Clinical Bacteriology (Lec) PDF

CLINICAL BACTERIOLOGY (LEC) STRUCTURE, METABOLISM, & GENETICS PROKARYOTIC CELL OF BACTERIA CHRISTINA A. JAMORA, RMT LEARNING OBJECTIVES 1. Differentiate prokaryotic cell from eukaryotic cell 2. Enumerate the different shapes and...

CLINICAL BACTERIOLOGY (LEC) STRUCTURE, METABOLISM, & GENETICS PROKARYOTIC CELL OF BACTERIA CHRISTINA A. JAMORA, RMT LEARNING OBJECTIVES 1. Differentiate prokaryotic cell from eukaryotic cell 2. Enumerate the different shapes and parts of a bacterial cell 3. Explain the method of Bacterial Classification 4. List nutritional and environmental requirements for bacterial growth 5. Describe importance of bacterial Cell Envelope Structures metabolism o The outermost structure of the bacterial 6. Discuss development and transfer of cell antibiotic resistance in bacteria o Composed of: ▪ Glycocalyx ▪ Outer membrane (for gram- PROKARYOTIC VS. EUKARYOTIC negative bacteria) ▪ Cell wall ▪ Plasma membrane Cell Wall o Referred to as the peptidoglycan or murein layer o Maintains the shape of the cell o Functions: ▪ Prevents rupturing of the cell ▪ Anchor for flagella ▪ Determines staining characteristics of a species TYPES OF CELL WALL Prokaryotic cell Gram Positive Cell Wall o Composed of a very thick protective peptidoglycan (murein) layer o Consists of alternating: ▪ N-acetyl-D-glucosamine (NAG) Eukaryotic cell ▪ N-acetyl-D-muramic (NAM) acid BSMT 3A FRANCISCO | LPU DAVAO Page 1 of 10 CLINICAL BACTERIOLOGY (LEC) Gram Positive Cell Wall B. Inner membrane / Wall o Contains a negatively charged teichoic - Composed of a thin peptidoglycan acid and lipoteichoic acid layer, which is the reason for its high o Main target of antimicrobial agents, like susceptibility to mechanical breakage penicillin, which prevents the synthesis of peptidoglycan Gram Negative Cell Wall o Has porins the contribute to the permeability of the cell wall o Does not contain teichoic acid o Has thinner peptidoglycan layer LAYERS OF THE GRAM-NEGATIVE CELL WALL A. Outer Membrane - Composed of: ▪ Proteins ▪ Phospholipids ▪ Lipopolysaccharide (LPS) - Contributes to the negative charge of the bacterial surface - Allows hydrophilic compounds to enter the cell through porins - Acts as a barrier to toxic substances that prevents movement inside the cell Fixation – to preserve or immobilize bacteria - Evades host defenses – strong o Best fixation: Heat fixation negative charge is an important factor in Crystal violet evading phagocytosis o Primary stain - Considered as an endotoxin (exception: Iodine treatment Listeria monocytogenes) o Mordant – used to set or stabilize stains or dyes to the cell wall Decolorizer o Composed of acetone and ethanol (1:1) o Helps us determine if the bacteria is gram positive or negative ▪ Gram positive – primary stain will retain its color, as the peptidoglycan Safranin o Counter stain BSMT 3A FRANCISCO | LPU DAVAO Page 2 of 10 CLINICAL BACTERIOLOGY (LEC) Absence of a cell wall o Prokaryotes that do not have a cell wall contain sterols in their cell membrane o Ex. Mycoplasma and Ureaplasma o Antimicrobials target protein or DNA synthesis Gram positive – stains purple Gram negative – stains pink PROKARYOTIC CELL (CONT’D) Plasma / Cell Membrane o Deepest layer of the cell envelope o Consists of a phospholipid bilayer that surrounds the cytoplasm Acid-fast cell wall o Acts as an osmotic barrier o Has a gram-positive cell wall structure o Functions as the mitochondria (ATP o Also has a waxy layer of glycolipids and production), Golgi complexes, and fatty acids (mycolic acid), which is a lysosomes strong hydrophobic structure, bound to exterior PROKARYOTIC CELL: o Mycolic acid – structure present in acid- CYTOPLASMIC STRUCTURES fast organisms which allows them to resist acid decolorization o Ex: Mycobacterium and Norcadia BSMT 3A FRANCISCO | LPU DAVAO Page 3 of 10 CLINICAL BACTERIOLOGY (LEC) Ribosome o Site of protein synthesis o Consists of RNA and proteins o It is 70S in size and separates into two subunits: 50S and 30S o 80S (60S + 40S) in eukaryotes o Svedberg units (S) – unit used which determines sedimentation rate Genome o Consists of a single, circular chromosome Plasmid o An extrachromosomal, double-stranded element of DNA that is associated with virulence o Located in the cytoplasm o Site for coding antibiotic resistance and toxin production o Not essential for growth; some