Mireille Ansaldi

Due to the high public health risk posed by antibioresistance, phage therapy - the use of bacteriophages as antibacterial agents - is experiencing renewed interest. As the combined administration of antibiotics and phages is a common practice in compassionate treatments, our research focuses on the effects of antibiotics on phage predation, which may be of crucial importance for phage therapeutic applications. A distinctive manifestation of phage infection in solid media is the appearance of lysis plaques, corresponding to the circular thinning of a bacterial lawn. During plaque formation, successive cycles of phage replication take place from a single point of infection and spread radially in a lawn of immobilized bacterial hosts. Many factors affect plaque size, such as the composition and the reticulation of the propagation matrix, the characteristics of the phage, but also parameters related to the physiology of the bacterial host. It has also been known for decades that some antibiotics enable phages to spread more rapidly, resulting in better bacterial eradication. This phenomenon, called Phage-Antibiotic Synergy (PAS), is evidenced by larger lysis plaques on solid media. Our previous experimental work has focused on the phage characteristics and pointed to enhanced adsorption as a key factor leading to more efficient predation. However, since sublethal antibiotic concentrations can drastically affect bacterial physiology - for instance halting cell division in the case of ciprofloxacin or ceftazidime - and since plaque formation is strongly influenced by host growth dynamics, a comprehensive model integrating both the host growth and phage infection parameters is required to investigate PAS. We characterized the epidemics of two different phages (T5 and T7) during E. coli MG1655 infection on semi-solid media in the presence of sublethal antibiotic concentrations that affect (or not) cell morphology in different ways (cell filamentation or cell bloating). We observed that in these conditions lysis plaque enlargement is linked to the host’s morphological changes. We conclude this work with a mathematical model that captures such observations and explains the increase in plaque size observed in the presence of antibiotics.

Les virus de bactéries, ou bactériophages, sont les virus les plus abondants sur Terre, et leurs hôtes sont les organismes vivants les plus répandus dans la biosphère. Ils sont retrouvés à l’état libre, sous forme de virions, et aussi très abondants dans les génomes de bactéries, à l’état de prophages. Les bactériophages sont présents dans tous les biotopes colonisés par les bactéries, eaux, sols, environnements extrêmes, microbiotes humains, animaux et végétaux, où ils participent aux échanges génétiques. Tous les facteurs qui impactent ces environnements ont donc des conséquences importantes sur la dynamique des populations de bactéries et de leurs virus.

Viral taxonomy is a challenging task due to the propensity of viruses for recombination. Recent updates from the ICTV and advancements in proteome-based clustering tools highlight the need for a unified framework to organize bacteriophages (phages) across multiscale taxonomic ranks, extending beyond genome-based clustering. Meanwhile, self-supervised large language models, trained on amino acid sequences, have proven effective in capturing the structural, functional, and evolutionary properties of proteins. Building on these advancements, we introduce HieVi, which uses embeddings from a protein language model to define a vector representation of phages and generate a hierarchical tree of phages. Using the INPHARED dataset of 24,362 complete and annotated viral genomes, we show that in HieVi, a multi-scale taxonomic ranking emerges that aligns well with current ICTV taxonomy. We propose that this method, unique in its integration of protein language models for viral taxonomy, can encode phylogenetic relationships, at least up to the family level. It therefore offers a valuable tool for biologists to discover and define new phage families while unraveling novel evolutionary connections.

A distinctive manifestation of phage infection in solid media is the appearance of lysis plaques, which correspond to the circular thinning of a bacterial lawn. During plaque formation, successive cycles of phage replication generally take place from a single point of infection and spread radially in a matrix of immobilized bacterial hosts. Many different factors affect plaque size, such as the composition and the reticulation of the propagation matrix, the characteristics of the phage, but also parameters related to the physiology of the bacterial host. Since combined administration of both antibiotics and phages is a common practice during compassionate treatments, our research focuses on the effects of antibiotics on phage predation, which can be of crucial importance for the therapeutic applications of phages. It has been previously observed that the presence of antibiotics at sublethal concentrations can affect drastically bacterial physiology, allowing phages to spread more rapidly and resulting in better bacterial eradication. Previous experimental work has focused on the phage characteristics that influence plaque size. However, as plaque formation is strongly influenced by host growth dynamics, a comprehensive model integrating both the host growth and phage infection parameters is required. In this work, we suggest that plaque enlargement is linked to morphological changes of the host that have an impact on the rate of epidemic propagation and certainly on phage diffusion into the matrix. To support this hypothesis, we characterized the growth parameters of two different phages and bacteria in semi-solid media in the presence of various antibiotics. By combining these data, we have produced a mathematical model that accounts for these observations and explains the increase in plaque size when the host morphology is affected. Significance Statement This study provides new insights into the phenomenon of Phage-Antibiotic Synergy (PAS) by demonstrating that antibiotic-induced morphological changes in bacterial hosts play a critical role in enhancing phage propagation in structured environments. By linking these morphological alterations—such as cell filamentation and bloating—to increased lysis plaque size, the research underscores the importance of host dynamics in phage therapy. The development of a mathematical model integrating both host growth and phage infection parameters offers a novel framework for understanding and optimizing phage-based treatments in the presence of antibiotics, potentially improving therapeutic outcomes.

