Microbial Physiology

INTRODUCTION TO BACTERIOLOGY

Cell organelles

Eukaryotic Cell Contain a membrane-bound nucleus and numerous membrane-enclosed organelles Mitochondria, lysosomes, golgi apparatus- not found in prokaryotes Animal, plants, fungi More complex Found in many different forms

Nucleus Contains most of the genetic material (DNA) of the cell Additional DNA: mitochondria and (if present) chloroplasts Nuclear DNA is complexed with proteins to form chromatin organized as a number of linear chromosomes Genetic control of the cell is carried out by the production of RNA in the nucleus (the process of transcription) Subsequent transfer of this RNA to a ribosome in the cytoplasm, where protein synthesis is directed (the process of translation) The resulting proteins carry out cell functions Also located in the nucleus is the nucleolus or nucleoli where ribosomes are assembled Nucleus is bounded by a nuclear envelope a double membrane perforated with pores connected to the rough endoplasmic reticulum membrane system

Both contains: DNA Plasma membrane Cytosome Ribosome

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Cytoskeleton Consists of microtubules, intermediate fibers, and microfilaments Maintain cell shape and anchor organelles Causes cell movement Frequently undergoes assembly and disassembly according to cellular needs

Plasma Membrane ayer Phospholipid bilayer with embedded proteins encloses every living cell Blocks uncontrolled movements of waterwater soluble materials into or out of the cell Various proteins embedded in the phospholipid bilayer penetrate into and through the bilayer Responsible for the specific functions of the plasma specif membrane 1. controlling the flow of nutrients and ions into and out of the cells 2. mediating the response of a cell to external stimuli (signal transduction) 3. interacting with bordering cells

Ribosome Site of protein synthesis in the cell Each ribosome consists of a large subunit and a small subunit each contains rRNA (ribosomal RNA) and ribosomal proteins In protein synthesis the mRNA (messenger RNA) moves through the ribosome amino acids attached to tRNAs (transfer RNAs) are brought to the ribosome Amino acids are joined to produce the protein Ribosomes exist free in the cytoplasm and bound to the endoplasmic reticulum (ER) Free ribosomes synthesize the proteins that function in the cytosol Bound ribosomes make proteins that are distributed by the membrane systems, including those which are secreted from the cell.

Mitochondria Site of cellular respiration generates ATP from substrates in reactions using oxygen Primary function: synthesize ATP (adenosine triphosphate) from ADP (adenosine diphosphate) and Pi (inorganic phosphate).

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Antibiotic resistance Resistance to heavy metals such as mercury Resistance to ultraviolet light Pili Exotoxins including several enterotoxins The walls of bacteria consist of peptidoglycans Murein, mucopeptide Gives rigid support Protects against osmotic pressure Site of action of penicillins and cephalosporins Some bacteria have flagella and/or pili For locomotion To pull two cells in close contact Facilitate the transfer of genetic material

Prokaryotic cell

Fundamentally different in their internal organization from eukaryotic cells lack a nucleus and membranous organelles The genetic material (DNA) is localized to a region called the nucleoid has no surrounding membrane Consists of a single circular molecule of loosely organized DNA lacking a nuclear membrane and mitotic apparatus The cell contains large numbers of ribosomes that are used for protein synthesis Periphery of the cell is the plasma membrane Outside the plasma membrane of most prokaryotes is a fairly rigid wall which gives the organism its shape Plasmid Contains a variety of genes for antibiotic resistance and toxins Extrachromosomal double stranded circular DNA molecules that are capable of replicating independently of the bacterial chromosome Carry genes for the following functions:
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Bacteria are held by peptidoglycan, protecting against osmotic rupture Gram-positive bacteria: substantial layer 50 to 100 molecules thickness Gram-negative bacteria: only 1-2 molecules thick Outer lipopolysaccharide layer is present on top of peptidoglycan

