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LECTURE 4: MICROBIAL NUTRITION AND METABOLISM

Introduction: Microorganisms require about ten elements in large quantities because they are used to construct carbohydrates, lipids, proteins, and nucleic acids. Several other elements are needed in very small amounts and are parts of enzymes and cofactors. Biochemical Components of Cells

- Water: 80% of wet weight - Dry Weight

o Protein: 40-70% o Nucleic Acid: 13-34% o Lipid: 9-15% o Polysaccharide: 5.0% o Lipopolysaccharide: 3.4% o DNA: 3.1% o RNA: 20.5% o Also monomers, intermediates, and

inorganic ions Macronutrients – cells make proteins, nucleic acids and lipids.

- Macromolecules, metabolism - Dry weight percentage: C (50%), O (17%), N

(13%), H (8.2%), P (2.5%), S (1.8%), Se (<0.01%)

- Other elements: K, Mg, Fe - Sources: organic compounds and inorganic

salts Micronutrients – elements needed in trace quantities

- Refer to Table 4.1 (Trace Metals) in Lecture 4, Slide 7

o Co: Vitamin B12; transcarboxylase (only in propionic acid bacteria)

o Cu: In respiration, cytochrome c oxidase; in photosynthesis, plastocyanin, some superoxide dismutases

o Mn: Activator of many enzymes; component of certain superoxide dismutases and of the water-splitting enzyme in oxygenic phototrophs (photosystem II)

o Zn: Carbonic anhydrase; alcohol dehydrogenase; RNA and DNA polymerases; and many DNA-binding proteins

o V: Vanadium nitrogenase; bromoperoxidase

- Enzymes and Tap water - Refer to Lecture 4, Slide 8

o Essential to all: H, C, N, O, P, S, Se o Essential cations and anions for most:

Na, Mg, Cl, K, Ca o Trace metals: Y, Mn, Fe, Co, Ni, Cu,

Zn, Mo, W o For special functions: B, F, Si, As, Sr,

Cd, Ba o Unessential but metabolized: the rest o Unessential, unmetabolized: Noble

gases, Zr, Nb, Hf, Ta, Fe, Ds, Ir, At, Rs

Growth Factors

- Biotin: carboxylation (Leuconostoc) - Cyanocobalaman or Vit B12: molecular

rearrangements (Euglena) - Folic Acid: one-carbon metabolism

(Enterococcus) - Pantothenic Acid: fatty acid mechanism

(Proteus) - Pyridoxine or Vit B6: transamination

(Lactobacillus) - Niacin: precursor of NAD and NADP

(Brucella) - Riboflavin or Vit B2: precursor of FAD and

FMN (Caulobacter)

- Thiamine or Vit B1: aldehyde group transfer (Bacillus anthracis)

Energy Sources

Phototrophs Light

Chemotrophs Oxidation of organic or inorganic compound

Hydrogen and Electron Sources

Lithotrophs Reduced inorganic molecules

Organotrophs Organic molecules

Carbon Sources

Autotrophs CO2 sole or principal biosynthetic carbon

source

Heterotrophs Reduced, preformed, organic molecules from

other organisms

Major Nutritional Types

Sources of Energy, Hydrogen/Electrons or Carbon

Examples

Photolithotrophic Autotrophy

Light energy Inorganic hydrogen/electron donor CO2 carbon source

Algae; Purple and green sulphur bacteria; Blue-green algae (cyanobacteria)

Photoorganotrophic Heterotrophy

Light energy Organic materials

Purple and green non-sulfur

Major Nutritional Types

Sources of Energy, Hydrogen/Electrons or Carbon

Examples

Chemolithotrophic Autotrophy

Chemical energy source (inorganic) Inorganic hydrogen/electron donor CO2 carbon source

Sulfur-oxidizing bacteria; Hydrogen bacteria; Nitrifying bacteria; Iron bacteria

Chemoorganotrophic Heterotrophy

Chemical energy source (organic) Organic materials

Protozoa; Fungi; Most non-photosynthetic bacteria

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Chemoorganotrophs are by definition heterotrophs. Most chemolithotrophs and phototrophs are autotrophs. Autotrophs are sometimes called primary producers. They synthesize new organic matter from CO2. Virtually all organic matter on Earth has been synthesized by primary producers, in particular, the phototrophs. MICROBIAL PHOTOSYNTHETIC SYSTEMS:

Property Cyanobacteria

Green and

Purple Bacteria

Purple Nonsulfur Bacteria

Photo – pigment

Chlorophyll Bacterio-

chlorophyll Bacterio-

chlorophyll O2 Prod. Yes No No e

- donors H2O H2, H2S, S H2, H2S, S

Carbon source

CO2 CO2 Organic /

CO2 Primary products

of energy cnvrs’n

ATP + NADPH ATP ATP

CHEMOAUTOTROPH

Bacteria Electron Donor

Electron Acceptor

Products

Alcaligens and

Pseudomonas sp.

