Intro_to_Bacterial_Cells_5116_M2P_2024.pptx

Introduction to Bacterial Cells July 29th, 2024 8:00 AM – 9:00 AM (FL) 10:00 AM – 11:00 AM (NM)...

Introduction to Bacterial Cells July 29th, 2024 8:00 AM – 9:00 AM (FL) 10:00 AM – 11:00 AM (NM) 5116: Molecules to People CDC image # 19267 CDC image # 1792 Marc Benson, Ph.D. Reading Material Office: 317 (NM) Medical Microbiology. Murray et al. Chapter 12 E-mail: [email protected] Link: Phone: (575) 674-2317 http://ezproxy.ad.bcomnm.org/login?url=https://www.clinicalkey Office hours: Open door policy or e-mail.com/dura/browse/bookChapter/3-s2.0-C20180000924 to schedule a time 1  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. Clicker Pre-Lecture Questions 2. Objectives 3. Eukaryotes versus Prokaryotes 4. Gram positive versus Gram negative Bacteria 5. Bacterial Morphology 6. Stain Methods 7. Clicker Post-Lecture Questions 8. Sample Questions (Learning Catalytics) 3  Pre-Lecture Clicker Questions Answer will not immediately be provided at first. The answers to these questions are in the lecture. Attempt to find the answer within the lectures as we go through it. At the end of the lecture, these questions will be asked again. Make it a game and see how well you do from your initial answer!  Lecture Outline 1. Clicker Pre-Lecture Questions 2. Objectives 3. Eukaryotes versus Prokaryotes 4. Gram positive versus Gram negative Bacteria 5. Bacterial Morphology 6. Stain Methods 7. Clicker Post-Lecture Questions 8. Sample Questions (Learning Catalytics) 5  Objectives 1. Compare and contrast prokaryotic and eukaryotic cells, particularly with respect to size, membranes, DNA structure, ribosomes, organelles, cell walls, and cell surface structures 2. Describe the classification of bacteria based on differences in Gram stain, cell wall structure, cell shape, cellular arrangements, as well as differential and selective media for bacterial isolation and identification 3. Explain the use of bacterial stains for identification 4. Define the different types of stains used to visualize bacteria (simple, differential, and structural) 5. Describe the Gram stain procedure and the purpose of each step 6. Compare and contrast the structure of Gram positive and Gram negative cell walls 7. Differentiate between the following bacterial species based on their appearance in a Gram stain: Escherichia coli, Streptococcus pyogenes, Bacillus anthracis, Treponema pallidum, Clostridium tetani, Neisseria gonorrhoeae, Staphylococcus aureus 8. Describe the following external bacterial structures and their roles in pathogenesis: slim layer, capsules, flagella, pili/fimbriae, F- pili, biofilms 9. Describe sporulation and germination, the structure of endospores and the medical significance of spore formation in the two major genera of bacteria which produce endospores (Clostridium, Bacillus) 6  Lecture Outline 1. Clicker Pre-Lecture Questions 2. Objectives 3. Eukaryotes versus Prokaryotes 4. Gram positive versus Gram negative Bacteria 5. Bacterial Morphology 6. Stain Methods 7. Clicker Post-Lecture Questions 8. Sample Questions (Learning Catalytics) 7  Eukaryotes versus Prokaryotes Characteristic Eukaryote Prokaryote Nucleus Present Absent Chromosomes Strands of DNA – generally diploid genome Single, circular DNA (nucleoid)- haploid genome Mitochondria Present Absent Golgi bodies Present Absent Endoplasmic reticulum Present Absent Ribosomes (S = sedimentation coefficient) 80S (60S + 40S) 70S (50S + 30S) Cytoplasmic membrane Contains sterols Does not contain sterols, hopanoids Cell wall Present for fungi and plants; otherwise absent Peptidoglycan Reproduction Sexual and asexual Asexual (binary