Sublethal interaction factor (SIF), a growth-based method to analyze antibiotic combinations at sub-inhibitory concentrations

Antibiotic resistance is a global concern with significant implications for healthcare, food production, and the environment. Therapies involving antibiotic combinations are frequently employed as a strategy to overcome antibiotic resistance. When present in combination, the efficacy of antibiotics may be enhanced or weakened, and as antibiotic interactions are <i>a priori</i> generally unpredictable, they need to be experimentally determined. Though antibiotics are regularly present at sublethal concentrations (e.g., in patients with suboptimal dosing regimens, late after the last dosage, difficult-to-penetrate tissues, and also in the natural environment), effects of antibiotic combinations are generally studied at lethal dosages. To address this, we developed the sublethal interaction factor (SIF) assay, based on the Bliss independence model, to quantify antibiotic combination effects at sublethal concentrations. SIF assay uses the whole growth curve, instead of only the growth rate, and determines reliably the outcome of the interaction between two antibiotics at sublethal concentrations. The SIF method was validated against the CombiANT assay and showed high sensitivity and specificity, attesting to its usability. IMPORTANCE Sublethal interaction factor (SIF), a method herein proposed, simplifies the analysis of antibiotic interactions at sub-inhibitory concentrations and shows a high correlation with the fractional inhibitory concentration index (FICi), the gold-standard measure used to classify antibiotic interactions when tested at lethal concentrations. The SIF can be easily incorporated into laboratories and has great potential for studying not only antibiotic combinations but also drug interactions and phage therapy.