LECTURE 4. MICROBIAL GROWTH - PowerPoint Presentation

1. LECTURE 4. MICROBIAL GROWTH1. Microbial survival strategies2. Microbial growth3. Effects of the environment4. Microbial cultures5. Growth measurements6. Antimicrobial agents7. Antibiotics

2. 1. Microbial survival strategies PROKARYOTES: MINIATURIZATION EUKARYOTES: COMPLEXITY Surface/VolumenMetabolic rates (osmotrophs) Rapid growth, short generation timesPopulation-level adaptationProkaryotes – r strategists: “the advantages of being small”Eukaryotes –K strategists: “the advantages of being complex”PhagocytosisComplex genomesRapid movementIndependence from the environmentComplex sensor and motor systemsIndividual-level adaptation

3. 2. MICROBIAL GROWTHGROWTH (IN MICROBIOLOGY)=INCREASE IN CELL NUMBERBinary fission vs otherDNA replication and cell elongationSeptum formationCell separationCompletion of septum with formation of distinct walls2.1. CELL DIVISION AND PARTITION OF CELL COMPONENTS

4. Partition of cell components: random (except DNA)DNA SEGREGATION:DNA binds to the cytoplasmic membranePROTEINS Fts Bidirectional replication (in one fork) Several simultaneous forks2. MICROBIAL GROWTH2.1. CELL DIVISION AND PARTITION OF CELL COMPONENTS

5. 3. EFFECTS OF THE ENVIRONMENT3.1. NUTRIENT CONCENTRATIONAt a very low nutrient concentration, permeases cannot keep high levels of nutrients inside the cell and growth rate decreases.However, high nutrient concentrations can be toxic for many microorganisms3.2. PREASUREAl sea level = 1 atmIn the oceans up to 600 -1.100 atm No barotolerant Barotolerant Barophile (or piezophile)

6. 3. EFFECTS OF THE ENVIRONMENT3.3. TEMPERATUREAdaptations to high temperaturesAdaptations to low temperaturesThermoresistent proteins (enzymes)Stable membranes ( saturated fatty acids)Archaea, special membranes (lipid monolayers)Proteins (enzymes) that function optimally in the coldModified active transport processesFluidity of membranes ( unsaturated fatty acids) Psychrophile ( 0 - 20ºC) Mesophile (10 - 50ºC) Termophile (50 - 70ºC) Hyperthermophile (80 - 121ºC*)Enzymatic reactions occurring at maximum possible rateProtein denaturation; collapse of the cytoplasmic membrane;thermal lysisMembrane gelling; transport processes so slow that growth cannot occurEnzymatic reactions occrring at increasingly rapid rates

7. 3. EFFECTS OF THE ENVIRONMENT3.4. OXYGEN CONCENTRATION1 2 3 4 5Aerobic (1)Microaerophilic (aerobic) (4)Facultative (3)Aerotolerant anaerobe(5)(Strict) anaerobic (2)TOXIC OXYGEN SPECIES:Singlet oxygen (1O2)Superoxide (O2-)Hydrogen peroxide (H2O2)Hydroxyl radical (OH-)Enzymes that destroy them

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9. 3. EFFECTS OF THE ENVIRONMENT3.5. pH

10. 3.6. OSMOLARITY Adaptations to high saltCounterbalance of external osmotic preasure by accumlation of:Inorganic ions (K+) . Acidic proteins! Archaea and some extremely halophilic Bacteria.Compatible organic solutes (either imported or synthesized): glycine betaine, proline, glycerol, etc.3. EFFECTS OF THE ENVIRONMENT

11. 3. EFFECTS OF THE ENVIRONMENT3.7. RADIATIONSPhotodynamic effect: light-mediated generation of singlet oxygen ( 1O2 ) Carotenoids: photoprotectant pigmentstransform 1O2 into non toxic speciesRadiotolerant microorganisms:Bacterial endosporesDeinococcus radiodurans

12. 3. EFFECTS OF THE ENVIRONMENT3.8. EXTREMOPHILISM AND EXTREMOPHILES

13. 4. MICROBIAL CULTURESCULTURE: a system used to allow the multiplication of a microbial population and reach a high microbial densityCulture components:Nutrients (medium)Inoculum(absence of contamination)Culture types:Pure (or axenic)Mixed(According to the metabolic categories):C sourceE sourceMacronutrients (N, O, P, S, salts, vitamins, etc.)Micronutrients (normally, present as salt contaminants)H2OpH (buffers)WARNING! Auxotrophs vs prototrophsInoculationPreparationSterilizationIncubationTypes of culture media:Liquid / solidDefined, syntheticComplexSelective“Test”…

14. 4. MICROBIAL CULTURES

15. 4. MICROBIAL CULTURESClosed system (only energy, and sometimes gases, are interchanged with the external environment; no cells or disolved products).Growth curve with 4 phases.4.1. BATCH (DISCONTINUOUS) CULTURE