fission) Respiration Via mitochondria Via cytoplasmic membrane Spore function Reproduction Survival  Eukaryotes versus Prokaryotes Ribosomes Bacterial ribosome Human ribosome Mitochondrial ribosome * * Ribosomal RNA Ribosomal RNA Ribosomal RNA (rRNA) (rRNA) (rRNA) * Relevance to antibiotics 30S subunit targeted by aminoglycosides, tetracyclines 50S subunit targeted by macrolides, clindamycin (Material in green font will be presented in detail elsewhere)  Lecture Outline 1. Clicker Pre-Lecture Questions 2. Objectives 3. Eukaryotes versus Prokaryotes 4. Gram positive versus Gram negative Bacteria 5. Bacterial Morphology 6. Stain Methods 7. Clicker Post-Lecture Questions 8. Sample Questions (Learning Catalytics) 10  Gram Positive versus Gram Negative Bacteria Summary Items in parenthesis are not in all bacteria Murray, Patrick R., PhD, F(AAM), F(IDSA), Medical Microbiology, 12, 114-126.e1, Copyright © 2021, Elsevier Inc. All rights reserved.  Gram Positive versus Gram Negative Bacteria Peptidoglycan (Murein) Peptidoglycan layer Peptidoglycan layer Murray, Patrick R., PhD, F(AAM), F(IDSA), Medical Microbiology, 12, 114-126.e1, Copyright © 2021, Elsevier Inc. All rights reserved.  Gram Positive versus Gram Negative Bacteria Peptidoglycan (Murein) Peptidoglycan, or murein, is the major component of the bacterial cell wall Rigid, mesh-like; composed of linear polysaccharide chains cross-linked by peptides mesh peptidoglycan  Gram Positive versus Gram Negative Bacteria Peptidoglycan (Murein) Peptidoglycan, or murein, is the major component of the bacterial cell wall Rigid, mesh-like; composed of linear polysaccharide chains cross-linked by peptides Polysaccharide chain is polymer of disaccharide: N-acetylglucosamine (NAG) * N-acetylmuramic acid (NAM) Linked by an  -1,4 glycosidic bond * 14 * Lysozyme (Specifics of green font taught elsewhere) Murray, Patrick R., PhD, F(AAM), F(IDSA), Medical Microbiology, 12, 114-126.e1, Copyright © 2021, Elsevier Inc. All rights reserved.  Gram Positive versus Gram Negative Bacteria Peptidoglycan (Murein) tetrapeptide A tetrapeptide (starts out as pentapeptide) is attached to NAM ** Cross-linking mediated by**transpeptidase and carboxypeptidase, a penicillin-binding protein Crosslink is different amongst bacteria *** 15 ** Penicillins, Cephalosporins *** Vancomycin (Specifics of green font taught elsewhere) Murray, Patrick R., PhD, F(AAM), F(IDSA), Medical Microbiology, 12, 114-126.e1, Copyright © 2021, Elsevier Inc. All rights reserved.  Gram Positive versus Gram Negative Bacteria Peptidoglycan (Murein) Thin Thick peptidoglycan peptidoglycan Gram negative Gram positive Purpose: Rigidity of peptidoglycan allows for shape and protection from harsh environmental conditions Elucidating Peptidoglycan Structure: An Analytical Toolset. Porfírio, Sara, Trends in Microbiology, Copyright © 2019  Gram Positive versus Gram Negative Bacteria Membranes Outer Membrane Cytoplasmic membrane Cytoplasmic membrane Murray, Patrick R., PhD, F(AAM), F(IDSA), Medical Microbiology, 12, 114-126.e1, Copyright © 2021, Elsevier Inc. All rights reserved.  Gram Positive versus Gram Negative Bacteria Membranes Gram negative Two Membrane Bilayers Outer membrane Permeability barrier Porin proteins: passage of small hydrophilic * molecules Secretory systems LPS (lipopolysaccharide) * Inner membrane (cell membrane) Energy production, membrane potential, Gram negative transport * Polymyxins Elucidating Peptidoglycan Structure: An Analytical Toolset Porfírio, Sara, Trends in Microbiology, Copyright © 2019 18 Gram Positive versus Gram Negative Bacteria Membranes Gram positive One Membrane Bilayer Cell membrane Energy