Periodic Reporting for period 1 - ACtIVAtE (The virulence potential of human pathogens: how Acinetobacter baumannii survives Acanthamoeba castellanii predation)
Reporting period: 2018-01-01 to 2019-12-31
Antibiotics resistance is one of the most important modern health issue. With the apparition of pan-resistant bacteria (meaning resistant to all the commercially available antibiotics), it is urgent and crucial to find new antibiotics, but new fighting alternatives as well. In this context, the bacterial pathogen Acinetobacter baumannii was ranked as a top priority pathogen due to its worrying resistance arsenal (WHO 2017 and CDC 2019). Besides its antibiotic resistances, A. baumannii bacteria are highly resistant to environmental stresses and the human immune system. The study of this Gram-negative bacterium is rendered even more difficult by the fact that these bacteria are very heterogeneous, characterized by a dynamic genome due to horizontal gene transfer by natural competence. In other words, they can take up extracellular DNA fragments and integrate them in their genome to evolve and quickly acquire resistance genes. Seeing this whole diversity amongst A. baumannii isolates (both at the phenotypic and genotypic levels), it is not surprising that, nowadays, the virulence and the mechanisms of stress resistance still remain to be understood.A better understanding of the resistances mechanisms (not only for antibiotics) is crucial to better fight this multidrug resistant pathogen.
In this context, we wanted to first determine if the few classical reference strains of A. baumannii available represent good bacterial models to study the current and modern multidrug clinical isolates. Then, based on our phenotypic observations, we generate an experimental strategy to work on the capsule production. We could also study this phenomenon using a new host-pathogen interaction based on the phagocytic pressure exerted by the amoeba called Acanthamoeba castellanii. The aim being to develop a new infection model to study common resistance mechanism, shared by the majority of the current and modern clinical isolates. A better understanding of the resistance mechanisms is important for the development of further antimicrobial strategies.Beside this fundamental part of the project, we wanted to follow a more applied strategy, and also screened a compounds library to find new antibiotics against multidrug-resistant A. baumannii bacteria.
In this context, we wanted to first determine if the few classical reference strains of A. baumannii available represent good bacterial models to study the current and modern multidrug clinical isolates. Then, based on our phenotypic observations, we generate an experimental strategy to work on the capsule production. We could also study this phenomenon using a new host-pathogen interaction based on the phagocytic pressure exerted by the amoeba called Acanthamoeba castellanii. The aim being to develop a new infection model to study common resistance mechanism, shared by the majority of the current and modern clinical isolates. A better understanding of the resistance mechanisms is important for the development of further antimicrobial strategies.Beside this fundamental part of the project, we wanted to follow a more applied strategy, and also screened a compounds library to find new antibiotics against multidrug-resistant A. baumannii bacteria.
The work performed followed different steps:
As we could show that A. baumannii strains are very heterogeneous, we generated a collection of A. baumannii isolates, including reference and current multidrug-resistant clinical isolates. We have now about 250 strains, and we obtain the whole genome sequencing of 48 of them.This allows us to know if the phenotype and new antimicrobial are specific of a few isolates or if they target the majority of the current clinical isolates.
We have developed a new method to assess the capsule production levels of each isolate, and did a proof-of-concept experiment using Transmission Electron Microscopy coupled with capsule labeling to validate our approach.
We showed a high heterogeneity amongst current clinical isolates in term of capsule production and virulence levels. Thus, former reference strains commonly used worldwide do not represent the whole heterogeneity observed for the current clinical isolates. This is the reason why we are working with dozens of isolates at the same time.
We implemented the amoeba Acanthamoeba castellannii as a new infection model as show that upon phagocytic pressure, the majority of the current clinical isolates become hyper-mucoid and resistant to phagocytic cells. This is a common trait shared by the majority of the isolates of our collection.
We also screened a small to medium library of compounds and found a new antimicrobial against multidrug-resistant A. baumannii bacteria. This compound is currently under characterization.
As we could show that A. baumannii strains are very heterogeneous, we generated a collection of A. baumannii isolates, including reference and current multidrug-resistant clinical isolates. We have now about 250 strains, and we obtain the whole genome sequencing of 48 of them.This allows us to know if the phenotype and new antimicrobial are specific of a few isolates or if they target the majority of the current clinical isolates.
We have developed a new method to assess the capsule production levels of each isolate, and did a proof-of-concept experiment using Transmission Electron Microscopy coupled with capsule labeling to validate our approach.
We showed a high heterogeneity amongst current clinical isolates in term of capsule production and virulence levels. Thus, former reference strains commonly used worldwide do not represent the whole heterogeneity observed for the current clinical isolates. This is the reason why we are working with dozens of isolates at the same time.
We implemented the amoeba Acanthamoeba castellannii as a new infection model as show that upon phagocytic pressure, the majority of the current clinical isolates become hyper-mucoid and resistant to phagocytic cells. This is a common trait shared by the majority of the isolates of our collection.
We also screened a small to medium library of compounds and found a new antimicrobial against multidrug-resistant A. baumannii bacteria. This compound is currently under characterization.
The in depth characterization of the new compound, active against A. baumannii, has a great impact for the generation of new antibiotics.
The fact that the reference strains do not reflect the whole diversity of modern clinical isolates is important and deserved to be taken into account to further study A. baumannii worldwide.
We can now use the amoeba Acanthamoeba castellanii as a new validated infection model to screen for anti-resistance factors, impairing the hyper-production of the bacterial capsule used to resist phagocytic cells
The fact that the reference strains do not reflect the whole diversity of modern clinical isolates is important and deserved to be taken into account to further study A. baumannii worldwide.
We can now use the amoeba Acanthamoeba castellanii as a new validated infection model to screen for anti-resistance factors, impairing the hyper-production of the bacterial capsule used to resist phagocytic cells