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MICR 201 Microbiology for Health Related Sciences
Lecture 3: Microbial metabolism, microbial growth, control of microbial growthEdith Porter, M.D.
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Lecture Outline Microbial metabolism
Overview Enzymes and cofactors Oxidation Reduction reactions ATP generation Respiration and fermentation Biosynthesis
Microbial growth Physical requirements Chemical requirements Biofilm Bacterial growth curve
Control of microbial growth Terminology Microbial death rate and actions of microbial control agents Physical methods Chemical methods Microbial resistance to control agents
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Microbial Metabolism
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Overview of cellular metabolism
Metabolism is the sum of all chemical reactions within a living cell
Includes catabolism and anabolism
Catabolism Complex organic
molecules converted to small simple compounds
Releases energy Anabolism
Simple compounds converted to complex organic molecules (biosynthesis)
Consumes energy Catabolism Anabolism
Metabolism=
+
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Enzymes
Chemical reactions accelerated by Temperature increase Enzymes
Enzymes (xxx-ase) Mostly proteins Specific for certain reactions Not changed upon the
reaction Typically re-usable Some require co-factor or co-
enzyme for activity▪ Co-factor: Ions
(magnesium,calcium)▪ Co-enzyme: organic molecule;
many are derivates from vitamines, e.g. NAD+ and NADP+
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Selected vitamins and their co-enzymatic function
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Factors influencing enzyme activity Temperature (if too
high: enzyme becomes denatured)
pH (if too extreme: enzyme becomes denatured)
Substrate concentration
Inhibitors E.g. cyanide, arsenic,
mercury Block enzymes that
require metal ions Tie up metal ion
activators of enzymes
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Classes of enzymes
Based on the chemical reaction Oxido-reductases: oxidation-reduction reaction in which
oxygen and hydrogen are gained or lost Transferases: transfer of functional groups Hydrolases: cleavage of molecules with hydrolysis (addition of
water) Lyases: removal of groups of atoms without hydrolysis Isomerases: rearrangement of atoms within a molecule Ligases: joining of 2 molecules
Based on the target Protease Lipase DNAse RNAse
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Oxidation-reduction reaction
: electron removal
: electron uptake Basic reaction
Biological reaction
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Generation of the Energy Currency ATP Adenosine Tri Phosphate
ADP + energy + phosphate ATP contains energy that can be easily released
(high-energy or unstable energy bond) Required for anabolic reactions Produced by
Substrate-level phosphorylation (fermentation): direct transfer of phosphate group from one molecule to the next
C-C-C~ + ADP C-C-C + ATP
Oxidative phosphorylation (respiration): involves electron transport chain, oxidation-reduction reactions and inorganic phosphate
C D
P
ADP + ATPP
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Electron transport chain
Important electron carriers NAD+ FMN FAD
Important oxidase Cytochrome oxidases E.g. cytochrome C oxidase
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Chemiosmosis and the proton motive force
The electron flow is coupled to H+ efflux via proton pumps
H+ accumulates outside and a chemical and charge based gradient is generated (potential energy)
Special protein channels allow H+ flux back into the cell
Re-entering of H+ into the cell generates energy for ATP Motility Active transport
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Overview of respiration and fermentation
2 ATP (energy entrapped in organic compounds)
36 ATP
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Fermentation products
CO2 and H2 gas production!Acid production will lower
pH!!!
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Industrial fermentation products
Grapes and yeast: wine Grain and yeast: beer Milk and lactobacilli: yogurt Milk and lactobacilli and
propionibacteria: swiss cheese Ethanol and Acetobacter: vinegar And many more…
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Diagnostic use of cytochrome C oxidase
Part of electron transport chain Membrane-bound, water soluble
enzyme Found in some bacteria like
Pseudomonas aeruginosa or Neisseria
Can be easily detected by adding to the grown cultures a substrate that changes color when oxidized “oxidase positive”
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Diagnostic use of fermentation Single sugar + protein + pH indicator+ Durham
tube Inoculate organism and incubate for 24- 48 h
Gas
Turbid = growthYellow/orange = acid
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Important sugars used in clinical microbiology
Glucose: to test for ability to conduct fermentation
Lactose: many intestinal pathogens are lactose negative!