Bacterial structures (outside the cell wall): Flagella Long whiplike appendages used for chemotaxis Process of moving the bacteria toward nutrients and other attractants Capsule Protects against phagocytosis Gelatinous layer covering the entire bacterium Important mainly for 4 reasons:

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Determinant of virulence of many bacteria since it limits the ability of phagocytes to engulf the bacteria Specific identification of an organism Capsular polysaccharides are used as antigens in certain vaccines for they are capable of eliciting protective antibodies May play a role in the adherence of bacteria to human tissue Pilus or fimbria Mediates attachment to cell surfaces Sex pilus mediates attachment of 2 bacterias during conjugation Hairlike filaments that extend from the cell surface Shorter and straighter than flagella Found mainly on gram negative bacterias Spore Provides resistance to dehydration, heat and chemicals Formed in response to adverse conditions Has no metabolic activity and can remain dormant for years As a result of their resistance to heat, sterilization cannot be achieved by boiling Glycocalyx (slime layer) Mediates adherence to surface Gram Stain developed in 1884 by Hans Christian Gram characterizes bacteria based on the structural characteristics of their cell walls thick layers of peptidoglycan in the "Grampositive" cell wall stain purple thin "Gram-negative" cell wall appears pink By combining morphology and Gram-staining, most bacteria can be classified as belonging to one of four groups Gram-positive cocci or bacilli Gram-negative cocci or bacilli Useful in: Identification of many bacteria Influencing the choice of many antibiotic because in general, gram (+) bacteria are more suspceptible to penicillin G than gram (-) Acid-Fast Cell Wall In addition to peptidoglycan, the acid-fast cell wall of Mycobacterium contains a large amount of glycolipids, mycolic acids Porins required to transport small hydrophilic molecules thru the outer membrane of the acidfast cell wall Because of its unique cell wall, it is stained by the acid-fast procedure will resist decolorization with acid-alcohol and stain red, the color of the initial stain, carbolfuchsin
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Common Features of Prokaryotic and Eukaryotic Cells DNA, the genetic material contained in one or more chromosomes and located in a nonmembrane bound nucleoid region in prokaryotes and a membranebound nucleus in eukaryotes Plasma membrane, a phospholipid bilayer with proteins that separates the cell from the surrounding environment and functions as a selective barrier for the import and export of materials Cytoplasm, the rest of the material of the cell within the plasma membrane, excluding the nucleoid region or nucleus, that consists of a fluid portion called the cytosol and the organelles and other particulates suspended in it Ribosomes, the organelles on which protein synthesis takes place Classification of Bacteria seeks to describe the diversity of bacterial species by naming and grouping organisms based on similarities Basis of Classification cell structure cellular metabolism (differences in cell components such as DNA, fatty acids, pigments) Serologic reactivity (antigens) Animal pathogenicity Antibiotic sensitivity Bacterial Morphology

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Difference between Gram (+) and Gram (-) Bacterias ) Gram Positive Gram Negative 2 layers: 3 layers: 1. inner cytoplasmic 1. inner cytoplasmic membrane membrane 2. outer thick 2. thin peptidoglycan layer peptidoglycan (5-10%) 3. outer membrane with layer (60-100%) lipopolysaccharide (LPS) Low lipid content High lipid content NO endotoxin (except Listeria Endotoxin (LPS) lipid A monocytogenes) No periplasmic membrane Periplasmic space NO porin channel Porin channel orin Vulnerable to lyzozyme and Resistant to lyzozyme and penicillin attack penicillin attack 6 gram-positive organisms that cause disease in humans 2 gram-positive cocci Streptococcus Staphylococcus 4 gram-positive bacilli Bacillus Produce spores Clostridium Corynebacterium Listeria Rest of other bacterial organisms are gram-negative gram Gram Positive Bacteria: Purple/ Blue

Gram positive bacilli (ex. Bacillus anthracis)

