Antimicrobial susceptibility testing (AST) involves standardizing test conditions to determine if bacteria are susceptible, intermediate, or resistant to antimicrobial agents. There are three main methods: broth microdilution directly measures minimum inhibitory concentrations; agar dilution tests bacteria on agar plates containing serial drug dilutions; and disk diffusion examines growth inhibition zones around disks containing drugs. Commercial systems automate these methods. AST guides effective antimicrobial selection and resistance monitoring.

1. Antimicrobial Susceptibility
Testing
Dr. Pooja Gupta

2. • The procedures used to produce antimicrobial
susceptibility profiles and detect resistance to
therapeutics are referred to as antimicrobial
susceptibility testing(AST) methods.
• AST is performed only for pathogenic bacteria
isolated from the specimen.
• Not for the commensal bacteria.
• e.g.:- E.coli isolated from urine specimen
should be subjected to AST, whereas E.coli
isolated from stool is a commensal.

3. Standardization
To control the impact of environmental factors,
the conditions for susceptibility testing are
standardized.
It serves three important purposes:
1. It optimizes bacterial growth conditions.
2. It optimizes conditions for maintaining
antimicrobial integrity and activity.
3. It maintains reproducibility and consistency
in the resistance profile of an organism.

4. • The standardized components of antimicrobial
susceptibility testing include:-
• Bacterial inoculum size
• Growth medium
• Incubation atmosphere
• incubation temperature
• Incubation duration
• Antimicrobial concentrations

5. Limitations of standardization
• Factors that not taken into account by
susceptibility testing are:
• Antibiotic diffusion into tissues and host cells
• Serum protein binding of antimicrobial agents
• Drugs interactions and interference
• Status of patient defense and immune systems
• Multiple simultaneous illness.
• Virulence and pathogenicity of infecting
bacterium
• Site and severity of infection.

6. TESTING METHODS
• Three general methods are available to detect and
evaluate antimicrobial susceptibility:
1. Methods that directly measure antimicrobial activity
2. Methods that directly detect specific resistance
mechanism.
phenotypic methods
genotypic methods
3. Special methods that measure complex antimicrobial-
organism interactions.
bactericidal tests
tests for activity of antimicrobial combinations

7. Methods that directly measure
antimicrobial activity.
• It involves bringing the antimicrobial agents of
interest and the infecting the bacterium together
in the same in vitro environment to determine
the effect of the drugs presence on bacterial
growth and viability.
It includes:-
• conventional susceptibility testing methods such
as broth dilution, agar dilution and disk diffusion.
• Commercial susceptibility testing systems
• Special screens and indicator tests.

8. Conventional testing methods
General Considerations
• Inoculum preparation
• Properly prepared inocula are the key to any
antimicrobial susceptibility testing method.
• Two important requirement
• Use of a pure culture
• Use of a standard sized inoculum

9. To obtain pure inocula:
• select four or five colonies of the same
morphology ,inoculate them on to a broth
medium, and allow the culture to achieve
active growth.
• Most organisms requires 3-5 hours of
incubation.
• Indicated by observable turbidity in the broth.

10. Standard inoculum size
• Standard inoculum size accomplished by
comparing the turbidity of the organism
suspension with a turbidity standard.
• McFarland turbidity standards: prepared by
1% sulfuric acid + 1.175% barium chloride to
obtain a solution with a specific optical
density.
• 0.5 McFarland standard provides an optical
density comparable to the density of a
bacterial suspension of 1.5 X 10(8) CFU/Ml.

11. Selection of antimicrobial agents for testing:
• The antimicrobial agents chosen for testing
against a particular bacterial isolate are referred
to as the antimicrobial battery or panel.
Criteria for antimicrobial battery content and use:
• Organism identification or group: e.g. vancomycin
versus gram negative bacilli, ceftazidime for use
against Pseudomonas aeruginosa
• Antimicrobial susceptibility testing method used:
some do not reliably detect resistance
• Site of infection: e.g. nitrofurantoin achieve
effective levels only in urinary tract
• Availability of antimicrobial agents in the
formulary: ability to detect bacterial resistance to
agents used by medical staff

12. • MIC : minimum inhibitory concentration
Lowest concentration of an antimicrobial agent that will
inhibit the visible growth of a microorganism after
overnight incubation
useful :
for confirming AST results obtain by disc diffusion tests.
for testing antimicrobial sensitivities(slow growing
bacteria, bacteria for which disk diffusion test not
standardized).
demonstration of very small degree of resistance
when therapeutic dose of drug has to be regulated
accurately(bacterial endocarditis).
• MBC : minimum bactericidal concentration
The tube containing the lowest concentration of the drug
that fails to show growth, on subculture onto a nutrient
agar plate without any antimicrobial agent, is the MBC of
the drug for that test strain.

13. Broth dilution
• Broth dilution testing involves challenging the organism
of interest with antimicrobial agents in a liquid
environment.
• Serial dilutions of antimicrobial agent in Mueller
Hinton broth are taken in tubes and each tube is
inoculated with fixed suspension of test organism.
• The concentration range based on the level of drug
required to reliably detect a particular resistance
mechanism.
• e.g.: to detect clinical significant resistance to cefepime
in s.pneumoniae in CSF isolates, uses maximum
concentration of 2microgram per ml, whereas in
nonmeningitis isolates, maximum concentration used
is 4mircog/mL
• For E.coli the required maximum concentration to
detect cefepime resistance is 16 microg/mLor higher.