H2 O2 H2O

Nitrobacter NO2-

O2 NO3-, H2O

Nitrosomonas NH4+

O2

NO2-, H2O

Desulfovibrio H2 SO42-

H2O, H2S

Thiobacillus denitrificans

S0, H2S NO3

- SO4

2-, N2

Thiobacillus ferroxidans

Fe2+

O2 Fe3+

, H2O

UPTAKE OF NURIENTS – nutrient molecules frequently cannot cross selectively permeable plasma membranes through passive diffusion and must be transported by one of three major mechanisms involving the use of membrane carrier proteins.

1. Phagocytosis – protozoa 2. Permeability absorption – most organisms

a. Simple transport i. Passive or simple diffusion –

process in which molecules move from a region of higher conc. to one of lower conc. as a result of random thermal agitation. A few substances, such as glycerol, can cross the plasma membrane by this process.

ii. Facilitated diffusion (against or with the conc. gradient) – rate of diffused increases by the use of carrier proteins (permeases), which are embedded in the plasma membrane. The rate of facilitated diffusion increses with the conc. gradient much more rapidly and at lower conc. of the diffusion molecule than that of passive diffusion.

Note: The membrane carrier can change conformation after binding an external molecule and subsequently release the molecule on the cell interior. It then returns to the outward oriented position and is ready to bind another solute molecule. (No energy input)

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iii. Symport and Antiport

iv. Active Transport – is the transport of solute molecules to higher conc., or against, with the use of metabolic energy input.

b. Group translocation – (against the conc. gradient) the best-known group translocation system is the phosphoenolpyruvate: sugar phophotransferase system (PTS), which transports a variety of sugars into prokaryotic cells while simultaneously phosphorylating them using phosphoenolpyruvate (PEP) as the phosphate donor.

( )

( )

c. ATP-Binding Cassette (ABC) transporter – (against conc. gradient)

for the uptake of organic compounds like sugars and amino acids; and inorganic compounds such as sulphate and phosphate; and trace metals. And this process includes the following proteins:

i. Periplasmic binding protein – high affinity to substrate even at low conc. (less than 1 µM).

ii. Membrane spanning transporter – forms the transport channel

iii. ATP hydrolysing protein – supply energy

SIMPLE COMPARISON OF TRANSPORT SYSTEMS

Items Passive

Diff. Facilitated Diffusion

Group Trans-

location

ABC Trans.

Carrier Proteins

None Yes Yes Yes

Transport Speed

Slow Rapid Rapid Rapid

Against Gradient

No Yes / No Yes Yes

Transport Molecule

No spec.^

Spec.^* Spec.^ Spec.^

Metabolic Energy

No need Need* Need Need

Solute Molecules

Not changed

Changed* Changed Not

Changed

^specificity *may or may not be

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CULTURE MEDIA – are the nutrient solutions used to grow microorganisms (mostly chemotrophs) in the laboratory. Note: Most microorganisms have yet to be cultured. MAJOR CLASSES OF MEDIA

1. Defined – precise amounts of highly purified inorganic or organic chemicals; exact composition (qualitative and quantitative sense)

2. Complex – employ digests of microbial, animal or plant products, such as casein (milk protein), beef (beef extract), soybeans (tryptic soy broth), yeast cells (yeast extract), or any of a number of other highly nutritious yet impure substances.

3. According to Use a. Enriched – enhances growth (e.g.

Chocolate and Blood Agar) b. Selective – inhibits growth of other

bacteria (e.g. EMBA, BGLB) c. Differential – differentiates two types

of microorganisms in a single medium (e.g. EMBA, BGLB)

d. General Purpose – all-purpose medium (e.g. Nutrient A/B)

4. Fluidity a. Solid – contain 1.5-1.7% agar b. Semi Solid – contain 0.5-0.7% agar;

for studying motile bacteria c. Liquid – no agar

DEVELOPMENT OF SOLID MEDIUM

- Before Agar – liquid medium - Potato Slices – Robern Koch (1881) used

boiled potato slices but not all bacteria grew well

- Gelatin – Frederick Loeffler; meat extract medium + gelatin; liquid at room temperature

- Agar – Fannie Hesse (1882); agar is used for jams and jellies; generally not metabolized by microbes; liquefies at 100

oC and solidifies at

40oC.

