Periodic Reporting for period 1 - DIMEvAR (Impact of medical drugs on the evolution of human microbiome function and antimicrobial resistance development)
Reporting period: 2021-04-01 to 2024-03-31
The human gastrointestinal tract harbors trillions of microbes, known as the gut microbiota. This microbial community helps modulate developmental, immunological, and metabolic functions of the human host and it plays an important role in medicinal drug response. Several works showed the bidirectional relationship between the gut microbiota and drugs. Medication modifies microbiome composition both in vitro and in the human gut. Human cohort studies revealed that commonly used drugs influence the microbiome’s metabolic function and increase the abundance of antimicrobial resistance (AMR) genes, suggesting that non-antibiotic drugs could contribute to the emergence of AMR. At the same time, microbiome-encoded enzymes can metabolise a wide range of medical drugs, participating in both beneficial and adverse effects. Considering that many drugs are used over extended periods of time to treat chronic diseases, long-term exposure to drugs may likely drive microbiome evolution. This could influence and evolve metabolic properties of gut microbes and lead to the emergence of novel AMR. This project aimed at a better understanding of the impact of medical drugs on human gut microbiome evolution and the underlying molecular mechanisms involved.
The experimental laboratory work was divided into three work packages (WP):
WP1. - “Impact of drug-induced evolution on microbiome antibiotic sensitivity and metabolism”
To investigate the extent and potential of gut microbial evolution upon the exposure of individual bacterial strains to non-antibiotic compounds, we performed a pilot in vitro evolution experiment with four representative gut bacterial strains in the presence of ten drugs that can act as cellular stressors. Throughout the drug exposure, we monitored changes in microbial drug resistance. The selection of the drugs and bacterial species was based on an extensive analysis from the existing literature and new experimental data generated for this project. Our results demonstrated that long exposure to subinhibitory concentration of non-antibiotic drugs can induce drug resistance in a diverse gut bacterial species. Additionally, we collected data of the in vitro growth parameters of ten representative human gut bacteria and determined the minimal inhibitory concentration of approximately 160 drug-bacteria combinations. The methods and the results established in this WP can be used for current and future projects on the topic in the hosting lab.
WP2- “Role of the SOS and RpoS response in microbiome evolution”
Based on previous studies on the gut bacteria sensitivity to human-targeted drugs, we hypothesized that these non-antibiotic drugs, similar to antibiotics, could act as stressors and activate two stress transcriptional programs named the SOS and RpoS responses. Both pathways can cause mutagenesis in bacterial genomes and they have been associated with microbial evolution acceleration and its consequences in the antimicrobial resistance development in bacterial pathogens. However, there is currently no understanding how these transcriptional programs function in gut bacteria, in particular in the most abundant and prevalent human commensal of Bacteroides spp. To gain insights in these molecular mechanisms, we studied the role of LexA and RpoS homologues (the stress responses regulators, respectively) in Bacteroides thetaiotaomicron as a model. Using molecular microbiology, culturomics and transcriptomics, we found that LexA may play a different role in this species. We are currently analyzing the transcriptomics data of the rpoS mutant in B. thetaiotaomicron. Furthermore, we developed various molecular biology tools for the Bacteroides spp. research, such as the mutagenesis system and the luminescent reporters, that are available to be used for current and future studies in these species.
WP 3. “Drug-induced microbiome evolution in vivo using gnotobiotic mouse models”
In this WP, we validated the in vitro results in vivo using a gnotobiotic mouse model. Briefly, we performed a competition assay of different B. thetaiotaomicron lexA mutants strains to gain insight into their role in mouse gut colonization. For strain identification, we used the newly barcoding method followed by sequencing. Currently the data is under processing.
The results of the three WPs were presented to the scientific community on various occasions including international conferences and seminars. Together the data is currently being written up for peer- review in an OA journal to be submitted in October 2024.
