definitions and description of the main procedures used for antimicrobial susceptibility testing; to support teaching general medicine students in their second year of study

1. Antimicrobial susceptibility
testing
- an overview of definitions and procedures -

2. Definition and Purpose
• In vitro tests that measure the growth of an
isolated microbe in the presence of particular
drug or drugs (antibiotics) in order
to predict the in vivo success or failure of
antibiotic therapy
• The results - guide the choice of antibiotics (+
clinical information and experience)

3. Bacterial Resistance to Antimicrobial Agents
• Natural:
– genetically determined for all members of a bacterial
species
• Acquired:
– Present only in certain strains of a naturally sensitive
species
Natural spectrum = list of microbial species
naturally sensitive to an antibiotic

4. Methods for antimicrobial susceptibility
testing (1)
• Disk difusion (Kirby Bauer):
– swab bacterial suspension on agar plate
– place small filter paper disks impregnated with
a standard amount of antibiotic
– incubate overnight
– zone of inhibition of bacterial growth =
measure of susceptibility (predefined cutoffs):
• large zone = susceptible
• small / no zone = resistant
• zone between the above = intermediate

5. Disc difusion antimicrobial susceptibility
testing (Kirby Bauer)
• Pure culture of the germ to be tested: 4-6 colonies raised
from culture plate with sterile bacteriological loop and
suspended in sterile saline solution
• 0.5 McFarland units turbidity (adjusted by adding more
colonies if required)

6. Disc difusion antimicrobial susceptibility
testing (Kirby Bauer)
• Bacterial suspension evenly
swabed on Müller Hinton agar,
blood agar, complex media
with swab or L-shaped
bacteriological loop

7. Disc difusion antimicrobial susceptibility
testing (Kirby Bauer)
• Place antibiotic disks
depending on known natural
spectrum of tested
microorganism

8. Disc difusion antimicrobial susceptɄbility
testing (Kirby Bauer)
• Antibiotics sets (e.g.):
– Current use: Penicillin G (P), Oxacillin (O), Ampicillin (A),
Streptomycin (S), Kanamycin (K), Chloramphenicol (C),
Tetracyclin (T), Erythromycin (E)
– Additional set for digestive infections: Bacitracin (B),
Furazolidone (Fu), Neomycin (Ne), Polymyxin B (Po)
– Additional set for urinary infections: Nalidixic Acid (Nx),
Colistin (Co), ...

9. Disc difusion antimicrobial susceptibility test
– Kirby Bauer -
Various sizes of
growth inhibition
around different
antibiotics
Interpretation:
zone size diameter
compared to cutoffs
for each
antibiotic-microbe
pair

10. Methods for antimicrobial susceptibility
testing (2)
• MIC (minimum inhibitory concentration) =
minimum concentration of antibiotic that will
inhibit the growth of a microorganism
Measurement methods:
- Broth dilution
- E-test (Epsilometer test)

11. MIC (continued): Measurement methods
Broth dilution:
- Bacteria inoculated in culture broth + antibiotic (various
concentrations) and incubated
- MIC = the lowest concentration of antibiotic which
inhibited bacterial growth

12. MIC (continued): Measurement methods
E-test:
- plastic strips impregnated with
decreasing antibiotic
concentrations (log scale)
- strips applied on agar
microbial culture and
incubated
- eliptic inhibition zone intersects
the MIC value scale (μg/ml) at
the level of MIC
- shape resembles Greek letter
”Epsilon” (ε)

13. Methods for antimicrobial susceptibility
testing (3)
• MBC (minimum bactericidal concentration) = minimum
concentration of antibiotic that will kill 99.9-100% of the
microbes of a tested bacterial strain
- Less used than MIC; more time consuming
- Indicated in:
- severe infections in immunocompromised patients,
- infections in anatomic sites hard to reach with antibiotics e.g.
endocarditis, osteomyelitis

14. Standardization of antimicrobial
susceptibility testing (AST)
• Methodology for performing the tests:
– Standard operational procedures (SOPs)
– Interpretation of results (cutoff diameters S / R)
– Recommended antibiotic sets for various pathogens
– .....
– .....

15. Standardization of antimicrobial
susceptibility testing
http://www.eucast.org/antimicrobial_susceptibility_testing/

16. Standardization of antimicrobial
susceptibility testing
http://www.clsi.org
International standards,
including antimicrobial
susceptibility testing
(AST)