Emergence of High-Level Colistin Resistance in an Acinetobacter baumannii Clinical Isolate Mediated by Inactivation of the Global Regulator H-NS

Abstract

Colistin is a crucial last-line drug used for the treatment of life-threatening infections caused by multidrug-resistant strains of the Gram-negative bacterium <i>Acinetobacter baumannii</i> However, colistin-resistant <i>A. baumannii</i> isolates can still be isolated following failed colistin therapy. Resistance is most often mediated by the addition of phosphoethanolamine (pEtN) to lipid A by PmrC, following missense mutations in the <i>pmrCAB</i> operon encoding PmrC and the two-component signal transduction system PmrA/PmrB. We recovered a pair of <i>A. baumannii</i> isolates from a single patient before (6009-1) and after (6009-2) failed colistin treatment. These strains displayed low and very high levels of colistin resistance (MICs, 8 to 16 μg/ml and 128 μg/ml), respectively. To understand how increased colistin resistance arose, we sequenced the genome of each isolate, which revealed that 6009-2 had an extra copy of the insertion sequence element IS<i>Aba125</i> within a gene encoding an H-NS family transcriptional regulator. To confirm the role of H-NS in colistin resistance, we generated an <i>hns</i> deletion mutant in 6009-1 and showed that colistin resistance increased upon the deletion of <i>hns</i> We also provided 6009-2 with an intact copy of <i>hns</i> and showed that the strain was no longer resistant to high concentrations of colistin. Transcriptomic analysis of the clinical isolates identified more than 150 genes as being differentially expressed in the colistin-resistant <i>hns</i> mutant 6009-2. Importantly, the expression of <i>eptA</i>, encoding a second lipid A-specific pEtN transferase but not <i>pmrC</i>, was increased in the <i>hns</i> mutant. This is the first time an H-NS family transcriptional regulator has been associated with a pEtN transferase and colistin resistance.