Download (MIC) Definition: The lowest antibiotic - Norazli@CUCST

ANTIBIOTICS SUSCEPTIBILITY
TESTING
Prepared by:
Miss Norzawani Jaffar
Bsc Hons Biomedical Science, UKM
MICROBIOLOGY II: TOPIC 2
Lecture’s Outcome
By the end of the lecture student should be
• able to discuss the importance and limitation of antibiotics
susceptibility test.
• able to define
–
–
–
–
Resistant
sensitive/susceptible
MIC
MBC
• Able to describe methods use for susceptibility test
– Disk diffusion
– Dilution methods
– E-TEST
MICROBIOLOGY II: TOPIC 2
Antimicrobial Susceptibility Testing
• In the treatment and control of infectious
diseases, especially when caused by
pathogens that are often drug resistant,
susceptibility (sensitivity) testing is used to
select effective antimicrobial drugs.
MICROBIOLOGY II: TOPIC 2
Limitation of Antimicrobial
Susceptibility Testing
• Susceptibility testing measure antimicrobial
activity against bacteria under laboratory
condition (in vitro), not in the patient (in vivo
activity).
• It cannot be assumed therefore, that an
antimicrobial which kills or prevents an
organism from growing in vitro will be a
successful treatment.
MICROBIOLOGY II: TOPIC 2
• Antimicrobial treatment- considering the
patient’s clinical condition (eg. Liver or kidney
disease), the type of infection, any history of
drug hypersensitivity, age of patient and
whether a patient is pregnant.
• Necessary to know the activity of the different
drugs including their rates of absorption,
diffusion in the tissues, metabolism, excretion,
and also possible toxicity and effects on the
patient’s normal microbial flora.
MICROBIOLOGY II: TOPIC 2
Laboratory antimicrobial susceptibility testing
can be performed using:
1. Disk diffusion technique
2. Broth / Agar Dilution technique
3. E-test
MICROBIOLOGY II: TOPIC 2
Minimum Inhibitory Concentration
(MIC)
• Definition:
–The lowest antibiotic concentration
of tested antibiotic that inhibit the
growth of the bacteria.
MICROBIOLOGY II: TOPIC 2
• Paper discs containing known concentrations of antibiotics are
applied to the surface, and the plate is incubated at 35°C for
16-18 hrs.
• The appearance of a zone of inhibition surrounding the disc is
indicative of sensitivity.
• Strains susceptible to antimicrobial are inhibited at a distance
from the disc whereas resistant strains have smaller zones of
inhibition or grow up to edge of the disc.
• By comparing the diameter of the zones to a standard table,
one may determine if the test organism is susceptible, or
resistant to the antibiotic.
• If the organism is susceptible, it is likely to be killed in the
blood stream of the patient if that concentration of the drug is
reached.
• Resistance indicates that the antibiotic will not be effective at
that concentration in the blood stream.
MICROBIOLOGY II: TOPIC 2
Modified Kirby-Bauer susceptibility
testing technique
• REQUIRED
– MUELLER HINTON AGAR
Prepare and sterilize the medium. The pH of the
medium should be 7.2-7.4. Pour into 90 mm
diameter sterile petri dishes to a depth of 4 mm
(about 25 ml per plate).
Care must be taken to pour the plates on a level
surface so that the depth of medium is uniform.
MICROBIOLOGY II: TOPIC 2
– ANTIMICROBIAL DISC
Paper antimicrobial discs are commercially available
from most manufacturers of culture media.
Most paper disc can be used for 1 yr or longer from
the date of manufacture providing they store
correctly (-20°C, or working stock at 2-8°C in an
airtight container with indicating desiccant.
Expired disc should not be used. Quality control of
disc is essential.
-About 1 hr before use, the working stock of disc
should be allowed to warm to room temperature,
protected from direct sunlight.
MICROBIOLOGY II: TOPIC 2
– STANDARD INOCULUM/BACTERIA SUSPENSION
5 colonies from an agar plate culture grown
overnight are inoculated into 5 ml of suitable
liquid broth and the culture is incubated at 35°C
until a turbidity equivalent to 0.5 McFarland
(approx 108 colony-forming-unit (CFU)/ml is
reached, which can occur within a few hours.
