Characterization of Acinetobacter baumannii Filamentous Cells by Re-Scan Confocal Microscopy and Complementary Fluorometric Approaches

Abstract

The spread of Acinetobacter baumannii in clinical settings is a great concern at the time being, given the epidemic potential and the capacity to elude the effects of drugs of this bacterial species. How A. baumannii achieves its resistance determinants is not yet exactly understood, hence the need for new studies aimed at resolving its structure and functions. In this work, we employ Re-scan Confocal Microscopy (RCM), an emerging super-resolution technique, for investigating the morphology of this pathogen. Interestingly, about 1.5% of the imaged cells were found to be filamentous without internal division septa, in both logarithmic and stationary phases, which, to our knowledge, has not been previously reported. Since filamenting represents one of the first mechanisms adopted by bacteria to resist antibiotic effects, we also focus on evaluating the sensitivity of A. baumannii to colistin. For this, the LIVE/DEAD ratio is assessed by means of a fluorometric approach based on SYTO9 and KK114 dyes, two stains that we also used for RCM imaging. Their dual role demonstrated here can potentially be exploited in-tandem in multimodal systems for imaging and fluorometry.