Assay technology and synergy readout.

(A) Diffusion map for the antibiotic MEC after 24 hours. Lines indicate equal surface concentrations. The chromatic scale represents concentration of MEC starting the input concentration of 4.8 mg/L denoted in red, down to negligible concentrations in blue. (B) Agar surface antibiotic diffusion landscape for 3 antibiotics. The chromatic scale represents concentrations of antibiotic starting from input concentrations with blue indicating antibiotic A, yellow indicating antibiotic B, and pink indicating antibiotic C, down to negligibly low concentrations expressed by white. (C) Description of CombiANT analysis using a representative assay result. The E. coli strain DA46056 that was isolated from a UTI was here tested against the antibiotics MEC, TMP, and NIT. Inhibition zones have formed around the antibiotic reservoirs because of the antibiotic diffusion. The black triangle indicates the interaction imaging area. Blue points outside of the interaction imaging area indicate the IC points in the agar surface of the antibiotics acting alone. As illustrated by the blue line and IC_B, the blue IC points are placed at the midpoint of the outer inhibition zone from the corresponding reservoir, here reservoir B. Red points inside the interaction imaging area indicate CPs of adjacent antibiotic pairs. As illustrated by the red lines and CP_BC, the red CP points are placed at the corner of the inner growth zone, in the position that is closest to the corresponding interaction are corner, here between the reservoirs B and C for CP_BC. The red dashed line indicates the radius of the solid red line circle segment. Darker shades below the insert are trapped air bubbles. (D) Quantification of antibiotic interactions expressed as FICi for the image in panel C. The dotted line indicates a theoretical additive interaction with FICi = 1. Numerical values for this figure are available in S1 Data. CP, combination inhibitory point; FICi, fractional inhibitory concentration index; IC, inhibitory concentration; MEC, mecillinam; NIT, nitrofurantoin; TMP, trimethoprim; UTI, urinary tract infection.