Chapter 4-5 Lecture PDF

Chapter 4 - 5 1  What nutrients do microbes need to grow? Essential nutrients: microbes need but cannot make Need of nutrients: Increase biomass Gain energy...

Chapter 4 - 5 1  What nutrients do microbes need to grow? Essential nutrients: microbes need but cannot make Need of nutrients: Increase biomass Gain energy 2 Building the biomass Source of Carbon Heterotrophy Autotrophy Organic compounds as carbon source CO2 as carbon source Gain of the energy Source of Energy Phototrophy Chemotrophy Absorption of light Chemolithotrophy Chemoorganotrophy 3  Nutritional Categories of Microbes by Energy and Carbon Source Category Energy Source Carbon Example Source Autotroph Photoautotroph Sunlight CO2 Photosynthetic organisms, such as algae, plants, cyanobacteria Chemoautotroph: Organic compounds CO2 Methanogens Chemoorganic autotrophs Chemoautotroph: Inorganic compounds CO2 Thiobacillus, “rock-eating” Chemolithoautotrophs (minerals) bacteria Heterotroph Photoheterotroph Sunlight Organic Purple and green photosynthetic Photoorganotroph bacteria Chemoheterotroph Metabolic conversion of Organic Protozoa, fungi, many bacteria, Chemoorganoheterotroph the nutrients from other animals organisms Chemoheterotroph: Metabolizing the Organic Fungi, bacteria (decomposers) Saprobe organic matter of dead organisms Chemoheterotroph: Parasite Utilizing the tissues, Organic Various parasites and pathogens; fluids of a live host can be bacteria, fungi, protozoa, animals 4  Energy gained by phototrophy or chemotrophy is stored for later use. ATP synthesis Chemical or Pump protons Electrochemical potential Active transport light energy outside the cell Proton motive force Rotation of flagella Cell Membrane Cytoplasm https://doi.org/10.1002/med.21946 5 Incorporation of Nitrogen in Biomass Denitrification Nitrogen fixation Nitrification 6 How do bacteria gather nutrients? Nutrient Transport 7 https://www.sciencefacts.net/active-and-passive-transport.html  Facilitated Diffusion Compounds too large or too polar to diffuse on their own Higher concentration Facilitated diffusion transporter Lower concentration Facilitated Diffusion of glycerol through GlpF 8  Active Transport Coupled transport (energy from chemical gradient) Symport Antiport Example: LacY (Lactose permease) Example: Na+/H+ antiporter In both symport and antiport, substrate (B, blue) is taken up against its gradient using the energy released by substrate (A, red) traveling down its gradient. 9  Maintaining ion gradients Ion transporters work collaboratively to maintain ion gradients 10  Active Transport ABC transporters (ATP-binding cassette superfamily ) Carry out import and export of large variety of compounds - Simple carbohydrate like maltose, arabinose, galactose - Long chain polysaccharides like alginate Multidrug Efflux pumps - Protect microbes from hazardous chemicals - Export antibiotics (resistance to those drugs) ABC transporter - Transmembrane domain (TMD) - Nucleotide binding domain (NBD) ATP biding cassette Uptake system has substrate binding protein (SBP) periplasm (gram negative), tethered to cell surface (gram positive) https://www.cusabio.com/Transmembrane/A-transport-machine-ATP-binding- cass ette.html ABC transporter in a Gram-negative organism 12  Scavenger Siderophores and Iron Transport Siderophores: very high affinity for soluble ferric ion in the environment Bacteria without siderophores might acquire iron from the receptors on their surface (Neisseria gonorrheae binds to human iron complex like lactoferrin or transferrin) Siderophores and Iron Transport in E. coli 13  Active Transport Group Translocation: Phosphotransferase system (PTS) Alters the substrate during transport by attaching a new group (example: Phosphate) - Uses energy to chemically alter the solute - Parent solute is always moving down its concentration gradient Group Translocation: Phosphotransferase system (PTS) of E. coli 14  Environmental Influences Basic environmental parameters ❖ Temperature ❖ pH ❖ Osmolarity ❖ Oxygen ❖ Pressure 15  How do microbes react to hot and cold? Temperature effects on physiology of microbes. Cardinal Temperatures Minimum temperature: the lowest temperature that permits a microbe’s continued growth and metabolism Maximum temperature: the highest temperature at which growth and metabolism can proceed Optimum temperature: temperature that promotes the fastest rate of growth and metabolism 16  Microbial Classification by Growth Temperature Mesophiles (most human pathogens) Optimal: 20oC – 40oC Minimum: 15oC, Maximum: 45oC Psychrophiles (Rarely pathogenic) Optimal: about 15oC Minimum: upto -20oC, cannot grow above 20oC Storage at refrigerator temperature causes them to Psychrotolerant (pshychrotrophs) grow rather than inhibiting them Cold resistant mesophiles Optimal: 20oC – 35oC, can grown below 7oC Listeria monocytogenes can grow at refrigeration temperature How do these organisms grow so well in the cold? ## proteins of psychrophiles are more flexible, require less energy (heat to function). Membrane more fluid at low temperature. 