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
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Methods for Determining Fractional Inhibitory Concentration (FIC)
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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
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.