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Understanding the evolution of plasmid-mediated antibiotic resistance in real life scenarios

Periodic Reporting for period 3 - PLASREVOLUTION (Understanding the evolution of plasmid-mediated antibiotic resistance in real life scenarios)

Reporting period: 2020-07-01 to 2021-12-31

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 PLASREVOLUTION we are bridging this gap by exploring the genetic basis underlying the evolution of plasmid-mediated AR in clinically relevant scenarios. We are studying, 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 are analysing the molecular mechanisms that determine the success of AR plasmids and bacterial clone associations (obj. 2). Finally, we are developing 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.
We have performed am important amount of work since the beginning of the project. We have advanced in all the different objectives proposed in the project producing high quality results.
We first established the ideas and concepts that we wanted to investigate during PLASREVOLUTION by publishing two perspective/opinion pieces in Trends in Microbiology and Science. In parallel, we started all the experimental work, with the main focus of understanding the evolution of plasmid-mediated antibiotic resistance in the Hospital Universitario Ramon y Cajal, which is a large public hospital in Madrid. Specifically, we have focused our efforts in understanding the evolutionary dynamics of the antibiotic resistance plasmid pOXA-48, which is the an extremely prevalent and relevant carbapenem resistance plasmid worldwide. We have performed an extensive series of epidemiological work, molecular analyses and mathematical modelling to characterize the “in-hospital” ecology and evolution of this plasmid, which we detailed below (work belonging to objectives 1 and 2).
First, we have used mechanistic models combining epidemiological data from more than 9,000 patients with whole genome sequence information from 250 enterobacteria clones to characterise the dissemination routes of a pOXA-48-like carbapenemase-encoding plasmid in our hospital setting over a two-year period. Next, we used these epidemiological and molecular analyses to discover events of in vivo compensatory evolution in the gut of hospitalised patients. Finally, we determined the distribution of fitness effects for the antibiotic resistance plasmid pOXA-48 in wild-type, ecologically compatible enterobacterial isolates from the human gut microbiota. The results of these works have produced two manuscripts which have been uploaded to bioRxiv (under review in Nature Microbiology and Nature Communications) and one more manuscript which is currently in preparation. In addition, we have also published a methods paper covering some of the techniques used in these experiments (in Methods in Molecular Biology).
Moreover, we have also experience important progress in objective 3 (plasmid dynamics). Specifically, we uncovered the central role of genetic dominance shaping genetic cargo in plasmids, using antibiotic resistance as a model system. Our results provide a new understanding of how mobile genetic elements evolve and spread, uncovering the neglected influence of genetic dominance on bacterial evolution. As a result, we published a paper on this topic (in PNAS).
In summary, the project is advancing at a good pace and over the next 2.5 years we will continue to accomplish the different objectives we established for PLASREVOLUTION, producing high impact publications as a result.
We have made several progresses beyond the state of the art in the field, such as:
-Describing the relative importance of between-patient and within-patient spread of pOXA-48-mediated carbapenem resistance in a hospital setting.
-Analysing the in vivo evolution of plasmid-mediated antibiotic resistance in the gut of hospitalised patients.
-Describing the distribution of plasmid fitness effects in wild-type, clinically relevant, enterobacteria isolates.
-Uncovering the central role of genetic dominance shaping genetic cargo and horizontal spread of mobile genetic elements in bacteria.

In the second half of the project we will continue characterising the evolution of plasmid-mediated antibiotic resistance, using animal models to complement the results we have obtained from the patients and the in vitro experiments.