lecture 3: microbial metabolism, microbial growth, control of microbial growth edith porter, m.d. 1

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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