Clinical Microbiology And Immunology PDF

CLINICAL MICROBIOLOGY AND IMMUNOLOGY FUNDAMENTALS OF MICROBIOLOGY INTRODUCTION TO MICROBIOLOGY  WHAT IS MICROBIOLOGY?  MICROBIOLOGY IS THE STUDY OF MICROORGANISMS  MICROORGANISMS ARE A LARGE & DIVERSE GROUP OF MICROORGANISMS THAT EXIST IN SINGLE CELLS OR CLUSTERS  VIRUSES ARE C...

CLINICAL MICROBIOLOGY AND IMMUNOLOGY FUNDAMENTALS OF MICROBIOLOGY INTRODUCTION TO MICROBIOLOGY  WHAT IS MICROBIOLOGY?  MICROBIOLOGY IS THE STUDY OF MICROORGANISMS  MICROORGANISMS ARE A LARGE & DIVERSE GROUP OF MICROORGANISMS THAT EXIST IN SINGLE CELLS OR CLUSTERS  VIRUSES ARE CONSIDERED TO BE MICROORGANISMS, EVEN THOUGH THEY ARE ACELLULAR  WHY STUDY MICROBIOLOGY ? Impact on Microbes They are human life are extremely ubiquitous adaptable POSITIVE AND NEGATIVE IMPACT ON HUMAN LIFE 1. RESPONSIBLE FOR CYCLING OF ELEMENTAL CARBON, NITROGEN, PHOSPHORUS, HYDROGEN, SULPHUR & OXYGEN  MORE PHOTOSYNTHESIS IS CARRIED OUT BY MICROBES THAN BY GREEN PLANTS Microorganisms as disease agents Bacteria, Fungi & Viruses cause many diseases that affect man, animals & plants Microorganisms & agriculture N fixation by Rhizobium in nodules on roots of leguminous plants Cellulose digesting bacteria in the rumens of herbivorous animals 4. MICROORGANISMS & FOOD FOOD PRODUCTION- CHEESES, YOGHURTS, PICKLED PRODUCTS, ALCOHOLOIC BEVERGARES, BREADS FOOD SPOILAGE DUE TO MICROBIAL GROWTH & FOODBORNE ILLNESS 5. ENERGY & THE ENVIRONMENT NATURAL GAS (METHANE) IS A PRODUCT OF METHANOGENIC BACTERIA MICROBES CAN BE USED TO CLEAN UP THE ENVIRONMENT IN A PROCESS CALLED BIOREMEDIATION; ORGANISMS CAN DEGRADE SPILLED OIL, SOLVENTS, PESTICIDES 6. ORGANISMS & THE FUTURE BIOTECHNOLOGY- IN LARGE SCALE INDUSTRIAL PROCESSES, GENETICALLY MODIFIED MICROBES ARE USED TO SYNTHESIZE PRODUCTS LIKE ANTIBIOTICS, INSULIN  EMPIRES: PROKARYOTES – NO NUCLEUS TWO EUKARYOTES – TRUE NUCLEUS EMPIRES DOMAINS: AND THREE ARCHAEA DOMAINS BACTERIA EUKARYOTA  Discipline did not develop until 19th century HISTORY OF MICROBIOLOGY Since then, it has developed rapidly, spawning new subdisciplines IMPORTANT MILESTONES: 1684: VAN LEEUWENHOEK, 1 ST CRUDE MICROSCOPE THAT COULD VISUALIZE MICROORGANISMS 1798: JENNER PRODUCES COWPOX VACCINATION FOR SMALLPOX 1847-1850: SEMMELWEIS INTRODUCES HAND HYGIENE. Pasteur disproves spontaneous generation theory Fleming discovers penicillin 1884 1933 1861 1929 Koch’s postulates Ruska develops electron published, Gram’s stain microscope developed 1953 1995 Watson & Crick propose DNA double helix Chicken pox vaccine approved for U.S. use; Haemophilus influenzae genome sequenced HIV isolated & identified by Gallo & Montagnier; Mullis develops PCR technique “Super resistant” HIV strain isolated in NYC 1983–1984 2005 FUTURE TRENDS MICROBIOLOGY IS REQUIRED TO FACE THREAT OF NEW AND REEMERGING HUMAN INFECTIOUS DISEASE & DEVELOP INDUSTRIAL TECHNOLOGIES THAT ARE MORE