control of microorganisms unit 7

7/30/2019 Control of Microorganisms Unit 7

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Control of Microorganisms

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Control of Microorganisms

Outline:

Definitions

Conditions Influencing Microbial Activities Physical Methods

Chemical Methods

Chemotherapeutic Agents

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Definitions

Sterilization: A treatment that kills or

removes all living cells, including viruses

and spores, from a substance or object.

Disinfection: A treatment that reduces the

total number of microorganisms on an

object or surface, but does not necessarily

remove or kill all of the microorganisms.

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Definitions

Sanitation: Reduction of the microbial

population to levels considered safe by

public health standards

Antiseptic: A mild disinfectant agent

suitable for use on skin surfaces

-cidal: A suffix meaning that the agent

kills. For example, a bacteriocidal agent

kills bacteria

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Definitions

static: A suffix that means the agent

inhibits growth. For example, a fungistatic

agent inhibits the growth of fungi, but

doesnt necessarily kill it.

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Conditions Influencing Antimicrobial

Activity

Population size

Types of organisms

Concentration of the antimicrobial agent

Duration of exposure

Temperature pH

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Antimicrobial Targets

Cell membrane

Enzymes & Proteins

DNA & RNA

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Established methods of microbial

control

1. Physical agents

used exclusively on objects outside the body

2. Chemical agents

used on inanimate objects as, well as on thebody surface

3. Chemotherapeutic agents

most often used inside the living body

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Physical Methods of Control

Moist Heat

Dry Heat

Low Temperatures Filtration

Irradiation

Drying Osmotic Strength

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Moist Heat

Mechanism: protein/nucleic acid

denaturation

and membrane disruption

Presence of spores- more difficult to kill

Effectiveness dependent on: type of cells present

environment (type of medium or substrate)

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Moist Heat

Measurements of killing:

Thermal Death Point (TDP)

lowest temp. at which all microorganism in a

liquid culture are killed in 10 minutes

Thermal Death Time (TDT)

time required to kill a known population of

microorganisms in a specific suspension ata particular temperature does not account forthe logarithmic nature of the death curve(theoretically not possible to get down to zero)

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Methods of Moist Heat

Methods:

Boiling at 100 degrees Celsius

Autoclaving Pasteurisation

Tyndallisation

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Boiling

Kills most vegetative bacteria and viruses

within 10 minutes( not ideal for heat

sensitive chemical etc). Generally done at

100 C for 30 minutes.

Bacterial endospores can survive boiling

temperatures

Some bacterial toxins are heat resistant

e.g. Staphylococcal enterotoxin

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Autoclaving (steam underpressure)

Preferred method of sterilization

Water boils at 100 C higher temp. may be

obtained under pressure.

Increasing the pressure:15 psi raises the

Temp. 121C

121 C for 15-30 min.

NB!! validated autoclave by testing with spores ofClostridium or

Bacillus stearothermophilus

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Schematic diagram of a laboratory autoclave in use to

sterilize microbiological culture mediumThe sterilization process is a 100% kill, and guarantees that the medium will

stay sterile unless exposed to contaminants.

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Pasteurization

Reduces microbial count in milk/beverages

Eliminates the transmission ofCoxiellaburnetti, Mycobacterium tuberculosis,

Brucella, Staphylococcus, Salmonella andE. coli .

Initially food was heated at 66C for 30minutes

Flash pasteurization 71 C for 15 seconds.140 150C for 1-3 sec (UHT)

Does not sterilize

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Tyndallisation

Boiling a solution for 30 minutes

boiled medium is cooled

incubated for a period of hours

boiled again and this cycle is repeated threetimes.

Cooling facilitates germination of endospores

into heat-sensitive vegetative cells.

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Dry Heat

Incineration

Burner flames, electric loop incinerators

Oven sterilization

glassware & heat-resistant metal equipment

generally 2 hr at 160C is required to kill

bacterial spores by dry heat-does not include

penetration nor cooling time

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Low Temperature

Refrigeration

Temperature: 4C

inhibits growth of mesophiles and

thermophiles but not psychrophiles Freezer:

ordinary freezer around -10 to -20C

ultracold laboratory freezer typically -80C Generally inhibits all growth; many survive

freezing temperatures

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Low temperature is not damaging to mostmicroorganisms

For most when brought up to suitable

temperatures, will begin growing again. Bacterial cells are too small for ice crystals toform within them, they are not killed by themechanical destruction of cellular structures.

