The recent nomination by the World Health Organization of Acinetobacter baumannii as the number one priority pathogen for the development of new antibiotics is a direct consequence of its fast evolution of pathogenicity, and in particular of multidrug resistance. While the development of new antibiotics is critical, understanding the mechanisms behind the crescent bacterial antibiotic resistance is equally relevant. Often, resistance and other bacterial virulence elements are contained on highly mobile pieces of DNA that can easily spread to other bacteria. Prophages are one of the mediators of this form of gene transfer, and have been frequently found in bacterial genomes, often offering advantageous features to the host. Here we assess the contribution of prophages for the evolution of A. baumannii pathogenicity. We found prophages to be notably diverse and widely disseminated in A. baumannii genomes. Also remarkably, A. baumannii prophages encode for multiple putative virulence factors that may be implicated in the bacterium’s capacity to colonize host niches, evade the host immune system, subsist in unfavorable environments, and tolerate antibiotics. Overall our results point towards a significant contribution of prophages for the dissemination and evolution of pathogenicity in A. baumannii, and highlight their clinical relevance.

Recently, phages have become popular as an alternative to antibiotics. This increased demand for phage therapy needs rapid and efficient methods to screen phages infecting specific hosts. Existing methods are time-consuming, and for clinical purposes, novel, quick, and reliable screening methods are highly needed. Flow cytometry (FC) allows a quick differentiation and enumeration of bacterial cell populations and has been used to assess in vitro the activity of antimicrobial compounds. In this work, we propose FC as a rapid and reliable method to assess the susceptibility of a bacterial population to phage infection. For that, the interaction of phages vB_PaeM_CEB_DP1 and vB_PaeP_PE3 with Pseudomonas aeruginosa PAO1 was characterized by FC. Synchronous infection assays were performed, and samples were collected at different time points and stained with SYTO BC and PI before analysis. Part of the collected samples was used to characterize the expression of early, middle, and late genes by qPCR. Both FC and qPCR results were correlated with phage propagation assays. Results showed that SYTO BC median fluorescence intensity (MFI) values increased in the first 25 min of PE3 and DP1 infection. The increase of fluorescence is due to the expression of phage genes observed by qPCR. Since SYTO BC MFI values increase with gene expression, it allows the determination of host susceptibility to a phage in a short period of time, avoiding false positives caused by lysis from without. In conclusion, this method may allow for a quick and high-throughput real-time screening of different phages to a specific host, which can be crucial for a quick phage selection in clinical practice.

The human pathogen Mycobacterium tuberculosis (Mtb) thrives in lipid-rich microenvironments. A strong body of evidence demonstrated that, during infection, Mtb utilizes long-chain fatty acids (LCFA) as a preferred carbon source. However, LCFA also have antimicrobial properties. Mtb must therefore employ mechanisms to utilize LCFA while mitigating their toxicity. Using transposon sequencing (TnSeq), we defined the Mtb LCFA resistome as comprising 38 genes. Surprisingly, LCFA resistance requires diverse metabolic pathways, indicating pleiotropic effects of LCFA on Mtb physiology. We investigated the function of the TnSeq top-hit, the universal stress protein TB15.3, and demonstrate that it participates in a “metabolic brake” mechanism restricting LCFA uptake and catabolism to prevent membrane hyperpolarization. TB15.3 absence caused Mtb to lose viability during chronic infection in mice and in an in vitro caseum model. Our work highlights Mtb LCFA resistance mechanisms as an important host adaptation and a promising target space for drug development.