EFFICIENT & ENVIRONMENTALLY FRIENDLY  PRODUCTION OF NEW DRUGS & VACCINES TO COUNTER THE SPREAD OF MULTIPLE ANTIBIOTIC RESISTANCE FURTHER DEVELOPMENT OF MOLECULAR TECHNIQUES TO FACILITATE STUDY OF MICROBES IN THEIR NATURAL ENVIRONMENT (MICROBIAL ECOLOGY)  PROTEOMICS & GENOMICS- SEQUENCING OF MICROBIAL GENOMES, STUDY OF PROTEIN STRUCTURE & FUNCTION. PROVIDE UNDERSTANDING OF HOW STRUCTURES DEVELOP, HOW CELLS COMMUNICATE & FUNCTION WITHIN THE ENVIRONMENT  BIOLOGICAL CHARACTERISTICS OF MICROORGANISMS  MICROORGANISMS Are classified according Some belong to the Include some eukaryotes to their structure, Protista biologic and prokaryotes, viruses, chemical composition, kingdom. viroids, and prions. and biosynthetic and genetic organization.  Eukarya Algae, Protozoa, Fungi, Slime molds Prokaryotes MAJOR Bacteria, Archaebacteria GROUPS Viruses Are not cells and are not visible with the light microscope. Are obligate intracellular parasites. Contain no organelles or biosynthetic machinery, except for a few enzymes. Contain either RNA or DNA as genetic material. Are called bacteriophages (or phages ) if they have a bacterial host.  PRIONS- INFECTIOUS PARTICLES ASSOCIATED WITH MAJOR SUBACUTE PROGRESSIVE, DEGENERATIVE DISEASES OF THE CENTRAL NERVOUS SYSTEM (E.G., CREUTZFELDT-JAKOB GROUPS DISEASE)  Prokaryotes Have 70S ribosomes composed of 30S and 50S subunits. PROKARYOTES Organelles not bound by a lipid membrane. VS No well-defined nucleus; a nucleoid instead. EUKARYOTES Prokaryotic cells are usually much smaller than eukaryotic cells. Smaller cells have a higher surface: volume ratio The cells of the Bacteria and Archaea are prokaryotic. Bacterial cell walls characteristically contain peptidoglycan, a polysaccharide that is not seen in eukaryotic cell walls.  Eukaryotes All those cells excepting Bacteria and Archaea. Have 80S ribosomes PROKARYOTES Contain organelles that are membrane-bound, as well as a well- VS defined nucleus. EUKARYOTES Tend to be larger than prokaryotic cells. Plant cells, most algal cells and some fungal cells contain cellulose. Fungal cell walls contain chitin. Animal cells do not contain chitin.   MICROBIAL EUKARYOTES ARE CALLED PROTISTS EUKARYOTES,  MEMBERS: ALGAE, PROTOZOA, CONT’D… FUNGI & SLIME MOLDS  ALGAE PHOTOSYNTHETIC EUKARYOTIC AQUATIC ORGANISMS UNICELLULAR & MULTICELLULAR FORMS CYANOBACTERIA (BLUE GREEN ALGAE) CONSIDERED PROKARYOTIC  PROTOZOA UNICELLULAR, NON PHOTOSYNTHETIC ANCESTORS WERE ALGAE THAT BECAME HETEROTROPHIC MOTILITY- PSEUDOPODIA, FLAGELLA, CILIA PARASITIC FORMS- PLASMODIUM  FUNGI ACHLOROPHYLLOUS, CONTAIN CHITIN IN CELL WALLS UNICELLULAR FORMS- YEASTS MULTICELLULAR- MOLDS. GROW AS A MASS OF INTERLACING, BRANCHING FILAMENTS (HYPHAE), THAT FORM A MYCELIUM EN MASSE SAPROPHYTIC, PARASITIC FORMS  HAVE AN AMEBOID, MULTINUCLEATE MASS OF CYTOPLASM LIFECYCLE STAGE CALLED A PLASMODIUM FOOD SOURCE IS BACTERIA