Gram positive cocci (Ex. Staphylococcus aureus) Gram Negative Bacteria: Pink/ Red

Gram negative diplococci (ex. Neisseria)

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Gram negative rods (ex. Escherichia coli) Gram Staining 1. Crystal Violet Dye Stains all cells blue/purple 2. Iodine solution Mordant Form crystal violet-iodine complex 3. Organic Solvent: acetone or ethanol Gram negative colorless Gram positive remain blue Solvent extracts the blue-dye complex from the lipid rich, thin-walled gram negative 4. Red dye Safranin Stains decolorized gram (-) red or pink Gram (+) remains blue If step 2 is omitted and Grams iodine is not added, gram (-) bacteria stain blue rather than pink. This is because the organic solvent removes the crystal violet-iodine complex but not the crystal violet alone. Gram (+) bacteria also stain blue when iodine is not added.

Culture Medium Nutrient material prepared for the growth of microorganisms in the laboratory Inoculum Microbes introduced into a culture medium to initiate their growth Culture Microbes that grow and multiply in or on culture medium Inoculation of Culture Media Inoculation of a liquid medium Involves adding a portion of the specimen to the medium

Inoculation of a solid or plated medium Involves the use of a sterile inoculating loop to apply a portion of the specimen to the surface of a medium

MICROBIAL PHYSIOLOGY A. ENCOURAGING GROWTH OF MICROORGANISMS IN VITRO Bacterial Growth Increase in the number of microorganisms Bacterial Colony Mound of bacteria containing millions of cells
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Sterile technique When it is necessary to exclude all microorganisms from a particular area Incubation Used in culturing human pathogens Incubator Chamber that contains the appropriate atmosphere, moisture level and temperature Bacterial Population Count Number of bacterial cells in a millilitre of liquid or in a gram of solid material To determine the degree of bacterial contamination in drinking water, food, etc.

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E.g. viable plate count- determines the number of viable bacteria in a liquid sample such as milk, water, ground food diluted in water or a broth culture Bacterial Population Growth Curve Shows the growth of a bacterial population over time 4 Phases Lag Phase Time for bacteria to absorb nutrients, synthesize enzymes and prepare for cell division Little or no cell division during this phase Log Phase Logarithmic growth phase or exponential growth phase Time when growth rate is greatest Period when microorganisms are particularly susceptible to adverse conditions (e.g. radiation, antimicrobial drugs) Stationary Phase Nutrient exhaustion Waste product accumulation Harmful pH changes slow rate of cell division No. of bacteria dividing=No. of bacteria dying Death Phase Logarithmic decline phase No. of cell deaths > No. of new cells formed

B. INHIBITING THE GROWTH OF MICROORGANISMS Sterilization Complete destruction of all microorganisms Disinfection Destruction or removal of pathogens from nonliving objects by physical or chemical methods Sanitization Reduction of microbial populations to levels considered safe by public health standards Sepsis Presence of pathogens in blood or tissues Antisepsis The absence of pathogens

Chemical Agents Disinfectants Chemicals used to disinfect inanimate objects Antiseptics Solutions used to disinfect living tissues Microbiostatic Agents Chemicals or drugs used to inhibit the growth and reproduction of microorganisms Bacteriostatic Agents Drugs which inhibit the growth ad reproduction of bacteria by interfering with bacterial protein production, DNA replication or other aspects of bacterial cellular metabolism Bactericidal Agents Any substance that kills bacteria ideally and nothing else either disinfectants, antiseptics or antibiotics Physical Methods Heat Cold Dessication Radiation Ultrasonic waves Filtration Gaseous atmosphere Broad-spectrum Antibiotics Active against several types of microorganisms Acts against both gram (+) and gram (-) bacteria E.g. tetracyclines are active against many gram (-) rods, chlamydiae, mycoplasmas and rickettsiae Narrow-spectrum Antibiotics Active against only specific families of bacteria E.g. vancomycin primarily used against certain gram (+) cocci 5 Sites of Action of Antimicrobials Cell Wall synthesis Cell membrane function Nucleic acid synthesis or DNA replication Protein synthesis Synthesis of essential metabolites