Instead they are killed by the high osmoticstrength that develops as water in theenvironment freezes

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Filtration

mechanical device for removingmicroorganisms from a solution.The organisms are trapped in the pores of the

filter, and the filtrate is decontaminated orpossibly sterilized.

culture media

enzymes

vaccines antibiotics

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Filtration

Three types of Filters:

Depth filters: fibrous sheet or mat forming arandomly arranged lattice of paper, asbestos

etc Traps particles in network

Membrane filters: most common type made

of polymers with high tensile strength,functioning like a sieve eg. Nitrocellulose,nylon, polyvinylidene difluoride

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HEPA filters: High efficiency particulate air

filters used in laminar flow biological safety

cabinets.

Filtration does not remove viruses from

solution -too small In essence the solution is nonsterile

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Irradiation

high energy electromagnetic radiation used in

the reduction of microbial load.

Types of electromagnetic radiation: UV light

X-rays

Gamma rays

Electrons beams

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Irradiation

Types of Radiation :

1. ionizing radiation

2. Non-ionizing radiation.

Ionizing Radiation; has enough energy to remove electrons from

a target molecule causing it to form ions. EgXrays, gamma rays and electron beam.

Powerful sterilizing agent; penetrates and damagesboth DNA and protein; effective against bothvegetative cells and spores

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Irradiation

2. Non-Ionizing Radiation

UV Light has, a wavelength between 100 and

400 nm, and the energy at about 265 nm is

most destructive to bacteria.

The spectrum of visible and invisible energies.

Exposure to UV damages the DNA. UV

radiations are used to reduce air contamination.

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Drying

removal of H2O

involve removal of water from product by

heat, evaporation, freeze-drying

Frequently used to preserve perishable

materials such as proteins, blood products

and reference cultures of microorganisms,

Often used to preserve foods (e.g. fruits,

grains, etc.).

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Osmotic Strength

Utilises high concentrations of salt or

sugar

High osmotic strength of salt and sugar

solution - damage cells by plasmolysis.

Method not used routinely in laboratory

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Chemical Methods of Control

Antimicrobial agents employs the use of naturalor synthetic chemicals that kills or inhibitmicrobial growth

Employs technique of selective toxicity

Denature protein and disrupt membranes Rarely achieve sterilization as in physical

methods. The process of removal is calleddisinfection.

If the object is non living, the chemical is knownas disinfectant, if the object is living, as a tissueof human body, then the chemical is anantiseptic.

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Chemical Agents

Phenolics

Alcohols

Halogens

Heavy metals

Quaternary Ammonium Compounds

Aldehydes

Sterilizing Gases

Evaluating Effectiveness of Chemical Agents

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Table showing: Antiseptics, sterilants,

disinfectants and sanitizers

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Chemical Agents: Disinfectants

Chemicals used to kill pathogenicmicroorganisms

May or may not kill endospores

Used on inanimate objects

Include: chlorine compounds such as hypochlorites,

copper sulfate

quaternary ammonium compounds.

Uses:

Important in infection control

Decontaminate surfaces eg tables, floors etc

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Chemical Agents: Antiseptics

inhibit or kill microorganisms and are safe touse on the skin and mucous membranes, butare normally not taken internally.

Examples include: Mercurials silver nitrate iodine solution Alcohol Used to reduce but not eliminate

microbial load to a safe number.

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Chemical Agents: Sterilants

Sterilizers or sporicides

Destroy vegetative cells and endospores

Ideally used when the use of heat or radiation

is not practical

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Types of sterilants

High level Germicides

These are generally alkylating agents, which kill by

adding alkyl groups to nucleic acids or proteins.

Intermediate-Level Germicides

little activity against endospores.