production, membrane potential, transport Gram positive Elucidating Peptidoglycan Structure: An Analytical Toolset Porfírio, Sara, Trends in Microbiology, Copyright © 2019 19  Gram Positive versus Gram Negative Bacteria Periplasmic Space Periplasmic space Transport proteins, hydrolytic enzymes that break down macromolecules In Gram negative, virulence factors: collagenase protease hyaluronidase β-lactamase Gram negative Gram positive Elucidating Peptidoglycan Structure: An Analytical Toolset Porfírio, Sara, Trends in Microbiology, Copyright © 2019 20  Gram Positive versus Gram Negative Bacteria Lipopolysaccharide (Endotoxin) LPS is comprised of different sugars amongst species but all composed of three parts: O-antigen – Polysaccharide repeat; used for serotyping (E. coli O157: H7) Core polysaccharide – various sugars Lipid A – Embedded in the outer membrane; toxic Gram negative Elucidating Peptidoglycan Structure: An Analytical Toolset 21 Porfírio, Sara, Trends in Microbiology, Copyright © 2019 Murray, Patrick R., PhD, F(AAM), F(IDSA), Medical Microbiology, 12, 114-126.e1, Copyright © 2021, Elsevier Inc. All rights reserved.  Gram Positive versus Gram Negative Bacteria Lipopolysaccharide (Endotoxin) O-antigen – Polysaccharide repeat; used for serotyping (E. coli O157: H7) Core polysaccharide – various sugars Lipid A – Embedded in the outer membrane; toxic Purpose: Lipid A stimulates proinflammatory cytokines: IL-1, IL-6, IL-8, and TNFα Activation of the complement cascade (C3a and C5a) and causes histamine release (inflammation) B cell mitogen, stimulates differentiation and multiplication of B cells Recognized by host Toll-like receptor 4 (TLR4) Gram negative LOS - Lipooligosaccharide - lacks O-antigen (Neisseria spp.) 22 Elucidating Peptidoglycan Structure: An Analytical Toolset Murray, Patrick R., PhD, F(AAM), F(IDSA), Medical Microbiology, 12, 114-126.e1, Copyright © 2021, Elsevier Inc. All rights reserved. Porfírio, Sara, Trends in Microbiology, Copyright © 2019 Gram Positive versus Gram Negative Bacteria (Lipo)teichoic acid Teichoic acid – provides cell wall rigidity Negative charge Antigenic - initiate immune responses Types: Lipoteichoic acid – Attached to the plasma membrane Wall teichoic acid – Attached to peptidoglycan cell wall Gram positive Elucidating Peptidoglycan Structure: An Analytical Toolset Porfírio, Sara, Trends in Microbiology, Copyright © 2019 23  Gram Positive versus Gram Negative Bacteria Capsule Sometimes referred to as glycocalyx or slime layer Capsule Capsule Typically composed of polysaccharides – target of certain vaccines Protein capsules (rare) B. anthracis Encapsulated strains are referred to as smooth (S) and non-encapsulated as rough (R) Major virulence factor – adherence, antiphagocytic 24 Murray, Patrick R., PhD, F(AAM), F(IDSA), Medical Microbiology, 12, 114-126.e1, Copyright © 2021, Elsevier Inc. All rights reserved.  Gram Positive versus Gram Negative Bacteria Pili/Fimbriae Pili/Fimbriae F-pilus Short appendages that protrude Pili Pili from bacteria Function: Adherence Motility: Twitching – grappling hook-like Major virulence factor in certain bacteria F-pilus (sex pilus) – specialized, longer fimbriae used to transfer DNA via conjugation Encoded on the F plasmid Specific to Gram negative bacteria Murray, Patrick R., PhD, F(AAM), F(IDSA), Medical Microbiology, 12, 114-126.e1, Copyright © 2021, Elsevier Inc. All rights reserved.  Gram Positive versus Gram Negative Bacteria Flagella Flagella Motility, propulsion Rigid structure Chemotaxis (directed movement towards attractants or away from repellents) Powered by proton motive force Highly antigenic (TLR-5) Flagellum Used for serotyping (E. coli Flagellum O157: H7) 26 Murray, Patrick R., PhD, F(AAM), F(IDSA), Medical Microbiology, 12, 114-126.e1, Copyright © 2021, Elsevier Inc. All rights reserved.  