New regulatory pathways activated by the stringent response in antimicrobial resistance

Resistance to antimicrobial agents can involve mutations affecting complex regulatory pathways rather than ‘conventional’ mechanisms affecting drug targets, uptake or degradative enzymes. Whole genome sequencing has the potential to improve our understanding of these complex mechanisms, including those affecting the diazabicyclooctane, OP0595, and the ?-lactam, mecillinam, which both target penicillin-binding protein 2. Using whole genome sequencing we identified the global stringent response signal, entailing induction of the RpoS sigma factor, as the main mediator of mutational resistance to OP0595 in Escherichia coli. Sequencing of twenty laboratory-selected mutants identified fifteen altered targets, of which thirteen encoded either (i) tRNA synthase and modification functions whose alterations induce expression of RpoS through activation of the stringent response or, (ii) known RpoS suppressors CydA and ArcAB. Our results provide new insights into the regulatory pathways governed by the stringent response alarmone ppGpp in bacterial antibiotic resistance and highlight the potential of WGS to rapidly define complex mechanisms of resistance. The results also elucidate the ability of low concentrations of OP0595 and mecillinam to still cause sphaeroplasting of cells that are resistant to inhibition of growth by these agents