17 ****** contain antifreeze proteins and other cryoprotectants so they can grow but will not freeze.  Microbial Classification by Growth Temperature Thermophiles Optimal: 50oC – 80oC Hyperthermophiles Optimal: 121oC or higher Thermoduric Specially adapted membranes and protein sequences: enzymes (Thermozymes) do not unfold easily and hold their shape at higher temperatures - contain chaperone proteins that help refold cellular proteins - numerous DNA binding proteins to stabilize DNA - more saturated linear lipids into their membrane (more stable membranes) - hyperthermophilic archaea have lipid monolayers not bilayers, isoprene chains instead of fatty acids Thermophilic DNA polymerase is used in PCR to amplify DNA (in thermocycler) 18 Microbial Classification: Adaptation to Pressure Barophiles or piezophiles (>380 atm) Can grow in range of 1-50Mpa (10 - 500 atm) Survival at high pressure: - membrane with high levels of polyunsaturated fatty acids to increase membrane fluidity - pressure adapted internal structure  Microbial Classification: Osmolarity Most microbes on land or freshwater require water activity > 0.95. Halophiles Require high salt concentration (water activity 0.94 - 0.75) to grow - Microbes increase their intracellular osmolarity with compatible solutes during their survival in hypertonic environment - special ion pumps to excrete sodium ion and replace with compatible solute like potassium and organic compatible solutes like proline, betaine, glutamic acid Halotolerant High salt concentration not required but able to grow 20 Microbial Classification: Optimal Growth pH Majority of enzymes tend to operate between pH 5 and 8.5 Neutralophiles (most human pathogens) Optimal: pH 5 to 8 Acidophiles (often lithotrophs) Optimal: pH 0 to 5 - altered membrane lipid profiles (tetraether lipids) Alkalophiles (often halophiles) Optimal: pH 9 to 11 - presence of acidic polymers and an excess of hexosamines in the peptidoglycan - high level of diether lipids in membrane 21  Microbial Classification: Oxygen Strict Aerobes Microaerophiles Grown only in the presence of O 2 Only small amount of O2 Facultative microbes Strict Anaerobes With or without O2 Grow only without O2 22  Oxygen Has Benefits and Risks Benefits Risks Aerobes use O2 as the terminal electron acceptor for their Electron Transport System Reactive Oxygen species (ROS) severely damage cells (ETS) during aerobic respiration. O2 as the terminal electron acceptor Microbes (aerobes) destroy ROS with enzymes like superoxide dismutase, peroxidase and catalase 23 Why do anaerobes perish in oxygen? Lack of enzymes to destroy ROS molecules produced by own metabolism ROS might not allow them to grow Dissolved oxygen interferes with the use of non-oxygen electron acceptors which is needed to make energy (anaerobic respiration or fermentative metabolism) Oxygen directly oxidizes metal cofactors of enzymes in anaerobes and inactivating them. Facultative anaerobes, aerotolerant anaerobes and microaerophilic microbes have enzymes to destroy ROS (levels of these enzymes vary) Anaerobic jar or Anaerobic chamber are used to grow anaerobic microbes in the lab 24  How do we capture and study the bacteria? Bacteria can be cultured using culture media at lab providing optimal growth environment Types of culture media Physical nature Chemical composition Function 1. Liquid media 1. Defined (synthetic ) media 1. Supportive media / Basal 2. Semi solid media 2. Complex media media 3. Solid media 2. Enriched media 4. Biphasic media 3. Enrichment / Selective media 4. Differential media 5. Indicator media 6. Transport media 7. Anaerobic media  Culture media: Physical nature Liquid Semi-solid Solid No agar Contains 0.5% agar Most commonly used Broth Soft consistency 1 – 2% agar Preparation of inoculum To demonstrate motility of Separate and isolate mixed High growth of bacteria bacteria bacteria Enrichment e.g. Sulfide Indole motility Colony morphology, e.g. Nutrient Broth (SIM) medium pigmentation, hemolysis can be observed e.g. Nutrient agar, Blood agar  Culture media: Chemical Composition Synthetic / Defined Complex Exact chemical composition is known Contains some ingredients of unknown composition