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Phenols

Aromatic organic compounds with attached-OH

Denature protein & disrupt membranes

Phenol, orthocresol, orthophenylphenol,hexachlorophene

Commonly used as disinfectants (e.g.Lysol); are tuberculocidal, effective inpresence of organic matter, remain onsurfaces long after application

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Phenol is the standard disinfectant, whichcoagulates the proteins, particularly cellmembrane enzymes.

especially useful against Gram-positivebacteria.

An alternative of phenol, cresol has become

more popular in modern medicine as it ischeaper than phenol.

Because of its toxicity, this compound isgenerally used as a solution between 2% to

5% in concentration

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Halogens

Act as oxidizing agents; oxidize proteins & othercellular components

Most commonly used halogens chlorine and

iodine Chlorine compounds

Used in disinfecting municiple water supplies (assodium hypochlorite, calcium hypochlorite, or

chlorine gas) Sodium Hypochlorite (Chlorine Bleach) used at 10 -

20% dilution as bench top disinfectant

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Iodine

lethal to all vegetative forms of microorganisms, caninactivate viruses.

Pure iodine is caustic to tissues, so it is diluted withother compounds:

Iodine Compounds Tincture of iodine (iodine solution in alcohol)

Potassium iodide in aqueous solution

Iodophors: Iodine complexed to an organiccarrier; e.g. Wescodyne, Betadyne

Used as antiseptics for cleansing skin surfaces andwounds

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Chemical Agents: Sterilants3. Low level Germicides-

ineffective against M. tuberculosis but areactive against other vegetative cells, fungi andsome viruses.

Hydrogen Peroxide

a weak acid, with strong oxidizing properties powerful bleaching agent, disinfectant,

antiseptic,

Mechanism of action: releases the peroxide ionwhich is a strong oxidizing agent, and the waterreleased provides hydroxide ions which striphydrogen from biological molecules (oxidizingthem).

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effective antiseptic applied to skin. The most common isethyl alcohol, though propyl, butyl and pentyl alcoholshave a greater germicidal ability.

Ethanol acts particularly on vegetative bacterial cells. It is

strong dehydrating agent. Ethyl alcohol (70%) is mostlyused.

Mechanisms: kill microorganisms by denaturing proteins,dehydration (100% concentration), and as solvents whichdisrupt the phospholipid structure of the cell membrane.

Also proteins are not soluble in high concentrations ofalcohol, hence wont coagulate hence do not use 100%alcohol as germicide.

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Alcohols


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Heavy Metals

The activity on microorganisms is termedoligodynamic action.

Metals as silver, mercury(as mercuric chloride)

(HgCI2) and copper are used. In products like mercurochrome, mercury is

combined with organic carrier compounds,that reduces its toxicity to skin.

Copper is particularly active against algae.

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Mechanism: combine with sulfur groups in theproteins of microorganisms, causing them to denature.

Silver is also toxic, and is applied as silver nitrate(AgNO3) in a 1% solution, was commonly used toinhibit the growth ofNeisseria gonorrhoeae in theeyes of newborn infants- antibiotics are used instead.

Copper, in the form of copper sulfate used to limit thegrowth of algae in ponds and lakes.

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Detergents and soaps

Detergents and soaps (surfactants)are compoundsthat have hydrophillic and hydrophobic parts.

Detergents are synthetic chemicals developed fortheir ability to be strong wetting agents and surface

tension reducers. They destabilize the plasma membrane of

microorganisms.

To some extent it also destroys microorganisms due

to alkalinity- about ph 8.0 Soap is used for mechanical washing of the skinsurface.

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Quartenary ammonium compounds

(QUATS)

A major class of surfactant germicides. Quats such as benzalkonium chloride

(Zephiran)

have broad-spectrum inhibitory activity againstbacteria, fungi, and protozoa

are mildly antiseptic and disinfecting when usedas cleaning agents for laboratory fomites and onthe surface of skin

remain active after drying, but lose much oftheir activity when mixed with soaps.

