1st Year MPharm Micro 4 (Growth) PDF

MPharm Programme Microbial growth and evolution Dr Callum Cooper [email protected]  Learning Objectives How microbes are cultured Different stages of bacterial and viral growth Introduce bacterial evolution and where it can be import...

MPharm Programme Microbial growth and evolution Dr Callum Cooper [email protected]  Learning Objectives How microbes are cultured Different stages of bacterial and viral growth Introduce bacterial evolution and where it can be important  Top 10 elements making up a bacterium Element % Dry Weight Source Function Carbon 50 Organics or CO2 Main constituent of cell Cell material and water; Oxygen 20 H2O, Organics, CO2, and O2 electron acceptor in aerobic respiration amino acids, nucleotides, Nitrogen 14 NH3, NO3, organics, N2 and coenzymes Organic compounds and cell Hydrogen 8 H2O, organics, H2 water Nucleic acids, nucleotides, Phosphorus 3 Inorganic phosphates phospholipids SO4, H2S, So, organic sulfur Sulfur 1 Proteins, several coenzymes compounds Main inorganic cations and Potassium 1 Potassium salts enzymatic cofactor Inorganic cations, enzymatic Magnesium 0.5 Magnesium salts cofactor Inorganic cations, enzymatic Calcium 0.5 Calcium salts cofactor, endospores Cytochrome component, Iron 0.2 Iron salts enzymatic cofactor  Sources of Carbon, Energy & Electrons Carbon Source Autotrophs CO2 sole or principle carbon source Heterotrophs Obtained from other organisms Energy Sources Phototrophs Light Chemotrophs Compound oxidation Electron Sources Lithotrophs Reduced inorganic compounds Organotrophs Organic molecules N.B. These terms may be joined together e.g.“chemoorganoautotroph”  In-vitro microorganism culture Two ways to culture microorganisms Liquid media (broth) Solid media (agar plates) Originally grown by Koch (late C19th) on potato slices and gelatine In liquid media bacteria grow as individual cells until available nutrients are exhausted Produces a suspension of cells (unable to distinguish between multiple cell types without further testing) On solid media bacteria and fungi form colonies with distinctive appearances In theory each colony derives from a single cell (makes culture purification easier) Formula of media can influence colony appearance (selective and differential media) Solid media formulas generally the same as liquid media with the addition of a gelling agent (agar)  Undefined Vs Defined media Undefined media contains chemically undefined yeast/vegetable/meat extracts and digested proteins Batch-batch variation and reproducibility Useful for routine growth applications Defined media (synthetic media) all components are chemically defined Highly reproducible Can be rich or minimal depending on requirements Undefined Media Defined Media TSA agar Sabouraud dextrose agar M9 minimal media Tryptone 15g Dextrose 40g M9 Salts (Na2HPO4, KH2PO4, NaCl, NH4Cl) Soytone 5g Peptone 10g MgSO4 NaCl 5g Agar 20g CaCl2 Agar 15g dH2O to 1L Carbon Source (e.g. glucose) dH2O to 1L pH 5.6 dH2O pH 7.5-8  Atmospheric requirements 1. Obligate aerobe e.g. Mycobacterium tuberculosis Cannot survive without oxygen 2. Obligate anaerobe e.g. Clostridium difficilie Cannot survive (killed) in the presence of oxygen 3. Facultative aerobe e.g. Staphylococcus aureus and E. coli Can use grow in the presence of oxygen or produce energy by fermentation 4. Microaerophile e.g. Camplylobacter jejuni Requires reduced oxygen content (increased CO2 content) in order to survive 5. Aerotolerant anaerobe e.g Streptococcus mutans Can tolerate oxygen in the air but produces energy by fermentation  Anaerobic microorganisms Several methods available for Anaerobic (or aerotolerant) culture: Anaerobic cabinet Basically big isolator cabinets 95% N2 5%H2 with a palladium catalyst Can be under positive pressure GasPak sachets Produces CO2 and H2 from breakdown of citric acid, cobalt chloride and sodium borohydride. Candle extinction Uses up oxygen by burning of a candle in the jar GasPak and Candle methods rarely produce a true anaerobic environment  Bacterial cell division Each cell is able to survive and reproduce independently Most bacteria reproduce by DNA binary fission Some reproduce by budding Time taken to reproduce is called generation time Varies wildly between species Escherichia coli ~30 min Mycobacterium leprae ~14 days 4 distinct phases to bacterial growth https://www.youtube.com/watch?v=gEwzDydc iWc  Bacterial growth Lag phase No immediate increase in cell number Old cells depleted & need time for synthesis of new cell components / metabolites Log number viable cells lag Time  Bacterial growth Exponential (log) phase Growth & division at maximum possible rate given genetic potential & environmental conditions Regular doubling time Log number viable cells log Time  Bacterial growth Stationary phase In closed system nutrients become depleted & waste products build-up Growth ceases (or is balanced by death) Morphological and metabolic changes (e.g. secondary metabolism) stationary Log number viable cells Time  Bacterial growth Death phase (senescence) Severe nutrient deprivation