جامعة لبنانية microbiology 2020-2021

Prof. Ali CHOKR B2230 2020-2021 MICROBIOLOGY 1 2  ‫حق المعلم‬ ‫حق سائسك بالعلم فالتعظيم له‪ ،‬والتوقير لمجلسه‪ ،‬وحسن االستماع إليه‪،‬‬ ‫وأ ّما ّّ‬ ‫واإلقبال عليه‪ ،‬والمعونة له على نفسك في ما ال غنى بك عنه من الع...

Prof. Ali CHOKR B2230 2020-2021 MICROBIOLOGY 1 2  ‫حق المعلم‬ ‫حق سائسك بالعلم فالتعظيم له‪ ،‬والتوقير لمجلسه‪ ،‬وحسن االستماع إليه‪،‬‬ ‫وأ ّما ّّ‬ ‫واإلقبال عليه‪ ،‬والمعونة له على نفسك في ما ال غنى بك عنه من العلم‪ ،‬بأنّ‬ ‫تفرغ له عقلك‪ ،‬وتحضره فهمك‪ ،‬وتزكي له قلبك‪ ،‬وتجلي له بصرك‪ :‬بترك‬ ‫ّ‬ ‫اللّذات‪ ،‬ونقص الشهوات‪.‬وأنّ تعلم أنّك ـ في ما ألقى إليك ـ رسولّه إلى من لَ ِق َيك‬ ‫فلز َمك حسنّ التأدية عنه إليهم‪ ،‬وال تخنهّ في تأدية رسالته‪،‬‬ ‫من أهل الجهل‪ِ ،‬‬ ‫التجيب أحداّ يسأله عن‬ ‫َّ‬ ‫القيام بها عنه إذا تقلّدتها وأن الترفع عليه صوتك‪ ،‬وأن‬ ‫تغتاب عنده‬‫َّ‬ ‫ث في مجلسه أحدا‪ ،‬وال‬ ‫يكون هو الذي يجيب‪ ،‬والتح ّد َّ‬ ‫َّ‬ ‫شيء حتّى‬ ‫أحدا‪ ،‬وأن تدف َّع عنه إذا ذكر عندك بسوء‪ ،‬وأنّ تستر عيوبه‪ ،‬وتظهر مناقبه‪،‬‬ ‫عدوا‪ ،‬وال تعادي له وليّاّ‪.‬‬ ‫والتجالس له ّ‬ ‫ل بأنّك قصدتَهّ وتعلّ َّ‬ ‫مت علمه هلل ج ّلّ‬ ‫شهدت مالئكةّ هللا ّّ‬ ‫عز وج ّّ‬ ‫ّ‬ ‫فعلت ذلك‬ ‫َّ‬ ‫فإذا‬ ‫ّّ‬ ‫وعز اسمه‪ ،‬ال للناس‪.‬‬ ‫‪3‬‬  ‫حق المتعلم‬ ‫أن هللا قد جعلك قيّماّ لهم في ما آتاك من العلم‪،‬‬ ‫حق رعيّـتك بالعلم فأن تعلم ّّ‬ ‫وأ ّما ّّ‬ ‫ووالّك من خزانة الحكمة‪.‬فإنّ أحسنت في تعليم الناس ما والّك هللا من ذلك‪،‬‬ ‫وقمت لهم مقام الخازن الشفيق الناصح لمواله‬ ‫َّ‬ ‫ولم تخرق بهم ولم تضجر عليهم‬ ‫في عبيده‪ ،‬الصابر المحتسب الذي إذا رأى ذا حاجة أخرج له من األموال التي‬ ‫كنت له‬ ‫َّ‬ ‫الّ‬ ‫وكنت لذلك آمالّ معتقداّ‪.‬وإ ّ‬ ‫َّ‬ ‫في يديه زادك هللا من فضله كنت راشدا‪،‬‬ ‫ل أنّ‬‫عزوج ّّ‬‫متعرضا ‪.‬كان حقّاّ على هللا ّ‬ ‫ّ‬ ‫خائنا‪ ،‬ولخلقه ظالما‪ ،‬ولسلبه و ِغ ِ ّره‬ ‫ط من القلوب محلّك‪.‬‬ ‫يسلبَك العلم‪ ،‬وبها َءه‪ ،‬ويسق َّ‬ ‫‪4‬‬  References 1. Bouchet, Ph, Guignard, J-L, Madulo-Leblond, G et Régli, P. 1989. Mycologie générale et médicale. 2. Christopher Herbert Collins, Patricia M. Lyne, John M. Grange. 2004. Microbiological Methods. 3. Elmer W. Koneman. 2005. Koneman's Color Atlas and Textbook of Diagnostic Microbiology. 4. Garrity, Bell. 2000. Bergey’s Manual of Systematic Bacteriology. 5. Gerard J. Tortora, Berdell R. Funke, Christine L. Case. 2016. Microbiology: An Introduction. 6. Greenwood, D, Slack, R and Peutherer, J. 1992. Medical microbiology. 7. Jacquelyn G. Black. 2008. Microbiology: Principles and Explorations. 8. Jeffrey C. Pommerville, I. Edward Alcamo. 2015. Alcamo's fundamentals of microbiology. 9. Michael J. Leboffe, Burton E. Pierce. 2011. A Photographic Atlas for the Microbiology Laboratory. 10. Michael T. Madigan, John M. Martinko, David Stahl, David P. Clark. 2012. Brock Biology of Microorganisms. 11. Seeley, H Jr. Vandermark, P and Lee, J. 1995. Microbes in action. 12. Seeley, H Jr. Vandermark, P and Lee, J. 1995. Selected exercises from Microbes in action. 