Periodic Reporting for period 2 - ECOSTRAT (Interrogating the diversity of gut colonization strategies in multidrug-resistant E. coli to deduce robust competitive exclusion-based treatments)
Reporting period: 2023-07-01 to 2024-12-31
Multi-drug resistant strains of Escherichia coli producing Extended Spectrum B-lactamases (ESBL E. coli) are the leading cause of death attributable to antibiotic resistant bacteria worldwide. Although it is acknowledged that ESBL E. coli can persist asymptomatically in the intestine of healthy humans without selection by an antibiotic treatment, the fundamental principles underlying the gut colonization stability are largely unknown. Moreover, certain ESBL E. coli sequence types (ST) are over-represented in the human population worldwide and the reason for this remains unclear. Our working hypothesis is that genetic traits specific to prevalent STs favor intestinal colonization and the transmission to other hosts from this reservoir.
Our objective is to reveal the fundamental aspects of the evolutionary success of ESBL E. coli as colonizers of the mammalian intestinal tract and to design robust strategy to limit the transmission of these strains in a community of hosts.
Our first aim is to identify and compare the genetic determinants that promote intestinal colonization in prevalent ESBL E. coli STs.
Our second aim is to assess the evolutionary dynamics of ESBL E. coli during long-term intestinal colonization and to identify the main adaptive pathways, if any.
Our third aim is to understand the transmission dynamics of ESBL E. coli in cohabiting hosts and to develop innovative approaches to prevent it.
The rise of antibiotic resistance represents a major threat to public health and new approaches to fight against resistant bacteria are urgently needed. Our research will provide proof-of-principle for the evolutionary robust exclusion of ESBL E. coli from entire populations of hosts (humans or animals) without the use of antibiotics.
Our objective is to reveal the fundamental aspects of the evolutionary success of ESBL E. coli as colonizers of the mammalian intestinal tract and to design robust strategy to limit the transmission of these strains in a community of hosts.
Our first aim is to identify and compare the genetic determinants that promote intestinal colonization in prevalent ESBL E. coli STs.
Our second aim is to assess the evolutionary dynamics of ESBL E. coli during long-term intestinal colonization and to identify the main adaptive pathways, if any.
Our third aim is to understand the transmission dynamics of ESBL E. coli in cohabiting hosts and to develop innovative approaches to prevent it.
The rise of antibiotic resistance represents a major threat to public health and new approaches to fight against resistant bacteria are urgently needed. Our research will provide proof-of-principle for the evolutionary robust exclusion of ESBL E. coli from entire populations of hosts (humans or animals) without the use of antibiotics.
Aim 1. We identified five clinical isolates of ESBL E. coli from different STs able to colonize the gut of conventional mice harboring a fully protective microbiota. We were able to generate libraries of mutants in these strains using transposon insertion mutagenesis. We are currently using these libraries in mice in order to identify genes that favor colonization in the presence of a competitive gut microbiota.
Aim 2. In parallel to genetic screening, we monitored the within-host evolution of the five clinical isolates in conventional mice. The experiment has been pursued over a year. We have discovered that the isolates behave differently regarding the long-term stability of the colonization, certain strains being more stable than the others. Interestingly, our model recapitulates the colonization dynamics observed in humans with a highly variable duration of carriage spanning from few weeks to several months, for yet unknown reasons that we can now investigate in a tractable animal model. Isolated E. coli clones from colonized mice were isolated from fecal samples and sequenced on a monthly basis. We found that genetic drift (i.e. non-adaptive evolution) dominates in this selection regime. This will be further confirmed by evaluating the fitness effect of non-synonymous mutations detected in evolved clones with competition experiments.
