Agricultural Microbiology MICR20010 Past Paper PDF

MICR20010 Agricultural Microbiology Dr. Tadhg Ó Cróinín  Praccal MCQ Exam Complete Praccal Online MCQ exam results will be released this evening. Output is a percentage which is converted to a grade using the alternave linear scale. Percentage to Grade 3 ...

MICR20010 Agricultural Microbiology Dr. Tadhg Ó Cróinín  Praccal MCQ Exam Complete Praccal Online MCQ exam results will be released this evening. Output is a percentage which is converted to a grade using the alternave linear scale. Percentage to Grade 3  Praccal MCQ Exam Complete Praccal Online MCQ exam results will be released this evening. Output is a percentage which is converted to a grade using the alternave linear scale. Remember you sll have 70% of the grade to make up in the *nal exam….. Study the lecture slides!!!…  MICR20010 - remaining lectures Lecture 10 – Microorganisms and Disease Lecture 11 – The Immune System Lecture 12 - Pathogenic Bacteria Lecture 13 – Pathogenic Fungi and Viruses Lecture 14 – Anbioc Resistant Microorganisms Lecture 15 – Microbiology in the Food Industry – The Fungi Lecture 16 – Microbiology in the Food Industry - Fermentaons Lecture 17 – The Nitrogen Cycle  Fermentaons – Food and Drink.. An ancient process which pre-dates the science of microbiology Crical for the producon of Beer, Wine and many Dairy products. Fermentaon pathways  Fermented Vegetables Soya bean fermenta on – Koji - Aerobic fermentaon with Aspergillus – Moromi - Anaerobic Tetragenococcus halophila lowers pH as acid tolerant yeasts produce avour SaurKraut – Lactobacilli fermentaon of shredded cabbage – Salt added to prevent Gram negave contaminants  Fermented Meat and Fish Various Dry Sausages – Salami, Pepperoni, Bologna etc. – Staphylococcus, Pediococcus, Micrococcus, Lactobacilli. Fish Sauces – Variety of sauces and pastes used in Asian cooking. – Incubaon of *sh and shrimps and salt into sealed vessels to allow natural micro?ora work (mostly S. carnosus and Staphylococcus piscifermentans)  CoBee and Cocoa Coee - Co ea arabica – Wet or dry process to obtain bean from berry – Wet process uses indigenous fungi and bacteria secreng proteolyc enzymes – Then an acidic fermentaon by lacc acid bacteria Cocoa - Theobrama cacao – To release the beans from the pods – A sequence of fermentaons involving yeasts, lacc acid and acec acid bacteria Saccharomyces cerevisiae  Brewing Ancient use of fermentaon and most important economically. Primarily S. cerevisiae but occasionally Zymomonas Zymomonas – African palm wine or Mexican pulque.  The Basics Aerobic pathway is preferred by yeast so need to keep oxygen low to force fermentaon and producon of alcohol. Carbon dioxide produced must be allowed to escape… Glucose substrate is needed to provide the starng material. DiBerent approaches give diBerent results.  Wine making Grapes used as the sugar source. Fructose + Glucose and natural acidity Soil, climate, grape variety, method of pressing, primary and secondary fermentaons. Red wine - made with skins - anthocyanins  Making the “Must” Grapes crushed mechanically or by hand (or foot). Fermentaon by indigenous yeasts or starter cultures. Rate decided by temperature, pH, inial sugar content and yeast strain.  The Fermentor Typically wooden fermentors but now stainless steel mostly Cooling required for fermentaon as heat produced (1.3oC per 10g/L) Reduce un-wanted yeasts using SO2. Add starter culture of S. cerevesiae. Secondary fermentaon? Clarifying agents, *ltraon and boJling. Wine Producon  Cider? Cider producon not unlike that of wine. OLen more sugar is added to help the reacon but apple juice does have high sugar levels. But what about Beer? Where’s the sugar?  The problem with Barley Barley unsuitable as sugar source as 65% Starch Starch must be accessed and converted to a usable sugar Malting Process - Partial Germination Malting process used to generate enzymes (amylases) and carbon source useful for fermentation Green malt, produced after ve days of germination, is kiln dried and partly cooked in a forced ow of hot air (C). Kilning used to preserve the malt  Making Malt - The Steps Soak or Steep for 2 days at 10-16oC. Occasionally aerate Germinate for 3-5 days at 16-19oC on malng ?oors. Aerated and turned mechanically Kiln by drying at 50-60oC and then cure at 80- 110oC - arrests development, stops enzyme denaturaon, adds ?avour Making Wort Malted grains provide both the enzymes and the substrate but adjuncts can be added. Starch converted to simple sugars. Wort prepared as the glucose source (mashing). Then *ltered  Mashing - Making the Wort Allowing the enzymes to work on the endogenous and added substrates Various types of Mashing – Decocon mashing - First 35-40oC (protein rest), then remove some of mash and boil and re- introduce to raise temp… and repeat – Infusion mashing  But what is happening? Amylase and other enzymes breaking starch down to fermentable sugars. Many proteases also present to break down cell walls to allow access to starch. If enzymes liming then commercial enzymes can be added Finally boil the sweet wort with Hops Wort is boiled Amylases inactivated Starch breakdown now stopped alpha acids and oils from hops Kills microbes in the wort Sugars are caramelized 12 Hops are added; Bitter herb grown on a vine Have a preservative effect in the beer Stabilize flavor Hop oils have Alpha acids – bitter taste and preservative. Brewing ALer preparaon of Wort add yeast. Allow to ferment for approx 3 days where large amounts of CO2 produced Then a further 10 days Brewing: types of brewing yeasts Yeast strain type: 1. Top fermenting yeast  Remain distributed in wort  Carried to top by CO2  14-23C fermentation  Ales  S.cerevisiae Yeast strain type: 2.Bottom yeasts  Settle to bottom  6-12C fermentation  Saccharomyces carlsbergensis (budding yeast)  Larger  Differentiation of an ale yeast from a lager yeast 27oC S.carlsbergensis S. cervisiae S. Cervisiae and S. carlsbergensis will grow at 27oC S. cervisiae 37 C o S.carlsbergensis S. cervisiae will grow at 37oC S. carlsbergensis will not Brewing Allow yeast to seJle to boJom Siphon oB beer into boJles and allow to age further.  Dislled Beverages? Alcohol boils at 78 degrees Malt – Whiskey Wine - Brandy Molasses – Rum Grain/potatoes – Vodka Colour due to aging in barrels  Economic importance? We are one of the largest consumers of alcohol in Europe. What is our history in brewing like? A signi*cant growth area.. Local breweries! Scaling up Ethanol producon 50 Billion litres from the fermentaon of various feedstocks. Industrial solvent Biofuels  Vinegar Not only a condiment but an important preservave A soluon with greater than 4% Acec acid America - Cider Europe - Wine Britain - Malt Key being Acec acid bacteria Vinegar Producon Changing ethanol to Vinegar Dislled vinegar Acec acid bacteria convert ethanol to acec acid Vinegar Producon Fermented Dairy products Milk 87% water Proteins: whey and casein Fat- flavour Carbohydrates- Lactose Vitamines Cheese and Yoghurt  Microorganisms involved in Dairy Fermentaons Lactococci, Lactobacilli and S. thermophilus are the main organisms. Can be single or mixed cultures Other organisms can be used to add ?avour or texture  Curdling Milk Acidi*caon of the milk leads to the coagulaon of milk proteins - curd This can be achieved by a pure starter culture or a mixed culture. Rennet – enzyme mix that promotes curdling Tradionally Calf Chymosin but now oLen using fungal proteases. Dairy Products  A second inoculum ALer curd salted a second microbial inoculum can be added and fermentaon proceeds – Swiss Cheese - Propionibacterium freundenreichii ?avour and gas bubbles – “Blue Cheeses” - Penecillium roquefor inoculated into cheese – Camembert - Penicillium camember gives the characterisc rind and ?avor DiBerent variaons in the procedure lead to a huge variety in cheeses. Huge variaon in Cheeses  Dairy Products Other Fermented milk products – Yoghurt – Buttermilk – Sour Cream Streptococcus thermophilus Yoghurt controlled fermentation of milk – Lactobacillus bulgaricus and Lactotococcus thermophilus (also known as Streptococcus thermophilus) Take pasteurised milk add Streptococcus thermophilus Lactobacillus bulgaricus incubate 42-45C..yoghurt Symbiotic event: L.bulgaricus: proteases—peptides (stimulate) and lactic acid S. Thermophillus: acids (folic and formic) used by L bulgaricus for purine synthesis.  