5  Useful websites For books: www.pdfdrive.com For articles: www.ncbi.nlm.nih.gov/pubmed www.sci-hub. TW 6  CONTENTS Chapter Number of hours 1-Introduction To Microbiology 5 2-Bacterial Metabolism 2.5 3-Bacterial Growth 7.5 4-The Control Of Microbial Growth 8 5-Host-Microbe Interactions 4 6-Viruses 3 Total 30 7  INTRODUCTION TO MICROBIOLOGY (Diversity of microorganisms) 1-Definition : study of the microorganisms 2-Basic groups of micro-organisms : a. Bacteria b. Fungi : yeasts and molds c. Viruses d. Protozoa e. Algae Staphylococcus aureus Bacteria: unicellular, microscopic, prokaryotic, reproduce by binary fission. Microscopy 1-Light→2D 2-Electron: 2.a-Scanning electron microscopy (SEM)→ 3D (shape) 8 2.b-Transmission electron microscopy (TEM)→ 2D (details) Fungi: yeasts and molds Molds: filamentous, eukaryotic , Yeasts: unicellular, microscopic, eukaryotic, reproduce by producing asexual Reproduce by budding. reproductive spores. Conidiospore Sterigmata Conidiophore Viruses: submicroscopic, acellular, genome (DNA or RNA), obligate parasites Protozoa: unicellular, microscopic, eukaryotic, cell wall free. They live as free entities or parasites Algae: eukaryotic, photosynthetic, Abundant in fresh and salt water. 9  THE PROKARYOTIC CELL: BACTERIA 1-SIZES, SHAPES, AND ARRANGEMENTS OF BACTERIA 2-Bacteria are : a. procaryotic b. single-celled, microscopic organisms c. smaller than eukaryotic cells d. very complex despite their small size 10 Most common shapes: coccus, rod or bacillus, and spiral  THE COCCUS Spherical or oval Several arrangements (planes of division) 0.5-1.0 µm in diameter Neisseria gonorrhoeae 11  Sexually transmitted diseases and their agents 1-Syphilis----Treponema pallidum 2-Cervix cancer --- HpV 3-Herpes-----HSV 4-Hepatitis-----(HBV, HCV, HDV) 5-AIDS------HIV 6-Gonorrhea-----Neisseria gonorrheae 7-Chlamydia---Chamydia trachomatis 12  Typical Bacterial Arrangements streptococci sarcina staphylococci 13 14 15 16 17 18 19 20 21 22  THE ROD or BACILLUS Rod-shaped (0.5-1.0 µm wide by 1.0-4.0 µm long) Arrangements: bacillus, diplobacillus, streptobacillus, coccobacillus or palissades bacillus: single bacilli A coccobacillus: oval and similar to a coccus Gram Stain of Escherichia coli 23 24  THE SPIRAL Vibrio, spirillum, spirochete 1 µm to over 100 µm in length. vibrio: a curved or comma-shaped rod spirillum: a thick, rigid spiral spirochete: a thin, flexible spiral 25 Leptospira interrogans (SEM) 26  Other shapes Trichome-forming, filamentous, spindle-shaped, square, pleomorphic, star-shaped. Filamentous Bacterium Trichome(cha3r)-Forming Bacterium 27 PLEOMORPHIC  Archaeobacteria Eubacteria (archae) Nuclear membrane No No Cell wall Yes Yes Peptidoglycan in cell wall No Yes extreme conditions Yes No Unusual metabolic processes Yes No 28 CELL STRUCTURE OF THE DOMAIN BACTERIA 29 CELL STRUCTURE OF THE DOMAIN BACTERIA 30 Anatomy of a bacterium cell (longitudinal section)  CELL STRUCTURE OF THE DOMAIN BACTERIA 1-The cytoplasmic membrane 2-The bacterial cell wall : a. the Gram-Positive cell wall b. the Gram-Negative cell wall c. the Acid-Fast cell wall 3-Structures located within the cytoplasm : a. cytoplasm b. nucleoid c. plasmids and transposons d. ribosomes e. endospores f. inclusions bodies 4-Structures located outside the cytoplasm : a. glycocalyx (capsule) and S-layer b. flagella 31 c. pili  THE CYTOPLASMIC MEMBRANE 7 nm thick Composition: phospholipid + protein molecules 2 dark bands separated by a light band With the exception of the mycoplasmas (no cw) prokaryotic membranes lack sterols. 