Pseudomonas spp., Acinetobacter spp. and miscellaneous Gram-negative bacilli. Infectious Diseases. Wisplinghoff, Hilmar; Seifert, Harald. Published January 1, 2010. Pages 1704-1727. © 2010. Magnetotactic Spirillum Rotation of Amphitrichous Flagella Governs Oriented Swimming and Reversals in a ASM Journals. Journal of Bacteriology. Vol. 197, No. 20. Opposite and Coordinated Single polar (monotrichous) Pseudomonas aeruginosa M. magneticum Flagella on each end - Amphitrichous polar Helicobacter pylori Types of Flagella Helicobacter Pylori. Feigin and Cherry's Textbook of Pediatric Infectious Diseases. Multiple polar (lophotrichous) Maspons, Aldo; Gilger, Mark A.. Published January 1, 2014. Pages 1691-1699.e4. © 2014. Gram Positive versus Gram Negative Bacteria Salmonella Typhi Morphology and nature of micro-organisms. Medical Microbiology. Barer, M.R.. Published January 1, 2012. Pages 9-23. © 2012. Multiple everywhere (peritrichous) 27  Gram Positive versus Gram Negative Bacteria Motility/Chemotaxis Run and Tumble Counterclockwise - positive - Run Clockwise - negative - Tumble Monotrichous Bacteria alters frequency of tumbles if attractant/repellant is recognized Flagellum not rotating - bacterium slows to a stop Role of flagella in pathogenesis Peritrichous Motility Adhesion to host cells 28 From Schaechter’s Mechanism of Microbial Disease, Fifth Edition Copyright ©2015 by Wolters Kluwer  Gram Positive versus Gram Negative Bacteria Genetic Elements Chromosome (Nucleoid) Circular Haploid Polycistronic (multiple genes on a single mRNA transcript) Plasmid Plasmid Circular, haploid Plasmid Extrachromosomal element Replicates independent of chromosome Can be transferred to another Chromosome bacteria via F-pilus 29 Murray, Patrick R., PhD, F(AAM), F(IDSA), Medical Microbiology, 12, 114-126.e1, Copyright © 2021, Elsevier Inc. All rights reserved.  Gram Positive versus Gram Negative Bacteria Summary Gram Positive Gram Negative Cell wall thickness Thick Thin Number of membranes 1 2 Peptidoglycan content >50% ~10% Teichoic acid Yes No LPS (lipopolysaccharide) No Yes F-pili No Yes 30 Murray, Patrick R., PhD, F(AAM), F(IDSA), Medical Microbiology, 12, 114-126.e1, Copyright © 2021, Elsevier Inc. All rights reserved.  Lecture Outline 1. Clicker Pre-Lecture Questions 2. Objectives 3. Eukaryotes versus Prokaryotes 4. Gram positive versus Gram negative Bacteria 5. Bacterial Morphology 6. Stain Methods 7. Clicker Post-Lecture Questions 8. Sample Questions (Learning Catalytics) 31  Bacterial Morphology CDC 14759 CDC 14602 CDC 14855 CDC 2296 bacillus coccobacillus coccus diplococcus staphylococcus llus spirochete i to bac CDC 20517 CDC 5324 tre p vibrio s streptococcus CDC 20496 Murray, Patrick R., PhD, F(AAM), F(IDSA), Medical Microbiology, 12, 114-126.e1, Copyright © 2021, Elsevier Inc. All rights reserved. 32  Bacterial Endospores Endospore (or spore) Produced by certain Gram positive bacteria (Bacillus and Clostridium) under unfavorable environmental conditions = survival mechanism Actively growing cells are considered vegetative Endospores are considered dormant Dehydrated multishelled structure Two membranes Two peptidoglycan layers Outer protein coat Contains one full chromosome High amounts of dipicolinic acid and calcium Highly resistant to harsh environmental conditions Heat, radiation, pH, pressure, chemicals Difficult to kill Water begins the process of germination 33 Murray, Patrick R., PhD, F(AAM), F(IDSA), Medical Microbiology, 12, 114-126.e1, Copyright © 2021, Elsevier Inc. All rights reserved.  