FIC is the measure to determine interaction between two or more drugs intended to be used in combination.

1. Gulam Mohd
&
Bhoj R Singh
Division of Epidemiology
Indian Veterinary Research Institute,
Izatnagar- 243 122, India
Gulam Mohd
&
Bhoj R Singh
Division of Epidemiology
Indian Veterinary Research Institute,
Izatnagar- 243 122, India
Methods for Determining Fractional
Inhibitory Concentration (FIC)
Test to determine interaction between two or more drugs intended to be
used in combination

2. • Fractional inhibitory concentration (FIC):- It is
the test to estimate the interaction between two
or more drugs intended to be used in
combination.
• Purpose: Testing new antimicrobial agents in
combination with existing for determining the
Synergistic effect, Additive effect and
Antagonism.
Why FIC is measured ?

3. Combination therapy and drug
interaction
Synergistic Effect: When two drugs are used in combination. At
least one of the two drugs must show minimum 4-fold increase in
antibacterial activities (or a decrease in minimum inhibitory
concentration, MIC to ¼).
Additive Effect: When two antimicrobial agents with the same
mechanisms of action are used the effect is usually additive .
Antagonism: Usually bacteriostatic antibiotics are antagonistic to
bactericidal agents. (e.g. Chloramphenicol antagonize the bactericidal
activities of penicillin in the treatment of Pneumococcal meningitis.

4. • To widen the antibacterial activity of the treatment.
• To reduce the probability of selection of resistant mutant.
• To get the advantage of synergy of different antibacterial
drugs, which may be helpful in reducing the toxic effects
associated with large doses of the drugs when used alone.
• Common Antibacterial drug combinations: Amoxicillin/
clavulinic acid, Ampicillin/sulbactam, Trimethoprim/
sulfonamide, Trimetoprim/ sulfadimethoxine, Florfenicol/
tylosin etc. (Escudero et al., 1996; Fernández-Varón et al.,
2005; Kim et al., 2008)
Why combination of drugs?

5. Selection of FIC methodology
 Ease of performance.
 Adaptability to automated or semi-automated
platforms.
 Cost (economy).
 Reliability (repeatability).
 Accuracy.

6. Methods of synergy testing
(White et al., 1996)
• Checkerboard dilution assays :- measure of the
inhibitory activity
• Time kill curve methods :- assesses bactericidal
activity
• Multiple combination bactericidal testing
(MCBT)
• Synergy testing using E (epsilometer) tests

7. Checkerboard dilution assays
Two antimicrobial agents are serially diluted in a two-
dimensional fashion to include all combinations

8. Interpretation of results

9. Advantages
 Easy test to perform; however, it is merely a gauge of
inhibitory activity.
 Convenience of having prepared panels
 The economy of reagents and space that occurs due to
the miniaturization of the test
 There is also assistance in generating computerized
reports if an automated panel reader is used

10. Disadvantages
Time-consuming
Labor-intensive
Gradient do not know because dilution in
twofold only
Not validated in clinical trial
The possibility of errors in preparation of the
antibiotic Solutions
 The relatively large amount of reagents and
space required for each test

11. Time kill curve method
• Killing effect of drug can be expressed as rate
of killing of microbes by fixed concentration
of drug under controlled conditions.
• The rate of killing is determined by counting
the viable bacteria at various time interval.
• Resulting graphical depiction is called as time-
kill curve
• Has been used in evaluation of antibacterial
drug interaction

12. Time kill method for synergistic
action of drug
 MIC of each antibacterial agent is determined
 Broth-macrodilution of agent.
Containing single agent ranging from 1/4X to 2X of MIC
Containing two agents with different concentration ranging
from 1/4X to 2X of MIC as in checkerboard
A defined inoculum of the strain (5×105
colony forming
units/ml) is then inoculated into the tubes.
Aliquots of the samples from 0 hours of incubation to 24
hrs are collected.
Aliquots are serially diluted, inoculated on plate & cfu/ ml
are calculated in all preparations.

13. Interpretation
Results are plotted on semi-log curve
• Synergy was defined as a ≥100-fold or 2 log10decrease
in colony count at 24 h by the combination compared
with that by the most active single agent and as a ≥
100-fold decrease in colony count compared with the
starting inoculum.
• Antagonism was defined as a ≥ 100-fold increase in
colony count at 24 h by the combination compared
with that by the most active drug alone.

14. Advantages
 The time-kill method of synergy testing
assesses bactericidal activity
 Tests bactericidal concentrations
 Methodology defined by National Committee
on Clinical Laboratory Standards
Disadvantages
Time-consuming
Labor-intensive
A limited number of agents can be tested.

15. E (epsilometer) test
 The Epsilometer, or E test:- Agar diffusion method for
performing antimicrobial susceptibility testing.
 It employs strips coated with a continuous concentration
gradient of a specific antimicrobial agent that is placed on
an agar plate inoculated with the bacterial strain of interest.
 After overnight incubation, elliptical zone of ‘no growth’
develops around the strip.
 Interpretation of the MIC:- Read at the intersection of the
zone of lysis with the strip.
 To assess synergy, two strips of different agents are placed
at 90° at the intersection of the MIC of each agent for the
bacterial strain of interest

18.  Commercially available
 Quantitative test
 Can be performed in clinical microbiology laboratories
 Simplicity that does not require any special equipment.
 The provision of categorical results easily interpreted by all
clinicians.
 Flexibility in selection of disks for testing.
Disadvantages
Advantages
 Tests bacteriostatic concentrations
 The lack of mechanization or automation of the test
 All fastidious and slow growing bacteria can not be
accurately tested by this method.

19.  MCBT combines two or three drugs in microtitre wells.
 The peak serum concentration of each agent is tested and the
bactericidal activities determined.
 To detect synergy, one, two or three agents are added to the
appropriate wells, and a standardized inoculum (5×105
colony
forming units/ml) of the bacterial strain of interest is added to
each well, the plates then being incubated. Wells without visible
turbidity are sampled by streaking a 10 µl aliquot on an agar
plate, incubating the plate for a day and observing for 99.9%
killing (bactericidal activity).
 Synergistic activity:- Those combinations with demonstrable
bactericidal activity reveals synergism.
Multiple combination bactericidal
testing (MCBT)

20. Advantages
Tests bactericidal concentrations.
Disadvantages
 Tests peak serum concentrations, which may not reflect
concentrations obtained in vivo.