32  FUNCTIONS OF THE CYTOPLASMIC MEMBRANE a. Osmotic or permeability barrier (passive and facilitated diffusion) b. Location of transport systems for specific solutes (active transport) c. Energy generating functions (location of ETS) d. Synthesis of membrane lipids e. Synthesis of murein f. Assembly and secretion of extracytoplasmic proteins and waste removal g. Coordination of DNA replication and segregation with septum formation, cell division and endospores formation h. Chemotaxis i. Location of specialized enzyme system (DNA polymerase, permease, …) and mesosome 33  Prof. Ali CHOKR B2205 2019-2020 MICROBIOLOGY 34  CELL STRUCTURE OF THE DOMAIN BACTERIA 1-The cytoplasmic membrane 2-The bacterial cell wall : a. the Gram-Positive cell wall b. the Gram-Negative cell wall c. the Acid-Fast cell wall 3-Structures located within the cytoplasm : a. cytoplasm b. nucleoid c. plasmids and transposons d. ribosomes e. endospores f. inclusions bodies 4-Structures located outside the cytoplasm : a. glycocalyx (capsule) and S-layer b. flagella 35 c. pili  Cell Wall & Peptidoglycan -Mycoplasmas = No CW -CW of eubacteria contains peptidoglycan (murein)(exception of the Chlamydias) -Peptidoglycan = {NAG—NAM—pentapeptide}n The Peptidoglycan Monomer of a Gram-negative The Peptidoglycan Monomer of a Gram-positive bacteria (Escherichia coli) bacteria (Staphylococcus aureus) 36 37  Roles 1-Characteristic shape 2-Osmoresistance (peptidoglycan) 3-Point of anchorage for flagella 4-PAMP (peptidoglycan) 5-Activate the alternative complement pathway and the lectin pathway (peptidoglycan) 6-Clinically : -virulence factor -site of action of some antibiotics 38  The Gram-Positive cell wall -Broad, dense wall (20-80 nm) -Composition: numerous rows of peptidoglycan (60 to 90%), lipoteichoic acid (LTA), cell wall teichoic acid (cw-TA) and surface proteins -TA = {glycerol—phosphate—ribitol}n -LTA = TA + lipids Electron Micrograph of a Gram-Positive cell wall Structure of a Gram-Positive cell wall 39 40  Roles of teichoic acid 1-Make the cell wall stronger 2-Bind and regulate the movement of cations into and out of the cell 3-Storing phosphorus 4-PAMP 5-Activate the alternative complement pathway and the lectin pathway 6-Role in cell growth 7-Site of fixation for some viruses 8-Antigenic properties 41  The Gram-Negative cell wall -Multilayered: 1-thin inner wall (2-3 nm) of peptidoglycan (10 to 20%) (1 or 2 rows) 2-OM (7 nm) consisting of: phospholipids, LPS, lipoproteins & surface proteins LPS = lipid A + O polysaccharide Electron Micrograph of a Gram-Negative cell wall Structure of a Gram-Negative cell wall 42 43  The lipopolysaccharides (LPS) -LPS = lipid A (endotoxin) + core oligosaccharide + [O polysaccharide (antigen)]n A schematic diagram of a lipopolysaccharide molecule LPS 44 45  Roles of the OM in Gram negative CW 1-Toxic properties (lipid A is an endotoxin) 2-semipermeability (nonspecific and specific channel proteins) 3-Add strength to the outer membrane 4-PAMP (LPS) 5-Activate the alternative complement pathway and the lectin pathway (LPS) 6-Selectivity (compounds retention or prevention) 7-Site of fixation for some viruses 8-Antigenic properties (O-polysaccharide) 9-Evasion of phagocytosis (strong negative charge of OM) 46  Roles of the periplasm 1-Enzymes for nutrient breakdown 2-Binding proteins 3-Enzymes for nutrient & waste movement facilitation 47 48  Damage to the cell wall (lysozyme, protoplast, & spheroplast) Lysozyme Metabolism + Division – Reversion – Gram + Protoplast Color of protoplast And in Gram stain??? Lysozyme Metabolism + Division – Reversion + Why reversion Gram - Spheroplast is possible? 