Minimal defined medium (contains only Used for fastidious microbes with complicated those nutrients that are essential for nutritional needs growing a given microbe Contains complex media contain peptone, meat extracts e.g. M9 medium (defined) and yeast products e.g. Nutrient broth, Blood agar , Tryptic Soy broth  Culture media: Function Supportive media Enriched media Support the growth of many microbes When blood, serum, egg yolk, and other nutrients Called general purpose media are added to supportive media e.g. Tryptic soy agar/ broth e.g. Blood agar, Chocolate agar etc. Selective media Enrichment media Allows growth of particular microorganisms Similar to selective media but designed to increase while suppresses the growth of others the numbers of desired organisms to detectable e.g. Thayer Martin Agar (N. gonorrhoeae) levels Mannitol Salt Agar (S. aureus) Culture in liquid form Potassium tellurite medium (C. diphtheriae) e.g. selenite F broth, tetrathionate broth (recover MacConkey’s Agar (Enterobacteriaceae pathogens from fecal sample) members – bile salt inhibit gram positive Bolton broth with antibiotics for Campylobacter bacteria enrichment from food products Lowenstein Jensen medium (M. tuberculosis)  Culture media: Function Differential media Differentiate bacteria from others growing on the same plate Differentiate bacteria based on their colony colors and even gives tentative identifications e.g. Blood agar (both differential medium and enriched media) e.g. MacConkey agar (both differential and selective media) MacConkey agar : contains lactose and neutral red dye – lactose fermenter (pink colonies, non lactose fermenter (colorless), inhibits the growth of most gram-positive bacteria MacConkey agar (both differential and selective media)  Culture media: Growth Factors Growth Factors: specific nutrients required for growth of specific microorganism but not required by others Growing uncultured microbes (iChip: isolation chip method) Growing bacteria in their natural habitat iChip method to culture previously uncultured soil bacteria  Some culture techniques: Isolation of bacteria Isolation of pure colonies Streak Plating ( Dilution Streaking) In most cases, each colony on an agar plate represents one viable organism (or colony-forming unit CFU) present in the sample https://microbenotes.com/streak-plate-method-principle-methods-significance-limitations/#what-is-streak-plate-method  Some culture techniques: Isolation of bacteria Isolation of pure colonies Plating Serial dilution method / Spread Plating / viable count Used for isolation of bacteria Bacterial growth measurement Viable Serial dilution Count  Techniques for counting bacteria 1. Direct counting of live and dead bacteria Using hemocytometer (Petroff-Hausser counting chamber) Fluorescence microscopy Flow Cytometry (bacterial cells labeled with fluorescent antibody or chemical) 2. Viable counts Pour plate method / Spread plate method 3. Biochemical Assays Assays to measure cell biomass, protein content, or metabolic rate measurement 4. Optical density measurement Estimation of bacterial population size based on optical density (light scattered by bacteria) Spectrophotometers OD600 / OD 600 nm 4. Molecular method Quantitative PCR for the quantification of DNA content Viability PCR (uses propidium dye that penetrates the membranes of dead cells and binds to their DNA so that DNA only from viable cells will be recognized and amplified)  Growth Cycle of Bacteria ❖ Planktonic cells : unicellular life phase, in which the cells are free-swimming (planktonic) ❖ Biofilm : an assemblage of surface-associated microbial cells that is enclosed in an extracellular polymeric substance matrix ❖ Exponential growth: (In optimal growth condition) - The growth of a bacterial population occurs in a geometric or exponential manner: with each division cycle (generation), one cell gives rise to 2 cells, then 4 cells, then 8 cells, then 16, then 32, and so forth. ❖ Generation time: - Within an environment of unlimited resources, bacteria divide at a constant interval called generation time - depends on various environmental parameters - also known as doubling time In optimal growth condition, E. coli has doubling time 20 minutes  Phases of Bacterial Growth in Batch Culture (Growth Curve) 3. Stationary Phase: Cells stop growing and shut down their growth machinery while turning 4. Death Phase: Cells die, on stress responses to help negative exponential curve retain viability Batch Culture ❖ Lag Phase ❖ Log Phase ❖ Stationary Phase ❖ Death Phase Theoretical growth curve of bacterial suspension 1. Lag Phase: Bacteria are preparing