LECTURE 4. MICROBIAL GROWTH - RUA: Principal · L. 4: Microbial Growth. ... 2013-2014 LECTURE 4....

L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014

LECTURE 4. MICROBIAL GROWTH

1. Microbial survival strategies

2. Microbial growth

3. Effects of the environment

4. Microbial cultures

5. Growth measurements

6. Antimicrobial agents

7. Antibiotics


1. MICROBIAL SURVIVAL STRATEGIES PROKARYOTES: MINIATURIZATION

EUKARYOTES: COMPLEXITY

↑ Surface/Volumen

−Metabolic rates (osmotrophs) Rapid growth, short generation times

Population-level adaptation

Prokaryotes – r strategists: “the advantages of being small”

Eukaryotes –K strategists: “the advantages of being complex”

Phagocytosis

Complex genomes

Rapid movement

Independence from the environment

Complex sensor and motor systems

Individual-level adaptation


2. MICROBIAL GROWTH

GROWTH (IN MICROBIOLOGY)=INCREASE IN CELL NUMBER

Binary fission vs other

DNA replication and cell elongation

Septum formation

Cell separation

Completion of septum with formation of distinct walls

2.1. CELL DIVISION AND PARTITION OF CELL COMPONENTS


Partition of cell components: random (except DNA)

DNA SEGREGATION:

DNA binds to the cytoplasmic membrane

PROTEINS Fts

Bidirectional replication (in one fork)

Several simultaneous forks

2. MICROBIAL GROWTH 2.1. CELL DIVISION AND PARTITION OF CELL COMPONENTS


3. EFFECTS OF THE ENVIRONMENT 3.1. NUTRIENT CONCENTRATION

At 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 microorganisms

3.2. PREASURE

Al sea level = 1 atm

In the oceans up to 600 -1.100 atm

• No barotolerant • Barotolerant • Barophile (or piezophile)


3. EFFECTS OF THE ENVIRONMENT 3.3. TEMPERATURE

Adaptations to high temperatures Adaptations to low temperatures

Thermoresistent proteins (enzymes)

Stable membranes (↑ saturated fatty acids)

Archaea, special membranes (lipid monolayers)

Proteins (enzymes) that function optimally in the cold

Modified active transport processes

Fluidity of membranes (↑ unsaturated fatty acids)

• Psychrophile ( 0 - 20ºC) • Mesophile (10 - 50ºC) • Termophile (50 - 70ºC) • Hyperthermophile (80 - 121ºC*)

Enzymatic reactions occurring at maximum possible rate

Protein denaturation; collapse of the cytoplasmic membrane;thermal lysis

Membrane gelling; transport processes so slow that growth cannot occur

Enzymatic reactions occrring at increasingly rapid rates


3. EFFECTS OF THE ENVIRONMENT 3.4. OXYGEN CONCENTRATION

1 2 3 4 5

Aerobic (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


3. EFFECTS OF THE ENVIRONMENT 3.5. pH


3.6. OSMOLARITY

Adaptations to high salt

Counterbalance 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


3. EFFECTS OF THE ENVIRONMENT 3.7. RADIATIONS

Photodynamic effect: light-mediated generation of singlet oxygen ( 1O2 ) Carotenoids: photoprotectant pigments transform 1O2 into non toxic species Radiotolerant microorganisms: Bacterial endospores Deinococcus radiodurans


3. EFFECTS OF THE ENVIRONMENT 3.8. EXTREMOPHILISM AND EXTREMOPHILES


4. MICROBIAL CULTURES

CULTURE: a system used to allow the multiplication of a microbial population and reach a high microbial density

Culture components: Nutrients (medium) Inoculum (absence of contamination)

Culture types: Pure (or axenic)

Mixed

(According to the metabolic categories): C source E source Macronutrients (N, O, P, S, salts, vitamins, etc.) Micronutrients (normally, present as salt contaminants) H2O pH (buffers) WARNING! Auxotrophs vs prototrophs Inoculation

Preparation

Sterilization

Incubation

Types of culture media: Liquid / solid Defined, synthetic Complex Selective “Test”…



Closed 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


N = N02n

N= Number of cells after n generations N0 = Number of cells at the beginning tg = Generation time (time needed to double cell number) µ= specific growth rate (time units -1) (number of generations per time unit) tg = ln 2 / µ (hours)

Exponential growth

4. MICROBIAL CULTURES 4.1. BATCH CULTURE


4. MICROBIAL CULTURES 4.1. BATCH CULTURE


Y = (X – Xo) / S Y = yield X, Xo = cells/ ml S = nutrient concentration at to

Escherichia coli Tg= 20 min 4000 X Earth weight

LIMITING SUBSTRATE

Net growth: final biomass – initial biomass (inoculum)

Yield: unit of biomass produced per unit of nutrient consumed

4.1. BATCH CUTURE

µ depends on nutrient

concentration

µ = µ max S

Ks + S

After 48 hours



Cultures can be kept for long periods of time. Medium is added and culture removed, keepin V constant

4.2. CONTINUOUS CULTURE (“steady state”)

V entrance constant, [nutrient] constant, [cell] constant. V changes, µ changes and a new [cell] is reached



Biomass (X) constant in time. dX/dt = 0 Vproduction (cells produced) = Vlosses (cells removed)

4. MICROBIAL CULTURES 4.2. CONTINUOUS CULTURE (“steady state”)


4.3. CULTURES ON SOLID MEDIA 4. MICROBIAL CULTURES


5. GROWTH MEASUREMENTS

Only balanced growth (ordered increase of all cell components) can be measured properly…

5.1. BIOMASS

Dry weight Absorbance (cell density)*

5.2. CELL COMPONENTS

Nucleic acids, proteins, enzymatic activities

5.3. CELL NUMBRES

Total cells

Viable cells (culturable)***

Counting chamber

Flow cytometer

Plate counts

Most probable number (MPN)

