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Dr.T.V.Rao MD
ANTIBIOTIC SENSITIVITY TESTING
MINIMUM INHIBITORY CONCENTRATION
SKILL BASED LEARNING
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The responsibility of the
microbiology laboratory includes
not only microbial detection and
isolation but also the
determination ofmicrobial
susceptibility to antimicrobial
agents. Many bacteria, in
particular, have unpredictable
susceptibilities to antimicrobial
agents, and their susceptibilities
can be measured in vitro to helpguide the selection of the most
appropriate antimicrobial agent.
WHY ANTIBIOTIC SUSCEPTIBILITY TESTING
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WHAT IS SUSCEPTIBILITY TO
ANTIBIOTICS
The term susceptible means that the microorganism isinhibited by a concentration of antimicrobial agent that canbe attained in blood with the normally recommended doseof the antimicrobial agent and implies that an infection
caused by this microorganism may be appropriately treatedwith the antimicrobial agent. The term resistant indicatesthat the microorganism is resistant to concentrations of theantimicrobial agent that can be attained with normal doses
and implies that an infection caused by this microorganismcould not be successfully treated with this antimicrobialagent.
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ANTIMICROBIAL RESISTANCE
RESULTS FROM .
Results from misuse, overuse, under/ inadequate use of
antimicrobials
Costs money, lives and undermines effectiveness of health delivery
programs
Threat to global stability and national security
WHO Global Strategy for Containment of Antimicrobial
Resistance: Intervention framework to slow emergence and reduce the spread of
antimicrobial resistant microorganisms
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ANTIBIOTIC RESISTANT INFECTIONS
Diseases Agent Resistances
Pneumonia S pneumoniae Penicillin
Dysentery S dysenteriae Multiple resistances
Typhoid S typhi Multiple resistancesGonorrhea N gonorrhea Penicillin and
tetracycline
Tuberculosis M tuberculosis Rifampicin and INH
Nosocomial infections S aureus Methicillin, vancomycin
E species Vancomycin
Klebsiella,Pseudomonas
Multiple resistances
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ANTIMICROBIAL SUSCEPTIBILITY
TESTS
Minimum inhibitory concentration [MIC]
The smallest concentration of antibiotic that inhibits the
growth of organism
Liquid media (dilution) allows MIC estimation
Solid media (diffusion)
Disk diffusion (Kirby-Bauer)
E-tests
Allows MIC estimation
Beta lactamase production: quick screening methodDR.T.V.RAO MD 6
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SUSCEPTIBILITY TESTING: THE BIG GAPS
MICs help, but hard to standardize
Correlations appear possible based on individual
isolates. Broad correlations based on multiple isolatesare still lacking
Understanding this helps a lot when trying to correlate
outcome with MIC
- Some patients get better despite MICs
- Some patients just dont get better despite MICs
No rule when it correlates/not correlates
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THE TUBE DILUTION TEST IS STANDARD
METHOD
The tube dilutiontest is the standard method fordetermining levels of microbial resistance to anantimicrobial agent Serial dilutions of the test agent aremade in a liquid microbial growth medium which isinoculated with a standardized number of organisms andincubated for a prescribed time. The lowest concentration(highest dilution) of test agent preventing appearanceof turbidity (growth) is considered to be the minimal /
minimum inhibitory concentration (MIC). At this dilutionthe test agent is bacteriostatic.
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The MICorminimum inhibitory
concentration test determines
antimicrobial activity of a
material against a specific
bacteria.
The most commonly employed
methods are the tube dilution
method and agar dilution methods.
Test products that are not clear or
precipitate the growth media are
tested by agar dilution methodswhich is about same as tube dilution
method except dilutions are plated
on agar
MINIMUM INHIBITORY CONCENTRATIONTEST
DETERMINES
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The Minimum Inhibitory
Concentration (MIC) is the
smallest concentration of an
antimicrobial agent that inhibits
the growth of bacteria. The value
is obtained in a highly
mechanized fashion, but this
procedure only provides interval
censored reading. It is often of
interest to use data collected
from complex experiments to seehow the mean MIC is affected by
different factors
MINIMUM INHIBITORY CONCENTRATION
(MIC)
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The minimal bactericidal
concentration (MBC) or the
minimum lethal concentration
(MLC) of an antibacterial which is
defined as the maximum dilution
of the product that will kill a test
organism can be determined by
sub culturing last clear MIC tube
onto growth medium and
examining for bacterial growth.