The turbidity of the culture is adjusted with sterile
saline serum or broth to reach 0.5-McFarland
standard.
This can be checked using an optical device or by
visual inspection.
MICROBIOLOGY II: TOPIC 2
MICROBIOLOGY II: TOPIC 2
Interpretation of zone sizes
• Using interpretative Chart, interpret the zones
sizes of each antimicrobial, reporting the
organism as;
–Resistant
–Intermediate
–Moderately Susceptible
–Susceptible.
MICROBIOLOGY II: TOPIC 2
MICROBIOLOGY II: TOPIC 2
• Purchase Mueller Hinton agar from a reliable source. Check its
pH. Ensure plates contain the correct amount of agar.
• Prepare carefully the inoculum of the test and control organism
to ensure growth is confluent. Renew the turbidity standard
every few months.
• Use disc containing the correct amount of antimicrobial. Store
disc correctly and do not use them beyond their expiry date.
• Place disc or tables correctly on the plate, not too close to each
other or to the rim of the plate.
• Use appropriate control strains
• Regularly check the temperature of the incubator to ensure test
are incubated at 35°C.
• Measure inhibition zones carefully..
• Zone diameters with control strains should be with in the limits
published by NCCLS
MICROBIOLOGY II: TOPIC 2
DILUTION METHOD
• A weakness of the diffusion method is that it
does not determine whether a drug is
bactericidal and not just bacteriostatic.
• A dilution method is often useful in determining
the MIC and Minimal Bactericidal Concentration
(MBC) of antimicrobial drug.
• The MIC is determines by making sequence of
decreasing concentration of the drug in a broth,
which is the inoculated with the test bacteria.
MICROBIOLOGY II: TOPIC 2
MICROBIOLOGY II: TOPIC 2
• After overnight incubation, the MIC is reported as
the lowest concentration of antimicrobial required
to prevent visible growth.
• The wells that do not show growth (higher
concentration than the MIC) can be cultured in
broth/agar free of the drug for MBC determination.
• If growth occurs in this broth, the drug was not
bactericidal, and the MBC can be determined.
• MIC and MBC determination is important because it
avoids the excessive use of expensive antibiotics and
minimizes the chance of toxic reactions that largerthan-necessary doses might cause.
MICROBIOLOGY II: TOPIC 2
MICROBIOLOGY II: TOPIC 2
MICROBIOLOGY II: TOPIC 2
E-TEST METHOD
• Confirmation of unusual resistance profiles can
be performed using the E-test method.
• E-test consists of a predefined gradient of
antibiotic concentrations on a plastic strip and is
used to determine the Minimum Inhibitory
Concentration (MIC) of antibiotics, antifungal
agents and antimycobacterial agents
• E-test is a more advance diffusion method that
enables a lab technician to estimate the MIC.
• MIC is read directly from the scale in micrograms
per milliliter (µg/ml) at the point where the
inhibition elipse edge intersects the strips.
MICROBIOLOGY II: TOPIC 2
MICROBIOLOGY II: TOPIC 2
• Etest is recognized as a cost-effective tool for
generating MICs across 15 dilutions.
• Over 100 antibiotics are now available in the
product range for testing of aerobic bacteria
and
fastidious
organisms
such
as
pneumococci, haemophilus, H. pylori,
meningococci, gonococci, anaerobes, fungi
and mycobacteria.
• Etest promotes the rational use of antibiotics
by providing results to guide the therapy of
individual patients and to validate empiric
drug regimens.
MICROBIOLOGY II: TOPIC 2
The E-test, a diffusion method that determines antibiotic sensitivity and estimates
minimal inhibitory concentration (MIC). The plastic strip, which is places on agar
surface inoculated with test bacteria,
containsII: increasing
gradient of antibiotic.
MICROBIOLOGY
TOPIC 2
END
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MICROBIOLOGY II: TOPIC 2