Gulam Mohd&

Bhoj R SinghDivision of Epidemiology

Indian Veterinary Research Institute, Izatnagar- 243 122, India

Gulam Mohd&

Bhoj R SinghDivision 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

• 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 ?

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.

• 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?

Selection of FIC methodology

Ease of performance.

Adaptability to automated or semi-automated platforms.

Cost (economy).

Reliability (repeatability).

Accuracy.

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

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-consumingLabor-intensiveGradient do not know because dilution in

twofold onlyNot validated in clinical trialThe 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 MICContaining two agents with different concentration ranging

from 1/4X to 2X of MIC as in checkerboardA 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.

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-consumingLabor-intensiveA 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

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.

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)

Advantages

Tests bactericidal concentrations.

Disadvantages

Tests peak serum concentrations, which may not reflect concentrations obtained in vivo.