Introduction Flashcards
Robert Hooke
First description microbes
Fruiting structure of moulds
Antoni van Leeuwenhoek
First description bacteria
Louis Pasteur
Bacteria --> fermentation Food sterilising Disprove spontaneous generation Develop methods to control growth Vaccines
Robert Koch
Microbes –> infectious diseases
Koch’s postulates
Develop techniques to culture microbes
Koch’s Postulates
- X needs to be present in every case of Y
- X must be grown in pure culture
- Cells from a pure culture of X must cause disease in a healthy animal
- X must be reisolated and shown to be the same as the original
All bacterial cells
Metabolism
Evolution
Growth
Some bacterial cells
Differentiation
Communication
Genetic exchange
Motility
Bacterial cytoplasmic membrane
fatty acids joined to glycerol via ester linkages
Archaeal cytoplasmic membrane
isoprene units joined to glycerol via ether linkages
Peptidoglycan
peptide bonds (amino acids) and glycosidic bonds (sugars):
- N-acetylglucosamine (G)
- N-acetylmuramic acid (M)
Gram + Cell Wall
-90% peptidoglycan Teichoic acids Lipoteichoic acids -Peptidoglycan = outer layer -Crosslinking through formation of peptide interbridge
Gram - Peptidoglycan crosslinking
- 10% peptidoglycan
- Sandwiched
- OM has LPS layer
- LPS: core polysaccharide. O-polysaccharide, lipid A (endotoxin)
- [LPS] = patient outcome
- Crosslinking through NH2 group of DAP of one glycan chain to COOH group of D-alanine on adjacent chain
Capsules
Polysaccharide Attachment (biofilm) Evasion of immune system Looks wet \+ and -
Periplasm
- Space between cytoplasmic and OM
- Gel-like
- Contain proteins
Gram stain
- Crystal violet + peptidoglycan (+) –> purple
- Crystal violet + less peptidoglycan (-) –> clear
- Clear (-) + counterstain –> pink
\+ = purple - = pink
Archaea cell walls
S layer Interlocking proteins and glycoproteins No OM or peptidoglycan Some have pseudomurein - N-acetylglucosamine (G) - N-acetyltalosaminuronic acid (M)
Fimbriae
Filamentous, linear projections
Adhesion
Multiple types
Some with adhesive domains along shaft that anchor cell by ‘zippering effect’
Bacterial Flagella
- Rotatable filamentous bacterial surface appendages involved in bacterial locomotion
- Different arrangements:
- Peritrichous (Starfish)
- Polar (Sperm)
- Lophotrichous (Jellyfish)
- Helical in shape, composed of flagellin
- Hook: single type of protein, connects filament to motor at base
- Motor (Mot proteins): anchored in the membrane and cell wall, drives rotation of flagella
- Fli proteins: motor switch, reversing direction of rotation in response to IC signals
- Around 50 genes: structural, chaperone, regulatory proteins
- Flagellin molecules synthesised in cytoplasm, move up through hollow core in filament
- 20,000 flagellin –> one filament
Archaeal Flagella
Most
Several different flagellin proteins
Amino acid sequence of archaeal flagellins is not related to bacterial flagellins
Gliding motility
Flagella-independent
Surface contact, slower
- Excretion of polysaccharide slime (cyanobacteria)
- Type IV pili, twitching motility by repeated extension and retraction (Myxococcus xanthus)
- Gliding-specific membrane proteins (Flavebacterium johnsoniae)
Cell motion as a behavioural response
Chemotaxis Phototaxis Aerotaxis Osmotaxis Hydrotaxis
- Taxis: directed movement in response to gradients
- Chemotaxis: chemicals
- Phototaxis: light
- Aerotaxis: oxygen
- Osmotaxis: osmolarity
- Hydrotaxis: water
Run and tumble
Chemoreceptors –> chemical concentration
More attractant –> more directed –> less tumbling, more running
Phosphate
- Some can accumulate inorganic phosphate PO43- for nucleic acid, phospholipid, ATP synthesis
- Accumulated in P-rich environments, used in limiting environments
Sulphur
- Some can oxidise H2S to produce energy in fixation of CO2
- Elemental sulphur S stored in sulphur globules in the periplasm
Magnetosomes
- Intracellular particles of magnetite (Fe3O4)
- Enable bacteria to orient themselves in a specific arrangement within a magnetic field
- Magnetosomes in aquatic bacteria –> orientation in water column
Gas Vesicles
- Confer buoyancy in planktonic cells
- Spindle-shaped gas-filled structures made of protein
- Gas vesicle membrane impermeable to water
- Allows photosynthetic Bacteria to optimise position in water column
Endospores
- Highly differentiated cells that is resistant to harsh conditions/environments
- “Dormant” stage of bacterial life cycle
- Ideal for dispersal via wind, water or animal gut (like a seed)
- Only present in some gram-positive bacteria
Microbial growth
Increase in number of cells
Binary fission
Cell elongation
Septum formation
Cell separation
Growth rate
Generation time
Batch culture
Growth curve
Growth rate- change in cell number/mass per unit time Generation time (doubling time)- interval for formation of two cells Batch culture- closed-system microbial culture of fixed volume Growth curve- growth as a function of time
Growth phases in batch culture
Lag
Exponential
Stationary
Death
Lag phase
- Cells adapted to stationary phase transferred to fresh medium
- Cells transferred from rich to minimal medium
- In both cases induction of new enzymes is required
Exponential Phase
- Number of cells doubles during a constant time interval
- Increase initially slow but increases at an ever faster rate
- Time taken for all components of the cell to double is the same (balanced growth)
- Growth is unrestricted (excess nutrients, no toxic products)
Stationary Phase
- 1 limiting nutrient or accumulation of inhibitory products