***VBNC: Viable but not culturable


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 counts

Plate count (“viables”)

SAMPLE

5.3. CELL NUMBERS 5. GROWTH MEASUREMENTS


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


6. CONTROL OF MICROBIAL GROWTH

ANTIMICROBIAL AGENTS: either (i) limit or inhibit microbial growth or (ii) destroy microorganisms

• Sterilization: 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


6. CONTROL OF MICROBIAL GROWTH

ANTIMICROBIAL AGENTS


6. CONTROL OF MICROBIAL GROWTH 6.1. EFFECTS OF ANTIMICROBIAL AGENTS

BACTERIOSTATIC BACTERICIDE

BACTERIOLYTIC


6. CONTROL OF MICROBIAL GROWTH 6.2. FACTORS THAT AFFECT THE EFFICIENCY OF ANTIMICROBIAL AGENTS

• Population 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

agents • Concentration and performance of the antimicrobial agent • Exposure time • Temperature • Local environment: pH, organic matter, biofilms, etc.


6. CONTROL OF MICROBIAL GROWTH 6.3. STERILIZATION AND DISINFECTION BY PHYSICAL AGENTS

MOIST HEAT

Boiling in water for 10 minutes destroys

vegetative cells and eukaryotic spores but

NOT bacterial endospores

Autoclave: temperatures higher than 100oC (pressure) with water saturated

steam. Time: 10-15 minutes. Depends on the sample volume.



PASTEURIZATION NO STERILIZATION

Food 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 HEAT

Oven at 160-170 oC from 2 to 3 hours Not suitable for thermosensitive materials Used for glass, oil and other materials Suele utilizarse para material de vidrio, aceite y otros

materiales

Clostridium botulinum endospores Moist heat: 5 min at 121 oC Dry heat: 2 hours at 160oC


LOW TEMPERATURES

Inhibit growth Used to preserve (not to sterilize or disinfect)

FILTRATION



RADIATION

UV (ceiling, biological safety hoods) Ionizing radiation: very good sterilizing agent.

Pharmaceutical companies Disposable clinical materials Meat and other foods (spices)

Comercial radiation of spices and

seasonings, world data



Germicides Disinfectant and antispetic Antibiotics SELECTIVE TOXICITY

NO SELECTIVE TOXICITY

• Phenols • Alcohols • Halogenated compounds • Heavy metals • Aldehydes • Hydrogen peroxide • Surfactant agents • Ethiylene oxides • ANTIBIOTICS

STERILIZING 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 GROWTH 6.3. STERILIZATION AND DISINFECTION BY CHEMICAL AGENTS


6. CONTROL OF MICROBIAL GROWTH 6.3. STERILIZATION AND DISINFECTION BY CHEMICAL AGENTS


6. CONTROL OF MICROBIAL GROWTH 6.5. MEASURING ANTIMICROBIAL ACTIVITY

MINIMUM INHIBITORY CONCENTRATION


7. ANTIBIOTICS

BACTERIOSTATIC BACTERICIDE BACTERIOLYTIC

MAIN ANTIBIOTIC TARGETS

Cell wall synthesis Protein synthesis

Cell membrane integrity Nucleic acids synthesis

Essential cofactors synthesis

A 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”


ANTIBIOTIC MECHANISMS

7. ANTIBIOTICS


7. ANTIBIOTICS


7.1. CELL WALL (PETIDOGLYCAN) SYNTHESIS INHIBITORS

PENICILLINS (β-LACTAMIC)

β-lactamic ring (degraded by β-lactamases or penicillinases)

Penicilina G

Synthesized by the fungus Penicillium (Fleming, 1928) Bacteriolytic (destroy growing cells) They inhibit transpeptidation Bacteria can be resistant to penicillins if they synthesize penicillinases (β-lactamases) They can be combined with clavulanic acid

7. ANTIBIOTICS



PENICILLINS (β-LACTAMIC)

7. ANTIBIOTICS



CEPHALOSPORINS

Cephalosporium acreminium

They inhibit transpeptidation

7. ANTIBIOTICS


7.2. PROTEIN SYNTHESIS INHIBITORS

AMINOGLUCOSIDES TETRACICLINES

The effect cannot be reverted Examples: streptomycin, kanamycin, etc.

The effect can be reverted

CHLORAMPHENICOL MACROLIDES

Erythromycin

7. ANTIBIOTICS


7.3. OTHER MECHANISMS

Changes in the properties of the cell membrane: - Polymixin B Interference with nucleic acids synthesis: - Rifampicin (inhibits RNA polymerase) - Quinolones (inhibit DNA topoisomerases) Inhibit essential cofactors synthesis: - Sulfamides (inhibit folic acid synthesis)

7.4. BACTERIOCINS

Agents produced by certain bacteria (or archaea) that inhibit or kill closely related species.

7. ANTIBIOTICS


7.5. ANTIFUNGAL AGENTS 7. ANTIBIOTIC


7.6. ANTIVIRAL 7. ANTIBIOTICS


7.7. ANTIBIOTIC RESISTANCE 7. ANTIBIOTICS


RESISTANCE MECHANISMS 7.7. ANTIBIOTIC RESISTANCE

1. The antibiotic cannot reach its target

2. The antibiotic is degraded or modified

3. The antibiotic target is modified

Antibiotic resitance can be chromosomic (mutation) or

plasmidic (transferable)

Plasmids R

Can we stop antibiotic

resistance?

7. ANTIBIOTICS

LECTURE 4. MICROBIAL GROWTH - RUA: Principal · L. 4: Microbial Growth. ... 2013-2014 LECTURE 4....

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