bacteria do not have this o 2 kinds: ▪ Large plasmid – production of Beta lactamases that provide resistance to Beta lactam antibiotics like penicillin and oxacillin ▪ Small plasmid – for tetracycline and chloramphenicol resistance Inclusion bodies o They serve as the energy source or food reserve of bacteria o They are composed mainly of polysaccharides, which lessen osmotic pressure o Examples: ▪ Babes-Ernst bodies (Corynebacterium diphtheriae) ▪ Bipolar bodies (Yersinia pestis) ▪ Much granules (Mycobacterium tuberculosis) ▪ Sulfur granules PROKARYOTIC CELL: (Nocardia and Actinomycetes) CELL APPENDAGES Endospores / Asexual spores (Resistant structures) o Small, dormant structures located inside the bacterial cell o They aid in the survival of bacteria against external conditions o They are produced within vegetative cells of some Gram positive bacteria o The locations of these structures aid in the microscopic identification of bacteria o Schaeffer-Fulton stain is used for visualization = Endospores appear green BSMT 3A FRANCISCO | LPU DAVAO Page 4 of 10 CLINICAL BACTERIOLOGY (LEC) Glycocalyx o Important in the survivability and the o An outward complex of polysaccharides pathogenic ability of bacteria on the bacterial surface and other cells o 5 different flagellar arrangement: o Helps the bacteria in attaching to the ▪ Atrichous – no flagella surface of tissues or solid objects ▪ Monotrichous – one flagellum at o Appears as a capsule or slime layer one end Glycocalyx: Capsule ▪ Amphitrichous – flagella at each o Polysaccharide structure surrounding a end bacterial cell and is external to the cell ▪ Lophotrichous – tuft of flagella at wall one end ▪ (exception: B. anthracis = poly-D- ▪ Peritrichous – flagella all over glutamic acid) o Immunoevasion – may increase virulence by preventing phagocytosis o Antigenic, basis of serotyping by Quellung reaction o India ink stain is used to determine its presence o Some Killer Bacteria Have Pretty Nice Capsules ▪ Streptococcus pneumoniae ▪ Klebsiella pneumoniae ▪ Bacillus anthracis ▪ Haemophilus influenzae Pili / Fimbriae ▪ Pseudomonas aeruginosa o Pili ▪ Neisseria meningitidis ▪ “Conjugation pili / sex pili” ▪ Cryptococcus neoformans ▪ Nonmotile, long, hollow protein tubes Glycocalyx: Slime Layer ▪ Connect two bacterial cells and o Unorganized material that is loosely mediate DNA exchange attached to the cell wall o Fimbriae / Common Pili o Makes up the biofilm ▪ Nonflagellar, sticky, proteinaceous, o Also consists of polysaccharides o Can either inhibit phagocytosis or aid in hair-like appendages the adherence of the bacteria to the host ▪ Adhere some bacterial cells to one tissue or synthetic implants another and to environmental o Ex: Staphylococcus epidermidis in surfaces (virulence factor) prosthetic heart valves o Ex: Neisseria gonorrhoeae and Pseudomonas Flagella (organ of locomotion) o Exterior protein filament (flagellin) that rotates and causes bacteria to be motile BSMT 3A FRANCISCO | LPU DAVAO Page 5 of 10 CLINICAL BACTERIOLOGY (LEC) BSMT 3A FRANCISCO | LPU DAVAO Page 6 of 10 CLINICAL BACTERIOLOGY (LEC) EUKARYOTES Contains a true nucleus These cells of higher plants, animals, fungi, protozoa, and other morphologically complex and large organisms Contains: o Nucleus o Endoplasmic Reticulum o Golgi Apparatus o Mitochondria 4 PHASES OF GROWTH o Lysosomes 1. Lag Phase - Bacterium prepares to divide; no increase BACTERIAL GENETICS in number of living bacterial cells 2. Log Phase / Exponential Phase - Exponential increase in number of living bacterial cells. Phase where bacteria are most susceptible to antimicrobials 3. Stationary Phase - Nutrients become limited; growth stops - Number of living bacteria = number of dead bacteria 4. Death Phase - Bacteria die faster than they multiply - Number of nonliving bacteria > number of living bacteria BACTERIAL GENETICS The mechanism by which genetic information is changed and exchanged among bacteria: Generation Time / Doubling Time o Time required for 1 cell to divide into 2 cells BSMT 3A FRANCISCO | LPU DAVAO Page 7 of 10 CLINICAL BACTERIOLOGY (LEC) Mutation 3. Conjugation o A change in the original nucleotide - Transfer of genetic material from a donor sequence of a gene or genes within an bacterial strain to a recipient strain via sex organism’s genome (genotype) pili Recombination o (Homologous Recombination) BACTERIAL