The Probioc World The “Good Bacteria” theory Begun by Elie MetchnikoB (1845-1916) Later pasteur instute produces “la Ferment” 1930s Minoru Shirota selects L. casei Shirota from Human feces… now produced by Yakult.  The Probioc World Bi*dobacteria and Lacc acid bacteria - Commensals found in gut (16 and 3% of normal ?ora) Said to be useful in treang anbioc induced diarrhea, IBS, IBD and even associated with Brain and behavior.. Others suggest eBects may be more placebo than eBect Sll much research needed to *nd out how these organisms aBect the host.  In Summary Fermentaons key in Industrial Microbiology. Crical for Dairy Industry – Cheese, Yoghurt, Probiocs Beer and Wine – Fermentaon by S. cerevesiae Acec acid bacteria and the producon of vinegar.  Final lecture on Friday The Nitrogen Cycle and Environmental Microbiology How to study for exam! Dr. Tadhg Ó Cróinín  MICR20010 Lecture 8 Microbial responses to unfavourable environments Dr. Jennifer Mitchell Microbiology School of Biomolecular and Biomedical Science  Learning Outcomes Environmental conditions Temperature pH Osmolarity Oxygen Spore formation Bacterial Biofilms What are ideal growth conditions for a microbe? A temperature where all their enzymes are folded properly and working at the optimum rate Plenty of food The correct atmosphere for their own type of respiration Available water Environmental conditions dictate microbial growth and the distribution and habitats of microbes Temperature pH Osmolarity (water concentration/availability) Oxygen  Effect of temperature on growth Perhaps the most important environmental factor controlling microbial growth Too hot or too cold can prevent growth However different microbes have evolved to growth at very different temperatures Cardinal temperatures Minimum Optimum Maximum Temperature controls chemical and enzymatic reactions Above the maximum temperature, enzymes and proteins are denatured Below the minimum temperature, the cell membrane may no longer function Cell Membrane is required for nutrient transport and for energy production. Cell Membrane composition is altered depending on growth media – Maximum and minimum temperatures supporting growth are different in rich media versus minimal media  Growth at cold temperatures Organisms adapted for growth at cold temperatures do better when the temperature is constant e.g. deep ocean (approx 2oC, arctic/antarctic waters. Environments with high summer temperatures and cold winter temperatures are less suited to growth. Psychrophiles have an optimal growth temperature of 15oC or lower Found in environments that are constantly cold and rapidly die at room temperature – difficult to isolate and grow in laboratory Enzymes in psychrophiles are denatured/inactivated at even moderate temperature Cold-active enzymes are structurally different to normal enzymes Membrane structure is different in psychrophiles – allows normal nutrient transport at cold temps.  Frozen but not dead Liquid water is required for microbial growth Freezing prevents microbial growth but does not always cause cell death Effects of freezing: 1. Dehydration 2. Ice crystal formation Water-miscible liquids (e.g. glycerol, DMSO – liquids which mix well with water) at a low concentration (10%) are protective: These penetrate the cell and reduce the severity of the effects if freezing Routinely used for storing bacterial cultures at -20oC and -80oC. Can occur to varying degrees in natural environments  Growth at high temperatures Microbial life flourishes at high temperatures up to and including boiling point of water Above 65oC only prokaryotic life (bacteria and archaea) exists Thermophiles – optimum growth temperature >45oC Hyperthermophiles – optimum growth temperature >80oC High temperature environments found in nature are associated with volcanic phenomena – hot springs, hydrothermal vents in deep oceans Archaea are more thermophilic than bacteria Protein/enzyme stability at high temperature Critical amino acid substitutions facilitate heat stable folding Membrane stability at high temperature Alternative membrane composition maintains structure and function DNA stability at high temperature Double stranded DNA molecule usually separates at high temperature In hyperthermophiles, an enzyme called reverse DNA gyrase prevents this from happening. This enzyme is absent in organisms that grow below 80oC. Introduces positive supercoils into DNA, resulting in increased stability  Bacterial DNA Gyrase Heat labile Heat stable  Effect of pH on growth pH refers to the concentration of hydrogen ions (H+) in a solution and is commonly expressed in terms of the pH scale, which is a log scale. A log scale is used because the large variations in H+ ion concentration in different solutions. Low pH corresponds to high hydrogen ion concentration High pH corresponds to low hydrogen ion concentration. pH values are calculated as (-) the log of the H+ concentration (- log is used to get positive values for the pH scale). pH = -log [H+]  Most microorganisms grow best at pH 6 – pH 8 (Neutrophiles) Acidity and alkalinity can greatly affect growth Acidophiles – acid loving Alkaliphiles – alkaline loving Tolerance of extreme pH may depend in part on altered membrane stability Important: Internal cell pH must be close to neutral (between pH 5 – pH 9) even if external pH is very acidic or alkaline At pH extremes – the cell macromolecules (enzymes, proteins, nucleic acid) are destroyed How does Helicobacter withstand Stomach acid?  Effect of osmolarity on growth Water is required for growth of all cells – water is the solvent of life Water availability is dictated not only by how moist or dry an environment is but is also dependent on the concentration of solutes (e.g. NaCl or sugar) in the water Why? Because dissolved solutes have an affinity for water and make it unavailable to the microbial cell Osmosis is the diffusion of water from high water concentration (low solute concentration) to low water concentration (high solute concentration. Controlled in cells by the cytoplasmic membrane  Osmosis Diffusion of water Water molecules diffuse easily across the CM Water molecules associated with other molecules in solution do not Dissolved sugar limits that availability of water to the cell In nature, absence of water inhibits life and thus biologically relevant water availability linked to solute (usually NaCl) concentration Halophiles – NaCl loving Osmophiles – can grow high sugar concentrations Food preservatives: Salt and sugar are commonly used as preservatives to inhibit microbial growth How do microbes grow under conditions of low water availability? NaCl NaCl NaCl NaCl H2O NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl H 2O NaCl NaCl NaCl NaCl Compatible solute H2O NaCl H2O NaCl Compatible solute H2O NaCl H2O NaCl NaCl Cell NaCl Cell NaCl NaCl NaCl H2O Compatible solute H2O NaCl H2O NaCl NaCl Compatible solute H 2O H2 O NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl H2O NaCl NaCl NaCl H2O Increase in internal solute concentration High NaCl, low H2O availability - Increased uptake of H2O Compatible Solutes Microbes in high solute, low water environments Obtain water by increasing intracellular solute concentration Synthesising or accumulating an organic solute This solute must be non-toxic to cellular metabolism – hence called compatible solute Compatible solutes are highly water soluble – thus they “attract” water into cell  Effect of oxygen on growth Because most higher animals require oxygen – doesn’t mean the same is true for microbes O2 is only weakly water soluble – hence many aquatic habitats are anoxic Aerobes – grow at full oxygen tensions (air is 21% O2) Microaerophiles – grow at reduced O2 concentrations (may have oxygen sensitive enzymes Anaerobes - cannot use oxygen for respiration – strict and aerotolerant Facultative anaerobes – can grow in presence or absence of oxygen Oxygen killing bacteria in neutrophils ROS  Bacterial Sporulation Some Gram-positive bacteria can form Spores which provide protection from adverse conditions Spores introduced into a wound site can germinate and cause infection Gram-negative bacteria cannot form spores  Spore Formation Adverse environmental conditions trigger spore formation. The spore is surrounded by a peptidoglycan-rich cortex layer and a keratin-like spore coat.  