WP1. - “Impact of drug-induced evolution on microbiome antibiotic sensitivity and metabolism”
To investigate the extent and potential of gut microbial evolution upon the exposure of individual bacterial strains to non-antibiotic compounds, we performed a pilot in vitro evolution experiment with four representative gut bacterial strains in the presence of ten drugs that can act as cellular stressors. Throughout the drug exposure, we monitored changes in microbial drug resistance. The selection of the drugs and bacterial species was based on an extensive analysis from the existing literature and new experimental data generated for this project. Our results demonstrated that long exposure to subinhibitory concentration of non-antibiotic drugs can induce drug resistance in a diverse gut bacterial species. Additionally, we collected data of the in vitro growth parameters of ten representative human gut bacteria and determined the minimal inhibitory concentration of approximately 160 drug-bacteria combinations. The methods and the results established in this WP can be used for current and future projects on the topic in the hosting lab.
WP2- “Role of the SOS and RpoS response in microbiome evolution”
Based on previous studies on the gut bacteria sensitivity to human-targeted drugs, we hypothesized that these non-antibiotic drugs, similar to antibiotics, could act as stressors and activate two stress transcriptional programs named the SOS and RpoS responses. Both pathways can cause mutagenesis in bacterial genomes and they have been associated with microbial evolution acceleration and its consequences in the antimicrobial resistance development in bacterial pathogens. However, there is currently no understanding how these transcriptional programs function in gut bacteria, in particular in the most abundant and prevalent human commensal of Bacteroides spp. To gain insights in these molecular mechanisms, we studied the role of LexA and RpoS homologues (the stress responses regulators, respectively) in Bacteroides thetaiotaomicron as a model. Using molecular microbiology, culturomics and transcriptomics, we found that LexA may play a different role in this species. We are currently analyzing the transcriptomics data of the rpoS mutant in B. thetaiotaomicron. Furthermore, we developed various molecular biology tools for the Bacteroides spp. research, such as the mutagenesis system and the luminescent reporters, that are available to be used for current and future studies in these species.
WP 3. “Drug-induced microbiome evolution in vivo using gnotobiotic mouse models”
In this WP, we validated the in vitro results in vivo using a gnotobiotic mouse model. Briefly, we performed a competition assay of different B. thetaiotaomicron lexA mutants strains to gain insight into their role in mouse gut colonization. For strain identification, we used the newly barcoding method followed by sequencing. Currently the data is under processing.
The results of the three WPs were presented to the scientific community on various occasions including international conferences and seminars. Together the data is currently being written up for peer- review in an OA journal to be submitted in October 2024.
So far, there is a significant gap regarding the processes that drive the functional adaptation of the human gut commensals to a changing environment. To address this, we focused on unravelling the fundamental basis of the stress responses pathways in Bacteroides, the most common and prevalent gut bacteria. By combining molecular microbiology, culturomics, transcriptomics and animal models, our work revealed key molecular mechanisms that may be underlying microbiome-drug interactions. Notably, we have developed several molecular biology tools that will help to address both fundamental and applied questions concerning the biology of human Bacteroides commensals. These contributions significantly advance the microbiome field and pave the way for future research in the specific research area of drug-gut bacteria interaction.
Additionally, in line with the proposed DoA, the project had a significant impact on my future career prospects. I expanded my background in molecular microbiology by gaining expertise in animal models, microbiome-related microbiology, high-throughput bacterial genetics and transcriptomics techniques. The findings from the research carried out thanks to MSCA funding will be published in an international peer-reviewed journal, which will have a strong impact on my scientific career. During my MSCA training, I had the opportunity to establish collaborations with European scientists. These collaborations, in combination with my participation at conferences and seminars, will facilitate the creation of new international projects in the future that can be seen as highly attractive when applying for funding. Additionally, by managing the project, I developed inclusive leadership skills through various high-quality courses and improved my soft skills in areas such as grant writing and scientific presentations.
Additionally, in line with the proposed DoA, the project had a significant impact on my future career prospects. I expanded my background in molecular microbiology by gaining expertise in animal models, microbiome-related microbiology, high-throughput bacterial genetics and transcriptomics techniques. The findings from the research carried out thanks to MSCA funding will be published in an international peer-reviewed journal, which will have a strong impact on my scientific career. During my MSCA training, I had the opportunity to establish collaborations with European scientists. These collaborations, in combination with my participation at conferences and seminars, will facilitate the creation of new international projects in the future that can be seen as highly attractive when applying for funding. Additionally, by managing the project, I developed inclusive leadership skills through various high-quality courses and improved my soft skills in areas such as grant writing and scientific presentations.