Serial dilutions are made of theproducts in bacterial growth
media.
THE MINIMAL BACTERICIDAL
CONCENTRATION
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A MINIMUM BACTERICIDAL
CONCENTRATION TEST
Figure 10.12DR.T.V.RAO MD 13
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MINIMUM INHIBITORY CONCENTRATION (MIC)PRACTICE THE EXERCISE FOR SKILLS
1. Grow cultures up overnight.
2. The following day, inoculate a fresh culture with a 1:10-20 dilution and
grow up to OD600 0.4.
3. Dilute to OD600 0.0005 (1:800) in THB (or relevant media).
4. Dilute this 1:200 and drop about 25l on THA (or relevant agar plates) to
confirm equal starting inoculums.
5. Put 50l of the 0.0005 cultures in a 96 well plate in triplicates.
6. Add 50l per well of the substance you want to test (ie. antimicrobial
peptide, antibiotic) to each well, keeping in mind the final concentration will
be half the original concentration you are adding. It is good to test 1:2
dilutions (in THB, or relevant media) of this substance.7. Incubate overnight and check by eye orOD600 24 hours later to
determine what concentration inhibited the growth of the different strains of
bacteria.
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STANDARD STRAINS FOR
QUALITY ASSURANCE
Precision and accuracy ensured through controlstrains
Known susceptibility to antimicrobial agents
Standard strains include
Staphylococcus aureus ATCC 25923
Escherichia coliATCC 25922
Pseudomonas aeruginosa ATCC 27853
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DIFFERENT STANDARDS
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Use standardized reference
National Committee for Clinical
Laboratory Standards (USA)
Other norms
Canadian
Chinese
National
Do not confuse the different tablesChoose one for everything
DIFFERENT STANDARDS
CLSI STANDARDS FOLLOWED ALL OVER THE
WORLD
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1. Culture media: Muller-Hinton
2. Reagents: disks
3. Size of the inoculums4. Incubation condition
5. Control with reference
strains
6. Reading inhibition
diameters (accurate
measurement)
7. Knowledge of staff
CRITICAL POINTS IN QUALITY ASSURANCE-
ORGANISE YOUR LABORATORY WITH
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PRINCIPLES OF MINIMUM INHIBITORY
CONCENTRATION
In minimum inhibitory concentration dilute on a log2 scale each
antimicrobial agent in broth to provide a range of concentrations
and to inoculate each tube or, if a micro plate is used, each well
containing the antimicrobial agent in broth with a standardized
suspension of the microorganism to be tested. The lowestconcentration of antimicrobial agent that inhibits the growth of
the microorganism is the minimal inhibitory concentration (MIC).
The MIC and the zone diameter of inhibition are inversely
correlated . In other words, the more susceptible themicroorganism is to the antimicrobial agent, the lower the MIC
and the larger the zone of inhibition. Conversely, the more
resistant the microorganism, the higher the MIC and the smaller
the zone of inhibition.DR.T.V.RAO MD 21
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Minimum Inhibitory Concentration(MIC) :
Principle: The tube dilution test is the standard method for determining
levels of resistance to an antibiotic.
Serial dilutions of the antibiotic are made in a liquid mediumwhich is inoculated with a standardized number of organismsand incubated for a prescribed time.
The lowest concentration of antibiotic preventingappearance of turbidity is considered to be the minimal
inhibitory concentration (MIC).DR.T.V.RAO MD 22
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Different concentrations of Gentamycin in Nutrient
broth:
Conc. in mcg/ml0.1 0.2 0.4 0.8 1.6 3.1
Gentamicin, generally considered a bacteriocidal antibiotic, for this
bacterium, has an MIC of 0.8 mcg/mlDR.T.V.RAO MD 23
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Different concentrations of Tetracycline in Nutrient
broth:
Conc. in mcg/ml
0.1 0.2 0.4 0.8 1.6 3.1 6.3 12.5
Tetracycline, generally considered a bacteriostatic antibiotic, for thisbacterium, has an MIC of 1.6 mcg/ml
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MINIMUM INHIBITORY CONCENTRATION TEST
Figu
re
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The main concept is the clinical categorisation" Strains are sorted according to level of Minimal Inhibitory
Concentration (MIC) versus reference breakpoints
c and C are the minor and major breakpointsSusceptible Intermediate Resistant
MIC < c MIC C Intermediate strain
c < MIC C Resistant strain
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Sometime the agent can still be used
Higher doses required to ensure
efficacy
Agent may be efficacious if
concentrated in vivo in an
infected body fluid (e.g., urine)
Sometimes there is uncertainty
Intermediate resistance may
represent a buffer zone that
prevents strains with borderline
susceptibility from beingincorrectly categorized as
resistant
INTERPRETING INTERMEDIATE
RESISTANCE
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COMMON INTERPRETATION
PROBLEMS
Results depends on the technique usedMany factors influence results
Lack of standardization of the inoculums
Thickness and quality of the culture media
Quality and conservation of the disks
Quality control with standardized strains
Condition and duration of incubation
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E-test is based on arraying aconcentration gradient of each
antibiotic on a polymer strip.