49  The Acid-Fast cell wall -Composition : peptidoglycan, arabinogalactan, mycolic acids (60%), lipoarabinomannan, phosphatidyinositol mannosides (PIM), polypeptides, glycolipids and peptidoglycolipids Structure of a Acid-Fast cell wall 50 51  Gram stain Smear & Heat fixation 52 53 The explanation of the gram staining in gram-positive bacteria 54 55 56 57 The explanation of the gram staining in gram-negative bacteria 58 59 60 61  CELL STRUCTURE OF THE DOMAIN BACTERIA 1-The cytoplasmic membrane 2-The bacterial cell wall : a. the Gram-Positive cell wall b. the Gram-Negative cell wall c. the Acid-Fast cell wall 3-Structures located within the cytoplasm : a. cytoplasm b. nucleoid c. plasmids and transposons d. ribosomes e. endospores f. inclusions bodies 4-Structures located outside the cytoplasm : a. glycocalyx (capsule) and S-layer b. flagella 62 c. pili  THE CYTOPLASM Composition: water (80%) Contains DNA, RNA, enzymes, amino acids, carbohydrates, lipids, inorganic ions… Cytosol Inclusion bodies (in some bacteria) Functions: the site of most bacterial metabolism. 63  THE NUCLEOID Genome = nucleoid = sum of an organism's genetic material (DNA) [orchestra of the cell] Nucleoid = one long single circular molecule of double stranded, helical, supercoiled DNA NO: NM, nucleoli, mitosis, meiosis Bacteria only reproduce asexually DNA → RNA → (r, tRNA) or proteins (enzymes, …) 64  PLASMIDS Small nonchromosomal circular DNA molecules 5 and 100 genes bacteria may lose or gain them without harm Synthesis of a few proteins, antibiotic resistance, tolerance to toxic metals, toxins, etc Conjugation 65  TRANSPOSONS Transposons = transposable elements or jumping genes Small pieces of DNA that encode enzymes that move DNA from one location to another. Found on nucleoid or in plasmids Antibiotic resistance or other traits Transmission of antibiotic resistance among a population of bacteria?? 66  RIBOSOMES rRNA + proteins Two subunits with densities of 50S and 30S → 70S ribosome about 25nm in diameter A typical bacterium may have as many as 15,000 ribosomes. Workbench for protein synthesis 67  ENDOSPORES Dormant forms produced by certain bacterial genera (Bacillus, Clostridium, Desulfotomaculum, etc) It is not a reproductive structure but rather a resistant, dormant survival form of the organism Resistant to high temperatures (including boiling), most disinfectants, low energy radiation, drying, antibiotics, etc 68  A-A vegetative bacterium about to enter the endospore cycle. A B-A spore septum forms as the cytoplasmic membrane invaginates. L C-Each nucleoid becomes surrounded by its own cytoplasmic membrane. D-The cytoplasmic membrane surrounds the isolated nucleoid, cytoplasm, and membrane from the previous step forming a forespore. E-The forespore is completed and the other molecule of DNA is eventually B degraded. K F-A thick protective layer of peptidoglycan called the cortex is synthesized between the inner and outer forespore membranes. Calcium dipicolinate is synthesized and incorporated in the forming endospore. C G-A second impermeable proteinaceous protective layer called the spore coat is then synthesized. J H-Sometimes a final layer called the exosporium may be