Fig 1.
Design of the assay and the experimental protocol.
(A) Technical drawing of the CombiANT insert with top view (top) and side projection along the red dashed line (bottom). The insert comprises 3 antibiotic reservoirs (marked ‘A,’ ‘B,’ and ‘C’) that surround an internal triangular area called the interaction imaging area. The outer diameter of the insert is 42 mm. (B) Examples of how CombiANT assays can be used, whether in multiples or single assays in standard agar plates. (C) Illustration of the assay protocol. The insert is loaded by adding 0.5 mL liquid agar (60°C) containing antibiotics into the reservoirs. The prepared inserts can be stored at 4°C–8°C. To activate an insert, a second layer of 25 mL agar is added as to enclose the insert and fill the plate, thereby permitting diffusion of the antibiotics to the agar surface and the reservoir periphery. After solidification, a bacterial cell suspension of 0.5 McFarland is inoculated on the agar surface using a sterile cotton swab and exposed to the antibiotic gradient landscape. The finished plates are incubated at 37°C, and stable zones of growth inhibition establish within 16–24 hours. Numerical values for this figure are available in S1 Data.
Fig 2.
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 ICB, 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 CPBC, 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 CPBC. 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.
Fig 3.
Technical validation of the CombiANT assay.
(A) Quantification of drug interactions in reference strains of E. coli, P. aeruginosa, and S. aureus. Synergy is expressed by FICis (mean ± SD, n = 10 replicates). (B) Analysis of assay precision depicted in violin plots. Repeatability is expressed as relative standard error for assays conducted on the same day; dashed lines represent median standard error, and dotted lines represent the upper and lower quartiles, n = 6 combinations. Reproducibility is expressed as relative differences in FICi between days; dashed lines represent median relative differences, and dotted lines represent the upper and lower quartiles, n = 6 combinations. (C) Coefficient of variation for the pooled data (n = 10 replicates) encompassing both different-day and same-day replicates to quantify biological and technical replicability. Results are grouped by antibiotic interaction and then further subdivided between the 3 strains. (D) Bland-Altman method comparison of CombiANT and checkerboard assays (performed in house, mean values, n = 18 strain–antibiotic pair combinations). Absolute difference of FICi of methods is plotted against their average values. The small scattering of the points is indicative of little bias between the 2 methods. One sample Wilcoxon signed rank test against FICi = 1, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Numerical values for this figure are available in S1 Data. AMP, ampicillin; CIP, ciprofloxacin; CTX, cefotaxime; FICi, fractional inhibitory concentration index; GEN, gentamicin; SD, standard deviation.
Fig 4.
Interaction screening of E. coli UTI clinical isolates.
(A) Schematic representation of the screening of 12 clinical isolates against a panel of 5 antibiotics. Ten pairwise interactions were quantified in every strain in n > 3 biological replicates, of which at least one was on a different day. (B) Antibiotic interactions expressed by FICi (mean ± SD). Clinically relevant levels of synergy (FICi < 0.5) are indicated in blue, while clinically relevant levels of antagonism (FICi > 4) are indicated in red. The upper measurement range in the screening was FICi = 8, which was exceed by several replicates and the MEC-NIT combination. These replicates were plotted as FICi = 8, and error bars that exceed this limit are trimmed. One-sample Wilcoxon signed rank test against FICi = 1, ***P < 0.001, **P < 0.01, *P < 0.05. Numerical values for this figure are available in S1 Data. CIP, ciprofloxacin; FICi, fractional inhibitory concentration index; FOF, fosfomycin; MEC, mecillinam; NIT, nitrofurantoin; SD, standard deviation; TMP, trimethoprim; UTI, urinary tract infection.