15. • Mueller-Hinton preparation is standard
medium used for most broth dilution testing.
• Media supplements are required to obtain
good growth and reliable susceptibility
profiles for bacteria like S.pneumoniae and
H.influenzae.
• Broth dilution testing divided into:
microdilution and macrodilution.

17. • The difference in both is the volume of broth
• Microdilution testing:0.05 to 0.1 mL
• Macrodilution testing: 1mL or greater.
• Most susceptibility test batteries require testing
of several antibiotics at several different
concentrations, so smaller volume used in
microdilution allows this to be conveniently
accomplished in a single microtiter tray.
• Final standard bacterial concentration of 5X10(5)
CFU/Ml required.
• Most commonly tested bacteria incubated at 35
degree C.
• Should not be incubate for prolong time since it
cause antimicrobial deterioration.

18. • Reading and interpretation of results
• Each tray should include growth control(pos.)
that does not contain antimicrobial agent and a
sterility control(neg.) that was not inoculate.
• The recorded MICs for the antimicrobials
interprets as three categories: susceptible(S),
intermediate(I), resistant(R).
• Nonsusceptible(NS) and susceptible dose
dependent(SDD).
• e.g. isolate of P. aerugenosa with imipenem
• MIC<= 2microg/mL - susceptible(S)
• MIC= 4microg/mL - intermediate(I)
• MIC= 8microg/mL - resistant(R)
• Broth dilution methods provide data for both
quantitative results(MIC) and qualitative results
(category interpretation).

19. • Susceptible: indicates that the antimicrobial
agent in question may be an appropriate choice
for treating the infection caused by organism
• Susceptible-dose dependent: implies
susceptibility of an isolate is dependent on dosing
regimen and that altering dosing results in higher
drug exposure than the dose that was used to
establish the susceptible breakpoint.
• Resistant: indicates that the antimicrobial agent
in question may not be an appropriate choice for
treatment since organism not inhibited with
serum achievable levels of drug or develop
resistance mechanism.
• Nonsusceptible: antimicrobial agents having zone
diameters below the susceptible breakpoint.
• Breakpoints: the specific concentration that
separate different categories

20. Agar Dilution
• The antimicrobial concentrations and
organisms to be tested are brought together
on agar based medium.
• The surface of each plate is inoculated with
1X10(4)CFU
• This method allows examination of one or
more bacterial isolates per plate.

23. Mueller-hinton agar
• MHA use for routine susceptibility testing of non
fastidious bacteria.
• It has minimal inhibitory effect on sulfonamide
and trimethoprime
• Satisfactory growth
• Acceptable batch to batch reproducibility for
susceptibility testing
• Lysed horse blood is added to MHA to support
growth of S. pneumoniae and S. pyogenes
• For testing MRSA isolates 2-4% NaCl is added.

24. DISK DIFFUSION
• Most widely used method.
• Suitable rapidly growing pathogenic bacteria
• Not suitable for slow growing bacteria.
• Test bacterium inoculated(as lawn culture) on
solid media by spreading with sterile swab
• Ideally incubate overnight (16-18 hours), for
MRSA incubate for 24hours.

25. Control strain:
• similar to test isolate
• Tested for AST
• e.g. S.aureus ATCC 25923
• P.aeruginosa ATCC 27853
Antibiotic disk:
• Uses filter paper disk impregnated with
appropriate concentration of antibiotic solution.
• 6mm diameter
• First line drugs tested first
• Second line drugs tested later if organism
resistant to all the first line antibiotics.

26. Kirby-bauer disk diffusion method
• A sterile cotton swab dipped into inoculum
and squeezed
• Than inoculated on MHA by streaking 3 times
over entire agar surface
• Dry surface of agar plate for 3-5 min
• Antibiotic disk(6) applied not closer than
20mm on 100mm plate
• Only use for category interpretation of
susceptible, intermediate, resistant.

29. Stokes disk diffusion method

30. Primary or direct disk diffusion test
• When results are required urgently
• Single pathogen is suspected in
specimen(sterile fluid, urine)
• Specimen directly inoculated uniformly and
antibiotic disk are applied on agar plate.
• Not useful when mixed growth present(pus,
stool, sputum)

31. COMMERCIAL SUSCEPTIBILITY
TESTING SYSTEMS
The goal of detecting resistance is same for all
commercial methods but principles and practices
vary with respect to:
• The format in which bacteria and antimicrobial
agents are brought together
• Extent of automation for inoculation, incubation,
interpretation and reporting
• Method used for detection of bacterial growth
inhibition
• Speed with which results are produced
• accuracy

32. • Broth microdilution methods
• Agar dilution derivations
• Diffusion in agar derivations :
combines the convenience of disk diffusion and
ability to generate MIC data.
Etest uses plastic strips, one side contains
antimicrobial agent concentration gradient and
other numeric scale.
• Automated antimicrobial susceptibility test
system:
VITEK 2 identification and antimicrobial
sensitivity system
Phoenix system
Micro scan walk away system

34. Special screens and indicator tests

37. Thank you