added. D I&J-As the vegetative portion of the bacterium is degraded, the completed endospore is released. K-With the proper environmental stimuli, the endospore germinates. I L-As the protective layers of the endospore are enzymatically broken down, E the endospore is released and a vegetative bacterium begins to form and emerge F G H 69  Sporulation Different types of spores: Location (terminally, subterminally, centrally) Size (deformant, not deformant) 70 71  Factors of endospores heat resistance 1-Calcium-dipicolinate→stabilize & protect the endospore's DNA 2-DNA-binding proteins→saturation and protection of endospore's DNA 3-Cortex→dehydration (resistance to heat and radiation) 4-DNA repair enzymes→repair damaged DNA during germination Depletion of calcium from the medium prior to endosporulation Leads to the heat sensitivity of the endospore and block germination 72  Elimination of endospores from fragile products “Tyndallisation” Vegetative cells Vegetative cells Destruction & Destruction & Vegetative cells Induction Induction Destruction & of germination of germination Waiting Waiting Waiting Heating Heating Heating Sterile 70°C 70°C 70°C object 30min 30min 30min 73 bacillus Endospore  INCLUSION BODIES Cytoplasmic Where found Composition Function inclusions Glycogen many bacteria e.g. E. coli polyglucose reserve carbon and energy source Polybetahydrox- polymerized yutyric acid many bacteria e.g. Pseudomonas reserve carbon and energy source hydroxy butyrate (PHB) Polyphosphate linear or cyclical reserve phosphate; possibly a reserve of high (volutin many bacteria e.g. Corynebacterium polymers of PO4 energy phosphate granules) phototrophic purple and green sulfur reserve of electrons (reducing source) in Sulfur globules bacteria and lithotrophic colorless sulfur elemental sulfur phototrophs; reserve energy source in bacteria lithotrophs protein hulls or buoyancy (floatation) in the vertical water Gas vesicles aquatic bacteria especially cyanobacteria shells inflated with column gases Parasporal endospore-forming bacilli (genus Bacillus) protein unknown but toxic to certain insects crystals magnetite (iron orienting and migrating along geo- magnetic Magnetosomes certain aquatic bacteria oxide) Fe3O4 field lines enzymes for Carboxysomes many autotrophic bacteria autotrophic CO2 site of CO2 fixation fixation lipid and protein Chlorosomes Green bacteria and light-harvesting pigments and antennae bacteriochlorophyll 74  CELL STRUCTURE OF THE DOMAIN BACTERIA 1-The cytoplasmic membrane 2-The bacterial cell wall : a. the Gram-Positive cell wall b. the Gram-Negative cell wall c. the Acid-Fast cell wall 3-Structures located within the cytoplasm : a. cytoplasm b. nucleoid c. plasmids and transposons d. ribosomes e. endospores f. inclusions bodies 4-Structures located outside the cytoplasm : a. glycocalyx (capsule) and S-layer b. flagella c. pili 75  THE GLYCOCALYX (CAPSULES AND SLIME LAYERS) Capsule : polypeptide and/or polysaccharide surrounding the cell wall →Virulence (pathogenecity) ECPS : Extracellular polymeric substances Glycocalyx : ECPS (polysaccharide) Slime : ECPS (polysaccharide, proteins, lipids…) Biofilm : Bacteria + Slime →Virulence (protection against phagocytosis, drying, …) →Environmental problems (adherence & proliferation) →Food industries problems →… 76 Unenhanced Attachment of Bacteria to Phagocytes Enhanced Attachment of Bacteria to Phagocytes Formation of Pseudopods by Rearrangement of Actin Molecules Placing the Bacterium in a Phagosome Fusion of Phagosome and Lysosome 77  Bacterial Capsule Preventing C3b Receptors on Phagocytes from Binding to C3b Attached Capsules Blocking the to a Bacterial Cell Wall Unenhanced Attachment of Bacteria to Phagocytes 78 Glycocalyx of Brucella (TEM) Biofilm of Staphylococcus epidermidis Capsule stain of Enterobacter aerogenes 79 Capsule stain of Streptococcus lactis  THE FLAGELLA Composition: a bacterial flagellum has 3 basic parts: a filament, a hook, and a basal body. 1) The filament: rigid, helical (flagellin) with a hollow core. 2) The hook is a flexible coupling between the filament and the basal body. 3) The basal body consists of a rod and a series of rings that anchor the flagellum to the cell wall and the cytoplasmic membrane. The basal body acts as a molecular motor (The motor of E. coli rotates 270 revolutions per second!) 80 Bacterial flagella are 10-20 µm long and between 0.01 and 0.02 µm in diameter and come in a number of distinct arrangements: 1. monotrichous: a single flagellum, usually at one pole 2. amphitrichous: a single flagellum at both ends of the organism 3. lophotrichous: two or more flagella at one or both poles 4. peritrichous: flagella over the entire surface 81 Monotrichous Flagellum of Vibrio cholerae Spirillum with Amphitrichous Arrangement of Flagella Spirillum with Lophotrichous 82 Flagella Stain of Proteus Showing Arrangement of Flagella Peritrichous Arrangement of Flagella  PILI Pili are thin, protein tubes originating from the cytoplasmic membrane and are found in virtually all gram-negative bacteria but not in many gram-positive bacteria. The pilus has a shaft composed of a protein called pilin. Adhesive tip enabling attachment to receptors on a host cell. There are two basic types of pili: 1) short attachment pili = fimbriae (numerous) 2) long conjugation pili = "F" or sex pili (very few in number) 83  PILI Bacteria are constantly losing and reforming pili as they grow in the body and the same bacterium may switch the adhesive tips of the pili in order to adhere to different types of cells and evade immune defenses. Conjugation or sex pilus Conjugation is the transfer of DNA from a donor or male bacterium with a sex pilus to a recipient or female bacterium to enable genetic recombination. 84  ATYPICAL PATHOGENIC BACTERIA Phagocytosis of the Rickettsia Orientia tsutsugamushi The Rickettsia Orientia tsutsugamushi Within an by a Mouse Peritoneal Mesothelial Cell Intact Endocytic Vacuole in a Mouse Peritoneal Mesothelial Cell 85  Ehrlichia Infecting Host Cells Ehrlichia Infecting Host Cells: Reticulate Cells and Dense-Core Cells hrlichia Inside an endocytic vacuole (arrows). A vacuole Ehrlichia inside endocytic vacuoles. The larger Ehrlichia within ontaining an ehrlichial microcolony is called a morula. the vacuoles are referred to as reticulate cells (RC); the smaller forms of the Ehrlichia are called dense-core cells (DC). A vacuole containing an ehrlichial microcolony is called a morula. 86 Ehrlichia Infecting Host Cells: Release from a Host Cell Ehrlichia being released from an infected host cell. 87 Life Cycles of Chlamydias 88