Methods for Determining Fractional

Inhibitory Concentration (FIC)

Test to determine interaction between two or more drugs intended to be
used in combination

Gulam Mohd
&
Bhoj R Singh
Division of Epidemiology
Indian Veterinary Research Institute,
Izatnagar- 243 122, India
 Why FIC is measured ?

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.
 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 ).

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.
 Why combination of drugs?
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; Fernndez-Varn et al., 2005; Kim et al., 2008)
 Selection of FIC methodology
Ease of performance.

Adaptability to automated or semi-automated

platforms.

Cost (economy).

Reliability (repeatability).

Accuracy.
 Methods of synergy testing
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
(White et al., 1996)
 Checkerboard dilution assays

Two antimicrobial agents are serially diluted in a two-

dimensional fashion to include all combinations
Interpretation of results
 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
 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
 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
 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 (5105 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.
 Interpretation
Results are plotted on semi-log curve
Synergy was defined as a 100-fold or 2 log10
decrease 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.
 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.
 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
 Advantages
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

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.
 Multiple combination bactericidal
testing (MCBT)
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 (5105 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 l 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.
 Advantages

Tests bactericidal concentrations.

Disadvantages

Tests peak serum concentrations, which may not reflect

concentrations obtained in vivo.