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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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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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