ANAEROBIC CONDITION FOR GROWTH

- Reducing Media – contain chemicals (thioglycollate or oxyrase) that combine O2; heated to drive off or use up O2.

- Anaerobic Jar – production of an anaerobic environment; used in culturing bacteria that die or fail to grow in the presence of oxygen.

METABOLISM – total of all chemical reactions occurring in the cell.

- Anabolism – nutrients from the environment or those generated from catabolic reactions are converted to cell components.

- Catabolism – energy sources from the environment are converted to waste products.

o Fermentation – anaerobic catabolism; organic compound is both an electron donor and acceptor; ATP is produced by substrate-level phospholyration; since many microbial habitats lack O2, this is the only option for energy conservation by chemoorganotrophs.

o Respiration – catabolism in which a compound is oxidized; O2 (or substitute) as the terminal electron acceptor; usually accompanied by ATP production by oxidative phospholyration; more ATP produced than in fermentation (preferred).

A simple view of cell metabolism depicts how catabolic degradative reactions supply energy needed for cell functions and how anabolic reactions bring about the synthesis of cell components from nutrients ENZYMES – biological catalysts; lowers activation energy of a reaction; generally much larger than the substrate(s).

- Active Site – portion of the enzyme to which substrate binds

GLYCOLYSIS (Embden-Meyerhof-Parnas Pathway)

- Stage I: Preparatory reactions o Not redox reactions and do not

release energy

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o Production of key intermediate of pathway.

- Stage II: Production of NADH, ATP, and Pyruvate

o Redox reactions o Energy is conserved in the form of

ATP, and two molecules of pyruvate are formed

o Redox balance has not yet been achieved

- Stage III: Consumption of NADH and Production of Fermentation Products

o Redox reactions occur once again o Fermentation products formed

Note: Refer to Slides 42 and 43 in Lecture 4 for flowchart of the Glycosis pathway and products formed

KREB CYCLE (Citric Acid Cycle)

RESPIRATION AND ELECTRON TRANSPORT

- the biochemical pathways involved in the transformation of organic carbon to CO2

- the way electrons are transferred from the organic compound to the terminal electron

acceptor, driving ATP synthesis at the expense of the proton motive force

ELECTRON TRANSPORT – composed of membrane associated electron carriers.

- To accept electrons from an electron donor and transfer them to an acceptor

- To conserve some of the energy released during electron transfer for synthesis of ATP

- Redox Enzymes involved in Electron Transport:

o NADH dehydrogenase o Riboflavin-containing electron carriers,

generally called flavoproteins o Iron-sulfur proteins o Cytochromes o Non-protein carriers, lipid-soluble

quinones ATP AND CELL YIELD – the amount of ATP produced by an organism has a direct effect on cell yield

- Cell yield is directly proportional to the amount of ATP produced

- Energy costs for assembly of macromolecules are much the same for all microorganisms

ENERGY STORAGE – most microorganisms produce insoluble polymers that can later be oxidized for the production of ATP

- Polymer formation: potential energy stored in stable form; little effect on the internal osmotic pressure of cells; do not interfere with other cellular processes; readily accessible

PROTON MOTIVE FORCE

- Carriers in the Electron Transport Chain (ETC) o Arranged in increasing positive

reduction potential o Oriented in such a way that as

electrons are transported, protons are separated

o The final donating the electrons plus protons to a terminal electron acceptor such as O2

- H+ are extruded to the other surface of the

membrane - From two sources:

o NADH – nicotinamide adenine dinucleotide

o The dissociation of water into hydronium ion and hydroxide

- The extrusion of hydronium ion to the environment results in the accumulation of hydroxide on the inside of the membrane

- As a result, the two sides of the membrane differ in both charge and pH

- Formation of an electrochemical potential across the membrane = Proton Motive Force

ATP SYNTHASE (ATPase) – the use of PMF to general ATP

- Catalysed a reversible reaction between ATP and ADP + P1

- Two components:

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o F1 complex – carries out the chemical functions; α3β3γδε

o F0 complex – ion-translocating functions; a, b2, c12

CATABOLIC DIVERSITY – Refer to Slide 54 in Lecture 4

ANABOLISM: Biosynthesis

- Sugar

- Amino Acids

- Fatty acids

REGULATION OF THE ACTIVITY OF ENZYMES

- Feedback Inhibition – reaction shut off because of an excess of the end product

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- Allosteric Inhibition – allosteric enzymes has two binding sites: the active site (where substrate binds) and the allosteric site, where the end product of the pathway binds

- Covalent Modification – when cells are

grown with excess ammonia, glutamine synthetase (GS) is covalently modified by adenylylation; as many as 12 AMP groups can be added.

