AbstractBackgroundBicyclic boronates are a new and potentially important class of β-lactamase inhibitor, with the ability to inhibit β-lactamases from all molecular classes, including mobile metallo-β-lactamases.ObjectiveOur objective was to identify mutants resistant to the actions of the bicyclic boronate inhibitor 2, when being used in combination with aztreonam.MethodsOvernight cultures were plated on to agar containing increasing concentrations of aztreonam with a fixed 10 mg/L concentration of the inhibitor. Resistant derivatives and parent strains were analysed by whole genome sequencing and LC-MS/MS proteomics to identify mechanism of resistance.ResultsWhen using a mixed overnight culture containing one Escherichia coli (TEM-1, CTX-M-15, CMY-4 producer) and one Klebsiella pneumoniae (SHV-12, CTX-M-15, NDM-1 producer) mobilisation of an IncX3 plasmid carrying blaSHV-12 from the K. pneumoniae into the E. coli generated an aztreonam/boronate resistant derivative.ConclusionsHigh-level production of three bicyclic boronate susceptible enzymes (CMY-4, CTX-M-15, SHV-12) capable of hydrolysing aztreonam plus TEM-1, which binds the inhibitor, overcomes the fixed inhibitor dose used. This was only identified when using a mixed culture for selection. It would seem prudent that to allow for coalescence of the myriad β-lactamase genes commonly found in bacterial populations colonising humans, this mixed culture approach should be the norm when testing the potential for generating β-lactamase inhibitor resistance in pre-clinical analysis.