Identificar, compreender e controlar a persistência e heterorresistência bacteriana em ambientes veterinários e humanos

Antimicrobial resistance (AMR) is a widely studied phenomenon; yet, there is still a lot to understand about the coexistence of different antimicrobial susceptibilities within a bacterial population, including the presence of persistent (pR) and heteroresistant (hR) cells (21). In fact, there are several factors that support the urgent need to develop new and effective strategies to control the dissemination of these bacterial phenotypes in all One Health settings, namely: a) the growing challenge posed by AMR and its global health and economic impacts (25,34,53,54); b) a lack of understanding on the processes involved in the formation of AMR relevant phenotypes, including of pR and hR strains (21); c) a lack of knowledge on the clinical relevance of these isolates, previously associated with treatment failure and increased mortality (10,37); d) a data gap on the environmental persistence of these phenotypes, especially in clinical settings, where reduced susceptibility to biocides may favour their endemism (33); and e) the inability of the EUCAST and CLSI protocols to identify these phenotypes, leading to the underestimation of their prevalence (10,37). Although a few studies have focused on these issues, there is still a lack of personalised, contextualized, economic and sustainable measures to control the occurrence of pR and hR strains in veterinary and human hospitals (25). Using a multidisciplinary approach to understand and control AMR spread, this project aims to contribute for unravelling the mechanisms associated with the development of pR and hR strains in veterinary and human medicine, to explore their clinical relevance and environmental dynamics, and to develop advanced diagnostic tools, to be further applied to the direct analysis of samples from veterinary and human hospitals. The project will be based on a collection of 40 enterococci of veterinary and human origin, thus ensuring its applicability in a One Health context. Specifically, it aims to: create a library of pR and hR bacterial populations derived from the available enterococcal collection, using selected antibiotics and conventional bacteriological techniques (task 1); evaluate the alterations in the bacterial genome and gene expression of pR and hR cells, through WGS and RNA-Seq, and to identify potential therapeutic and diagnostic targets (task 2); assess the clinical relevance of pR and hR states using a G. mellonella in vivo model, studying infection development, histological effects and expression of marker genes related to larvae growth, infection, reproduction, and humoral and cellular immunities (task 3); evaluate biocides efficacy in the environmental control of pR and hR phenotypes, and determine the optimal biocide/concentration to eliminate these strains at clinical settings (task 4); and develop an advanced, rapid, specific and sensitive diagnostic method for the detection of pR and hR isolates based on a unique biotechnology, flow cytometry (task 5), to be directly applied to clinical and environmental samples from veterinary and human hospitals, in a One Health perspective. As this diagnostic tool has a translational applicability, following the project's completion, we aim to register the standardized diagnostic protocol established as a patent, to be supported by our research centre. Since AMR reduction is one of the 5 priority action tracks of the One Health Joint Plan of Action 2022-2026, the project’s aims are of major relevance.