LECTURE 5: MICROBIAL REPRODUCTION AND GROWTH

GROWTH – increase in cellular constituents; leads to a rise in cell number

- Budding: is a form of asexual reproduction in which a new organism develops from an outgrowth or bud due to cell division at one particular site.

- Binary Fission: meaning "division in half", refers to a method of asexual reproduction. It is the most common form of reproduction in prokaryotes and occurs in some single-celled eukaryotes.

- Coenocytic organisms (multinucleate): growth results in increased cell size not in number

GROWTH CURVE – population growth is studied by analysing the growth curve of microorganisms

- Batch Culture: microorganisms are cultivated in a liquid medium; closed system; incubated in a closed culture vessel with a single batch of medium; no fresh medium provided during incubation; nutrient concentration decline and concentrations of waste increase during incubation period

- Plotted as the logarithm of cell number versus incubation time

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LAG PHASE – no immediate increase in cell mass or number (synthesizing new components)

- Necessity of a lag phase: o Cells may be old and ATP, essential

cofactors and ribosomes depleted (synthesized first before growth can begin)

o Medium may be different from the one the microorganism was growing previously (new enzymes would be needed to use different nutrients)

o Microorganisms have been injured and require time to recovery

- Cells retool, replicate their DNA, begin to increase in mass and finally divide

- Long Lag Phase: inoculum from old culture; from refrigerated source; into a chemically-different medium

- Short Lag Phase (or even absent): young, vigorously growing exponential phase culture is transferred to fresh medium of same composition

EXPONENTIAL PHASE/LOG PHASE – microorganisms are growing and dividing at the maximal rate possible even their genetic potential, nature of medium and conditions under which they are growing

- Rate of growth is constant - Microorganism doubling at regular intervals - Population is most uniform in terms of

chemical and physiological properties - Smooth curve because each individual divides

at a slightly different moment STATIONARY PHASE – population growth ceases and the growth curve becomes horizontal (around 10

9

cells on the average) - Due to: nutrient limitation (slow growth);

oxygen limitation; accumulation of toxic waste products

DEATH PHASE – detrimental environmental changes like nutrient depletion and build-up of toxic wastes lead to the decline in the number of viable cells

- Usually logarithmic (constant every hour) - Death: no growth and reproduction upon

transfer to new medium - Death rate may decrease after the population

has been drastically reduced due to resistant cells

MATHEMATICS OF GROWTH

- Generation Time: time required for a microbial population to double in number

- Mean Growth Rate Constant (k): rate of microbial population growth expressed in terms of the number of generations per unit time

- Mean Generation Time (g):

Example: Given an initial density of 4 x 10

4. After 2

hours, the cell density became 1 x 106. Compute for

the generation time.

( ) ( )

GENERATION OF SOME COMMON MICROBES

Microorganisms Temperature(oC)

Generation Time (hr)

Escherichia coli 40 0.35 Bacillus subtilis 40 0.43 Mycobacterium

tuberculosis 37 12

Euglena gracilis 25 10.9 Giardia lamblia 37 18 Sacharomyces

cerevisiae 30 2

MEASUREMENT OF CELL NUMBERS AND MASS

- Direct o Total Cell Count or Direct

Microscopic Counts

o Viable Cell Count

o Membrane Filtration Method: used to test large volumes of sample; size exclusion principle; usually used for coliform detection

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- Indirect o Biomass Determination: Biomass is

biological material derived from living, or recently living organisms.

o Turbidity Measurements

o Most Probable Number: count positive tubes and compare to statistical MPN table

CONTINUOUS CULTURE SYSTEM – a culture system with constant environmental conditions maintained through continual provision of nutrients and removal of wastes.