METABOLISM o Genes are transferred or exchanged RESPIRATION: efficient ATP-generating between homologous regions on 2 DNA process in which molecules are oxidized and molecules results in an inorganic molecule as the final electron acceptor o Glycolysis (Embden-Meyerhof-Parnas Pathway) ▪ 1st stage in CHO metabolism ▪ Oxidation of glucose to pyruvic acid o Krebs Cycle (Tricarboxylic Acid or TCA cycle) LAYERS OF THE GRAM-NEGATIVE CELL WALL ▪ Most important process for the complete oxidation of a substrate under aerobic conditions ▪ An enzyme converts pyruvate into CO2 and an acid FERMENTATION: does not require oxygen (anaerobic process), the use of Krebs cycle, or an electron transport chain o Alcoholic Fermentation: turns sugar into ethanol and CO2 o Homolactic Fermentation: pyruvate is 1. Transformation reduced to lactate, which is used to make - Uptake and incorporation of naked DNA yogurt and pickles into a bacterial cell o Heterolactic Fermentation: produces substances other than lactate, such as alcohol, CO2, formic acid and acetic acid o Mixed Acid Fermentation: production of ethanol and acids (lactic, acetic, succinic, formic acid) o Butanediol Fermentation: pyruvate is converted into acetoin then reduced to 2,3- butanediol with NADH; small amounts of ethanol and mixed acids are also synthesized 2. Transduction o Butyric Acid Fermentation: conversion - Transfer of bacterial genes by a of pyruvate into butyric acid along with bacteriophage (virus-infected bacterium) acetic acid, CO2 and H2 from one cell to another MICROBIAL GROWTH AND NUTRITION Major nutritional need for growth: o Source of CARBON ▪ For making cellular constituents ▪ 50% o Source of NITROGEN ▪ For making proteins ▪ 14% o Source of ENERGY ▪ ATP, for performing cellular functions BSMT 3A FRANCISCO | LPU DAVAO Page 8 of 10 CLINICAL BACTERIOLOGY (LEC) PHYSIOLOGIC REQUIREMENTS OF BACTERIA ACC. TO TEMPERATURE REQUIREMENT ACCORDING TO OXYGEN REQUIREMENT Psychropiles / Cryophiles o “Cold-loving” Aerobes o Can grow at 0-20°C o Air contains 15-21% O2 and 1% CO2 Mesophiles Anaerobes o “Middle loving” o Obligate anaerobes o Best growth between 25-45°C ▪ Does not require O2 ▪ Dies after prolonged exposure to Thermophiles / Hyperthermophiles air o “Heat loving” o Facultative anaerobes o Optimum growth between 50-60°C ▪ Most clinically significant bacteria Extremophiles ▪ Considered as aerobes but can o Prokaryotes that are able to survive grow anaerobically unusual conditions o Aerotolerant anaerobes ACCORDING TO Ph REQUIREMENTS ▪ Can survive in the presence of O2 Neutrophiles but unable to perform processes o Grow best at neutral pH unless placed in anaerobic o 0-5.5 pH conditions o Ex: Lactobacillus Microaerophiles o SESH o 2-10% O2 for growth ▪ Salmonella spp. ▪ Escherichia coli ENVIRONMENT ▪ Staphylococcus spp. GROUP AEROBIC ANAEROBIC O2 EFFECT ▪ Helicobacter pylori Acidophiles Required o Grow best under acidic conditions Obligate (utilized for aerobes Growth No growth aerobic o 5.5-8.5 pH respiration) o Most clinically significant Alkaliphiles Obligate anaerobes No growth Growth Toxic o Grow best under alkaline conditions o 8.5-11.5 pH Required for Facultative growth but can o Ex: Vibro cholerae Growth Growth aerobes grow in its absence OTHER REQUIREMENTS Halophiles Not required for Facultative growth but o Can grow and often thrive in areas of Growth Growth anaerobes utilized when available high salt (NaCl) concentration Capnophiles Not required o Grow better at high CO2 levels and low and not utilized. Aerotolerant Growth Growth Inhibits some O2 levels anaerobes metabolic o Requires 5-10% CO2 processes Barophiles o High pressure Growth if level Required but at Microaerophiles not too high No growth levels 2-10% o 600-1100 atm pressure COMMON STAINS FOR MICROSCOPIC VISUALIZATION Gram Stain – differential stain to differentiate gram negative and gram positive Acid Fast Stains – diagnostic stain for Microbacterium spp. Acridine Orange – fluorescence stain Calcoflour White – fluorescence stain Methylene blue – direct stain to identify morphology BSMT 3A FRANCISCO | LPU DAVAO Page 9 of 10 CLINICAL BACTERIOLOGY (LEC) Lactophenol Cotton Blue (LPCB) – used for fungi India Ink – for capsules; esp. for yeast and hyphae Endospore – for bacterial spores BACTERIA MORPHOLOGY Cocci (spherical) Bacilli (rod-shaped) Spirals BSMT 3A FRANCISCO | LPU DAVAO Page 10 of 10