Spores are resistant to: Heat, Drying, Radiation, Freezing, Toxic chemicals Antibiotics & Can be difficult to eradicate with standard disinfectants. The existence of bacterial spores highlights the need for proper sterilisation - 121oC, 15psi Spores can persist for hundreds and possibly thousands of years, before germinating under the right conditions Although harmless themselves until they germinate, they are involved in the transmission of some diseases to humans including: *anthrax, caused by Bacillus anthracis; *tetanus, caused by Clostridium tetani; *botulism, caused by Clostridium botulinum; and *gas gangrene, caused by Clostridium perfringens  B. anthracis - Anthrax Spores persist in soil - animals and animal products are usual source of human infection Woolsorters were often infected - woolsorters’ disease Disease: Treatment: High fever, bacteraemia Penicillin, ciprofloxacin Massive swelling (oedema) Systemic affects Death Anthrax endospores as bioterrorism agents ? Bacterial Biofilms In all natural environments, microbes grow in complex communities called biofilms. Biofilms offer safety in numbers and provide increased resistance to adverse environmental conditions The majority of bacterial infections treated by clinicians involve biofilms Pseudomonas aeruginasa infections in cystic fibrosis patients Staphylococcus epidermidis catheter related infections Coagulase-negative staphylococci form biofilms or slime on implanted biomaterials and catheters Biofilms form when bacteria adhere to surfaces and excrete slimy glue-like substances which anchor the cells  Why are biofilm infections difficult to treat? Antibiotic Doses 1 1000 Planktonic Biofilm cells cells  Further Reading Brock Biology of Microorganisms Chapter 6 “Microbial Growth”  MICR20010 Agricultural Microbiology Dr. Tadhg Ó Cróinín  Praccal MCQ Exam Praccal Online MCQ exam is on this Friday Nov 22nd from 2-3pm Please try the sample quesons under quiz tab in brightspace and note instrucons in e-mail to follow. Separate e-mail for DSS students. Sample MCQ quesons for *nal exam in RDS also available on Brightspace.  MICR20010 - remaining lectures Lecture 10 – Microorganisms and Disease Lecture 11 – The Immune System Lecture 12 - Pathogenic Bacteria Lecture 13 – Pathogenic Fungi and Viruses Lecture 14 – Anbioc Resistant Microorganisms Lecture 15 – Microbiology in the Food Industry – The Fungi Lecture 16 – Microbiology in the Food Industry - Fermentaons Lecture 17 – The Nitrogen Cycle Single cell protein + Microbial Biomass  Single Cell Protein Began in WW2 to produce protein cheaply - S. cerevisiae and Candida ulis. Rapid development in 1960s and 1970s with emergence of phrase SCP. Most used as animal feed rather than human consumpon Producon of yeast from whey or Quorn producon Protein increase in global population…search for alternative protein source Single cell protein (SCP) – SCP= microbial cells grown and harvested for animal/ human food WHY SCP? Choice of m/os due to rapid growth high protein content use cheap organic substrates to grow Protein production by cattle + yeast bullock.. Wt 500Kg..yields 0.4kg protein/24h Yeast.. Wt 500kg..yields> 50,000kg/24h The Fermentor Applicaons for Yeast in the Food Industry  Producon of Yeast Biomass Key to high yield is aeraon – avoid fermentaon Cheap media –historically media from cereal grains, now predominantly molasses. Gradual scale up of culture from bench-top to fermentor! Special feeding regime to avoid fermentaon (fed batch)  Producon of Yeast Biomass Ripening stage to encourage producon of trehalose and reduce protein producon Centrifugal separators before washing, drying and packing Maximising Biomass is the key aim Yeast Producon  What makes a good Bakers Yeast? High growth rates Good storage characteriscs including cryotolerance Osmotolerance – to funcon within dough Rapid ulizaon of maltose – main sugar of dough High Glycolyc acvity and “gassing power” SCP-can be produced from – Algae..spirulina – fungi… – bacteria Biomass can be produced by – submerged fermentation – solid state fermentation – After fermentation: biomass is harvested, washed, disrupted, and protein extracted Substrates for micro-organism production: Algae: use CO2 + sunlight Fungi: cheap wastes supply C + N source Bacteria: wastes or by-products of industrial processes Substrates for micro-organism production: molasses from sugar manufacture/ starch hydrolysis spent sulphite liquor acid hydrolysates of wood Agricultural wastes methane methanol +ethanol gas oil Nutritive value of micro-organism Algae: rich in proteins, fats + vitamins A,B,C,D +E 40-60% protein 7% mineral Fungi: B-complex group of vit. aa content of A.niger: well balanced Yeast: have thiamine, riboflavin, biotin Bacterial SCP: high in protein + certain essential aa methionine: 2.2-3% > algal (1.4-2.6%) and fungi (2.5-1.8%) SCP: limitations for use Algal SCP cell wall- cellulytic component cannot be digested by humans..walls must be pre-digested (not for cattle feed) production is weather dependent Fungal SCP: Aspergillus parasiticus, A.flavous:.. Mycotoxins Bacterial SCP use is limited to cost harvesting can be expensive bacterial cells have high nucleic acid content (uric acid accumulates in body..kidney stones + gout) SCP in solving protein malnutrition source of human + animal feeds m/o’s accepted for human consumption: – Saccharmyces cerevisiae – Candida utilis – Chlorella spp. SCP example: Quorn myco-protein Fusarium graminearum Schwabe A3/5-used in UK Quorn myco-protein for humans meat alternative: – taste,textures of meat products (vegi alternative) myco-protein: cells grown on glucose harvested High nutritional value once produced it is mixed with a binder to give desired shape + flavour  Indirect Fermentations M/O in food production direct direct Mushrooms Single cell protein Mushrooms Mushrooms: Filamentous fungi that form fruiting bodies known as mushrooms ~12,000 fungal species (~2000 have some edibility) – Agaricus bisporus (button mushroom) – Lentinus edodes – Pleurotus spp (oyster mushroom)  Mushrooms A long tradion of use of mushrooms as a food source (or narcoc) Very cheap to produce and can readily be grown on plant waste Excellent source of protein Easy to harvest the fruing bodies Agaricus Bisporis  Mushroom producon Preparaon of inoculum in liquid culture Preparaon of beds - composng Inoculaon into compost and mycelial growth at 25oC for 2-3 weeks Applicaon of casing of peat (covering layer) Fruing body producon in about four Fushes (successive crops) over a period of 4-6 weeks. Lennus edulus Other benefits of mushrooms: Medicinal mushrooms Extracts of species from genera: Auricularia Flammulina, Ganoderma, Hericium And more Employed medicinally In medicine: Immunomodulators – Some have anti-cancer action Source of rare minerals and amino acids – Copper, zinc, selenium, iron. Problems of fungi: Mycotoxins Mycotoxins secondary metabolite of fungi: – mycotoxicoses in animals + humans – some linked with certain types of cancer – present in mycelium and in some cases in the spores of filamentous fungi – not all are harmful In human food chain problems with – Aspergillus, Penicillium: contaminate foods during drying and storage – Fusarium: plant pathogen- produces mycotoxin before/ immediately after harvesting Alflatoxins: first discovered in 1960 – 100,000 turkey poults died suddenly in England – 14,000 ducklings – 9 out breaks in calves – common factor: Brazilian peanut meal in animal feed. Produced mainly by: Aspergillus flavus, A.parasiticus Alflatoxins: 4 main alflatoxins: B1,B2,G1, G2 (&M1) difuranocoumarin derivatives-B &G = Blue (B) & green (G) fluorescent colours produced under UV light Aflatoxins found in: maize, animal products, peanuts.  