Periodic Reporting for period 1 - MAREE (Mechanisms of Antibiotic Recalcitrance in ESKAPE pathogens)
Berichtszeitraum: 2023-07-01 bis 2025-06-30
The discovery of antibiotics in the late 1920s was one of the biggest achievements in medicine. However, the rise of antimicrobial resistance (AMR) now threatens this progress. The World Health Organization (WHO) warns that by 2050, antibiotic-resistant infections could cause 10 million deaths per year and cost the global economy 1.2 trillion US dollars.
A particular group of bacteria called ESKAPE pathogens—which include Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species—are especially dangerous. These bacteria often cause hospital-acquired infections and have developed resistance to multiple antibiotics.
One major problem is antibiotic (AB) recalcitrance—a situation where bacteria that seem sensitive to antibiotics survive treatment and cause the infection to return once the treatment ends. This contributes to the failure of antibiotic therapies and can lead to the development of even more resistant bacterial strains. Unfortunately, the biological mechanisms behind this phenomenon are still poorly understood.
The MAREE project aims to understand how this recalcitrance works by focusing on two ESKAPE bacteria : Enterococcus faecium (Efm) and Enterobacter cloacae complex (Ecc)
The overall biological question how these bacteria survive antibiotic treatment at the protein level, as proteins play a crucial role in changing the bacteria’s metabolism and helping them survive treatment.
The project has three main goals: (i)identify the stress conditions and antibiotics that trigger recalcitrance and isolate specific subpopulation, (ii)analyze the proteins and phosphorylated proteins (those that regulate many cellular functions) involved in this state, (iii)test the role of key proteins through genetic modification and lab experiments, in collaboration with Prof. Helaine at Harvard Medical School.
The expected outcome is a better understanding of how bacteria survive antibiotic treatments. This knowledge could lead to new targeted therapies to fight recurring infections and slow down the spread of antibiotic resistance.
A particular group of bacteria called ESKAPE pathogens—which include Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species—are especially dangerous. These bacteria often cause hospital-acquired infections and have developed resistance to multiple antibiotics.
One major problem is antibiotic (AB) recalcitrance—a situation where bacteria that seem sensitive to antibiotics survive treatment and cause the infection to return once the treatment ends. This contributes to the failure of antibiotic therapies and can lead to the development of even more resistant bacterial strains. Unfortunately, the biological mechanisms behind this phenomenon are still poorly understood.
The MAREE project aims to understand how this recalcitrance works by focusing on two ESKAPE bacteria : Enterococcus faecium (Efm) and Enterobacter cloacae complex (Ecc)
The overall biological question how these bacteria survive antibiotic treatment at the protein level, as proteins play a crucial role in changing the bacteria’s metabolism and helping them survive treatment.
The project has three main goals: (i)identify the stress conditions and antibiotics that trigger recalcitrance and isolate specific subpopulation, (ii)analyze the proteins and phosphorylated proteins (those that regulate many cellular functions) involved in this state, (iii)test the role of key proteins through genetic modification and lab experiments, in collaboration with Prof. Helaine at Harvard Medical School.
The expected outcome is a better understanding of how bacteria survive antibiotic treatments. This knowledge could lead to new targeted therapies to fight recurring infections and slow down the spread of antibiotic resistance.
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
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
This project has delivered validated bacterial strains and antibiotics for recalcitrance studies, identified effective stress conditions to generate persisters, and adapted innovative tools to isolate and analyze these subpopulations. It has also provided new genetic and physiological insights into bacterial persistence, including the discovery of unusual antibiotic responses. These achievements represent important progress toward a deeper understanding of how bacteria endure antibiotic treatment, paving the way for more effective antimicrobial strategies in the future.