This project set out to understand how bacteria survive antibiotic treatment by identifying stress conditions that promote survival, known as recalcitrance, and by developing tools to study persistent bacterial populations.
We successfully identified antibiotics that induced recalcitrance in E. cloacae complex (Ecc) and E. faecium (Efm). Stress conditions relevant to healthcare environments were tested, and while single stresses did not significantly increase bacterial persistence, complex conditions proved much more effective. In E. faecium, biofilm growth in combination with antibiotics led to a marked rise in persisters, while in E. cloacae complex, infection models using host cells such as macrophages and osteoblasts provided suitable conditions for persistence to emerge.
A dual-fluorescent plasmid system, originally developed for Salmonella, was successfully adapted to distinguish growing from non-growing cells in E. cloacae complex. Progress in adapting the tool to E. faecium has been slower due to technical limitations, but encouraging steps forward have already been made.
Alongside this, alternative approaches were introduced to deepen the understanding of persistence mechanisms. Preparations for transposon sequencing are well advanced, with conditions optimized for both bacterial species. In parallel, experimental evolution strategies produced hyperpersistent clones. Genome sequencing of these clones revealed new candidate genes potentially involved in persistence, and follow-up analyses are underway. Importantly, an unexpected persistence behavior was also observed in E. cloacae complex when treated with carbapenems, linked to the beta-lactamase resistance gene, a finding that challenges current assumptions about these antibiotics
