Antimicrobial susceptibility testing

(AST)

Disc and gradient diffusion methods

– principles, methods, interpretive
reading, error sources
 Remember – Definition of AST
Clinical antimicrobial resistance =

survival and/or multiplication of bacteria in

the presence of an antibiotic concentration
higher than the concentration that can be
achieved at the site of infection
 Susceptibility test usefulness

–Guide for treatment

–Epidemiological tool
•The emergence of resistant strains of major
pathogens
•Continued surveillance of the susceptibility
pattern of the prevalent strains
 When to do AST for guiding the
treatment?
• It is mandatory for microorganisms which are
able to easily get resistance mechanisms (e.g.
Staphylococcus aureus, Escherichia coli etc.)
S. aureus on blood agar E. coli on McConkey agar
 First condition for AST

• AST (antimicrobial susceptibility testing)

requires pure culture

Remember - 3 methods
to obtain pure cultures
 Routine TSA methods
• Dilution method
–amount of antimicrobial substances incorporated
into liquid or solid media is variable (dilutions)
–followed by inoculation of test bacteria
• Diffusion method
–Put a filter disc containing measured quantity of
drugs on the a solid medium that has been
seeded with test bacteria
 Dilution method
Broth dilution/ Agar dilution methods
•Permits quantitative results:
–Indicating amount of a given drug necessary to
inhibit (bacteriostatic activity) or kill (bactericidal
activity) the microorganisms tested

•Minimum Inhibition Concentration (MIC)

•Minimum Bactericidal Concentration (MBC)
 Remember – MIC and MBC
• MIC – minimum concentration of antimicrobial substance
measured in mg/L that inhibits visible bacterial growth (no
turbidity of broth in bacterial broth dilution method)

• MBC – minimum concentration of antibacterial substance

measured in mg/L that kills bacteria as demonstrated by
inoculation on solid culture media of samples from tubes
without visible growth in broth dilution method

• MBC higher than MIC if the antimicrobial is bacteriostatic

Broth dilution method – day 1

CFU = Colony Forming Unit

Broth dilution method – day 2 and 3
 Microbroth dilution method
Visualize turbidity
–Light box/ mirror reader
–Automated reader
 Bacterial disc diffusion (Kirby Bauer)
method
• Prepare the saline dilution of bacteria, as recommended
by the chosen AST disc diffusion standard
– Pick 3-5 isolated colonies from plate
– Adjust the turbidity to the same as the McFarland No. 0.5
standard.

• Streak the swab on the surface of the Mueller-Hinton agar

(3 times in 3 quadrants)
 Bacterial disc diffusion (Kirby Bauer)
method (cont.) Bacterial growth
• Place the appropriate
drug-impregnated discs
on the surface of the
inoculated agar plate
• Invert the plates and
incubate them at 35
oC, o/n (18-24 h)
• Measure the diameters
of inhibition zone in
mm
Inhibition zone
 Bacterial disc diffusion (Kirby Bauer)
method (cont.)
• Compare zone diameter
with tables offered by
S =Susceptible
the used AST standard I = Intermediate susceptible
•Low toxic antibiotics:
• Declare the Moderate susceptible
microorganism as S, I, R •High toxic antibiotics: buffer
against the zone btw resistant and
antimicrobials used for susceptible
AST, after validating the R = Resistant
test using bacterial
reference strains
Interpretive reading
• Additional protocols for
Beta-lactamase inhibitor
special resistance
mechanisms Cephalosporin

• Interpreting rules Extended Spectrum Beta-lactamases - ESBLs

• Modifying the raw
results Erythromycin
• Result communication
rules
Clindamycin
Macrolids Lincosamides Streptogramins B inducible resistance –
D test
Principles of disc diffusion method
• Microorganism is inoculated onto Mueller Hinton agar surface
• Bacterial agent is incorporated into filter paper discs which will be
placed on the agar after inoculating it with the tested
microorganism
• Bacterial agent is radially diffusing into the Mueller-Hinton culture
medium; if the agar medium’s thikness is standardized at 4 mm, the
radius of the antimicrobial diffusion will be the same in all plates
• After incubation at 37 o C, the microorganism growth will stop
where the antimicrobial reaches the MIC
• The microorganism relationship with the antimicrobial can be
qualified as S (Susceptible), I (Intermediate), or R (Resistant) by
comparing the zone diameter with tables as stipulated by the
same AST standard (e.g. CLSI standard – clinical Laboratory
Standards Institute- U.S.A. or EUCAST standard – European
Antimicrobial Susceptibility Testing Committee)
Factors Affecting Size of Zone of Inhibition
Muller-Hinton agar: composition, pH,
thickness
• Too thick  smaller zones
Inoculum density
Factors Affecting Size of Zone of Inhibition
Inoculum density Larger zones with light
inoculum and vice versa

Timing of disc application If after application of disc, the

plate is kept for longer time at
room temperature, small
zones may form

Temperature of incubation Larger zones are seen with

temperatures < 35 oC

Incubation time Ideal 16-18 hours; less time

does not give reliable results
Distance between discs – too small
Contamination
 Gradient diffusion method
• Antimicrobials are incorporated in paper
filter strips in a concentration gradient (E-
test);
• We can read the MIC directly on the paper
filter strips.