Investigation of Novel <i>pmrB</i> and <i>eptA</i> Mutations in Isogenic Acinetobacter baumannii Isolates Associated with Colistin Resistance and Increased Virulence <i>In Vivo</i>

Abstract

Colistin resistance in <i>Acinetobacter baumannii</i> is of great concern and is a threat to human health. In this study, we investigate the mechanisms of colistin resistance in four isogenic pairs of <i>A. baumannii</i> isolates displaying an increase in colistin MICs. A mutation in <i>pmrB</i> was detected in each colistin-resistant isolate, three of which were novel (A28V, I232T, and ΔL9-G12). Increased expression of <i>pmrC</i> was shown by semi-quantitative reverse transcription-PCR (qRT-PCR) for three colistin-resistant isolates, and the addition of phosphoethanolamine (PEtN) to lipid A by PmrC was revealed by mass spectrometry. Interestingly, PEtN addition was also observed in some colistin-susceptible isolates, indicating that this resistance mechanism might be strain specific and that other factors could contribute to colistin resistance. Furthermore, the introduction of <i>pmrAB</i> carrying the short amino acid deletion ΔL9-G12 into a <i>pmrAB</i> knockout strain resulted in increased <i>pmrC</i> expression and lipid A modification, but colistin MICs remained unchanged, further supporting the strain specificity of this colistin resistance mechanism. Of note, a mutation in the <i>pmrC</i> homologue <i>eptA</i> and a point mutation in IS<i>Aba1</i> upstream of <i>eptA</i> were associated with colistin resistance and increased <i>eptA</i> expression, which is a hitherto undescribed resistance mechanism. Moreover, no cost of fitness was observed for colistin-resistant isolates, while the virulence of these isolates was increased in a <i>Galleria mellonella</i> infection model. Although the mutations in <i>pmrB</i> were associated with colistin resistance, PEtN addition appears not to be the sole factor leading to colistin resistance, indicating that the mechanism of colistin resistance is far more complex than previously suspected and is potentially strain specific.