16. N = N02nN= Number of cells after n generationsN0 = Number of cells at the beginningtg = Generation time(time needed to double cell number)= specific growth rate (time units -1)(number of generations per time unit)tg = ln 2 /  (hours) Exponential growth4. MICROBIAL CULTURES4.1. BATCH CULTURE

17. 4. MICROBIAL CULTURES4.1. BATCH CULTURE

18. Y = (X – Xo) / S Y = yield X, Xo = cells/ ml S = nutrient concentration at to Escherichia coli Tg= 20 min 4000 X Earth weightLIMITING SUBSTRATENet growth: final biomass – initial biomass (inoculum)Yield: unit of biomass produced per unit of nutrient consumed4.1. BATCH CUTUREµ depends on nutrient concentrationµ = µ max SKs + SAfter 48 hours4. MICROBIAL CULTURES

19. Cultures can be kept for long periods of time. Medium is added and culture removed, keepin V constant4.2. CONTINUOUS CULTURE (“steady state”)V entrance constant, [nutrient] constant, [cell] constant. V changes, µ changes and a new [cell] is reached4. MICROBIAL CULTURES

20. Biomass (X) constant in time. dX/dt = 0Vproduction (cells produced) = Vlosses (cells removed)4. MICROBIAL CULTURES4.2. CONTINUOUS CULTURE (“steady state”)

21. 4.3. CULTURES ON SOLID MEDIA4. MICROBIAL CULTURES

22. 5. GROWTH MEASUREMENTSOnly balanced growth (ordered increase of all cell components) can be measured properly…5.1. BIOMASSDry weightAbsorbance (cell density)*5.2. CELL COMPONENTSNucleic acids, proteins, enzymatic activities5.3. CELL NUMBRESTotal cellsViable cells(culturable)***Counting chamberFlow cytometer Plate countsMost probable number (MPN) ***VBNC: Viable but not culturable

23. Description of natural microbial communities How many microbes are present in a natural sample?“Who” are they? What do they do? (niche) How do they relate to each other and to other organisms? (competition, antagonisms, symbioses, etc.)Direct countsPlate count (“viables”)SAMPLE5.3. CELL NUMBERS5. GROWTH MEASUREMENTS

24. Problems encountered when counting “viables” (i.e. when culturing) Are they dead?Are they viable but not culturable (VBNC)*?They do not grow on standard culture media*Important in public health

25. 6. CONTROL OF MICROBIAL GROWTHANTIMICROBIAL AGENTS: either (i) limit or inhibit microbial growth or (ii) destroy microorganismsSterilization: a process that destroys all living organisms and their viruses from an object or habitat.Disinfection: partial elimination or inhibition of microbes, normally pathogens Disinfectant: (chemical) agents used to disinfect; used on inanimate objects.Antisepsis: prevention of sepsis or infection (antiseptic agents are used over tissues to prevent infections, normally less toxic than disinfectant agents).Germicide: destroy germs (pathogens) and non-pathogens, but not necessarily spores (bactericide, algaecide, fungicide, virocide...)IMPORTANT CONCEPTS

26. 6. CONTROL OF MICROBIAL GROWTHANTIMICROBIAL AGENTS

27. 6. CONTROL OF MICROBIAL GROWTH6.1. EFFECTS OF ANTIMICROBIAL AGENTSBACTERIOSTATICBACTERICIDEBACTERIOLYTIC

28. 6. CONTROL OF MICROBIAL GROWTH6.2. FACTORS THAT AFFECT THE EFFICIENCY OF ANTIMICROBIAL AGENTSPopulation size: the same fraction of the microbial population is destroyed in each time interval; a larger population needs more time to be completely eliminated than a smaller one.Population composition: different microbes have different sensitivity to antimicrobial agentsConcentration and performance of the antimicrobial agentExposure timeTemperatureLocal environment: pH, organic matter, biofilms, etc.

29. 6. CONTROL OF MICROBIAL GROWTH6.3. STERILIZATION AND DISINFECTION BY PHYSICAL AGENTSMOIST HEATBoiling in water for 10 minutes destroys vegetative cells and eukaryotic spores but NOT bacterial endosporesAutoclave: temperatures higher than 100oC (pressure) with water saturated steam. Time: 10-15 minutes. Depends on the sample volume.