- Some metabolic activity slow down
- E. coli cells get smaller and contain more glycogen
- Adapt by activation expression of certain genes
- RNA polymerase composed of core enzyme and sigma factor (holoenzyme)
- Holoenzyme recognises promoter and initiates transcription
- After initiation of transcription, sigma factor dissociates
- RpoD is major sigma factor in bacteria
- RpoD directs transcription of most genes in the cell
- RpoD is active in exponential and stationary phase
- RpoS directs RNA polymerase to transcribe genes involved in stationary phase adaptation
- RpoS binds to distinct promoter consensus sequence
- Majority of transcription still depends on RpoD
Death phase
-Cells begin to die (cell lysis)
Total cell count
- Direct counting of cells under microscope in a chamber of known volume
- Rapid estimate of cell number
- Can’t distinguish live and dead cells
- Difficult to see under microscope
- Precision difficult to achieve
- Phase contrast required when not stained
- Suitable for density greater than 106 per ml (accuracy)
Viable Count
- Spread plate method
- Assumption that each viable cell with produce a single colony
- 10-fold dilutions to make sure a suitable number is plated
- Number of colonies may depend on conditions
- Small colonies could be overlooked
- Replicate plates (accuracy)
- Cell clumping could reduce counts
- Highly sensitive
Turbidimetric Measurements
- Spectrophotometer
- Measures light not scattered by bacteria
- Calibration of OD vs viable cell count
- High cell density –> backscattering –> deviation from linearity
- Rapid measurements without disturbing culture
Primary Metabolite
-Produced during exponential growth (alcohol)
Secondary Metabolite
- Produced during stationary phase (antibiotics)
- Not essential for growth
- Often significantly over-produced
Penicillin Production
- Stationary phase
- Excreted into media
- Extracted using organic solvents
Sterilisation
Inhibition
Decontamination
Disinfection
Sterilisation- killing or removal of all viable organisms within a growth medium
Inhibition- effectively limiting microbial growth
Decontamination- treatment of abject to make it safe to handle
Disinfection- directly targets removal of all pathogens, not necessarily all microbes
Heat Sterilization
Decimal reduction time
Thermal death time
Autoclave
Pasteurisation
- Most widely used
- High temperatures –> denature macromolecules
- Endospores can survive heat
- Decimal reduction time- time required for 10-fold reduction in viability
- Thermal death time- time it takes to kill all cells at a given temperature
- Autoclave- sealed heating device that uses steam under pressure (moist heat sterilization)
- 121 degrees, 10-15 minutes
- Pasteurisation- precisely controlled heat to reduce the microbial load in heat-sensitive liquids
- Doesn’t kill all organisms (not sterilisation)
- Controls pathogens and spoilage organisms
- Milk: 71 degrees for 15 seconds
- UHT milk: 135 degrees for 1-2 seconds
Radiation Sterilization
Microwaves, UV, gamma rays, electrons
UV has sufficient energy to cause modifications and breaks in DNA
-decontamination of surfaces
- Cannot penetrate solid surfaces (limited to exposed surfaces)
-Ionising radiation
- Electromagnetic radiation of sufficient energy to produce ions and other relative molecular species
Electrons, hydroxyl radicals, hydride radicals
- Cathode ray tubes, X-rays, radioactive nuclides
- Used in medical and food industry
- Approved by WHO and is used in USA for decontamination of foods
Filter Sterilization
-Avoids use of heat for sensitive liquids and gases
- Pores of filter are too small for organisms to pass through
- Large enough to allow liquid or gas to pass through
-Depth filters
- High efficiency particulate air (HEPA) filters
-Membrane filters
Function more like a sieve
Chemical Growth Control
-Bacteriostatic, bacteriocidal and bacteriolytic
-MIC
- Synthetic agents (growth factor analogues)
- Sulfanilamide (inhibits production of folic acid)
- Isoniazid (interferes with synthesis of myolic acid, a mycobacterial cell wall component)
- Antibiotics (including semi-synthetic agents)
Microbially produced
Antibiotics
- Targets properties not present in humans
- Cell wall synthesis
- Protein synthesis (50S and 30S inhibitors)
B-lactam Antibiotics
- Penicillin G active mainly against Gram +
- Chemically modified semi-synthetic antibiotics to change properties
- Transpeptidase enzymes in cell wall bind to penicillin (PBP)
- When PBP bind to penicillin, cross linking does not occur
- Cell wall synthesis continues –> weakened cell wall
- Penicillin-PBP complex stimulates release of autolysins (enzymes that degrade the cell wall) –> degraded cell wall
Antimicrobial Resistance Mechanisms
-Lacks structure that the antibiotic inhibits
Mycoplasmas lack a cell wall –> resistant to penicillin
-Impermeable to antibiotic
Most G- bacteria impermeable to penicillin
-Can inactivate antibiotic
B-lactamases cleave B-lactam ring (plasmids)
-Modify target of antibiotic
Mutations in PBPs, ribosomes
-Develop resistant biochemical pathway
Folic acid taken up from environment instead of being synthesised
-Efflux
Tetracycline efflux pathway
Staphylococcus aureus
Vancomycin is an antibiotic against S. aureus
- Glycopeptide antibiotic –> binds to D-Ala D-Ala on pentapeptide to block cross linking
- Also targets cell wall –> more difficult to develop resistance
Can develop resistance
-Replaces D-Ala D-Ala with D-Ala D-Lac
Peptidase cleaves D-Ala from D-Ala D-Ala and ligase adds lactate
-D-Ala D-Lac is still recognised for cross linking