- Chemostat: feeds medium into the culture vessel at the same rate as medium containing microorganisms is removed; contains one essential nutrient in limiting quantities

o Dilution Rate = Nutrient Exchange:

o Rate if nutrient exchange

Expressed as the dilution rate (D)

Rate at which medium flows through the culture vessel to the vessel volume

D = f/V; where f is the flow rate (mL/hr) and V is the vessel volume (mL)

- Turbidostat: equipped with a photocell that adjusts the flow of medium through the culture vessel so as to maintain a constant cell density or turbidity

o Difference from Chemostat: dilution rate in a turbidostat varies rather than remaining constant and its culture medium lacks nutrients

o Operates best at high dilution rates (chemostat most stable stable and effective at low dilution rates)

ENVIRONMENTAL GROWTH FACTORS

- Solutes and Water Activity o Water Activity (aw = Psol/Pwater; P =

vapour pressure): degree of water availability; inverse proportional to osmotic pressure; 1/100 the relative of the solution when expressed as percent; example: osmotolerant yeasts and bacteria

o Compatible Solutes: solutes that are compatible with metabolism and growth when at high intracellular concentrations; example: Halobacterium (require high salt concentration for normal activity)

o Types of Microbes According to Solutes and Water Activity:

Osmophilic, osmophobic, osmotolerant (osmotic pressure)

Halophilic, halophobic, halotolerant (salt content)

Xenophilic, xenophobic, xenotolerant (dry environment)

Water Activity

Source Bacteria Fungi Algae

1.00 (pure water)

Blood

Most Gram (-) and non-halophile

None None

0.90 Ham

Most cocci and

Bacillus

Fusarium, Mucor,

Rhizopus

0.60 chocolate None S. rourii None

0.55 (DNA disordered)

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- Osmotic Pressure o Hypertonic environments, increase

salt or sugar, cause plasmolysis (loss of water of the cell)

o Extreme or obligate halophiles require high pressure

o Facultative halophiles tolerate high osmotic pressure

- pH – most bacteria grow between 6.5 & 7.5 o Molds and Yeasts grow between 5&6 o Types: acidophiles, alkalophiles,

neutrophils - Temperature – cardinal temperature:

minimum, optimum, maximum o Dependence on enzyme activity: as

the temp. rises, chemical and enzymatic reactions in the cell proceed at more rapid rates and growth becomes faster

o Above a certain temp., proteins, nucleic acids, and other cellular components may irreversibly damaged

o Dependence on lipid content Psychrophily: high content of

unsaturated fatty acids; help maintain a semi-fluid membrane state at low temp.

Thermophily: proteins or enzymes = increased number of salt bridges (resist unfolding in the aqueous milieu; membranes = rich in saturated fatty acids (stable at high temp.)

o Temperature Range Stenothermal Microbes:

small range; e.g. Neisseria gonorrhoea

Eurythermal Microbes: wide range; e.g. Enterococcus faecalis

o Spoilage: microbes grow between 0

oC and 20-30

oC, causing food

spoilage

- Oxygen Concentration o Obligate aerobes: top o Faultative anaerobes: mostly on top o Obligate anaerobes: bottom o Aerotolerant anaerobes: mostly

bottom o Microaerophiles: middle

o Toxic Forms of Oxygen Single Oxygen: O2 boosted to

a higher energy state

Peroxide anion: O2

2-

Hydroxyl radical: OH·

- Pressure: 1 atm (atmosphere)

o Barotolerant: increased pressure does adversely affect them but not as much as it does non-tolerant bacteria

o Barophilic: grow more rapidly at high pressures

TRIVIA: One barophile has been recovered from the Mariana Trench near the Philippines (10,500 m depth). It can only grow at pressure greater than 400-500 atm (at 2

oC).

ADDITIONAL NOTES ON TEMPERATURE

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LECTURE 6: MICROBIAL CONTROL

HISTORY – Bubonic Plague or the Black Death - Epidemic swept thru Europe in the Middle

Ages (13th and 14

th centuries)

- 40 million people were killed (about 1/3 of the population of the continent)

- Etiological agent: Yersinia pestis Gram (-) rod - 2 vectors (carriers): flea and rat - Infection:

o Flea bite with Yersinia pestis o Bacteria multiply in the bloodstream

(Bacteremia) o Bacteria localize in lymph nodes,

especially axillary and groin areas o Hemorrhaging occurs in lymph nodes,

resulting in reddish rash that will eventually become “black and blue” swellings or Buboes (hence the name, Black Death or Bubonic Plague)

o If untreated, about 50% mortality rate o If bacteria spread to the lungs, it

becomes Pneumonic Plague and is now highly contagious (almost 99% mortality rate)

- In relation to the song, a pouch of sweet smelling herbs or posies (other versions use flowers) was carried due to the belief that the disease was transmitted by bad smells (or sneezes).

- Other versions use “Ashes! Ashes!” since the

corpses of the infected were incinerated to kill the microbe.