Bacterial Endospores May be used to help identify bacteria or differentiate between spore-formers Central Bacillus anthracis 2007 Kenneth Todar PhD Subterminal Clostridium difficile www.uphs.upenn.edu/bugdrug Terminal/Swollen sporangium Clostridium tetani CDC 14348 CDC image # 1792 Blue = endospores Red = vegetative bacteria CDC image # 1932 Green = endospores Red = endospores 34  Lecture Outline 1. Clicker Pre-Lecture Questions 2. Objectives 3. Eukaryotes versus Prokaryotes 4. Gram positive versus Gram negative Bacteria 5. Bacterial Morphology 6. Stain Methods 7. Clicker Post-Lecture Questions 8. Sample Questions (Learning Catalytics) 35  Stain Methods Microorganisms are generally too small to visualize directly Simple Stain – usually a single stain to visualize organisms Differential stain – colored stains/dyes that differentiate organisms or structures within organisms Gram stain: Acid-fast stain: Purple = Gram positive Purple = mycolic acids (mycobacteria) Red = Gram negative Green = bacteria without mycolic acids Structural stain – stain that targets bacterial structures (e.g. endospore, flagella, capsule) CDC image # 1932 CDC image # 19267 Capsule Stain Protocols Roxana B. Hughes, Ann C. Smith. American Society for Microbiology © 2016 Endospore stain Flagella stain Capsule stain  Stain Methods Gram Stain Developed by Hans Christian Gram (1853-1938) - Danish microbiologist Modified multiple times over the years to enhance efficiency Gram positive cocci Gram negative bacilli (rod) Medical Bacteriology. Wojewoda, Christina M., Henry's Clinical Diagnosis and Management by Laboratory Methods, Chapter 57, 1130-1165.e4. Copyright © 2022 Elsevier Inc. All rights reserved.  Stain Methods Gram Stain Methodology bacterial suspension https://laboratoryinfo.com/ Heat fix Heat fix the slide – Bacterial suspension smeared and dried on slide. The underside of the slide is passed through a flame to heat fix  Stain Methods Gram Stain Methodology Crystal violet bacterial suspension 1 min https://laboratoryinfo.com/ Heat fix Add primary dye (crystal violet) Heat fix the slide – Bacterial suspension smeared and dried on slide. The underside of the slide is passed through a flame to heat fix Crystal violet solution – A purple primary stain that is positively charged. Enters bacterial cell and complexes with anionic molecules  Stain Methods Gram Stain Methodology bacterial suspension 1 min 1 min https://laboratoryinfo.com/ Heat fix Add primary dye Add iodine (crystal violet) (mordant) Heat fix the slide – Bacterial suspension smeared and dried on slide. The underside of the slide is passed through a flame to heat fix Crystal violet solution – A purple primary stain that is positively charged. Enters bacterial cell and complexes with anionic molecules Gram's iodine solution – Iodine acts as a mordant. It interacts with the crystal violet and forms a larger complex  Stain Methods Gram Stain Methodology bacterial suspension 1 min 1 min 30-45 sec https://laboratoryinfo.com/ Heat fix Add primary dye Add iodine Wash with alcohol (crystal violet) (mordant) (decolorization) Heat fix the slide – Bacterial suspension smeared and dried on slide. The underside of the slide is passed through a flame to heat fix Crystal violet solution – A purple primary stain that is positively charged. Enters bacterial cell and complexes with anionic molecules Gram's iodine solution – Iodine acts as a mordant. It interacts with the crystal violet and forms a larger complex Gram's decolorizer – Alcohol that dehydrates the bacterial cells. The peptidoglycan cell wall shrinks and