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Factors affecting effectiveness of

disinfectants

Biofilms, which are populations of microorganisms that growon surfaces and encase themselves in excreted layers ofpolysaccharide. Biofilms can greatly retard or even preventthe diffusion of disinfectants to the microorganisms,eliminating the effectiveness of the compound.

The composition of the item being disinfected can also alterthe activity of a disinfectant. High concentrations of organiccompounds decrease the potency of disinfectants and it isusually prudent to clean a surface before adding disinfectant.

Endospores are characteristically more resistant todisinfectants than are vegetative cells. However, certainagents kill spores.

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ibi i d h h i


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Antibiotics and chemotherapeutic

agents

Antibiotics are low-molecular weight substances that are

produced as secondary metabolites by certain groups of

microorganisms, especially Streptomyces, Bacillus, and a

few molds (Penicillium and Cephalosporium) that are

inhabitants of soils.

Many are now synthetic or semi-synthetic compounds

produced by the pharmaceutical industry.(that is, natural metabolites are isolated and then

chemically modified)

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A standard assay often used to gauge the

effectiveness of an antimicrobial is the minimum

inhibitory concentration (MIC) test.

The MIC is the lowest concentration of a compound

that still inhibits the growth of a microorganism.

The MIC of a given compound for a certain bacterial

species is determined using a series of test tubes

containing medium in which the microbe will

normally grow.

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Each tube contains progressively lowerconcentrations of the test compound.

Each tube is inoculated with the microbe and after

incubation the tubes in which growth does not occurare noted.

The lowest concentration of the antimicrobial

compound that prevents growth defines the MIC.

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In some situations it is important to determine the

minimum concentration at which a compound is lethal

to a microorganism and this is defined as the minimal

lethal concentration (MLC).

Antimicrobials that are cidal will normally kill amicroorganism at two to four times their inhibitory

concentration.

A static agent will require a much higher concentration

and may not ever be lethal.

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Another commonly used assay to assess the potency


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Another commonly used assay to assess the potencyof an agent is the agar diffusion method.

A microbial culture is spread evenly on the top of anagar plate containing medium that will support itsgrowth.

Disks impregnated with antimicrobial compounds arethen placed onto the agar and the plate incubated atan appropriate temperature for that microbe.

During incubation the antimicrobial compounddiffuses away from the disk and into the agar creatinga concentration gradient that is highest near the diskand decreases as one moves away from the disk.

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If a microbe is inhibited by the agent, it will be unable

to grow near the disk, which we see as a zone ofclearing in the lawn of growth.

Farther away from the disk, where the concentration

of the antimicrobial compound is much lower, growthwill be evident.

The size of the zone of clearing around the disk is an

indication of the potency of the antimicrobial for thetested microbe.

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Antibiotics


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Antibiotics

The range of bacteria or other microorganisms thatis affected by a certain antibiotic is expressed as itsspectrum of action.

Antibiotics effective against prokaryotes which kill orinhibit a wide range of Gram-positive and Gram-negative bacteria are said to be broad spectrum.

If effective mainly against Gram-positive or Gram-negative bacteria, they are narrow spectrum.

If effective against a single organism or disease, they

are referred to as limited spectrum. 53

A clinically-useful antibiotic should have as many


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A clinically useful antibiotic should have as manyof

these characteristics as possible:

It should have a wide spectrum of activity withthe ability to destroy or inhibit many different

species of pathogenic organisms.

It should be nontoxic to the host and without

undesirable side effects.

It should be nonallergenic to the host.

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It should not eliminate the normal flora of the host.

It should be able to reach the part of the humanbody where the infection is occurring.

It should be inexpensive and easy to produce.

It should be chemically-stable (have a long shelf-life).

Microbial resistance is uncommon and unlikely todevelop

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Mode of Action of antibiotics

Cell wall synthesis inhibitors:

include two widely used classes of antibiotics, thepenicillins and cephalosporins. Both contain a -lactam ring.

They act on various Gram positive and Gram negativerods and cocci, responsible for various diseases.

They inhibit the formation of peptide cross linkageswithin the peptidoglycan backbone of the cell wall.

