation (pH indicator that turns pink when pH decreases during fermentation of lactose) Mannitol salt agar – selective for Gram-positive Staphylococci (high salt); differential for S. aureus (mannitol fermentation - yellow) Blood agar – differential for erythrocyte lysis Lowenstein-Jensen agar – selective for Mycobacteria spp. Eosin-methylene blue agar – selective for Gram-negative bacteria; differential for lactose-fermentation (pink) Hektoen enteric agar – selective for Gram-negative bacterial; differential for hydrogen sulfide production (black) and lactose/sucrose/salicin fermentation (yellow) MacConkey agar Mannitol salt agar Lowenstein-Jensen agar Eosin-methylene blue agar Hektoen enteric agar Salmonella spp. E. coli P. aeruginosa Klebsiella Enterobacter E. coli CDC Image 2904  Culture Medium Specialized Specialized – ingredients support the growth of specific organisms Thayer martin – Neisseria gonorrhoeae Buffered charcoal yeast extract (BCYE) agar – Legionella Thiosulfate citrate bile salts sucrose (TCBS) agar – Vibrio spp. Regan-Lowe agar and Bordet-Gengou agar – Bordetella pertussis And others as presented in future lectures BCYE agar TCBS agar Regan-Lowe agar CDC Image 6549 CDC 3906 https://www.thermofisher.com/order/catalog/product/R01298  Lecture Outline 1. Pre-Lecture Clicker Questions 2. Objectives 3. Bacterial Growth 4. Oxygen Tolerance 5. Summary of Metabolic Processes 6. Biochemical Tests 7. Growth Medium 8. Pathogenesis 9. Post-Lecture Clicker Questions 10. Sample Questions 37  Pathogenesis Definitions Pathogen Organism that causes disease Opportunistic pathogen Organism that causes disease in immunocompromised individuals Infectious disease The disease caused by an organism Virulence Ability of organism to cause disease (severity of disease) Virulence factor Trait that enhances disease Contagious disease Transmissibility of disease from individual to individual Virulence factors/mechanisms: Antigenic variation Nucleases Adhesins Proteases (IgA protease, elastase) Biofilm Siderophores Chemotaxis Capsule Toxins Lipases Motility 38  Pathogenesis Do not memorize specifics General Characteristics Entry into host Adherence Adhesins Colonization, invasion, growth Toxins and effector proteins Siderophores Secretion systems Motility Endospore Immune evasion Biofilm Toxins and effector proteins Secretion systems 39  Pathogenesis Toxins Type General description Example Endotoxin (LPS) Glycolipid that elicits a robust immune response – inflammation Gram negative bacteria Exotoxin A protein that is released from the bacteria cell - AB toxin A domain is catalytic; B domain is receptor binding Cholera toxin - Pore forming toxin Forms holes/channels in host membrane Listeriolysin O Toxic shock syndrome - Superantigen Nonspecifically activates T cells to produce a robust immune response toxin Intoxication versus infection Intoxication – a toxin is directly causing the disease (Staphylococcal enterotoxin) Infection – the pathogen is causing the disease 40  Pathogenesis Bacterial Secretion Do not memorize table, just understand orange Host Cell Membrane Extracellular Type I Type II Type III Type IV Type V Type VI Type VII Outer Membrane Mycolic acid Periplasmic space Plasma Membrane Intracellular Type I Type II Type III Type IV Type V Type VI Type VII Gram Type Negative Negative Negative Negative Negative Negative Positive Two step: One step: Two step: cytoplasm One step: Cytoplasm to One step: Cytoplasm to host tothe periplasm, then One step: Two step: Cytoplasm to periplasm Process Cytoplasm to periplasm to Cytoplasm to or bacterial protein itself Cytoplasm to host or and through outside outside host cytoplasm cytoplasm forms a channel in bacterial cytoplasm peptidoglycan/mycolic acid outer membrane Yersinia, Neisseria Proteases, gonorrohoeae (gene Substrates lipases, Cholera Toxin, Shigella, acquisition), IgA protease in Hydrolase adhesins Exotoxin A Pseudomonas Helicobacter Neisseria toxins effectors Injectisome Injectisome (protein Autotransporter Notes ABC Sec/Tat (unfolded and nucleotides) - system/ Spear gun, Bacterial Secretion transporters components protein) related to Sec/Tat communication conjugation system components 41  Pathogenesis Immune Evasion Complement evasion (Staphylococcus aureus) Antibody evasion/destruction – (Protein A - Staphylococcus aureus / IgA protease - Neisseria spp. / antigenic variation) Phagocytosis evasion Resist reactive oxygen species (Catalase and SOD) Prevent phagosome from fusing to lysosome (Mycobacterium tuberculosis) Escape from phagosome (Listeria monocytogenes) Prevent phagocytosis (capsule) Kill phagocytes Intracellular behavior and cell-to-cell spread (Shigella dysenteriae) Biofilm production (Streptococcus mutans, Pseudomonas aeruginosa) pH survival (Helicobacter pylori) 42  Lecture Outline 1. Pre-Lecture Clicker Questions 2. Objectives 3. Bacterial Growth 4. Oxygen Tolerance 5. Summary of Metabolic Processes 6. Biochemical Tests 7. Growth Medium 8. Pathogenesis 9. Post-Lecture Clicker Questions 10. Sample Questions – Learning Catalytics 43 Post-Lecture Clicker Questions 44 Practice Questions will be on Learning Catalytics 45 Thank you 46