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Screening for an antibiotic

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Screening for an antibiotic
Screening for new
antibiotics
Bacteriostatic and bactericidal
antibiotics
• Antibiotics are categorized as bactericidal if they kill the
susceptible bacteria or bacteriostatic if they reversibly
inhibit the growth of bacteria.
• In general the use of bactericidal antibiotics is preferred
but many factors may dictate the use of a bacteriostatic
antibiotic.
• When a bacteriostatic antibiotic is used the duration of
therapy must be sufficient to allow cellular and humoral
defense mechanisms to eradicate the bacteria. If possible,
bactericidal antibiotics should be used to treat aggressive
infections.
Methods for antibiotic detection
• Diffusion methods:
-These methods based on the diffusion of
antibiotics in agar inoculated with sensitive
microorganisms.
- The effects of antibiotics are revealed by the
production of zones of inhibition.
• Three categories of diffusion are employed:
1- One-dimentional (linear) diffusion in test
tubes or capillaries.
2-Two- dimentional diffusion e.g., radial
diffusion from cups cut in agar layers.
3- Three dimentional diffusion from cylinders
or disc located on the surface of the agar layer.
Sensitivity of microorganisms to
antibiotics
Measurement of the antibiotic sensitivity of
an organism in the laboratory is designed
to predict whether an infection will respond
to treatment with that antibiotic or not.
Role of laboratory
Sensitivity Testing:
•Disc
diffusion
•MIC/ MBC measurement
• The MIC is the lowest concentration of the antibiotic
that results in inhibition of visible growth (i.e.
colonies on a plate or turbidity in broth culture) under
standard conditions.
• The MBC is the lowest concentration of the
antibiotic that kills 99.9% of the original inoculum in
a given time. OR
The lowest concentration of antibiotic that allows
less than 0.1% of the original inoculum to survive.
Measurement of Sensitivity of
microorganisms to antibiotics
• In the laboratory, sensitivity is most often
measured using a disk diffusion test.
• Antibiotic
solutions
of
particular
concentrations are dried onto filter paper disks.
• These are then applied to a lawn of the
microbe under examination which has
previously been inoculated onto an appropriate
solid medium.
• The effectiveness of
the
antibiotic
is
relative
to
the
inhibition zones of the
bacterial growth, the
more the diameter, the
more potent the tested
antibiotic.
Stokes controlled sensitivity test
• a control sensitive bacterium is
inoculated on part of a plate
and the tested bacterium is
plated on the remainder. Disks
of antibiotics are placed at the
interface and the zones of
inhibition are compared. The
use of a sensitive control
shows that the antibiotic is
active, so that if the test
organism grows up to the disk
it may safely be assumed that
the test organism is resistant to
that drug.
• The bacterium in the
diagram is susceptible
to drug "x" but
resistant to drug "y".
The disc containing
drug "y" contains
active antibiotic as
shown by the zone of
inhibition it causes in
the control bacterium.
Determination of the minimum inhibitory
concentration of antibiotics using broth
dilution method:
• A series of broths are mixed with 2 fold serially
diluted antibiotic solutions and a standard
inoculum is applied.
• After incubation, the minimum inhibitory
concentration MIC is the first broth in which
growth of the organism has been inhibited.
• The more resistant an organism is, then the
higher will be the MIC.
The MIC test of a bacteriostatic drug.
If the bacteria removed from the drug can
grow on drug free medium at highest
concentrations, the drug is known to have
bacteristatic action.
If the bacteria removed from the drug can not grow
on drug free medium at most concentrations, the drug
is known to have bactericidal action. One tube
difference is allowed in this test
Disadvantages
– Only one antibiotic & one organism can be tested
each time
– Time-consuming
Solutions??
– Disc diffusion method
– Microbroth dilution method
Microbroth Dilution Method
–
Microdilution plates:
• “Microdilution/ Microbroth
dilutions”
• 96 wells/ plate: simultaneously
performed with many tests
organisms/ specimens, less
reagent required
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