Mannitol: used to screen for Staphylococcus aureus which is able to ferment mannitol
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Non-Fermenters
Group of important organisms that are under no circumstances able to perform fermentation
Only respiration is possible Example: Pseudomonas aeruginosa Non-fermenters play a role as
opportunistic pathogens in the hospital setting
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Lipids and proteins are funneled into glucose catabolism
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Anabolic reactions: go backwards…
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Important to remember
Respiration Complete oxidation of glucose to carbon dioxide and
water while ATP is generated Involves glycolysis, krebs cycle and extensive electron
transport chain Higher energy yield, faster growth
Fermentation Anaerobic process Involves glycolysis and production of organic compound Low energy yield, slower growth
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Microbial Growth
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Requirements of microbial growth
Physical Temperature Osmotic pressure
▪ Salt: halophil ▪ High salt (Halobacterium spec. requires 30% !!)
pH▪ Low pH (1.0 -2.0): acidophil▪ High pH (> 8.0): alkaliphil
Chemical Elements:
▪ Macroelements: C, N, S, P▪ Trace elements: iron, copper, zinc
Atmosphere▪ Oxygen▪ CO2
-PHIL MEANS MUST HAVE!!!
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Microbes differ in their temperature optimum
Psychrophiles: -10 to 20C Psychrotrophs: 0 to 30 C Mesophiles: 10 to 48C Thermophiles: 40 to 72C Hyperthermophile: 65 to
110C
Only Archaea can grow above 95C!
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How does optimal growth temperature relate to your daily live?
Some pathogens can multiply in the refrigerator: Listeria monocytogenes
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Effect of salt on cells
Similar effect with sugars
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Oxygen is toxic
Oxygen is readily converted into radicals (singlet oxygen, superoxide, hydrogen peroxide, hydroxyl radical)
Most important detoxifying enzymes are superoxide dismutase and catalase
Cells differ in their content of detoxifying enzymes and hence, ability to grow in the presence of oxygen
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Effect of Oxygen on Microbial Growth
Type of Bacteria
Catalase
Superoxide
Dismutase
Oxygen and Growth
Obligate aerobes + + Require oxygen
Facultative anaerobes
+ + Can proliferate with and without oxygen
Obligate anaerobes
- - Cannot survive oxygen, must have anaerobic conditions
Aerotolerant anaerobes
- + Survive oxygen but cannot use it for growth
Microaerophiles (+) (+) Require low levels of oxygen
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Catalase is an important diagnostic enzyme
Classification of gram-positive cocci Staphylococci are catalase + Streptococci are catalase -
Staphylococci
Streptococci
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Other atmosphere requirements
Enhanced CO2 concentration (5%) Capnophile Many mucosal pathogens are
capnophile
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Food preservation
Most pathogens Are mesophiles Require moderate
pH Require
physiological salt concentrations
Require an atmosphere
To prevent spoilage Refrigeration Acidity Add salt or high
concentrations of sugar
Vacuum package
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Bacterial cell division under optimal conditions
Average reproduction rate of E. coli: ~ 20 minutes
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Bacterial growth curve
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Bacterial growth phases
Lag phase Bacteria adjust to new medium
Log phase: Logarithmic growth, all cells in the same growth phase
Stationary phase: Nutrients limited, population very inhomogeneous, Bacilli/Clostridia: sporulation; Some pathogens upregulation of virulence factors Biofilm production
Decline phase: Accumulated toxic products, nutrients exhausted
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Bacterial biofilm
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Important to remember
XX-phile Requires XX for growth
Ability to survive oxygen depends on the presence of enzymes that detoxify oxygen radicals
The typical growth curve of bacteria includes lag, log, stationary and decline phase
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Control of Microbial Growth
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Definition of key terms
Disinfection: removal of potential vegetative pathogens on in-animated objects (disinfectants)
Antisepsis: removal of potential vegetative pathogens on tissues (antiseptics)
Sterilization: eliminates all forms of microbial life (and prions)
Commercial sterilization: killing of C. botulinum endospores
Sanitization: generates safe conditions for the public
Degerming: modified antisepsis, mechanical removal of microbes with alcohol patch
Pasteurization: eliminates pathogens and spoilage microbes
40Time [h]
CFU
/ml
Addantimicrobi
al
Antimicrobial effects
-cidal: to kill, reduce numbers of viable microbes
-static: to prevent growth and proliferation
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Modes of action of antimicrobials Alteration of Membrane permeability Damage to Proteins
Disulfide bridges Hydrogen bonds
Damage to Nucleic acids Strand brakes Dimerization
Loss of activity
Errors in proteins with loss of function or no protein at all
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Factors affecting efficacy of antimicrobials
Microbial population number, composition
Concentration of agent Exposure time Environment
Temperature pH Pressure Presence of organic material
70% EtOH is more effective than 95% EtOH
Heat works better at low pH!
