Tazobactam selects for multidrug resistance

Piperacillin-Tazobactam is a {beta}-lactam/{beta}-lactamase inhibitor combination which is amongst the most prescribed antimicrobials in hospital medicine. Piperacillin is inactivated by commonly carried resistance enzymes, but tazobactam inhibits these allowing successful treatment. The effect of piperacillin on Gram-negative bacteria has been widely studied, but less attention has been paid to the effects of tazobactam. We used a massive transposon mutagenesis approach (TraDIS-Xpress) to determine the genes in Escherichia coli that affect survival when exposed to piperacillin and tazobactam, separately and together. We found significant differences in the selective pressure of the two drugs: a striking finding was that multiple efflux pump families and regulators were essential for survival in the presence of tazobactam, but only one efflux system was beneficial for piperacillin. Additionally, we identified the shikimate kinase AroK as a potential target for tazobactam. This method also found that genes involved in DNA replication and repair reduced E. coli susceptibility to a combination of piperacillin and tazobactam, not seen from either drug treatment alone. Treatment of E. coli and Klebsiella pneumoniae with piperacillin and/or tazobactam selected for mutants with reduced susceptibility, and SNP analyses supported the TraDIS-Xpress findings that tazobactam selects for changes in membrane permeability and maintenance associated with multidrug-resistance. Increased efflux activity is an important foundation of multidrug resistance in human pathogens, therefore the finding that tazobactam can select for this is concerning. These findings could have consequences for antibiotic prescription and should inform the development of future {beta}-lactamase inhibitors to reduce the global increase in multidrug-resistant infections.