HUMANS VS. MICROBES – most of history, microbes have been winning the battle; in the last 100 years or so the battle swung in our favour because of our increasing knowledge of how to control Microbial Growth (e.g. Smallpox (Variola virus) eradicated in 1977 (Somalia)) DEFINITION OF TERMS STERILIZATION – destroying all forms of life DISINFECTION – destroying pathogens or unwanted organisms DISINFECTANT – antimicrobial agent used on inanimate object ANTISEPTIC – antimicrobial agent used on living tissue BACTERICIDAL – kills bacteria BACTERISTATIC – inhibits bacterial growth ASEPTIC TECHNIQUE – use of specific methods to exclude contaminating microorganisms from an environment

DECONTAMINATION – treatment used to reduce the number of disease-causing microbes to a level that is considered safe to handle DEGERM – treatment used to decrease the number of microbes in an area PASTEURIZATION – a brief heat treatment used to reduce the number of spoilage organisms and to kill disease-causing microorganisms PRESERVATION – process of inhibiting the growth of microorganisms in products to delay spoilage ANITIZE – reduce the number of microorganisms to a level that meets public health standards implies cleanliness as well TERILANT – chemical used to destroy all microorganisms and viruses in a product, rendering it sterile STERILE – completely free of all microorganisms and viruses; an absolute term VIRICIDE – inactivates viruses GERMICIDE – kills microorganisms and inactivates viruses COMMERICIAL STERILIZATION: killing C. botulinim endospores MICROBIAL DEATH: a microbe is defined dead if it does not grow when inoculated into culture medium that would normally support its growth; defined as the inability of the organisms to form a visible colony

Bacterial poulations die at a constant logarithmic rate. It takes more time to kill a large population of bacteria than it does to kill a small population, because only a fraction of organisms die during a given time interval. CONDITIONS INFLUENCING EFFECTIVENESS OF ANTIMICROBIAL AGENT ACTIVITY

- Population Size: larger population requires a longer time to die

- Population Composition: microorganisms vary markedly on susceptibility; vegetative versus spores; young versus mature cells

- Concentration or Intensity of an Antimicrobial Agent: the more concentrated an agent the more rapidly microbes can be destroyed; but sometimes an agent may be

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more effective at lower concentrations (e.g. 70% alcohol)

- Duration of Exposure: the longer the exposure to an agent the more they will be killed

- Temperature: an increase in temperature at which chemical acts often enhances its activity (e.g. acids used in high temp. = more effective)

- Local environment: pH, organic matter, etc.; controls or protects the pathogen

TARGETS OF ANTIMICROBIAL AGENTS

- Cell membrane - Enzymes & Proteins - DNA & RNA

METHODS OF MICROBIAL CONTROL

- Physical: heat, filtration, radiation - Chemical

MICROBIAL CONTROL: HEAT

- Thermal Death Point (TDP): lowest temperature at which a microbial suspension is killed in 10 minutes

- Thermal Death Time (TDT): shortest time needed to kill all organisms in a microbial suspension at a specific temperature and under defined conditions

- Decimal Reduction Time or D value: time require to kill 90% of the microorganisms or spores in a sample test at a specified temperature; time required for the line to drop by one log cycle or tenfold; used to estimate the relative resistance of a microbe to different temperatures

Note: However, such a destruction is logarithmic and it is theoretically not possible to “completely destroy” microbes in a sample.

- Z value: increase in temperature required to reduce D to 1/10 of its value or to reduce it by one log cycle

- F value: time in minutes at a specific temperature needed to kill a population of cells or spores; usually at 121

oC

PHYSICAL METHODS: MOIST HEAT

- Calculations using D and z value o Given: For Clostridium botulinum

spores suspended in phosphate buffer, D121 = 0.204 min

o How long would it take to reduce a population of C. botulinum spores in phosphate buffer from 10

12 spores to

100 spores (1 spore) at 121

oC?

o Answer: Since 1012

to 100 is 12

decimal reductions, then the time required is 12 x 0.204 min = 2.45 min

o Given the D value at one temperature and the z value, we can derive an equation to predict D value at a different temperature:

(

)

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o Given: For Clostridium botulinum spores suspended in phophsate buffer, D121 = 0.204 min and z = 10

oC

o How long would it take to reduce a population of C. botulinum spores in phosphate buffer from 10

12 spores to

100 spores (1 spore) at 111

oC?

o Answer: To answer the question we need to know D111, which we can calculate from the formula:

(

)

( )

( ) o Given: For Staph. aureus in turkey

stuffing, D60 = 15.4 min and z = 6.8oC

o How long would it take to reproduce a population of Staph. aureus in turkey stuffing from 10

5 cells to 10

0 cells at

55oC, 60

oC, and 65

oC?

o Answers: Work it out for yourself. 55

oC: 419 min

60oC: 77 min

65oC: 14.2 min

APPLICATION: FOOD INDUSTRY

- If the z value for Clostridium spores is 10oC, it

takes a 10oC change in temp. to alter the D

value tenfold - Thus, if the cans are to be processed at 111

oC

rather than 121oC, the D value would increase

by tenfold t 2.04 minutes; 12D value = 24.5 minutes

MOIST HEAT STERILIZATION

- Horizontal and vertical autoclaves have the same efficiency, according to Ma’am Suyom.

