Horizontal gene transfer (HGT) –the movement of genetic material between individuals– is a significant force fueling bacterial evolution. Through HGT, bacteria acquire new traits, develop new metabolic capabilities and learn to withstand harsh environmental conditions. However, in some cases, HGT brings genetic information that is not advantageous to its host. Despite its crucial relevance for bacterial ecology and evolution, understanding the selective forces that drive the success (or failure) of HGT remains a major challenge. Previous studies addressing this challenge ignored the fact that not all HGT events are alike: incoming DNA can be integrated into the host genome (e.g., transposons, integrons), or it can stand as a physically separated, autonomous DNA molecule (e.g., plasmids). This difference in genomic context poses several mechanistic constraints that are likely to alter the evolutionary outcome of HGT. Here, I will present a conceptually novel approach that explicitly considers genomic context to uncover the selective drivers of HGT in bacterial populations. First, I will develop a new genetic technology to obtain high-throughput fitness measurements of thousands of HGT events. Then, I will use these data to identify and quantify the constraints that determine the success of HGT, both considering the intrinsic effects of the transferred DNA and the role of genomic context on host fitness. Specifically, I will measure the fitness effects of genetic transfers mediated by plasmids (Obj. 1) or integrated into the chromosome and, in the latter case, in different regions of the chromosome (Obj. 2). Finally, I will leverage the rules derived from these analyses to reconstruct the role of HGT in the evolution of a relevant human pathogen (Obj. 3). This project will provide a quantitative and mechanistic understanding of the selective forces driving HGT, expanding horizons in evolutionary microbiology.

Antibiotics are essential tools in modern medicine and are indispensable not only for the treatment of infectious diseases but also to support other key interventions such as surgery and cancer chemotherapy. However, the extensive and inappropriate use of antibiotics has fuelled the spread of resistance mechanisms in pathogenic bacteria, leading to the dawn of a post-antibiotic era. Plasmids play a pivotal role in the evolution of antibiotic resistance (AR) because they drive the horizontal transfer of resistance genes between pathogenic bacteria by conjugation. Some of these plasmid-bacterium associations become particularly successful, creating superbugs that spread uncontrollably in clinical settings. The rise of these clones is mainly constricted because plasmids entail a fitness cost when they arrive in a new bacterial host. This cost can be subsequently alleviated through compensatory adaptation during plasmid-bacterium coevolution. Despite the importance of this cost-compensation dynamic in the evolution of plasmid-mediated AR, it remains completely unexplored in clinical contexts. In this project I plan to bridge this gap by exploring the genetic basis underlying the evolution of plasmid-mediated AR in clinically relevant scenarios. We will study, for the first time, the intra-patient transmission, fitness cost and adaptation of AR plasmids in the gut microbiome of hospitalized patients (obj. 1). We will analyse the molecular mechanisms that determine the success of AR plasmids and bacterial clone associations (obj. 2). Finally, we will develop new technology to test how antibiotic treatments affect AR plasmids dynamics in the gut microbiome at an unprecedentedly high-resolution (obj. 3). This ground-breaking project will allow a new understanding of the evolution of plasmid-mediated AR in real life, opening new research avenues and providing a major step towards meeting one of the central challenges facing our society: controlling the spread of AR.

In bone tissue engineering, UAveiro aims to address unmet needs and challenges in evaluating biomaterials for clinical translation. They propose to bioengineer mini-bone structures using cryogels made from human-derived proteins, specifically amniotic membranes. These cryogels have unique properties that support cell growth and nutrient diffusion, closely mimicking bone architecture and fostering osteogenic and osteoclastogenic differentiation and mineralization. UAveiro's approach involves co-culturing alloMSC, endothelial cells, and CD14+ monocytes on these cryogels to replicate key aspects of the bone cellular environment in vitro. We anticipate that the precisely balanced biochemical and cellular interactions within this tri-culture system will sustain a highly regulated bone remodelling, resembling the natural bone niche's homeostasis. Furthermore, we expect that the growth factors from the human-derived cryogel, dopamine domains, and endothelial cells will autonomously stimulate a differentiation environment toward bone-like tissue. To achieve a more realistic simulation of the bone's physiological conditions, we will design a novel microfluidic Bone-on-Chip (BoC) system with an integrated inflammatory environment to incorporate the bioengineered miniaturized bone structure. This BoC model will enable the assessment of the osteogenic regeneration capacity of biomaterials and ATMPs into simulated bone fractures and offer valuable insights into osteointegration, osteoconduction, and inflammatory responses. By replicating the bone-like tissue, this approach will provide a reliable and thorough method for evaluating the effectiveness of biomaterials in bone tissue engineering and regenerative medicine, potentially advancing our understanding of these processes.