SLIME MOLDS SOME MEMBERS AFFECT MAN   ACELLUAR, OBLIGATE INTRACELLULAR PARASITES  KNOWN TO INFECT ALL CELLS INCLUDING MICROBIAL CELLS  CONSISTS OF EITHER DNA OR RNA ENCLOSED IN A VIRUSES CAPSID, SOMETIMES ENVELOPED  PROTEINS ASSOCIATED WITH THE CAPSID OR ENVELOPE DETERMINE VIRAL TROPISM, MEDIATE ATTACHMENT TO HOST CELL  ONCE INSIDE CELL, VIRUS DIRECTS HOST CELL TO REPLICATE IT BEFORE ITS EVENTUAL EXIT  VIRAL REPLICATION  Proteinaceous infectious particles Prion diseases: PRIONS Scrapie, degenerative CNS disease of sheep BSE ( Bovine Spongiform Encephalopathy) in man, thought to result from the ingestion of feeds & bone meal prepared from sheep offal  Members- Bacteria & Archaebacteria Relatively small size (1µm diameter) Absence of nuclear membrane PROKAYOTES Circular DNA within nucleoid Ability to exchange genetic info via plasmids Diverse forms- Aerobes, Anaerobes Quorum sensing- regulates transcription of genes in processes like bioluminescence, production of virulence determinants  Have cell walls composed of peptidoglycan. BACTERIA: SIZE, SHAPE, It is the cell wall that gives the bacterium its characteristic shape. ARRANGEMENT Bacteria may be classified according to shape: cocci, bacilli, spirilla, vibrio, etc.  Bacilli: bacillus, diplobacilli, streptobacilli, coccobacilli. Examples M.tuberculosis, M.leprae, B. anthracisis MAIN Cocci: coccus, diplococcus (N. meningitidis), streptococcus (Enterococcus), staphylococcus (S. GROUPS: aureus). Spirilla: Vibrio- curved (Vibrio cholorae), spirillum- thick, rigid spiral (Leptospira), spirochaete- thin, flexible spiral (Treponema pallidum). Image:Bacterial morphology diagram.svg   HANS CHRISTIAN GRAM (1853-1938) IN 1884 GRAM STAINING DEVELOPED GRAM STAINING. AND DIFFERENCES IN  THE TECHNIQUE DIVIDES BACTERIA INTO TWO GROUPS: CELL WALLS OF GRAM NEGATIVE AND GRAM POSITIVE. GRAM POSITIVE AND GRAM NEGATIVE  GRAM NEGATIVE AND GRAM POSITIVE BACTERIA DIFFER BACTERIA. WITH RESPECT TO THE NUMBER OF LAYERS THAT MAKE UP THEIR ENVELOPES.  Outer membrane (selectively permeable) THE GRAM NEGATIVE BACTERIAL Cell wall (thin, made of peptidoglycan) ENVELOPE CONSISTS OF: Cytoplasmic membrane (selectively permeable) THE GRAM POSITIVE Cell wall (thick Cytoplasmic membrane BACTERIAL layer, made of (selectively ENVELOPE peptidoglycan) permeable CONSISTS OF: THESE DIFFERENCES IN THE CELL ENVELOPE ARE IMPORTANT BECAUSE THEY CAUSE THE BACTERIA TO STAIN DIFFERENTLY (OR PRODUCE DIFFERENT COLOURS UPON STAINING).  STRUCTURAL DIFFERENCES IN BACTERIAL CELL WALLS  STEPS IN GRAM STAINING  1. PRIMARY STAIN- CRYSTAL VIOLET IS ADDED TO FIXED SPECIMEN OF BACTERIA. BOTH GRAM NEGATIVE THE GRAM BACTERIA AND GRAM POSITIVE BACTERIA BECOME PURPLE. STAIN PROCEDURE 2. MORDANT- IODINE IS ADDED TO SET (FIX) THE STAIN.  