Potential efflux pump inhibition by the cyclic peptide MV6 restores netilmicin activity in Acinetobacter baumannii

Introduction

Acinetobacter baumannii is a critical global health threat due to its opportunistic nature and ability to evolve multidrug resistance. To combat the lack of effective treatments, adjuvants such as the cyclic peptide MV6 are being explored. While MV6 lacks intrinsic antimicrobial activity, it enhances the activity of aminoglycosides such as netilmicin. This project aims to elucidate the mechanism by which MV6 potentiates the activity of netilmicin.

Methods

Spontaneous mutants were generated under selective pressure from netilmicin alone or in combination with MV6. Whole-genome sequencing (WGS) and a custom variant-calling pipeline identified the mutational distribution. A representative subset of mutants, with the wild-type reference, underwent transcriptomic analysis. To validate the role of efflux systems, Minimum Inhibitory Concentrations (MICs) were determined in the presence and absence of the efflux pump inhibitor PaβN.

Results

WGS and variant-calling analysis revealed a convergent mutational profile across both spontaneous mutant groups; no differences were detected between mutants generated with or without MV6. Key mutations were identified in a tetR-family transcriptional regulator located in cis to a multidrug efflux pump, alongside a recurrent deletion in the intergenic region downstream of an adeABC operon. Additionally, diverse mutations were detected in the coding sequence or promoter region of an ATP-binding protein. Remarkably, transcriptomic analysis confirmed that these mutations resulted in the overexpression of the adeABC operon and the multidrug efflux pump. Preliminary phenotypic assays with PaβN corroborated that, as efflux inhibition partially restored netilmicin susceptibility, efflux systems are the primary driver of resistance.

Conclusion

Our results demonstrate that netilmicin resistance is primarily driven by overexpression of efflux systems. Interestingly, MV6 did not alter the mutational or transcriptomic profile of the mutants. This suggests that MV6 enhances netilmicin without exerting additional selective pressure, supporting its potential role as a “silent” adjuvant that may interfere with efflux function.