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ANTIMICROBIALS ANDSENSITIVITY TESTING

AntimicrobialsModes of action of antimicrobials

Kirby Bauer methodBacterial resistance

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Antimicrobial vs antibiotic Antimicrobial is the name for a chemical

that either kills or prevents the growth

of microbes ('bugs' or 'germs') such asbacteria, viruses, fungi or protozoa.

An antibiotic is "a chemical substanceproduced by microorganismswhich has

the capacity to inhibit the growth ofbacteria and even destroy bacteria andother microorganisms in dilutesolution."

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Contd

Antibiotics that kill the bacteria thatthey are exposed to are said to be

bacteriocidal

Some antibiotics/disinfectants/chemicalsjust prevent the bacteria from growing,

these are bacteriostatic.

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How do antibiotics kill bacteria Five modes of antimicrobial action ARE:

Inhibition of cell wall synthesis: drugs formcomplexes with the peptidoglycan layer

precursors and prevents cell wall maturity Disruption of cell membrane metabolic

functions: drug peptides break open themembrane and allows for selective receptivity

Inhibition of protein synthesis: drugs interferewith elongation of the 70s ribosomes andmessenger RNA cleavege.

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Inhibition of protein synthesis

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Contd

Inhibition of RNA/DNA synthesis: (i.e.,inhibition of replication of genetic material or

transcription), drug molecules bind to DNAgyrase and prevent super coiling of the DNA,they also bind to DNA-dependent RNApolymerase

Action as antimetabolites: disrupt metabolismprocesses e.g. inhibition of folic acid synthesis

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KIRBY BAUER TEST

PROCEDURE Take about 3-5 isolated colonies

Inoculate into Mueller Hinton broth or

Normal saline The turbidity should match 0.5 McFarland

turbidity standard (about 1.5 x 108CFU/ML)

Inoculate into agar by dipping sterile swabinto suspension, rotate against inside of tube

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Contd

Evenly streak the surface of agar plate

Place disk on agar

Incubate at 35-37 decrees for 18-24hours

Measure zone of inhibition - diameter

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Advantages vs disadvantages It is inexpensive

It is easy to perform

DISADVANTAGES

- Limited space for antibiotics ( 6)

- Takes long to read ( 24 HRS) Qualitative not quantitative (no MIC)

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Contd

Problems with agar or set up affectstest

- Agar too deep small zone size

- Agar too shallow large zone size

- Over inoculation small zones

- Under inoculation large zones

- Failure of organism to grow

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CONTD

An organism is regarded sensitive to acertain antibiotic if it shows a zone of

inhibition (no growth) around thatparticular antibiotic disc

An organism is regarded resistant to a

certain antibiotic if it shows no zone ofinhibition (growth) around thatparticular antibiotic disc

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Contd

http://en.wikipedia.org/wiki/Image:KB_test.jpg

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Contd

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Bacterial Drug resistance Caused by bacterial mutations change in a

base sequence of the DNA/RNA strand. Amutant an organism whose genotype ( itsDNA/RNA base sequence) differs from thatfound in nature

By altered protein binding sites

By altered DNA gyrase, RNA polymerase By decrease in entry or accumulation, by

bacteria changing permeability to the drug

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Contd

By inactivation of drug due toproduction of enzymes

Acquired Resistance: this is plasmid- (an extra chromosomal DNA moleculecapable of replicating independently)

mediated resistance that can be passedfrom organism to organism

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