DECOLOURIZATION- ETHANOL IS ADDED. GRAM NEGATIVE BACTERIA LOSE THEIR COLOUR BECAUSE THERE IS RELATIVELY LITTLE PEPTIDOGLYCAN TO HOLD THE STAIN. GRAM POSITIVE BACTERIA RETAIN THE STAIN BECAUSE OF THE THICK LAYER OF PEPTIDOGLYCAN. COUNTERSTAIN- SAFRANIN IS ADDED. THIS TURNS DECOLOURIZED GRAM NEGATIVE BACTERIA PINK AND GRAM POSITIVE BACTERIA DEEPER PURPLE.  Envelope (Outer portion of cell) BACTERIAL May consist of up to 3 CELL layers: STRUCTURE Outer membrane Cell wall Cytoplasmic membrane Gram negative bacteria and Gram positive bacteria differ with respect to the number of layers comprising their envelopes: Gram negative bacteria possesses all 3 layers; the space between the outer membrane and cytoplasmic membrane is called the periplasm. Gram positives contain only 2 layers. They contain no periplasm. a. OUTER MEMBRANE ONLY SEEN IN GRAM NEGATIVE BACTERIA CONSISTS OF PHOSPHOLIPIDS AND LIPOPOLYSACCHARIDES (LPS). LPS IS AN ENDOTOXIN; HARMFUL TO ANIMALS  PERIPLASM Only seen in Gram negative bacteria Contains proteins & enzymes that break down certain nutrients so that they can enter the cytoplasm of the cell.   COMPOSED OF PEPTIDOGLYCAN- A MOLECULE THAT IS PART PROTEIN & PART POLYSACCHARIDE. MUREIN IS THE SPECIFIC TYPE OF PEPTIDOGLYCAN FOUND IN BACTERIAL CELL WALL CELL WALLS  MUREIN CONSISTS OF TWO SUGARS NAG (N- ACETYLGLUCOSAMINE) AND NAM (N- ACETYLMURAMIC ACID) THAT ARE CROSS-LINKED BY PEPTIDES TO FORM A RIGID STRUCTURE.  GRAM NEGATIVE BACTERIA HAVE RELATIVELY THIN CELL WALLS, WHEN COMPARED TO GRAM POSITIVE BACTERIA.  GRAM POSITIVE BACTERIA HAVE THICK LAYER OF PEPTIDOGLYCAN, AS WELL AS TEICHOIC ACIDS THAT FURTHER STRENGTHEN THE WALL.  BACTERIAL CELL WALLS FUNCTION TO GIVE THE CELL ITS SHAPE AND TO CONTAIN TURGOR PRESSURE.  CYTOPLASMIC MEMBRANE ◦ PHOSPHOLIPID BILAYER, TRANSMEMBRANE & SURFACE PROTEINS. ◦ FUNCTIONS TO CONTAIN THE CYTOPLASM AND REGULATE WHAT ENTERS AND LEAVES THE CELL. FLUID MOSIAC MODEL.  1. SIMPLE DIFFUSION TRANSPORT 2. OSMOSIS ACROSS 3. ACTIVE TRANSPORT CELL 4. FACILITATED DIFFUSION MEMBRANE: 5. GROUP TRANSLOCATION 6. ENGULFMENT CYTOPLASM MATERIAL INSIDE CYTOPLASMIC MEMBRANE 90% WATER CONTAINS: NUCLEOID- IRREGULAR MASS OF DNA, NOT MEMBRANE BOUND RIBOSOMES- MANUFACTURE PROTEINS. PROKARYOTIC RIBOSOMES ARE CALLED 70 S RIBOSOMES; EUKARYOTIC RIBOSOMES ARE CALLED 80S RIBOSOMES. INCLUSION BODIES- I) STORAGE GRANULES- WHERE CELLS STORE NUTRIENTS VARY FROM II) MAGNETOSOMES- IRON CONTAINING STRUCTURES SPECIES TO III) GAS VACUOLES- SEEN IN AQUATIC BACTERIA, ENABLING THEM TO FLOAT. SPECIES:  APPENDAGES OUTSIDE OF ENVELOPE PRESENCE OR ABSENCE DEPENDS ON SPECIES: PILI, FLAGELLA, CAPSULE  Short, hairlike structures composed of proteins called pilins Pili aka fimbriae PILI Function to attach bacteria to other cells, or other bacteria during mating (sex pilus) Pili were identified in the gram positive bacteria in the last decade (e.g. streptococcal pathogens).  