The document outlines methods for determining fractional inhibitory concentration (FIC) to assess drug interactions in combination therapies, highlighting the importance of synergistic, additive, and antagonistic effects. It details various synergy testing methods such as checkerboard dilution assays, time-kill curve methods, E tests, and multiple combination bactericidal testing (MCBT), along with their advantages and disadvantages. The primary objectives are to enhance antibacterial activity while minimizing the risk of resistance development and toxic effects associated with high drug doses.

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.