captures the crystal violet- iodine complex within the cell Gram positive bacteria have a thick cell wall that does not let the crystal violet-iodine solution through (purple bacteria) Gram negative bacteria have a thin cell wall that allows the crystal violet-iodine solution through (unstained)  Stain Methods Gram Stain Methodology bacterial suspension 1 min 1 min 30-45 sec 1 min https://laboratoryinfo.com/ Heat fix Add primary dye Add iodine Wash with alcohol Add secondary dye (crystal violet) (mordant) (decolorization) (counter stain) (safranin) Heat fix the slide – Bacterial suspension smeared and dried on slide. The underside of the slide is passed through a flame to heat fix Crystal violet solution – A purple primary stain that is positively charged. Enters bacterial cell and complexes with anionic molecules Gram's iodine solution – Iodine acts as a mordant. It interacts with the crystal violet and forms a larger complex Gram's decolorizer – Alcohol that dehydrates the bacterial cells. The peptidoglycan cell wall shrinks and captures the crystal violet- iodine complex within the cell Gram positive bacteria have a thick cell wall that does not let the crystal violet-iodine solution through (purple bacteria) Gram negative bacteria have a thin cell wall that allows the crystal violet-iodine solution through (unstained) Safranin - A red/pink counter stain; a reagent that stains whatever the primary stain has not - Gram negative (red bacteria)  Stain Methods Gram Stain Methodology (Summary) bacterial suspension 1 min 1 min 30-45 sec 1 min https://laboratoryinfo.com/ Heat fix Add primary dye Add iodine Wash with alcohol Add secondary dye (crystal violet) (mordant) (decolorization) (counter stain) (safranin) Heat fix the slide – Bacterial suspension smeared and dried on slide. The underside of the slide is passed through a flame to heat fix Crystal violet solution – A purple primary stain that is positively charged. Enters bacterial cell and complexes with anionic molecules Gram's iodine solution – Iodine acts as a mordant. It interacts with the crystal violet and forms a larger complex Gram's decolorizer – Alcohol that dehydrates the bacterial cells. The peptidoglycan cell wall shrinks and captures the crystal violet- iodine complex within the cell Gram positive bacteria have a thick cell wall that does not let the crystal violet-iodine solution through (purple bacteria) Gram negative bacteria have a thin cell wall that allows the crystal violet-iodine solution through (unstained) Safranin - A red/pink counter stain; a reagent that stains whatever the primary stain has not - Gram negative (red bacteria)  Stain Methods Gram Stain Samples Murray, Patrick R., PhD, F(AAM), F(IDSA), Medical Microbiology,, CDC # 2112 CDC #2105 Copyright © 2021, Elsevier Inc. Escherichia coli CDC #1967 Streptococcus pyogenes Bacillus anthracis CDC #12056 CDC #15018 CDC #5144 Treponema pallidum *not Gram stain Clostridium tetani Neisseria gonorrhoeae Staphylococcus aureus  Lecture Outline 1. Clicker Pre-Lecture Questions 2. Objectives 3. Eukaryotes versus Prokaryotes 4. Gram positive versus Gram negative Bacteria 5. Bacterial Morphology 6. Stain Methods 7. Clicker Post-Lecture Questions 8. Sample Questions (Learning Catalytics) 45  Post-Lecture Clicker Questions Check to see how well you do from your initial answers  Lecture Outline 1. Clicker Pre-Lecture Questions 2. Objectives 3. Eukaryotes versus Prokaryotes 4. Gram positive versus Gram negative Bacteria 5. Bacterial Morphology 6. Stain Methods 7. Clicker Post-Lecture Questions 8. Sample Questions (Learning Catalytics) 47 Sample Questions TBD on Learning Catalytics Thank you