-

HEAT – flaming; incineration; hot-air sterilization

Hot-air Autoclave

Equivalent treatments

170oC, 2 hours 121

oC, 15 min

HOW DOES HEAT KILL MICROBES?

- Moist Heat: kill effectively by degrading nucleic acids and by denaturing enzymes and other essential proteins; may also disrupt cell membranes

- Dry Heat: microbial death results from the oxidation of cell constituents and denaturation of proteins

CHEMICAL CONTROL - Chemical Agents

Chemical Agent

Effectiveness Against

Endospores Mycobacteria Phenolics Poor Good

Quats None None Chlorines Fair Fair Alcohols Poor Good

Glutaraldehyde Fair Good

o Phenolics: first widely used

antisepctic and disinfectant; Joseph Lister (1867) reduced the risk of infection during operations; act by denaturing proteins and disrupting cell membranes; Lysol and bisphenols: hexachlorophene, Triclosan

Advantages: effective in the presence of organic material and remain active on surfaces long after application

Disadvantages: disagreeable odor and can cause skin irritation and in some instances brain damage (hexachlorophene)

o Alcohols: ethanol, Isopropanol (70-80% concentration); act by denaturing proteins and possibly by dissolving membrane lipids; 10-15 soaking in alcohol is sufficient to disinfect thermometers and small instruments

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o Halogens Iodine: kills by oxidizing cell

constituents and iodinating cell proteins; kills spores at high conc.; disadvantage: a stain may be left (answer = iodophor)

Chlorine: usually for water supply; kills by oxidization of cellular materials and destruction of vegetative bacteria and fungi; will not kill spores; death within 30 mins

o Heavy Metals: Hg, As, Zn, Cu used as germicides; heavy

metals combine with proteins, often with their sulhydryl group and inactivate them; may also precipitate cell proteins

o Quartnary Ammonium Compounds (Detergents): amphiphatic (both polar and non-polar ends); kill by disrupting microbial membranes and denature proteins

Advantage: stable, non-toxic Disadvantage: inactivated by

hard water

Soap Acid-anionic

Degerming

Detergents Sanitizing

Quats Cationic detergents

Bactericidal, denature proteins, disrupt plasma

membrane

o Aldehydes: formaldehyde; very

reactive molecules that combine with proteins and inactivate them; sporicidal and can be used as sterilants

o Sterilizing Gases EVALUATION OF ANTIMICROBIAL AGENT EFFECTIVENESS

- Phenol Coefficient Test o Best-known disinfectant screening

test o Potency of a disinfectant is compared

with that of phenol o The highest dilution that killed

bacteria after a 10 minute exposure are used to calculate phenol coefficient

o The higher the phenol coefficient value, the more effective the disinfectant under these conditions; directly proportional

o The reciprocal of the appropriate test disinfectant dilution is divided by that for phenol to obtain the coefficient

o Example: phenol dilution = 1/90 and the maximum effective dilution for disinfectant X = 1/450

o Phenol coefficient = 5

- Use Dilution Test o Metal rings dipped in test bacteria are

dried o Dried cultures placed in disinfectant

for 10 min at 20oC

o Rings transferred to culture media to determine whether bacteria survived treatment

- Disk Diffusion

CHEMOTHERAPHY: Chemotherapeutic Agent

- Antibiotic: a product produced by a microorganism or a similar substance produced wholly or partially by chemical synthesis, which in low conc., inhibits the growth of other microorganisms

o Antibiotics are medicines used to treat infections caused by bacteria only

o Infections are usually caused by bacteria or viruses

o Antibiotics, therefore, do not cure all infections

o Many infections like the common cold, flu, mild sore throat, or diarrhea are caused by viruses

- Broth Dilution – Minimum Inhibitory Concentration (MIC) Test

- Agar Dilution – MIC Test

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- Agar Diffusion – Kirby-Bauer Disk Diffusion

- Diffusion depends on: o Concentration o Molecular weight o Water solubility o pH and ionization o Binding to agar

- Zones of Inhibition (~antimicrobial activity) depend on:

o pH of environment o Media components (agar depth,

nutrients) o Stability of drug o Size of inoculum o Length of incubation o Metabolic activity of organisms

When bacteria are exposed to an antibiotic, they either die or adapt. Those that survive carry genes that protect them against the antibiotic and pass those genes to other bacteria. Since bacteria multiply very quickly and can be easily spread among people, resistant bacteria can easily occur in places like hospitals and nursing homes, where a lot of people are gathered and antibiotic use is high.