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The beta lactam antibiotics are stereochemically

related to D-alanyl-D-alanine which is a substrate for

the last step in peptidoglycan synthesis, the finalcross-linking between peptide side chains.

Penicillins bind to and inhibit the carboxypeptidaseand transpeptidase enzymes that are required for

this step in peptidoglycan biosynthesis.

Beta lactam antibiotics are normally bactericidal and

require that cells be actively growing in order to

exert their toxicity.

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Semi Synthetic Penicillins

In the late 1950s, the betalactum nucleus of

the penicillin molecule was identified and

synthesised.

Various groups then could be attached to thisnucleus, creating a number of new penicillins.

At present thousands of penicillins are

prepared by this semi-synthetic process.

E.g. Ampicillin, Amoxicillin

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Cephalolsporins

Are beta lactam antibiotics with a similar mode of

action to penicillins that are produced by species of

Cephalosporium.

They have a low toxicity and a somewhat broader

spectrum than natural penicillins.

They are often used as penicillin substitutes, against

Gram-negative bacteria

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Cell membrane inhibitors


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Cell membrane inhibitors

These antibiotics disorganize the structure orinhibit the function of bacterial membranes.

The integrity of the cytoplasmic and outermembranes is vital to bacteria, andcompounds that disorganize the membranesrapidly kill the cells.

However, due to the similarities inphospholipids in eubacterial and eukaryotic

membranes, this action is rarely specific 60


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The only antibacterial antibiotic of clinical

importance that acts by this mechanism ispolymyxin, produced by Bacillus polymyxis

Polymyxin is effective mainly against Gram-negative bacteria and is usually limited totopical usage.

Polymyxin binds to membrane phospholipidsand thereby interferes with membranefunction.

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Protein synthesis inhibitors


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Protein synthesis inhibitors

Many therapeutically useful antibiotics owe theiraction to inhibition of some step in the complex

process of protein synthesis.

Their attack is always at one of the events occurring

on the ribosome and never at the stage of amino

acid activation or attachment to a particular tRNA.

Most have an affinity or specificity for 70S (as

opposed to 80S) ribosomes, and they achieve their

selective toxicity in this manner.62


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The most important antibiotics with this mode of

action are the tetracyclines, chloramphenicol, the

macrolides (e.g. erythromycin) and the

aminoglycosides (e.g. streptomycin).

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Effects on Nucleic Acids


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Effects on Nucleic Acids

Some antibiotics and chemotherapeutic agentsaffect the synthesis of DNA or RNA, or can bind toDNA or RNA so that their messages cannot be read.

They block the growth of cells.

The majority of these drugs are unselective,however, and affect animal cells and bacterial cellsalike and therefore have no therapeutic application.

Two nucleic acid synthesis inhibitors which haveselective activity against prokaryotes and somemedical utility are the quinolones (eg. Nalidixic acid

and rifamycins (eg. Rifampicin ). 64

Competitive Inhibitors


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p

Many of the chemotherapeutic agents are

competitive inhibitors of essential metabolites orgrowth factors that are needed in bacterial

metabolism.

Hence, these types of antimicrobial agents aresometimes referred to as anti-metabolites or

growth factor analogs, since they are designed to

specifically inhibit an essential metabolic pathway in

the bacterial pathogen. At a chemical level, competitive inhibitors are

structurally similar to a bacterial growth factor or

metabolite, but they do not fulfil their metabolic

function in the cell. 65


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Someare bacteriostatic and some are bactericidal.

Their selective toxicity is based on the premise thatthe bacterial pathway does not occur in the host.

The sulfonamides (e.g. Gantrisin) are examples ofinhibitors of the bacterial enzymes required for the

synthesis of tetrahydofolic acid (THF), the vitamin

form of folic acid essential for 1-carbon transfer

reactions.

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Reference

http://www.textbookofbacteriology.net/contr

ol.html

http://microbiology.suite101.com/article.cfm/

control_of_microorganisms

http://www.microbiologyprocedure.com/micr

obial-control/microbial-control.htm

http://www.microbiologyprocedure.com/micr

obial-control/microorganisms-controlling-by-

heavy-metals.htm
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