Concentration values are marked
on the other side of the strip so
that one can easily locate
corresponding concentrations. E-
strips, also known as
epsilometers, are commercially
prepared by micro dispersing
robotic machines that can deliverNano liter volumes of antibiotic
concentration along the strip.
WHAT IS E-TEST
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E-TEST
Plastic strips with a predefinedgradient of
One antibiotic
One antifungal
Only one manufacturer
One strip per antibiotic
Wide range of antibiotics
Easy to use
Storage at -20C
Short shelf life, expensive
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AN E-TESTCOMBINES ASPECTS OFKIRBY-BAUER AND MIC TESTS
Figure 10.11DR.T.V.RAO MD 35
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READING E-TESTS
Susceptible < 1
Resistant > 4 ug/ml
Ciprofloxacin forYersinia pestis
Intermediate 1-4 ug/ml
Upper readingDR.T.V.RAO MD 36
COMMON INTERPRETATION
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Problems with E-test reading
COMMON INTERPRETATION
PROBLEMS
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The Liofilchem MIC Test
Strip is a quantitative assay
for determining the
Minimum Inhibitory
Concentration (MIC) ofantimicrobial agents
against microorganisms to
indicate appropriate patient
treatment and foridentifying resistance
patterns
NEW LIOFILCHEM MIC TEST STRIPS
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When the Liofilchem MIC Test
Strip is applied onto an
inoculated agar surface, the
preformed exponential gradient
of antimicrobial agent is
transferred into the agar matrix.After 18 hours incubation or
longer, a symmetrical inhibition
ellipse centered along the strip is
formed. The MIC is read directly
from the scale in terms of g/mL,at the point where the edge of
the inhibition ellipse intersects
with the MIC Test Strip.
NEW LIOFILCHEM MIC TEST STRIPS
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M I C EVALUATORS (M I C E )
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Oxoid has l improvedM.I.C.Evaluator
(M.I.C.E.) strips, a
product range for the
accurate determination ofminimum inhibitory
concentration (MIC) values.
The new distinctive
gradient format of M.I.C.E.
strips provides an excellentcontrast with agar and the
increased font size makes
reading easier.
M.I.C.EVALUATORS (M.I.C.E.)SIMPLE, CONVENIENT METHOD FOR ACCURATE
MIC VALUES
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ROLE OF NCDC ( INDIA )IN CONTAINMENTOF ANTIMICROBIAL RESISTANCE (AMR)
Antimicrobial resistance in pathogens causing important
communicable diseases has become a matter of great public
health concern globally including our country. Resistance has
emerged even to newer, more potent antimicrobial agents like
carbapenems. The factors responsible for this are widespreaduse and availability of practically all the antimicrobials across
the counter meant for human, animal and industrial
consumption. There are definite policies /guidelines for
appropriate use of antimicrobials at national level in specificnational health programme being run in the country
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IMPLEMENTATION OF WHONET CANHELP TO MONITOR RESISTANCE
Legacy computer systems,
quality improvement teams,
and strategies for
optimizing antibiotic use
have the potential tostabilize resistance and
reduce costs by
encouraging
heterogeneous prescribingpatterns and use of local
susceptibility patterns to
inform empiric treatment.
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ANTIBIOTIC RESISTANCE A CONCERN TO
HUMANITY ???
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FOLLOW ME FOR ARTICLES OF
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FOLLOW ME FOR ARTICLES OF
INTEREST ON MICROBIOLOGY ..
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Created by Dr.T.V.Rao MD for e learning
resources for Medical Microbiologists in the
Developing World
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