Background: Polymyxins are a last-line defense treating recalcitrant multidrug-resistant (MDR) Gram-negative infections. Polymyxins bind to lipid A, causing increased permeation of the Gram-negative outer membrane (OM). There are a variety of mechanisms of polymyxin resistance including lipid A modification, and in rare cases, complete loss of lipid A. This study aimed to investigate the mechanisms of polymyxin resistance and contributes to the growing understanding of OM remodeling in A. baumannii.
Methods: We generated polymyxin-resistant A. baumannii derivatives from two clinical isolates using repeated selection on agar containing 10 mg/L polymyxin E. Whole genome resequencing (WGS) was used to identify mutations in polymyxin-resistant strains. Polymyxin-resistant and -susceptible strains were compared by phenotypic assays, scanning/transmission electron microscopy (SEM/TEM) and proteomics.
Results: Resistant strains were 8-fold more resistant to polymyxins based on MICs. WGS identified mutations in the lpx biosynthesis cluster, similar to those previously reported. However, one strain contained an in-frame insertion in the gene encoding mlaF, a component of the mlaABCDEF retrograde phospholipid trafficking pathway. Analysis of lipid A by PAGE and Limulus amebocyte lysate assay demonstrated that LPS was absent in the mlaF mutant strain. SEM/TEM analysis indicated that both mlaF and lpx mutant strains were hyper-blebbing their OMs. Comparison of the proteome of the paired polymyxin-resistant and ‑susceptible cell envelopes indicated that 188 and 246 OM proteins were under-represented in lpx and mlaF mutant strains, respectively; only 7 and 9 proteins were over-represented correspondingly. We are currently characterising the mlaF mutant using biochemical methods and metabolomics.
Conclusion: Our results support the increasing evidence that the mla apparatus is important for OM homeostasis and polymyxin resistance in A. baumannii. These results are discussed in the context of A. baumannii OM plasticity in response to lipid A loss.