High‐Density <i>E. Coli</i> Cultivation Arrays for Combinatorial Drug Testing With Subsequent Lipid Profiling by MALDI‐MS

The increasing resistance of bacteria to current drugs underscores the urgent need for new antibiotics. Combination therapies offer an alternative strategy by enhancing efficacy and delaying resistance, with synergies assessed in checkerboard assays using systematically varied drug concentrations. Here, we introduce a miniaturized checkerboard assay for high-throughput combinatorial antibiotic testing against E. coli. Our platform reduces sample volume significantly, achieves high sample densities of 20832 droplets per microscopy slide, and allows generation of checkerboard arrays in under 25 min prior to incubation and analysis. We established the platform by comparing E. coli growth across different culture volumes and performed assays to determine the minimum inhibitory concentration (MIC) for various drugs. Further, we replicated the synergistic effect of amoxicillin/clavulanate against an amoxicillin-resistant strain. Testing various antibiotic combinations allowed us to determine fractional inhibitory concentration indices (FICI) of up to 1.86 ± 0.42, suggesting indifferent effects with a tendency towards antagonism. Lipidomic profiling of E. coli revealed the characteristic lipid composition of primarily phosphatidylethanolamines (PE) and phosphatidylglycerol (PG). We identified PE32:0 that showed enhanced signal intensities at the MIC region, hinting at stress-induced lipid changes. Our platform holds promise for exploring combinatorial drug effects across cell types, with unique capabilities for simultaneous cell monitoring and MALDI-MS analysis.