their cell machinery for growth 2. Log Phase: Exponential growth of bacteria (straight line on a logarithmic scale)  Chemostats and continuous culture Maintaining bacterial poupulations in exponential phase Batch Culture ❖ Chemostat - a continuous culture system - ensures lograrithmic growth by constantly adding and removing equal amounts of culture media Chemostats and continuous culture  Bacterial Growth ❖ Biofilm : an assemblage of surface-associated microbial cells that is enclosed in an extracellular polymeric substance matrix Biofilm Life Cycle ❖ Initiation ❖ Maturation ❖ Maintenance ❖ Dissolution Biofilms that forms on teeth is called plaque  Bacterial Growth: Biofilm Once population reaches to quorum, chemical signal achieves a concentration Quorum Sensing: communication of which triggers genetically regulated changes bacterial cells with each other by sending that cause cells to bind tenaciously to the and receiving chemical signals surface and to each other Formation of thick extracellular polymeric substances or Bacterial initiate exopolysaccharides (EPS) that entrap organic (DNA and proteins) biofilms when and inorganic materials nutrients are plentiful Biofilm formation induced - different environmental signals pH, iron concentration, temperature, oxygen availability, presence of certain amino acids Environmental signal induces genetic Planktonic cells start to attach to program in planktonic Surface coating with organic Mature Biofilm: complex 3D nearby inanimate surfaces by means of monolayer of polysaccharides shape with channels for cells flagella, pili, lipopolysaccharides, cell appendages or charge interactions or glycoproteins nutrient flow Bacterial Growth: Biofilm Beginning of starvation or oxygen depletion, some cells start making enzymes that dissolve matrix  Biofilm formation: a challenge Biofilm formation: - increased antibiotics tolerance - decreased immune system - persistent inflammation and tissue damage - diagnostic problems  Endospore formation (Heat and desiccation resistant) Spore formers (Pathogenic) - Clostridium tetani (tetanus) - Clostridium difficile (pseudomembranous colitis) - Bacillus anthracis (anthrax) - Starvation initiates endospore formation from vegetative cells - Proper nutrient conditions initiates germination of endospores The seven stages of endospore formation.  Control of Microbes Common terms used to describe antimicrobial control measures 1. Sterilization: process by which all living cells, spores, and viruses are destroyed on an object 2. Disinfection: killing, or removal of disease producing organisms from inanimate surfaces, (not necessarily result in sterilization) 3. Antisepsis: removing pathogens from surface of living tissues, such as the skin 4. Sanitation: reducing the microbial population to safe levels and usually involves both cleaning and disinfecting an object  Control of Microbes Germicide “-cidal”: killing cells “- static”: (inhibiting growth) Germicidal: kill pathogens (many nonpathogens), not necessarily kill spores e.g. Bactericide, fungicide, algicide, virucide Chemical agents that prevent the growth but do not kill e.g. bacteriostatic, fungistatic The D-value (D100) is the time required for an agent or condition to kill 90% of cells (that is, the time it takes for the viable cell count to drop by one log10 unit).  Control of Microbes Physical Chemical Biological 1. Heat Chemical agents 1. Probiotics 2. Cold a) Phenolics 2. Phage therapy 3. Filtration b) Metals 4. Pressure c) Halogens 5. Desiccation d) Alcohols 6. Irradiation e) Surfactants 7. Sonication f) Aldehydes 8. Filtration g) Bisbiguanides h) Alkylating liquids and gases i) Peroxygens j) Food preservatives Steam Autoclave  Pasteurization Milk Pasteurization (U.S. government approved methods) ❑ LTLT (Low temperature, long time): 63oC for 30 minutes ❑ HTST (High temperature, short time): also called flash pasteurization: 72oC for 15 seconds ❑ UHT (Ultra high temperature): 138oC for 1-2 seconds (produces nearly sterile milk, shelf life up to 6 months) https://openstax.org/books/microbiology/pages/13-2-using-physical-methods-to-control-microorganisms  https://openstax.org/books /microbiology/pages/13-2-using-physical-methods-to-control-microorganisms (a) UV radiation causes the formation of thymine dimers in DNA, leading to lethal mutations in the exposed microbes. (b) Germicidal lamps that emit UV light are commonly used in the laboratory to disinfect equipment.  