M1= hydroxylated derivative of B1 M2=.... of B2 – M1 & 2 – formed + excreted in milk of lactating animals – less toxic than B1 or 2 – BUT M1= toxic & carcinogenic Next Monday on MICR20010 Microbiology in the Food Industry – Alcohol, Dairy Industry and Vinegar Remember Online Praccal MCQ exam on Friday at 2pm! MICR20010 Lecture 7 Bacterial Genecs Dr. Jennifer Mitchell Microbiology School of Biomolecular and Biomedical Science  Lecture 6 Metabolic diversity Chemical basis of energy producon Simpli&ed model of energy producon Energy storage and release Chemotrophs Phototrophs Chemotrophs – Chemoorganotrophs – Chemolithotrophs Autotrophs Heterotrophs Photosynthesis Learning Outcomes DNA DNA replicaon Gene structure – Transcripon Protein synthesis – Translaon Anbiocs The Genec Code Mutaon Genec Exchange  DNA Deoxyribonucleic Acid (DNA) Monomer building blocks called deoxyribonucleodes: – 5-carbon sugar deoxyribose – a nitrogenous base – a phosphate group 5 4 1 3 2  Bases There are four nitrogenous bases found in DNA – Adenine (A) – Guanine (G) – Cytosine (C) – Thymine (T) DNA strand  DNA DNA consists of two complementary strands (double stranded) Complementary base pairing A=T G≡C G≡C  DNA Replica#on The process of generang an idencal set of genes during cell division Very accurate process carried out by DNA polymerases Occasional inaccuracies give rise to a slightly altered nucleode sequence – a mutaon  DNA Replica#on Iniaon Elongaon Proofreading Terminaon  DNA Polymerase DNA polymerase can add free nucleodes to only the 3' end of the newly-forming strand. This results in elongaon of the new strand in a 5'-3' direcon. No known DNA polymerase is able to begin a new chain (de novo). DNA polymerase can add a nucleode onto only a preexisng 3'-OH group DNA strand Gene#c Code DNA contains the genec informaon (genes) required for all cellular processes Genes can occur individually or in groups (operons) Gene Expression: Transcripon Iniated at the promoter region upstream of the gene RNA polymerase copies the DNA and produces an RNA transcript (mRNA) Translaon mRNA is decoded by ribosomes and tRNA molecules to specify the exact sequence of amino acids in a Gene Structure Protein Expression  The Gene#c Code Codon A set of three adjacent nucleodes that encode a parcular amino acid. Specifying the type and sequence of amino acids for protein synthesis.  An#bio#cs and DNA/RNA/Protein Some anbiocs target DNA replicaon, transcripon and translaon Rifampicin a=ects RNA polymerase Macrolides (erythromycin), Kanamycin, Tetracycline a=ect ribosome & protein synthesis Mutaons in the anbioc target can lead to bacterial resistance Ciprofoxacin Ciprofoxacin targets DNA gyrase, the enzyme which unwinds bacterial DNA during replicaon. Ciprofoxacin prevents cell division Quinolone anbioc Plasmids Circular extrachromosomal DNA Replicate independently and can move between cells Phenotypic advantage for the host cell Plasmid genes: – Anbioc resistance genes (oBen mulple) – Virulence genes (e.g. toxins) – Metabolic genes Hospital-acquired Infec#ons Plasmids with mulple anbioc resistance genes predominate within hospital bacteria Infecons caused by such bacteria (nosocomial or hospital-acquired infecons) are therefore parcularly serious and diCcult to treat. Anbioc resistance genes existed before the era of anbioc treatment but have become prevalent due to selecve pressure. Highlights bacterial adaptability. Transmission of AMR genes between species Muta#on Most common source of genec variaon Spontaneous or induced (mutagens) Three types – Substuon – Deleon – Inseron Codons - Muta#on  Gene#c Varia#on Important implicaons for microbial virulence: Resistance to anbiocs New virulence factors (e.g. E. coli 0157)  Mutagenesis Mutagens Physical - Radiaon Chemical mutagenesis – Base analogues – Intercalang agents – Metals - ROS Biological agents – Virus – Transposon Gene#c Exchange Modes of Genec Transfer between Bacterial Cells  Transforma#on DNA fragments can be taken up directly by bacterial cells Normally degraded Somemes integrated into host genome Some bacteria are naturally competent e.g. Streptococcus pneumoniae  Conjuga#on Describes plasmid transfer between bacterial cells Requires cell-to-cell contact & can occur between di=erent bacterial species and even between G+ve and G-ve tra genes encode pilus (channel) between the cells through which the plasmid moves Plasmids replicate in the donor cell prior to transfer into the recipient cell  Transduc#on DNA transfer between bacteria via infecon with a bacteriophage Phage infect the bacterial cell and replicate Involves incorporaon of phage DNA into phage capsids (heads) Occasionally host genomic DNA is also packaged  Transposi#on Transposons are DNA sequences that can ‘jump’ within the bacterial genome and from the genome to plasmids within the same cell Transposons carry the enzymes required for their own transposion (homology not needed). This can result in gene disrupon Transposons oBen contain anbioc resistance genes Transposion into broad host range plasmids has facilitated rapid disseminaon of anbioc resistance genes among di=erent bacterial species  Genec Variaon and Anbioc Resistance Mutaon – (e.g. drug resistance in tuberculosis) Transformaon/transposion – (e.g. Penicillin-resistant gonorrhea) Conjugaon – (e.g. mul-resistant shigella)  Further Reading Brock Biology of Microorganisms Chapter 10 “Bacterial Genecs”  MICR20010 Agricultural Microbiology Dr. Tadhg Ó Cróinín  MICR20010 – Assessments Remember to get your prac#cal reports submi$ed on #me, watch plagiarism. (15%) Prac#cal online MCQ exam on 2-3pm Friday Nov 22nd (15%) Sample MCQs to appear next week. Final MCQ exam in RDS on 12th December 9.30 – Con6rm on exam #metable (70%) Sample MCQs to appear next week.  MICR20010 - remaining lectures Lecture 10 – Microorganisms and Disease Lecture 11 – The Immune System Lecture 12 - Pathogenic Bacteria Lecture 13 – Pathogenic Fungi and Viruses Lecture 14 – An#bio#c Resistant Microorganisms Lecture 15 – Microbiology in the Food Industry – The Fungi Lecture 16 – Microbiology in the Food Industry - Fermenta#ons Lecture 17 – The Nitrogen Cycle Commercial as well as Health Implicaons! Transmission of Disease Figure 14.6a, d  Vehicle Transmission Transmission by an inanimate reservoir (food, water, air) Figure 14.7b Vectors Figures 14.8, 12.30  Nosocomial Infec#ons Are acquired as a result of a hospital stay A?ect 5–15% of all hospital pa#ents Figure 14.6b  Mechanisms of Pathogenicity Pathogenicity: The ability to cause disease Virulence: The extent of pathogenicity Mechanisms of Pathogenicity Figure 15.9  Infec#on and Adherence Adhesins/ligands bind to receptors on host cells – Fimbriae: Escherichia coli – M protein: Streptococcus pyogenes Form biolms Adherence Figure 15.1  Penetra#on into the Host Cell Cytoskeleton Invasins Salmonella alters host ac#n to enter a host cell Listeria Uses ac#n to move from one cell to the next Figure 15.2  Direct Damage by bacteria Disrupt host cell func#on Produce waste products Toxins – Toxin: Substance that contributes to pathogenicity – Toxigenicity: Ability to produce a toxin – Toxemia: Presence of toxin in the host's blood Figure 15.4 Exotoxin type A-B toxin, Membrane disrup#ng toxin, Superan#gens Exotoxin Corynebacterium A-B toxin diphtheriae Membrane-disrupting Streptococcus pyogenes erythrogenic toxin Clostridium botulinum A-B toxin; neurotoxin C. tetani A-B toxin; neurotoxin Vibrio cholerae A-B toxin; enterotoxin Staphylococcus aureus Superantigen  Endotoxins Source Gram Relation to Microbe Outer membrane Chemistry Lipid A Fever? Yes Neutralized by Antitoxin? No LD50 Relatively large Figure 15.4b The Stages of a Disease Figure 14.5 Bacterial Diseases Chronic vs Acute  Pseudomonas and the Pseudomonads Colony morphology Rods or curved rods with polar Eagellae Burkholderia, Pseudomonas etc. A par#cular challenge for those with Cys#c Fibrosis The Human Lung  Developing Chronic Infec#on Intermi$ent coloniza#on 6rst Followed by a persistent chronic infec#on Accompanied by a higher degree of InEamma#on.  B. Pertussis – An acute Infec#on Also a gram-ve rod shaped organism. Thus a similar cell envelope to that of P. aeruginosa. Also infects the lungs but this #me not opportunis#c… Highly contagious due to coughing spasms. Uses an array of virulence factors to cause disease. Whooping Cough  What is Happening The bacterial infec#on is a?ec#ng the ability of the lungs to clear mucus. An inability of the cilia to clear the mucus sends the host into a 6t of coughing. This in itself allows the bacteria to be spread rapidly by aerosol. Huge array of virulence factors!  The Symptoms Paroxysmal cough, inspiratory “whoop” Rib fractures, hernias, loss of conciousness Infec#on in newborns can be par#cularly severe and result in death in approx 1% 7-10 days incuba#on period followed by catarrhal stage, then two weeks later uncontrollable 6ts set in.  Treatment/Preven#on? Vaccina#on with pertussis toxin rela#vely successful. An e?ec#ve vaccine… but….. Uptake of vaccine cri#cal.  H. pylori– Commensal or Pathogen? Also a gram-ve organism but this #me spiral shaped Thus a similar cell envelope to that of the other Gram- nega#ve organisms. Only known to reside in the human stomach/duodenum. Gastri#s in most individuals More serious disease in others….  