30. 6. CONTROL OF MICROBIAL GROWTH6.3. STERILIZATION AND DISINFECTION BY PHYSICAL AGENTSPASTEURIZATIONNO STERILIZATIONFood treatment (milk...)Old method: 63oC for 30 minutes.Fast pasteurization(HTST: high-temperature short-term): 72oC for 15 seconds.Sterilization at ultrahigh temperature (UHT: ultra-high temperature): 140-150oC for 1-3 seconds.DRY HEATOven at 160-170 oC from 2 to 3 hoursNot suitable for thermosensitive materialsUsed for glass, oil and other materials Suele utilizarse para material de vidrio, aceite y otros materialesClostridium botulinum endosporesMoist heat: 5 min at 121 oCDry heat: 2 hours at 160oC

31. LOW TEMPERATURESInhibit growthUsed to preserve (not to sterilize or disinfect)FILTRATION6. CONTROL OF MICROBIAL GROWTH6.3. STERILIZATION AND DISINFECTION BY PHYSICAL AGENTS

32. RADIATIONUV (ceiling, biological safety hoods)Ionizing radiation: very good sterilizing agent.Pharmaceutical companiesDisposable clinical materialsMeat and other foods (spices)Comercial radiation of spices and seasonings, world data6. CONTROL OF MICROBIAL GROWTH6.3. STERILIZATION AND DISINFECTION BY PHYSICAL AGENTS

33. GermicidesDisinfectant and antispeticAntibiotics SELECTIVE TOXICITYNO SELECTIVE TOXICITYPhenolsAlcoholsHalogenated compoundsHeavy metalsAldehydesHydrogen peroxideSurfactant agentsEthiylene oxidesANTIBIOTICSSTERILIZING AGENTS Ethylene oxide Formaldehyde Glutaraldehyde H2O2 30%DISINFECTANT AGENTS Alcohols Chlorinated compounds Phenolic compounds H2O2 6% ANTISEPTIC AGENTS Mercury-containing compounds Iodine H2O2 3% 6. CONTROL OF MICROBIAL GROWTH6.3. STERILIZATION AND DISINFECTION BY CHEMICAL AGENTS

34. 6. CONTROL OF MICROBIAL GROWTH6.3. STERILIZATION AND DISINFECTION BY CHEMICAL AGENTS

35. 6. CONTROL OF MICROBIAL GROWTH6.5. MEASURING ANTIMICROBIAL ACTIVITYMINIMUM INHIBITORY CONCENTRATION

36. 7. ANTIBIOTICSBACTERIOSTATICBACTERICIDEBACTERIOLYTICMAIN ANTIBIOTIC TARGETSCell wall synthesisProtein synthesisCell membrane integrityNucleic acids synthesisEssential cofactors synthesisA chemical substance produced by a microorganism (fungi or bacteria) that kills or inhibits the growth of another microorganism.Normally, they have selective toxicity*: the ability of a compound to inhibit or kill pathogenic microorganisms without adversely affecting the host. Thus, they can be used as chemotherapeutical agents.Some antibiotics are semi-synthetic.“*The magic bullet”

37. ANTIBIOTIC MECHANISMS7. ANTIBIOTICS

38. 7. ANTIBIOTICS

39. 7. ANTIBIOTICS

40. 7.1. CELL WALL (PETIDOGLYCAN) SYNTHESIS INHIBITORSPENICILLINS (b-LACTAMIC)b-lactamic ring (degraded by b-lactamases or penicillinases) Penicilina GSynthesized by the fungus Penicillium (Fleming, 1928)Bacteriolytic (destroy growing cells)They inhibit transpeptidationBacteria can be resistant to penicillins if they synthesize penicillinases (b-lactamases)They can be combined with clavulanic acid7. ANTIBIOTICS

41. 7.1. CELL WALL (PETIDOGLYCAN) SYNTHESIS INHIBITORSPENICILLINS (b-LACTAMIC)7. ANTIBIOTICS

42. 7.1. CELL WALL (PETIDOGLYCAN) SYNTHESIS INHIBITORSCEPHALOSPORINSCephalosporium acreminiumThey inhibit transpeptidation7. ANTIBIOTICS

43. 7.2. PROTEIN SYNTHESIS INHIBITORSAMINOGLUCOSIDESTETRACICLINESThe effect cannot be revertedExamples: streptomycin, kanamycin, etc.The effect can be revertedCHLORAMPHENICOLMACROLIDESErythromycin7. ANTIBIOTICS

44. 7.3. OTHER MECHANISMSChanges in the properties of the cell membrane: - Polymixin BInterference with nucleic acids synthesis: - Rifampicin (inhibits RNA polymerase) - Quinolones (inhibit DNA topoisomerases)Inhibit essential cofactors synthesis: - Sulfamides (inhibit folic acid synthesis)7.4. BACTERIOCINSAgents produced by certain bacteria (or archaea) that inhibit or kill closely related species.7. ANTIBIOTICS

45. 7.5. ANTIFUNGAL AGENTS7. ANTIBIOTIC

46. 7.6. ANTIVIRAL7. ANTIBIOTICS

47. 7.7. ANTIBIOTIC RESISTANCE7. ANTIBIOTICS

48. RESISTANCE MECHANISMS7.7. ANTIBIOTIC RESISTANCEThe antibiotic cannot reach its targetThe antibiotic is degraded or modifiedThe antibiotic target is modifiedAntibiotic resitance can be chromosomic (mutation) or plasmidic (transferable)Plasmids RCan we stop antibiotic resistance?7. ANTIBIOTICS