Chemical agents Factors influencing the efficacy of a given chemical agent 1. The presence of organic matter 2. The kinds of organisms present 3. Corrosiveness 4. Stability, odor, and surface tension The phenol coefficient: is determined by comparing the highest dilution of a test agent capable of killing pathogens to that of phenol Chemical Disinfectants Chemical Mode of Action Example Uses Phenolics Cresols Disinfectant in Lysol o-Phenylphenol Prevent contamination of crops (citrus) Denature proteins and disrupt membranes Hexachlorophene Antibacterial soap Triclosan pHisoHex for handwashing in hospitals Metals Topical antiseptic Mercury Treatment of wounds and burns Silver Prevention of eye infections in newborns Copper Bind to proteins and inhibit enzyme activity Antibacterial in catheters and bandages Nickel Mouthwash Zinc Algicide for pools and fish tanks Containers for long-term water storage Halogens Topical antiseptic Hand scrub for medical personnel Iodine Water disinfectant Oxidation and destabilization of cellular Chlorine Water treatment plants macromolecules Fluorine Household bleach Food processing Prevention of dental carries  Chemical Disinfectants Chemical Mode of Action Example Uses Alcohols Ethanol Denature proteins and disrupt Disinfectant Isopropanol membranes Antiseptic Surfactants Soaps and detergent Lowers surface tension of water to Disinfectant Quaternary ammonium salts help with washing away of microbes, Antiseptic and disruption of cell membranes Mouthwash Bisbiguanides Chlorhexidine Oral rinse Disruption of cell membranes Alexidine Hand scrub for medical personnel Alkylating Agents Formaldehyde Disinfectant Glutaraldehyde Tissue specimen storage Inactivation of enzymes and nucleic o-Phthalaldehyde Embalming acid Ethylene oxide Sterilization of medical equipment β-Propionolactone Vaccine component for sterility  Chemical Disinfectants Chemical Mode of Action Example Uses Peroxygens Hydrogen peroxide Antiseptic Peracetic acid Oxidation and destabilization of cellular Disinfectant Benzoyl peroxide macromolecules Acne medication Carbamide peroxide Toothpaste ingredient Ozone gas Supercritical Gases Food preservation Penetrates cells, forms carbonic acid, lowers Carbon dioxide Disinfection of medical devices intracellular pH Disinfection of transplant tissues Chemical Food Preservatives Sorbic acid Benzoic acid Propionic acid Potassium sorbate Decrease pH and inhibit enzymatic function Preservation of food products Sodium benzoate Calcium propionate Sulfur dioxide Nitrites Natural Food Preservatives Nisin Preservation of dairy products, meats, and Inhibition of cell wall synthesis (Nisin) Natamycin beverages  Biological Control of Microbes 1. Probiotics – (Use of microbial competition) 2. Phage Therapy – possible alternative to antibiotics https://doi.org/10.3390/antibiotics11101406 https://foodmicrobiology.academy/2020/08/04/where-to-obtain-maximum-probiotic-benefits/comment-page-1/  Self Review 1. Define Generation time. What are the four phages in bacterial growth in batch culture. 2. Describe about the biofilm life cycle of bacteria. Why is biofilm formation by pathogenic bacteria a challenge in clinical field? 3. Bacterial endospore formation during starvation. 4. What are the physical methods of microbial control? 5. What are the chemical disinfectants (groups) used for microbial control? 6. What are the influencing factors for the efficacy of chemical disinfectants? 7. What are the biological control of microbes?  Self Review Define essential nutrients, macronutrients, and micronutrients with examples. Classify microbes according to their source of carbon and source of energy. Differentiate between passive and active transport in bacteria. Difference between symport and antiport. Working mechanism of ABC transporters, siderophores, and group location. 63  Self Review What are the common environmental influences that affect microorganism? Differentiate mesophile, thermophile and psychrophile. Define barophile and barotolerant. Classify bacteria according to their growth in variable pH and osmolarity. Differentiate among strict anaerobe, facultative, microaerophiles, and strict aerobes with examples. What are the enzymes responsible to protect microbes from ROS? Why do anaerobes have difficulty growing in aerobic condition? Identify the different types of culture media according to physical nature, chemical composition, and function. Differentiate selective, enrichment, and differential media with examples. What is growth factor? Give some examples. How iChip method is helping to grow previously uncultured bacteria? Different culture techniques for isolation of bacteria (Streak plate method, serial dilution, viable count after spread plate or pour plate method) Different techniques for quantification of bacterial population. 64