What is antibiotic synergy testing?

Antibiotic Synergy Testing Definition

Antibiotic synergy testing is laboratory evaluation that determines whether combining two or more antimicrobial agents produces an effect that exceeds the additive effects of the individual drugs when used alone. 1

Core Definition

Synergistic action occurs when the combined antimicrobial effect of multiple antibiotics is greater than simply adding together the effects of each drug individually. 2 This fundamentally differs from:

• Additive effects: The combination equals the sum of individual drug effects 1
• Indifference: The combination equals only the most active component 1
• Antagonism: The combination produces a reduced effect compared to the most effective individual drug 1

Mathematical Quantification Methods

Fractional Inhibitory Concentration (FIC) Index

The European Society of Clinical Microbiology and Infectious Diseases defines synergy quantitatively using the FIC Index (ΣFIC). 1

For two antibiotics A and B:

• FIC_A = (MIC_A in presence of B) / (MIC_A alone) 1
• FIC_B = (MIC_B in presence of A) / (MIC_B alone) 1
• ΣFIC = FIC_A + FIC_B 1

Synergy is typically defined as ΣFIC ≤ 0.5, though specific threshold criteria exist in standardized tables. 2

Alternative Quantification Methods

• Fractional Bactericidal Concentration (FBC): Calculated identically to FIC but using minimum bactericidal concentration (MBC) values instead of MIC values 1
• Time-kill curves: Graphical representation of bacterial killing over time with antibiotic combinations 1
• Isobologram analysis: Figurative representation of the interaction between two antibiotics 1

Laboratory Testing Methods

Checkerboard Assay

The checkerboard array is a modification of standard MIC testing where bacteria are incubated with antibiotics at different concentration combinations and evaluated for growth inhibition after overnight incubation. 3 This method:

• Results in multiple FIC indices from various antibiotic concentration combinations 1
• Can be automated using inkjet printer technology to increase efficiency and accuracy 3
• Serves as a high-throughput screening assay 3

Time-Kill Assay

In the time-kill synergy assay, bacteria are incubated with antibiotics both together and individually, then sampled at intervals over 24 hours for quantitative culture. 3 This method:

• Determines whether a combination is synergistic and bactericidal 3
• Provides data on inhibition and killing of bacteria over time 3
• Is considered the most promising method to detect synergistic combinations, followed by checkerboard assay and E-test 4

Critical Clinical Caveats

In Vitro vs. Clinical Significance

These synergistic effects are demonstrated in vitro, and their clinical significance remains unclear. 1 However:

• In one study of 148 severe gram-negative infections in cancer patients, combinations that were synergistic in vitro were associated with significantly better clinical response (P < 0.01) compared to non-synergistic combinations. 5
• Clinical studies are needed to determine therapeutic responses of synergistic combinations in severe infections 2

Common Pitfalls to Avoid

• Antagonism detection: Antagonism was detected in 25% of double antibiotic combinations in some studies, but never with triple combinations 6
• Resistance mechanisms: The presence of synergy or antagonism correlates with the MIC of the agent and molecular mechanisms involved in resistance 4
• Drug interaction studies: In vitro synergy studies with vaborbactam-meropenem demonstrated no antagonism with levofloxacin, tigecycline, polymyxin, amikacin, vancomycin, azithromycin, daptomycin, or linezolid 7

Clinically Relevant Synergistic Combinations

Promising synergistic results have been demonstrated for:

• Meropenem plus colistin and rifampicin plus colistin against Acinetobacter baumannii 4
• Colistin plus carbapenem and carbapenem plus fluoroquinolones against Pseudomonas aeruginosa 4
• Colistin/polymyxin B plus rifampicin/meropenem against Klebsiella pneumoniae 4

Triple antibiotic combinations generally show greater synergy than double combinations, with modal degree of synergy being higher and organisms multiply resistant to three antibiotics never being resistant to all three combined. 6