A Brief History Discovered in 1982 Before this stomach thought to be sterile Ulcers thought to be stress related. A paradigm shiQ in the treatment of gastric disease.  Pathology of Infec#on The only known bacteria to colonize the human gastric mucosa. Gastri#s induced in all individuals colonized but typically asymptoma#c. Some can develop into more serious disease such as – Duodenal Ulcers, MALT Lymphoma, Gastric Cancer A class 1 carcinogen? Disease Progression  Chlamydia – An intracellular pathogen 15-25 are most at risk. Steady rise in Chlamydia cases Phylum 5 - Chlamydia  Phylum 5 : Chlamydia Organisms are obligate parasites – C. trachomas - STD and trachoma – C. psiaci – Psi$acosis – C. pneumoniae – Respiratory syndromes Clearly limited metabolic pathways Lack of some genes (QsZ) Presence of some eukaryo#c-like genes  Life Cycle of Chlamydia Elementary and Ri#culate bodies Thus a more complex life cycle. Key is the obligate intracellular life cycle.  Di?erent Niches – Di?erent Outcomes The male and female reproduc#ve organs contain very di?erent environments. Also di?erent epithelial surface cell molecules. This has a big e?ect on the progression of disease. Can cause permanent damage to fallopian tubes and sterility in women  Bacterial Diseases of the Eye Chlamydia trachomas – Causes trachoma – Leading cause of blindness worldwide – Infec#on causes permanent scarring; scars abrade the cornea leading to blindness  Anthrax Bacillus anthracis Endospores enter through minor cut – 20% mortality Gastrointesnal anthrax – Inges#on of undercooked, contaminated food – 50% mortality Inhalaonal (pulmonary) anthrax – Inhala#on of endospores – 100% mortality  Biological Weapons 1346: Plague-ridden bodies used by Tartar army against Ka?a 1937: Plague-carrying Eea bombs used in the Sino- Japanese War 1979: Explosion of B. anthracis weapons plant in the Soviet Union 1984: S. enterica used against the people of The Dalles 1996: S. dysenteriae used to contaminate food 2001: B. anthracis distributed in the United States  Biological Weapons Bacteria Viruses “Eradicated” polio and Bacillus anthracis measles Brucella spp. Encephalitis viruses Chlamydophila psittaci Hermorrhagic fever viruses Clostridium botulinum toxin Influenza A (1918 strain) Coxiella burnetii Monkeypox Francisella tularensis Nipah virus Rickettsia prowazekii Smallpox Shigella spp. Yellow fever Vibrio cholerae Yersinia pestis  Typhoid Fever Salmonella typhi Bacteria spread throughout body in phagocytes 1–3% of recovered pa#ents become chronic carriers 200,000 deaths/yr The Importance of asymptomac Infecon  Next on MICR20010 Pathogenic Fungi and Viruses Dr. Tadhg Ó Cróinín  MICR20010 Lecture 6 Bacterial Physiology and Metabolic Diversity Dr. Jennifer Mitchell Microbiology School of Biomolecular and Biomedical Science  Lecture 5 Microbial Growth and Physiology Growth of Bacteria – Bacteria Divide by Binary Fission – Growth of Bacteria on Solid Medium – Growth of Bacteria in Liquid Medium Growth Phases of liquid Bacterial Culture Measurements of Bacterial Growth Direct Measurements of Bacterial Growth: Indirect Measurements of Bacterial Growth: Growth Requirements  Learning Outcomes Metabolic diversity Chemical basis of energy produc*on Simpli+ed model of energy produc*on Energy storage and release Chemotrophs Phototrophs Chemotrophs – Chemoorganotrophs – Chemolithotrophs Autotrophs Heterotrophs Photosynthesis  Carbon and Energy All cells need carbon and energy sources for their metabolic ac*vi*es Di/erent microorganisms have evolved every conceivable means of obtaining carbon and energy This results in signi+cant metabolic diversity Hence microbes have been able to colonise environmental habitats which are too extreme for other life forms. h2ps://www.teagasc.ie/news--events/daily/other/the-soil-microbiome-and-soil-health.php  Chemical Basis of Energy Producon 1. Chemical reac*ons used to generate energy 2. Speci+cally chemical reac*ons involving the release of electrons 3. Electrons have stored energy and when an atom or molecule loses that electron (becomes oxidized) that energy is released 4. Oxida*on: atom or molecule loses one or more electrons 5. Reduc*on: atom or molecule gains those electrons. 6. Energy sources are oxidised to release electrons which have stored energy 7. Energy generated is stored in the form of ATP OIL RIG Oxida*on Is Loss of electrons Reduc*on Is Gain of electrons  Simpli!ed Model of Energy Producon Light e- e- ATP oxidised Iron Enzymes e- e- e- e- idised s ox yme Glucose Enz Example of simple oxida*on reac*on: Fe2+ ⇋ Fe3+ + e- Ferrous iron Ferric iron  ENERGY Energy Storage: Used to trap energy released from chemical reac*ons Energy stored as high-energy phosphate bond Inorganic phosphate group a2ached to adenosine diphosphate (ADP) Adenosine triphosphate (ATP) The energy currency of the cell Energy Release: Phosphate enzyma*cally removed from ATP to release energy  How to get Energy? Chemotrophs: Derive energy from chemicals Chemotrophs – Chemoorganotrophs (Use organic chemicals) – Chemolithotrophs (Use inorganic chemicals) Phototrophs – Derive energy from light  Chemoorganotrophs Derive energy from organic chemicals Organic chemicals are compounds containing carbon All cells require carbon as a major nutrient, hence these chemicals are a good source of carbon and energy for chemoorganotrophs 1000’s of di/erent organic chemicals present on Earth ALL can be broken down by microorganisms to derive energy  How do chemoorganotrophs derive energy from organic chemicals? Answer: Oxida*on of the compound releases electrons, which are ul*mately used to generate ATP. Electrons have stored energy and when an atom or molecule loses that electron (becomes oxidized) that energy is released Oxida*on: atom or molecule loses one or more electrons Reduc*on: atom or molecule gains those electrons. Energy sources are oxidised to release electrons which have stored energy Aerobes Some chemoorganotrophs can only produce energy in the presence of oxygen Anaerobes Microbes that can only produce energy in the absence of oxygen Facultave anaerobes Microbes that produce energy in the presence or absence of oxygen  Methanogens Livestock produce signicant amounts of methane as part of their normal digestive processes. Some feed additives can inhibit the microorganisms that produce methane in the rumen and subsequently reduce methane emissions. Ruminant livestock – cattle, sheep, bu alo, goats, deer and camels – have a fore-stomach (or rumen) containing microbes called methanogens, which are capable of digesting coarse plant material and which produce methane as a by-product of digestion (enteric fermentation): this methane is released to the atmosphere by the animal belching. h2ps://www.agric.wa.gov.au/climate-change/carbon-farming-reducing-methane- emissions-ca2le-using-feed-addi*ves#:~:text=Feed%20addi*ves%20or%20supplements%20can  Supplements Methane-reducing feed additives and supplements inhibit methanogens in the rumen, and subsequently reduce enteric methane emissions. Methane-reducing feed additives and supplements are most e ective when grain, hay or silage is added to the diet, especially in beef feedlots and dairies.  Reducing Methane Methane-reducing feed additives and supplements can be: synthetic chemicals natural supplements and compounds, such as tannins and seaweed fats and oils. Feeding one type of seaweed at 3% of the diet has resulted in up to 80% reduction in methane emissions from cattle.  Ac*ve inhibitors trihalomethanes, such as bromoform, which is an active ingredient that decreases methane emissions Tannins  Bene+ts: The reduced volume of methane formation may lead to better e)ciency of feed utilisation, given that methane emissions represent a gross energy loss from feed intake of about 10%.  