Identification of colistin resistance mechanisms in pmrC-, pmrA-, and pmrB-deficient Acinetobacter baumannii

[摘要] Acinetobacter baumannii is a menacing nosocomial pathogen that readily develops resistance to antibiotics. Colistin, a polymyxin, is currently the last-line choice for treating infections caused by multidrug-resistant A. baumannii. This cationic peptide is attracted to the negatively charged lipopolysaccharide (LPS) on Gram-negative bacterial membranes and acts by destabilizing and permeabilizing it. Unfortunately, there is a disturbing trend of strains developing resistance to colistin in recent years. A. baumannii is known to modify its LPS in order to interrupt the initial charge interaction with colistin. The most common mechanism implicated in this resistance strategy is the upregulation of the pmrAB genes encoding a two-component regulatory system, which in turn upregulates pmrC that encodes phosphoethanolamine transferase. PmrAB have also been shown to be global regulators of cellular growth processes and virulence. Gain-of-function mutations in pmrAB are the most commonly reported colistin resistance mechanism. However, it is not known if and how A. baumannii strains deficient in any of the pmrCAB genes develop colistin resistance. We therefore aim to determine secondary mechanisms of colistin resistance in pmrCAB-deficient strains.Using A. baumannii strain AB5075 and inactivation mutants of pmrC, -A, and -B, we generated genetically stable colistin-resistant mutants on agar containing increasing colistin concentrations. We conducted whole genome sequencing to identify genetic changes responsible for colistin resistance. We also characterized their phenotypes, including resistance profiles to Gram-positive antimicrobials, LPS structure, in vitro growth fitness, and in vivo virulence using a Galleria mellonella model. Colistin-resistant mutants possessed mutations in lpx and mla genes, which are involved in lipid biosynthesis and membrane composition, respectively. Most mutants were susceptible to Gram-positive antimicrobial agents, likely resulting from severely compromised outer membranes. Furthermore, colistin-resistant mutants displayed reduced fitness in vitro and decreased virulence in vivo.A. baumannii is adept at evading colistin by modifying its membrane structure, even outside of the control of pmrCAB. However, mutations that disrupt membrane integrity are costly and unlikely to persist in clinical settings. Therefore, colistin-resistant mutants with such mutations seemingly carry low public health significance. Even if these mutants do emerge in the clinic, various Gram-positive agents can be included as treatment options.

[发布机构] the University of Pittsburgh