Growth Inhibitors of Pathogens

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Bacteria are held by peptidoglycan, protecting against osmotic rupture

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Gram-positive bacteria: substantial layer 50 to 100 molecules thickness Gram-negative bacteria: only 1-2 molecules thick Outer lipopolysaccharide layer is present on top of peptidoglycan Mechanism of Action of Anti-Microbial Agents Inhibition of Cell Wall Synthesis Can inhibit peptidoglycan synthesis in 2 different manners: a. Inhibit synthesis of the linear strands (e.g. vancomycin) b. Inhibit cross-linking of the strands (e.g. Beta-lactams - penicillins and cephalosporins) Disrupt Cell Membrane Function Phospholipids of gram (-) bacteria Sterols of eukaryotic fungi ergosterol Inhibit Nucleic Acid Synthesis Quinolones inhibit DNA gyrase Prevents relaxation of supercoiled DNA during replication Rifampicins inhibits bacterial RNA polymerase mRNA synthesis Inhibit Protein Synthesis Targets of antimicrobial agents that inhibit protein synthesis: o 1. 30S subunit (small subunit) a. Aminoglycosides and Streptomycin bind irreversibly to the 30S subunit and prevent formation of the initiation complex - stop protein synthesis o 2. 50S subunit (large subunit) a. Erythromycin and Chloramphenicol bind reversibly to 50S - inhibit formation of growing polypeptide chain Inhibit Folic Acid Synthesis Two antimicrobial agents that inhibit folic acid synthesis: Sulphonamide- sulfa drug Trimethoprim

MICROBIAL ECOLOGY Symbiosis Close and long-term relationships between different biological species Neutralism relationship between individuals of different species where both individuals derive a benefit. Commensalism Relationship between two living organisms where one benefits and the other is not significantly harmed or helped. Mutualism Parasitism Relationship is one in which one member of the association benefits while the other is harmed Synergism NORMAL MICROFLORA Role of Indigenous Flora Denotes the population of microorganisms that inhabit the skin and mucous membranes of healthy persons Roles: Plays role in maintaining health normal function (ex. GIT flora synthesizes Vit.K and aids in absorption of nutrients) Prevents colonization of pathogens and disease thru bacterial inteference Bacterial Interference Microorganisms compete for receptors or binding sites on host cells Competition for nutrients Mutual inhibition by metabolic or toxic products

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When do normal flora become pathogenic? Normal flora may produce disease when introduce into the bloodstream or tissues Streptococcus viridans (resident of upper respiratory tract) Large #s are introduced into the bloodstream following tooth extraction or tonsillectomy May settle on deformed or prosthetic valves infective endocarditis Predominant normal flora at various sites Skin Staphylococcus epidermidis Corynebacterium Proprionibacterium Micrococcus Yeasts Conjunctivae Staphylococcus epidermidis Nose and nasopharynx Staphylococcus epidermidis Staphylococcus aureus Streptococcus species Mouth and Oropharynx S. epidermidis Non-group A streptococci Streptococcus pneumoniae Streptococcus mitis Stretococcus salivarius Neisseria Haemophilus Veillonella Bacteroides Fusobacterium Treponema Lactobacillus Yeasts Small intestine Lactobacillus Streptococcus species Enterococcus Veillonella Actinomycetes Yeasts Large intestine Bacteroides Clostridium Fusobacterium Eubacterium Bifidobacterium Lactobacillus Peptostreptococcus Enterococcus Enterobacteriaceae Gut Corynebacterium Alpha-hemolytic & Nonhemolytic streptococci Staphylococcus epidermidis
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Enterococcus Lactobacillus Mycobacterium smegmatis Enterobacteriaceae Bacteroides Fusobacterium

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