Chemolithotrophs Derive energy from inorganic chemicals Inorganic chemicals are compounds which do not contain carbon, e.g. H2, H2S, Fe2+ These inorganic compounds are oxidised to release electrons for ATP synthesis However all cells require carbon as a major nutrient  Chemolithotrophs Name Examples Source of energy and electrons Respiraon electron acceptor Iron bacteria Acidithiobacillus ferrooxidans Fe2+ (ferrous) → Fe3+ (ferric) + e- O2 → H2O Nitrosifying bacteria Nitrosomonas NH3 (ammonia) → NO2- (nitrite) + e- O2 → H2O Nitrifying bacteria Nitrobacter NO2- (nitrite) → NO3- (nitrate) + e- O2 → H2O Chemotrophic purple sulfur S2 (sul+de) → S0 (sulfur) + e- O2 → H2O bacteria Halothiobacillaceae Sulfur-oxidizing bacteria Chemotrophic S0 (sulfur) → Sulfate (SO2−4) + e- O2 → H2O Rhodobacteraceae Aerobic hydrogen bacteria Cupriavidus metallidurans H2 (hydrogen) → H2O (water) + e- O2 → H2O Thiobacillus denitri+cans Thiobacillus denitri+cans S0 (sulfur) → Sulfate (SO2−4) + e- NO3- (nitrate) Sulfate-reducing bacteria: H2 (hydrogen) → H2O (water) + e- Sulfate (SO2−4) Hydrogen bacteria Sulfate-reducing bacteria: Desulfo*gnum PO3−3 (phosphite) → PO3−4 Sulfate (SO2−4) Phosphite bacteria phosphitoxidans (phosphate) + e- Methanogens Archaea H2 → H2O + e- CO2 (carbon dioxide) Carboxydothermus carbon monoxide (CO) → carbon Carboxydotrophic bacteria dioxide (CO2) + e- H2O (water) → H2 (hydrogen) hydrogenoformans  Chemolithotrophs Chemolithotrophs obtain carbon from CO2 – autotrophy Ecological niche and compe**on Lithotrophy is advantageous because organisms deriving energy from inorganic compounds do not have to compete with chemoorganotrophs. In addi*on some of their energy sources (H2, H2S) are waste products from the chemoorganotrophs  Heterotrophs and Autotrophs Heterotrophs Microbial cells which use one or more organic compounds as their carbon source are called heterotrophs Autotrophs Microbial cells which use CO2 as their carbon source are called autotrophs – CO2 +xa*on or Calvin cycle  Autotrophs Autotrophs are called primary producers because they produce organic ma2er from CO2 in the air. Chemoorganotrophs and other organisms can ul*mately use this organic ma2er by feeding on the autotrophs or their waste products All organic ma2er on the planet has been synthesised from CO2 by autotrophs  Phototrophs Use light as energy source Phototrophs contain pigments which allow them to use light as an energy source These pigments give the cells colour  Photosynthesis Phototrophs obtain energy from light using photosynthesis Photosynthesis involves reac*ons in which ATP is generated Oxygenic photosynthesis – oxygen is produced as a bi-product Anoxygenic photosynthesis – no oxygen is produced What are the Pigments in Phototrophic Cells? Chlorophylls Carotenoids  Chlorophylls Green colour Similar to the pigments responsible for photosynthesis in plants Phototrophic bacteria contain chlorophylls called bacteriochlorophylls In plant cells photosynthesis takes place in chloroplasts In bacteria photosynthesis takes place in a specially developed cytoplasmic membrane.  Carotenoids Yellow, red, brown and green colours Carotenoids are closely associated with bacteriochlorophyll but play no direct role in photosynthesis Transfer light energy to bacteriochlorophyll Carotenoids have a photoprotec*ve role Photosynthec Bacteria  Further Reading Brock Biology of Microorganisms Chapter 5 “Nutri*on, Laboratory Culture and Metabolism of Microorganisms”  MICR20010 Agricultural Microbiology Dr. Tadhg Ó Cróinín  MICR20010 - remaining lectures Lecture 10 – Microorganisms and Disease Lecture 11 – The Immune System Lecture 12 - Pathogenic Bacteria Lecture 13 – Pathogenic Fungi and Viruses Lecture 14 – An+bio+c Resistant Microorganisms Lecture 15 – Microbiology in the Food Industry – The Fungi Lecture 16 – Microbiology in the Food Industry - Fermenta+ons Lecture 17 – The Nitrogen Cycle Immune System vs Pathogens  Immunity Concepts Suscepbility: Lack of resistance to a disease Immunity: Ability to ward o5 disease Innate immunity: Defenses against any pathogen Adapve immunity: Immunity, resistance to a speci8c pathogen  Pathogens, agents that cause disease, infect a wide range of animals, including humans The immune system recognizes foreign bodies and responds with the produc+on of immune cells and proteins – All animals have innate immunity, a defense ac+ve immediately upon infec+on – Skin – Chemical secre+ons that trap/kill microbe Vertebrates also have adapve immunity/ acquired immunity Innate immunity is present before any exposure to pathogens and is e5ec+ve from the +me of birth It involves nonspeci8c responses to pathogens Innate immunity consists of external barriers plus internal cellular and chemical defenses Microbiology an introducon Skin Mucus membranes trap bacteria and move it away from lungs Adapve immunity, or acquired immunity, develops a=er exposure to agents such as microbes, toxins, or other foreign substances It involves a very speci8c response to pathogens  Immune system: Microbes and Defenses The immune system recognizes bacteria and fungi by structures on their cell walls Pathogens entering the mammalian body are subject to phagocytosis – Phagocy+c cells recognize groups of pathogens by TLRs, Toll-like receptors  Formed Elements in Blood Red Blood Cells Transport O2 and CO2 (Corpuscles) White Blood Cells: Neutrophils Phagocytosis Eosinophils Kill parasites Microbiology an introducon  Formed Elements in Blood Monocytes Phagocytosis Dendri+c cells Phagocytosis Natural killer cells Destroy target cells Microbiology an introducon  Formed Elements in Blood T cells Cell-mediated immunity B cells Produce an+bodies Platelets Blood cloBng Microbiology an introducon A white blood cell engulfs a microbe, Phagocytosis There are di5erent types of phagocy+c cells – Neutrophils engulf and destroy pathogens – Macrophages are found throughout the body – Dendric cells s+mulate development of adap+ve immunity – Eosinophils discharge destruc+ve enzymes Neutrophils  Figure 43.3 Pathogen Phagocytosis. PHAGOCYTIC Phago: From Greek, meaning CELL eat Cyte: From Greek, meaning cell Vacuole Lysosome containing enzymes Extracellular killing by leukocytes Eosinophiles… natural killer cells Neutrophils… can carry out extracellular killing Natural killer lymphocytes (NK cells) Secret toxin onto surface of viral infected cells But can distinguish normal from infected cells via membrane proteins  2nd line of defence Neutrophils… can carry out extracellular killing..ways Have enzymes that generate O2- and H2O2… converted to hypochlorite Produce nitric oxide.. Inflammation inducer Can make NETS (neutrophil extracellular traps).. Kill via immobilisation  Bacterial Rod entrapped Budding yeast entraped in NETS Urban et al., Cellular microbiology (2006), 8, 1687.  Figure 43.8-1 Pathogen Splinter Macro- Signaling Mast molecules phage cell Capillary Red Neutrophil blood cells  Figure 43.8-2 Pathogen Splinter Macro- Movement Signaling phage of fluid Mast molecules cell Capillary Red Neutrophil blood cells  Figure 43.8-3 Pathogen Splinter Macro- Movement Signaling phage of fluid Mast molecules cell Capillary Phagocytosis Red Neutrophil blood cells  Adapve immunity receptors provide pathogen-speci8c recogni+on The adap+ve response heavily relies on two types of lymphocytes, or white blood cells Lymphocytes that mature in the thymus above the heart are called T cells, and those that mature in bone marrow are called B cells Angens elicit a response from a B or T cell  Angens are substances that can elicit a response from a B or T cell Exposure to the pathogen ac+vates B and T cells with angen receptors speci8c for parts of that pathogen The small accessible part of an an+gen that binds to an an+gen receptor is called an epitope Antigen receptors Mature B cell Mature T cell  Binding of a B cell an+gen receptor to an an+gen – an early step in B cell ac+va+on This gives rise to cells that secrete a soluble form of the protein called an anbody or immunoglobulin (Ig) Secreted an+bodies are similar to B cell receptors – but lack transmembrane regions that anchor receptors in the plasma membrane  Anbody Funcon An+bodies do not kill pathogens; instead they mark pathogens for destruc+on and/or phagocytosis - Opsonisa+on In neutralizaon, an+bodies bind to viral surface proteins preven+ng infec+on of a host cell An+bodies may bind to toxins in body Euids and prevent them entering cells Figure 43.10a  B cells can express 8ve di5erent forms (or classes) of immunoglobulin (Ig) with similar an+gen-binding speci8city but di5erent heavy chain C regions – IgD: Membrane bound – IgM: First soluble class produced – IgG: Second soluble class; most abundant – IgA and IgE: Remaining soluble classes  Acve and Passive Immunizaon Acve immunity develops naturally when memory cells form clones in response to an infec+on, Passive is when an+bodies are directly donated. It can also develop following immunizaon, also called vaccinaon In immuniza+on, a nonpathogenic form of a microbe or part of a microbe elicits an immune response to an immunological memory Figure 19.9 humans from pigs; “swine :u” 1 m (a) 2009 pandemic H1N1 (b) 2009 pandemic influenza A virus screening Killed 40 million, 1918 (c) 1918 flu pandemic  Vaccines are harmless deriva+ves of pathogenic microbes that s+mulate the immune system to mount defenses against the harmful pathogen Vaccines can prevent certain viral illnesses Measles in the United States, 1960–2007 Microbiology an introduc+on Clinical Focus, p. 505 Vaccine hesitancy and e5ec+veness A recent challenge A brief History Success?