Resistance to aztreonam, in combination with a bicyclic boronate β-lactamase inhibitor in Escherichia coli identified following mixed culture selection

Background Bicyclic 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. Objective Our objective was to identify mutants resistant to the actions of the bicyclic boronate inhibitor 2 , when being used in combination with aztreonam. Methods Overnight 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. Results When 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 bla SHV-12 from the K. pneumoniae into the E. coli generated an aztreonam/boronate resistant derivative. Conclusions High-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.