minimum inhibitory concentration, antibiotic sensitivity testing

8/6/2019 Minimum Inhibitory Concentration, Antibiotic Sensitivity Testing

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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



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Problems with E-test reading


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 ???

DR.T.V.RAO MD 43

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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

Email

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minimum inhibitory concentration, antibiotic sensitivity testing

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