… Next on MICR20010 Pathogenic Bacteria Dr. Tadhg Ó Cróinín  MICR20010 Lecture 5 Growth and Physiology Dr. Jennifer Mitchell Microbiology School of Biomolecular and Biomedical Science  Lecture 5 Prokaryo&c cell morphology Bacterial cell structure The gram stain – Gram stain mechanism Bacterial shapes – Di)erent morphological shapes Bacterial cell structure – G+ve G-ve Archaea – Cell membrane – Cell wall – Outer membrane – Cell appendages  Learning Outcomes Microbial Growth and Physiology Growth of Bacteria – Bacteria Divide by Binary Fission – Growth of Bacteria on Solid Medium – Growth of Bacteria in Liquid Medium Growth Phases of liquid Bacterial Culture Measurements of Bacterial Growth Direct Measurements of Bacterial Growth: Indirect Measurements of Bacterial Growth: Growth Requirements Microbial growth and physiology In the laboratory Liquid broths and Nutrient Agar plates  GROWTH OF BACTERIA: ASEPTIC TECHNIQUE STERILE GROWTH MEDIA BOIL – KILL ALL CELLS 100⁰C / 30 MIN AUTOCLAVE – KILL ALL CELLS & SPORES 120 ⁰ C / 30 MIN DRY HEAT – 150 ⁰ C / 120 MIN  Bacteria divide by Binary Fission Binary Fission Chromosome divides to produce two iden&cal copies These copies segregate to opposite ends of the cell Cell wall is laid down the middle of the cell to ul&mately produce two new cells which are iden&cal Binary Fission  Bacterial growth is Exponen'al 1->2->4->8->16->32->64->128->256->512 etc Bacterial growth proceeds exponen&ally Genera&on &mes (&me for bacterial mass to double) can be as fast as 20 minutes Contributes to the remarkable adaptability of bacteria Growth in a hos&le environment can create a selec&ve pressure for mutant cells which can persist. One mutant cell which can survive will rapidly grow and take over. GROWTH OF BACTERIA ON SOLID MEDIUM AGAR: MELTS AT 100⁰C, SOLIDIFIES AT 40⁰C STERILIZED BY AUTOCLAVING BACTERIA GROW AS COLONIES SINGLE COLONY PURIFICATION GROWTH IN LIQUID MEDIUM COTTON WOOL BUNG GAS EXCHANGE KEEP CONTENTS STERILE INCUBATE STANDING OR AGITATED TURBID CULTURE ~ 109 CELLS/ML Growth Phases of a Bacterial Culture 1. Lag Phase – Adapta&on 2. Logarithmic Phase – Cells mul&ply at the maximum rate 3. Sta&onary Phase – Lack of nutrients and build up of toxic metabolic intermediates means mul&plica&on is balanced by cell death 4. Phase of decline  Genera'on Times of Bacteria Bacterium Medium Genera'on Time (minutes) Escherichia coli Glucose-salts 17 Bacillus megaterium Sucrose-salts 25 Streptococcus lac&s Milk 26 Streptococcus lac&s Lactose broth 48 Staphylococcus aureus Heart infusion broth 27-30 Lactobacillus acidophilus Milk 66-87 Rhizobium japonicum Mannitol-salts-yeast extract 344-461 Mycobacterium tuberculosis Synthe&c 792-932 Treponema pallidum Rabbit testes 1980  Measurements of bacterial growth Direct measurements of bacterial growth: I. Total cell count. Using microscope and coun&ng chamber II. Total viable count. Cells in culture are diluted and spread on nutrient agar plates. Only viable cells will reproduce to give rise to a colony. Direct measurements of bacterial growth: Coun&ng chamber Direct measurements of bacterial growth: The area and volume under each square is known. Can determine the number of cells in sample volume. Total Viable Count Serial 10-fold dilu&ons  Total Viable Count: Spread plate method and pour plate method Indirect measurements of bacterial growth Turbidity (Cloudiness) Measures live and dead cells How a spectrophotometer measures turbidity (cloudiness)  Chemostat culture 1. Cell density controlled by nutrient conc. 2. Growth rate controlled by Oow rate of nutrient  Growth requires Energy, The building blocks required for the construc&on of cellular machinery Appropriate environmental condi&ons  Growth Requirements Nutrient Requirements – Water – Carbon (carbohydrate) – Nitrogen (protein) – Inorganic salts Iron - siderophores – Oxida&on of organic compounds – (carbohydrates, lipids, proteins) Temperature pH Atmosphere 20⁰C- 110 ⁰ C !! 4.0 - 9.0 O₂ / No O₂  Energy Derived from the enzyma&c breakdown of organic substrates (carbohydrates, lipids or proteins) in a process called Catabolism Energy generated from catabolism is used to synthesise cellular cons&tuents in a process called Anabolism Catabolism + Anabolism = Metabolism Bacterial growth in diverse environments In addi&on to carbohydrates, lipids and proteins bacteria can also derive energy from plas&c, rubber and toxic compounds like phenol. Important implica&ons for decontamina&on of environmental pollu&on Exxon Valdez Oil Spill in Alaska: Engineered bacteria that “eat” hydrocarbons” were fer&lised onto beaches contaminated with oil. Known as bioremedia&on, this method was successful on several beaches where the oil was not too thick.  Auxotrophs The ability of individual bacterial species to produce their own cellular components will dictate its nutri&onal requirements E.g. some species can synthesis all essen&al amino acids whereas others need amino acids to be added to their growth media (auxotrophs).  Oxygen Obligate aerobes – grow only in presence of O2. Obligate anaerobes – grow only in absence of O2, killed by O2 Faculta&ve anaerobes – grow in presence & absence of O2  Temperature Psychrophile - cold loving bacteria (10-20⁰C) Mesophile (20-40 ⁰ C) – human body temperature – pathogens – opportunists Thermophile - heat loving (>60 ⁰ C)  pH Many bacteria grow best at neutral pH Some can survive/grow – acid – alkali  Extremophiles Antar&ca Hot geyser at Yellowstone Na&onal Park  Further Reading Brock, Biology of Microorganisms, Madigan, Mar&nko and Parker 10th Ed. Chapter 5 “Nutri&on, Laboratory Culture and Metabolism of Microorganisms” Chapter 6 “Microbial Growth”  MICR20010 Agricultural Microbiology Dr. Tadhg Ó Cróinín Assessments Praccal accounts for 30% 15% on the two Praccal reports to be submi#ed online a$er the praccals. Note these include write ups on praccals as well as online material. 15% on the Praccal Exam online to be held Friday Nov 22nd 2-3pm. 70% on an end of term MCQ exam in the RDS. Microbiology The study of microorganisms. Bacteria/Viruses which cause disease Bacteria which help – anbiocs, probiocs Biotech Industry Why is Microbiology Important? Industrial Microbiology Food and Beverage Industry Health Industry Environmental Microbiology Bacteria and their role in the ecosystem Polluon and Bioremediaon Clinical Microbiology Developing vaccines, anbiocs new treatments Diagnoscs MICR20010 - remaining lectures Lecture 10 – Microorganisms and Disease Lecture 11 – The Immune System Lecture 12 - Pathogenic Bacteria Lecture 13 – Pathogenic Fungi and Viruses Lecture 14 – Anbioc Resistant Microorganisms Lecture 15 – Microbiology in the Food Industry – The Fungi Lecture 16 – Microbiology in the Food Industry - Fermentaons Lecture 17 – The Nitrogen Cycle Microorganisms and Disease Microorganisms play a variety of di?erent roles in disease and we have complex relaonships with these organisms. Mutualism Bene@cial associaons – bacteria providing vitamin precursors in gut Commensalism Passive associaons – non pathogenic Staphylococci Parasism Microorganism causes harm – pathogenic bacteria Key is understanding the relationship How do we prove a pathogen Koch’s postulates 1. The m/o must be present in the diseased and not in a healthy animal 2. M/O must be cultivated in pure culture 3. Pure culture inoculated into 2nd animaldisease 4. Pure culture from 2nd animal should be same as 1st. Koch’s Postulates What about exceptions?  Some pathogens difficult to culture.  