Long, thin structures extending from surface of envelope. FLAGELLA Function is locomotion, not adhesion Different bacteria have different numbers and arrangements of flagella: Monotrichous, amphitrichous, lophotrichous, peritrichous  Aka glycocalyx, Found exterior to the envelope Both Gram negative and Gram positive CAPSULE bacteria may secrete a slimy substance that becomes the outermost layer of the cell Functions to protect cell from dessication, allows cell to adhere to other cell surfaces   SOME BACTERIA FORM THICK-WALLED STRUCTURES CALLED ENDOSPORES WHEN CONDITIONS ARE UNFAVOURABLE FOR GROWTH. PROCESS IS CALLED SPORULATION.  ENDOSPORES ARE THICK-WALLED RESTING (NON- ENDOSPORE GROWING) STRUCTURES THAT ARE RESISTANT TO HEAT, FORMATION DEHYRATION, TOXIC CHEMICALS AND RADIATION.  THEY TYPICALLY CONTAIN LESS THAN 15% WATER. CLOSTRIDIUM TETANI IS KNOWN TO FORM ENDOSPORES, AS WELL AS MANY BACILLUS SP.  STEPS IN ENDOSPORE FORMATION  DYES THAT INCREASE CONTRAST BY BINDING SELECTIVELY TO CERTAIN CELLS OR TO CERTAIN PARTS OF THEM, SO THAT THEY MAY BE BETTER VISUALIZED UNDER THE MICROSCOPE. STAINS MOST STAINS ARE ONLY EFFECTIVE AFTER THE MICROORGANISMS HAS BEEN FIXED (KILLED & ATTACHED TO MICROSCOPE SLIDE)  HEAT- EMULSIFY IN WATER OR SALINE, AIR DRY, PASS FIXING IS THROUGH FLAME DONE BY: CHEMICAL- ADD 1 DROP FORMALDEHYDE, OSMIC ACID, GLUTALDEHYDE. CHEMICAL FIXATION DOES LESS DAMAGE TO CELL.  1. SIMPLE STAINS: ◦ BASIC DYES (POSITIVELY CHARGED) THAT BIND TO TYPES OF MICROBIAL SURFACES (TEND TO BE NEGATIVELY CHARGED). STAINS ◦ THESE DYES STAIN CELLS THE SAME COLOUR E.G. METHYLENE BLUE 2. Differential stains: Used to distinguish between different types of microbes. Procedure usually has three steps: Primary staining- same as simple staining Destaining- a treatment that removes stain from certain cells Counterstaining- application of another dye to reveal parts of the cell that have been destained. Examples: Gram-stain, Acid-Fast stain  ACID FAST STAIN WAS DEVELOPED BY EHRLICH IN 1882 TO IDENTIFY M. TUBERCULOSIS. ONLY COLOURS MYCOBACTERIA AND SOME ACTINOMYCETES. ACID-FAST BACTERIA RESIST DESTAINING BECAUSE OF A WAX LIKE SUBSTANCE (MYCOLIC ACID) IN THEIR ENVELOPES (BACTERIA RETAIN RED CARBOLFUCHSIN, HOST TISSUE STAINS BLUE).  ABLE TO STAIN SPECIFIC PARTS OF THE CELL- CELL WALL, NUCLEOID, ENDOSPORES, FLAGELLA ◦ LEIFSON FLAGELLA STAIN- STAINS FLAGELLA USING FIRST TANNIC ACID TO THICKEN FLAGELLA AND THEN ROSANILINE TO COLOUR THEM. SPECIAL STAIN NEGATIVE STAINING USED FOR STAINING ENCAPSULATED YEASTS & BACTERIAL CAPSULES, WHICH TEND TO BE COLOURLESS AND DIFFICULT TO SEE. PRESENCE OF A CAPSULE CAN BE REVEALED BY STAINING WITH INDIA INK TO BLACKEN THE BACKGROUND SO THAT CAPSULE AND CELL WALL ARE REVEALED AS A CLEAR ZONE. NEGATIVE STAINING  BROCK BIOLOGY OF MICROORGANISMS- MADIGAN, MARTINKO & PARKER REFERENCES MEDICAL MICROBIOLOGY- JAWETZ, MELNICK & ADELBERG PRESCOTT, HARLEY & KLEIN’S MICROBIOLOGY- WILLEY, SHERWOOD & WOOLVERTON