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Methods to Control Microbial Growth
Physical Heat Cooling Filtration Pressure Desiccation Radiation
Chemical Liquids Gas
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Heat Inactivation of Microbes: Autoclave Moist sterilization under pressure Exposure time: 15 min 121° C at 15 psi High pressure and high heat Special training is required to use an
autoclave
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Critical Thinking…
How can you prove that the autoclave is properly functioning and fulfills the requirements to Eliminate ALL microbial life forms?
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Heat inactivation of microbes: dry heat
Dry-heat sterilization 2 – 3 hrs 160 – 170° C
Prevents corrosion Suited also for powders
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Pasteurization
Kills pathogens, reduces spoilage organisms
Introduced by L. Pasteur in 1860s in wine production 30 min 55 – 60 ° C
Today: 30 min 63° C Flash: 15 sec 72° C
Better taste Ultra high temperature treatment for milk
1 – 3 sec 140 – 150 ° C
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Removal of microbes by filtration
0.2 (0.45) mm pore size Limitations
Cell wall less microbes (e.g. mycoplasma) are not removed
Problem in cell culture laboratories Viruses, nanobacteria not removed
Specialty filters with 0.01 mm pore size
Other material may adhere to the filters
HEPA filter with 0.3 mm pore size
filter air that go into special rooms
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Control of microbes through low Temperature
Most pathogenic bacteria do not replicate at 4C (static effect) Exception: Listeria monocytogenes
Freezing: most damage occurs during thawing Some worms are killed during storage at
subzero
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Control of microorganisms through pressure
High atmospheric pressure Prevent spoilage and preserve taste Fruit juices
Osmotic pressure Hypertonic High salt or high sugar Used in food preservation However, often molds can still grow
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Desiccation
Prevents typically proliferation but does not kill Exception:
Neisseria gonorrhoeae
Bacterial spores in particular resistant to desiccation Survive for
thousands of years Problem: dried pus,
urine, feces in hospital setting (mattresses…)
http://prokariotae.tripod.com/Neisseria_gonorrhoeae.jpg
http://www.acmp.com.au/portfolios/mischkulnig/images/hospital-bed.jpg
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Control of microbes through radiation
Ionizing (< 1nm wavelength) Gamma-rays (used for spices), x-rays, high-energy
electron beams Ionize water hydroxyl radicals damage of DNA and other
molecules Non-ionizing
UV light (1 – 400 nm, 260 nm!) Thymine dimerization
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Control of microbes through gas Ethylene oxide
Denatures proteins, attacks SH-, COOH- OH- groups
Highly penetrating Sterilize in closed chamber 4 – 18
hours Medical supplies, space crafts,
mattresses Caution: cancerogenic
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Control of microbes through liquid chemical agents
Agent Mechanism Preferred Use ExamplesPhenol based Disruption of membrane,
protein denaturationHospitals, work well in the presence of organic material, Mycobacteria
AmphylTriclosan
Biguanide Disruption of membrane Surgical scrubs Chlorhexidine
Halogenes Strongly oxidizingCellular function and structures altered
Wound treatment (I2)Household (CL2)
PovidoneiodineChlorox
Alcohol Protein denaturationDissolution of membrane
ThermometerSkin scrubbing (alcohol pads)
EthanolIsopropanol
Aldehydes Protein cross linker FixativeSurgical Instruments
FormalinGlutaraldehyde
Peroxygenes Oxidation Deep wounds with anaerobes
Peracetic acid
Detergents Membrane disruptionProtein denaturation
Industrial Instrument sanitizers
SoapZephiran
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Antimicrobial additives
Organic acids in food and cosmetics Sorbic acid Benzoic acid
Heavy metals (copper, silver, zinc) Copper: as algicide, copper coated cell
incubators Silver nitrate: prevention of ophthalmica
neonatarum, wound treatment Zinc chloride: wound treatment
Antibiotics (NOT as DRUG!!) Nisin and natamycin in cheese
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Problem: sensitivity of microbes varies
Major concern Endospores Mycobacteria Prions
▪ 134C autoclaving and sodium hydroxide not 100% effective
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Important to remember
You try .....
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Extra credit opportunity!
Look in your household and identify antimicrobial additives.
Bring a list describing 2 items, the antimicrobial additives incorporated (must be 2 different ones), and their mode of action in table format (as shown to the left) to class.
Complete tables will be worth 5 points
Item Antimicrobial Additive
Mode of Action