Build-up of toxic waste products Viable cell numbers decline at an exponential rate Log number viable cells death Time  Why does this matter? Secondary metabolism Log number viable cells stationary death log lag Time  Microbial metabolism Primary metabolism Includes major metabolic pathways Energy production and release Cell component synthesis Enzyme production Secondary metabolism Non-essential metabolic pathways Includes production of natural products e.g. antibiotics Production of secondary metabolites in disease states which can increase pathogenicity e.g. pyocyanin  Laboratory scale culture- batch culture Flask cultures- closed systems Used for optimisation of steps Nutrient availability limited Atmosphere limited due to diffusion at liquid surface Limited product production Not suitable for industry Lab scale only  Laboratory scale culture- Continuous culture Chemostats- Open systems Allows for highly controlled growth Nutrients supplied at constant rate When at steady state  = D = F/V Scaling up production  Bulk culturing Three growth modes: Batch - full at start Fed batch - fill until vessel full Continuous - fill and overflow Important criteria: Maintain adequate mixing Maintain high oxygen levels - if aerobic Control pH Control temperature Control foam Initial starting concentration  Viral replication Viral replication relies on the subversion of host replication machinery True in both prokaryotic and eukaryotic viruses In bacteriophages, subversion leads to cell destruction Lytic replication In Eukaryotes, viruses often bud rather than destroy the cell Influenza HIV  Viral replication Some bacteriophages can also integrate their genome into the host Lysogenic replication Replicates alongside host Can enter lytic replication in favourable conditions Viruses which can lie dormant can also cause human disease; Herpes simplex virus Human papillomavirus Important in the spread of bacterial genes (horizontal gene transfer)  Bacterial evolution Evolution: changes in population over time Can arise due to a variety of causes Acquisition of new genes Mutation of existing genes Results of bacterial evolution can be a good or bad thing; Increased product yield Become pathogenic Increase in resistance to treatment Rate of evolution varies by organism More complex the organism, slower the rate of evolution  Bacterial evolution: Mutation Mutation: Permanent change in a single cell, does not necessarily cause any noticeable change or get passed on; UV irradiation Chemical exposure Poor genome copy Different types of mutation; A harmful, or deleterious, mutation decreases organism fitness. A beneficial, or advantageous mutation increases organism fitness. Mutations that promote desirable traits (e.g increased product yield) Can include things which are harmful to other organisms A neutral mutation has no harmful or beneficial effect. Such mutations occur at a steady rate  Bacterial evolution: DNA acquisition Three main mechanisms by which bacteria can evolve; Transformation: Direct uptake of DNA through cell membrane Transduction: Introduction of genetic material via a viral vector Conjugation: Transfer of genetic material between two directly connected bacteria WHY SHOULD I CARE?? Bacterial evolution: Antimicrobial resistance Bacterial evolution: Antimicrobial resistance  Bacterial evolution: Antimicrobial resistance Antibiotic usage can act as a selective pressure on bacteria Removes competition for resistant cells by killing susceptible cells Unlikely to be due to a single mutation; Harvard Med School: https://www.youtube.com/watch?v=plVk4NVIUh8  Strain Improvement Initial strains may produced products at low concentrations Need to boost efficiency of production More cost effective Bacterial strains can improve naturally Spontaneous mutation (usually random and infrequent) Can also make things worse! Exposure to mutagens can increase frequency of mutation UV/Chemical exposure Random mutagenesis Mutants can be picked and assessed for increased production Genetic modification of organisms Targeted mutagenesis: Add/remove/alter genes to improve overall yield  Improvement in yields: Penicillium chrysogenum 1943 S X UV NRRL 1951 NRRL 1951 B25 X-1612 Q176 UV, N, S 53-399 S = Spontaneous mutants X = X-ray mutagenesis UV = UV mutagenesis N = mustard gas Today Used a combination of spontaneous and random mutagenesis to increase production by a factor of 20 Production is in units of activity/mL  Heterologous Gene Expression Insulin originally derived and purified from animal sources First human-identical insulin (humulin) produced in 1978 by Genetech then licenced to Eli Lilly Cloned human insulin gene into E.coli GMO Human insulin E. coli gene Plasmid Humulin Humulin and similar products been in use > 25 years  Summary Requirements for bacterial growth Different stages of bacterial and viral replication How bacteria can evolve Examples of why bacterial evolution is important Link between evolution and antibiotic resistance  Extra Reading Prescotts Microbiology: Part II; Section 6 & 7 Brock Microbiology: Part I; Section 3 and 5