EMERGENCE OF ANTIMICROBIAL RESISTANCE

SELECTION FOR ANTIMICROBIAL RESISTANT STAINS

RESISTANCE: Physiological Mechanism

- Lack of entry – tet, fosfomycin - Greater exit – efflux pumps, tet (R factors) - Enzymatic inactivation – bla (penase),

hydrolysis, CAT – chloramphenicol acetyl transferase, aminoglycosides & transferases

- Altered target o RIF – altered RNA polymerase

(mutants) o NAL – altered DNA gyrase o STR – altered ribosomal proteins o ERY – methylation of 23S rRNA

- Synthesis of resistant pathway – TMPr

plasmid has gene for DHF reductase; insensitive to TMP

CELL WALL SYNTHESIS INHIBITORS

- Resistance to β-Lactams – Gram (+)

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- Resistance to β-Lactams – Gram (-) [more resistant than (+)]

MECHANISM OF RESISTANCE

CONSEQUENCE OF RESISTANCE: Ecology of Pathogenesis

- Bacteria grow fast high population densities great competition for resources

- Pathogens = normal bacteria that has gained access to a new resource through new genes competitive advantage

Antibiotics revolutionized medicine. The first antibiotic, penicillin, was discovered by Alexander Fleming in 1929. It took less than 20 years for bacteria to show signs of resistance. Staphylococcus aureus, which causes blood poisoning and pneumonia, started to show resistance in 1950s. Today, there are different strains of S. aureus resitant to every form of antibiotic in use (MRSA = Multiple Resistance S. aureus).

WHERE DO WE GET ANTIOBIOTIC RESISTANT BACTERIA

- If a patient taking a course of antibiotic treatment does not complete it or forgets to take the doses regularly, then resistant strains get a chance to build up.

- When antibiotics are used on a person, the numbers of antibiotic resistant bacteria increase in other members of the family.

- In places where antibiotics are used extensively e.g. hospitals and farms, antibiotic resistant bacteria increase in number.

MYTHS AND FACTS ABOUT ANTIBIOTICS AND RESPIRATORY ILLNESS Myth: Taking antibiotic means I or my child can return to work or childcare sooner. Fact: Antibiotics do not shorten the duration of viral illnesses. Myth: Cold and flu symptoms will feel better or get better faster on antibiotics. Fact: Antibiotics cannot ease the symptoms of viral illnesses; these infections resolve on their own. Myth: Illnesses with the same symptoms require antibiotics. Fact: Illnesses with similar symptoms can be caused by different germs. Myth: If I take an antibiotic, I won’t spread my illness to others. Fact: Viral illnesses (cold, flu, etc.) usually spread from person to person before the onset of symptoms; before a person becomes ill. Antibiotics cannot stop the spread of viral illnesses. HOW TO STOP ANTIBIOTIC MISUSE?

- Don’t ask for antibiotics – let your doctor decided if you need them.

- Always take antibiotics exactly as prescribed. - Finish the whole prescription – do not stop

even when you feel better. - Never save antibiotics for a future illness – or

share with others. ANTIMICROBIAL RESISTANCE: Key Prevention Strategies

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12 STEPS TO PREVENT ANTIMICROBIAL RESISTANCE: HOSPITALIZED ADULTS

- Prevent Infection 1. Vaccinate 2. Get the catheters out

- Diagnose and Treat Infection Effectively 3. Target the pathogen 4. Access the experts

- Use Antimicrobials Wisely 5. Practice antimicrobial control 6. Use local data 7. Treat infection; not contamination 8. Treat infection, not colonization 9. Know when to say “no” to vanco

10. Stop treatment hen infection is cured or unlikely

- Prevent Transmission 11. Isolate the pathogen 12. Contain the contagion

ADDITIONAL FIGURES AND FLOW CHARTS

GLYCOLYSIS (In reference to Page 5)

STAGES I-III OF GLYCOLYSIS

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PRODUCTS OF PYRUVATE

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FERMENTATION VS. RESPIRATION