Aim 3. Mutation profiling of isolated clones from long-term colonization experiments in different mice indicated transmission events. We observed that ESBL E. coli clones isolated from co-housed mice sometimes shared the exact same mutations demonstrating that transmission occurred in this model despite the low colonization levels, high colonization resistance from the microbiota and the presence of the same strain in the recipient mouse. Interestingly, we also observed that re-infection can occur via transmission, since mice in which E. coli was cleared were then re-infected with bacteria from another mouse. Our experimental system therefore mimics transmission within a household also documented in humans. We are currently testing if strain replacement events were caused by a set of adaptive mutations by performing controlled transmission experiments with evolved clones compared to ancestors.
In addition, we isolated virulent bacteriophages able to infect and kill the five E. coli isolates. We are assessing the impact of these phages on the population of E. coli in the gut and on the transmission dynamics of these strains. Our goal is to use these phages to accelerate the exclusion of ESBL E. coli from the gut of colonized hosts.
Aim 2. In parallel to genetic screening, we monitored the within-host evolution of the five clinical isolates in conventional mice. The experiment has been pursued over a year. We have discovered that the isolates behave differently regarding the long-term stability of the colonization, certain strains being more stable than the others. Interestingly, our model recapitulates the colonization dynamics observed in humans with a highly variable duration of carriage spanning from few weeks to several months, for yet unknown reasons that we can now investigate in a tractable animal model. Isolated E. coli clones from colonized mice were isolated from fecal samples and sequenced on a monthly basis. We found that genetic drift (i.e. non-adaptive evolution) dominates in this selection regime. This will be further confirmed by evaluating the fitness effect of non-synonymous mutations detected in evolved clones with competition experiments.
Aim 3. Mutation profiling of isolated clones from long-term colonization experiments in different mice indicated transmission events. We observed that ESBL E. coli clones isolated from co-housed mice sometimes shared the exact same mutations demonstrating that transmission occurred in this model despite the low colonization levels, high colonization resistance from the microbiota and the presence of the same strain in the recipient mouse. Interestingly, we also observed that re-infection can occur via transmission, since mice in which E. coli was cleared were then re-infected with bacteria from another mouse. Our experimental system therefore mimics transmission within a household also documented in humans. We are currently testing if strain replacement events were caused by a set of adaptive mutations by performing controlled transmission experiments with evolved clones compared to ancestors.
In addition, we isolated virulent bacteriophages able to infect and kill the five E. coli isolates. We are assessing the impact of these phages on the population of E. coli in the gut and on the transmission dynamics of these strains. Our goal is to use these phages to accelerate the exclusion of ESBL E. coli from the gut of colonized hosts.
Intestinal colonization by pathogenic bacteria like ESBL E. coli is usually studied in experimental mouse models in which the gut microbiota is suppressed in order to favor stable and reproducible colonization levels. Here we took a different approach by studying these bacteria in a more realistic context were the gut microbiota is present and compete for space and resources as it does in a healthy intestinal tract. By doing so, we were able to recapitulate key aspects of the biology of ESBL E. coli observed in human hosts in the absence of antibiotics and to study them experimentally. We found that:
- The ability to stably colonize the healthy intestinal tract differs between strains and STs of ESBL E. coli and we discovered that it is not dependent on the presence/absence of other E. coli strains in the gut.
- Genetic drift is the major evolutionary force in the healthy mouse gut.
- ESBL E. coli transmission happens among co-housed mice and competing clones can replace each other.
We expect to identify the genetic bases that could favor prevalent STs of ESBL E. coli when colonizing a healthy gut. Rapid transmission came as a surprising observation that we will capitalize on to design bacteriophage-enhanced competitive exclusion of ESBL E. coli from communities of hosts.
- The ability to stably colonize the healthy intestinal tract differs between strains and STs of ESBL E. coli and we discovered that it is not dependent on the presence/absence of other E. coli strains in the gut.
- Genetic drift is the major evolutionary force in the healthy mouse gut.
- ESBL E. coli transmission happens among co-housed mice and competing clones can replace each other.
We expect to identify the genetic bases that could favor prevalent STs of ESBL E. coli when colonizing a healthy gut. Rapid transmission came as a surprising observation that we will capitalize on to design bacteriophage-enhanced competitive exclusion of ESBL E. coli from communities of hosts.