Some diseases are caused by combinations of  Pathogens  Physical, environmental  Genetic factors  Animal models and ethics: inoculation of healthy susceptible host not always possible (the postulate could never be fully applied to HIV) Virulence Factors A key di?erenal in pathogens is the presence of virulence factors which help the organisms cause disease. Evoluon allows these toxins to o$en be host speci@c Toxins which have very speci@c targets Adhesins which recognise speci@c receptors Other less so – Endotoxins (e.g. LPS) Virulence factors can thus de@ne host speci@city  Extracellular Enzymes Secreon of enzymes allows microorganisms to alter their environment Avoiding the Immune system– Some blood borne pathogens have the ability to secrete coagulase which leads to coagulaon allowing microorganisms to form clots which in turn can provide a physical hiding place from the immune system. Leukocidins can be used to destroy white blood cells. Catalase can be used to protect from reacve oxygen species in the Macrophage  Toxins  Exotoxins 1. Cytotoxins: kills or affects the functions of host cells 2. Neurotoxins: interferes with nerve cells 3. Enterotoxins: affects cells lining gut tract (clostridia, pathogenic strains of S.aureus and E.coli) Cytotoxins Neurotoxins Clostridum botulinum and Clostridium tetanii secrete extremely potent neurotoxins which lead to two very di?erent forms of fatal paralysis Related toxins Botulinum toxin inhibits the release of acetylcholine which smulates contracon therefore leading to relaxaon Tetanus toxin inhibits the release of Glycine which induces relaxaon of a contracted muscle which thus leads to contracon. Tetanus not a food poisoning toxin! Enterotoxins Clostridium perfringens secretes an enterotoxin which can induce gastroenters on its own Inoculaon with the toxin alone has the same e?ect as inoculang with the bacteria Virulence vs Colonization factors A virulence factor is directly involved in causing disease Toxins etc A colonizaon factor may be necessary for disease to progress but is not directly involved Adhesins and Jagella for molity Clostridium perfringens entertoxin (CPE) clearly a virulence factor Endotoxins Primarily found in Gram negave organisms Key is the constuents of the Outer membrane Lipopolysaccharide (LPS) Released on cell death or through membrane blebbing but has dramac e?ect on Immune system Anti-phagocytic factors  Capsules: many are made of chemicals found in body : no immune responses made  Anti-phagocytic compounds: some m/o make compounds that prevent fusion of lysosomes with phagocytic vesicles… (Neisseria gonorrhea)  Previously mentioned enzymes such as catalase Immune Evasion Phagocytosis blocked by capsule Surviving phagocytosis How is disease transmitted Microorganisms do not simply appear They can be already present and taking advantage of a change in environment – Opportunisc pathogens S. aureus commonly found on skin but pathogenic in blood infecons C. dicile takes advantage of anbioc treatments changing microbiome How are organisms transmi#ed - epidemiology Modes of Transmission A. Contact transmission Direct contact-person to person Indirect contact-needles, toothbrushes Droplet transmission- spread via droplet nuclei B. Vehicle transmission Air, drinking water, food C. Vector transmission Biological and mechanical Basic protections from infection Skin: barrier…tight layer of packed cells…entrance through cuts Mucous membranes: that line the body cavities open to the outside world (nose etc..) To infect: adherence of parasite to cells to allow for establishment of colonies Adaptive immunity Acquired immunity Develops from birth, as we encounter various pathogens Antigens trigger specific response Components of bacterial cells Cell walls, capsules Flagella Proteins (internal + external) Toxins Food may have antigens that provoke allergic reactions What are Antigens? Properties of antigens Antigens recognised by antigenic determinants (epitopes) >5-100KDa better than smaller antigens Proteins, glycoproteins etc… 500 different media for growing bacteria – reflects bacterial diversity COLONIES OF BACTERIA  Inspection Examination of colonies to determine if culture is pure – Each colony forms from a single cell – therefore the colony is an isolated population of an individual bacterial species The appearance of the colony is useful in identifying the bacterial species – Therefore in isolating a bacterial species, it is important that all the colonies appear the same Different colony type means that the culture is not pure or is mixed. – If culture is contaminated or mixed, a single colony of the desired species is subcultured In broth culture, not possible to determine if growth of more than one bacterial species has occurred.  Identification Macroscopic or colony morphology Microscopic morphology Biochemical characteristics Genetic characteristics  Disposal of cultures Sterilisation: Removal and destruction of all microbes in or on an object Physical Methods: Heat - Moist Heat. Boiling water, flowing steam. - Cells & most viruses. Not spores (Tyndallisation –intermittent boiling) – Steam (Autoclaving) 121oC - 15-30min. All spores/viruses/cells. Media & equipment - Dry Heat (Hot air), 1 hour at 171oC, – Incineration (burning) 1 sec or more at 1000oC) How an autoclave works  Disposal of cultures Radiation – Ionising e.g. X rays, gamma rays, secs-hrs. OH- radicals, damage to DNA. – Sterilize pharmaceuticals, medical supplies – Nonionising e.g. UV light. DNA damage. Operating theatres, kitchens  Disinfection Related process Reduction in bioload including the removal of pathogens Methods – Chemical – heat, e.g. pasteurisation – filtration Often easier to achieve than sterilisation and adequate for instruments in contact with mucous membranes, e.g. endoscopes Pasteurisation: Use of heat (e.g. 75°C, 15 seconds) to kill pathogens and reduce the number of spoilage micro-organisms in food and beverages (milk, fruit juice, wine, beer). Balance between removing microbes and affecting taste or quality of product  Antiseptics and disinfectants Antiseptics: microbicidal agents harmless enough to be applied to the skin and mucous membrane – should not be taken internally. Examples: mercurials, silver nitrate, iodine solution, alcohols, detergents.  Antiseptics and disinfectants Disinfectants: Agents that kill microorganisms, but not necessarily their spores, not safe for application to living tissues; they are used on inanimate objects such as tables, floors, utensils, etc. Examples: chlorine, hypochlorites, chlorine compounds, copper sulfate, quaternary ammonium compounds.  Antiseptics and disinfectants Note: disinfectants and antiseptics are distinguished on the basis of whether they are safe for application to mucous membranes. Often, safety depends on the concentration of the compound. For example, sodium hypochlorite (chlorine), as added to water is safe for drinking, but "chlorox" (5% hypochlorite), an excellent disinfectant, is hardly safe to drink.  Appropriate handwashing facilities Antiseptic hand wash  Further Reading Microbiology an Introduction, Tortora, Funke and Case 12th Ed. Chapter 6 “Microbial Growth” Chapter 7 “The control of Microbial Growth”  MICR20010 Agricultural Microbiology Dr. Tadhg Ó Cróinín Important Cycles for life on earth Carbon cycle Oxygen Cycle Nitrogen Cycle Others are “minor” but sll crical Microorganisms play key roles in all 2 Carbon Cycle 3 Oxygen Cycle 4 Importance of Microorganisms Microorganisms such as Prochlorococcus account for a huge percentage (20% by some esmates) of photosynthec oxygen producon on the planet This is more than all the tropical rain forests combined. Other organisms such as marine Algae play a crical role Need to also remember the historical context and endsymbiosis. 5 The arrival of Oxygen based metabolism Iron oxide bands from cyanobacterial photosynthesis Makes a huge di*erence to energy producon and evoluon of life Time Line Oxygen available as an electron carrier Much greater potenal for energy than in the anoxic environment Ozone layer protects from UV allowing for more stable DNA From Prokaryote to Eukaryote So what about Nitrogen? 9 Micro-organisms and the Nitrogen cycle. Healthy/normal Phosphate-deficient Potassium-deficient Nitrogen-deficient Nitrogen: Basic element for life- proteins, nucleic acids An element that is plenful in the atmosphere (78%) However, we and most other life forms cannot use this form of Nitrogen. Micro-organisms play a key role in providing us access to usable nitrogen sources. Brock 10th ed, Fig 19.29 Nitrogen: Brock 10th ed, Fig 17.36 Nitrogen: Brock 10th ed, Fig 17.36 Nitrogen: Brock 10th ed, Fig 17.36 Nitrogen: Brock 10th ed, Fig 17.36 Nitrogen: Brock 10th ed, Fig 17.36 Nitrogen: 3 major processes of m/o transformaon 1. Nitrogen