Phages are promising tools to fight antibiotic-resistant bacteria, and as for now, phage therapy is essentially performed in combination with antibiotics. Interestingly, combined treatments including phages and a wide range of antibiotics lead to an increased bacterial killing, a phenomenon called phage-antibiotic synergy (PAS), suggesting that antibiotic-induced changes in bacterial physiology alter the dynamics of phage propagation. Using single-phage and single-cell techniques, each step of the lytic cycle of phage HK620 was studied in E. coli cultures treated with either ceftazidime, cephalexin or ciprofloxacin, three filamentation-inducing antibiotics. In the presence of sublethal doses of antibiotics, multiple stress tolerance and DNA repair pathways are triggered following activation of the SOS response. One of the most notable effects is the inhibition of bacterial division. As a result, a significant fraction of cells forms filaments that stop dividing but have higher rates of mutagenesis. Antibiotic-induced filaments become easy targets for phages due to their enlarged surface areas, as demonstrated by fluorescence microscopy and flow cytometry techniques. Adsorption, infection and lysis occur more often in filamentous cells compared to regular-sized bacteria. In addition, the reduction in bacterial numbers caused by impaired cell division may account for the faster elimination of bacteria during PAS. We developed a mathematical model to capture the interaction between sublethal doses of antibiotics and exposition to phages. This model shows that the induction of filamentation by sublethal doses of antibiotics can amplify the replication of phages and therefore yield PAS. We also use this model to study the consequences of PAS on the emergence of antibiotic resistance. A significant percentage of hyper-mutagenic filamentous bacteria are effectively killed by phages due to their increased susceptibility to infection. As a result, the addition of even a very low number of bacteriophages produced a strong reduction of the mutagenesis rate of the entire bacterial population. We confirm this prediction experimentally using reporters for bacterial DNA repair. Our work highlights the multiple benefits associated with the combination of sublethal doses of antibiotics with bacteriophages.

Bacterial genome diversity is influenced by prophages, which are viral genomes integrated into the bacterial chromosome. Most prophage genes are silent but those that are expressed can provide unexpected properties to their host. Using as a modelE.coliK-12 that carries 9 defective prophages in its genome, we aimed at highlighting the impact of genes encoded by prophages on host physiology. We focused our work on AppY, a transcriptional regulator encoded on the DLP12 prophage. By performing RNA-Seq experiments, we showed that AppY production modulates the expression of more than 200 genes. Among them, 11 were identified by ChIP-Seq as direct AppY targets. AppY directly and positively regulates several genes involved in the acid stress response including the master regulator genegadEbut alsonhaRandgadY, two genes important for biofilm formation. Moreover, AppY indirectly and negatively impacts bacterial motility by favoring the degradation of FlhDC, the master regulator of the flagella biosynthesis. As a consequence of these regulatory effects, AppY increases acid stress resistance and biofilm formation while also causing a strong defect in motility. Our research shed light on the importance to consider the genetic interactions occurring between prophages and bacteria to fully understand bacterial physiology. It also highlights how a prophage-encoded transcriptional regulator integrates in a complex manner into the host regulatory network and how it benefits its host, allowing it to cope with changing environmental conditions.

Phages are promising tools to fight antibiotic-resistant bacteria, and as for now, phage therapy is essentially performed in combination with antibiotics. Interestingly, combined treatments including phages and a wide range of antibiotics lead to an increased bacterial killing, a phenomenon called phage-antibiotic synergy (PAS), suggesting that antibiotic-induced changes in bacterial physiology alter the dynamics of phage propagation. Using single-phage and single-cell techniques, each step of the lytic cycle of phage HK620 was studied in E. coli cultures treated with either ciprofloxacin or cephalexin, two filamentation-inducing antibiotics. In the presence of sublethal doses of antibiotics, multiple stress tolerance and DNA repair pathways are triggered following activation of the SOS response. One of the most notable effects is the inhibition of bacterial division. As a result, a significant fraction of cells forms filaments that stop dividing but have higher rates of mutagenesis. Antibiotic-induced filaments become easy targets for phages due to their enlarged surface areas, as demonstrated by fluorescence microscopy and flow cytometry techniques. Adsorption, infection and lysis occur more often in filamentous cells compared to regular-sized bacteria. In addition, the reduction in bacterial numbers caused by impaired cell division may account for the faster elimination of bacteria during PAS. We developed a mathematical model to capture the interaction between sublethal doses of antibiotics and exposition to phages. This model shows that the induction of filamentation by sublethal doses of antibiotics can amplify the replication of phages and therefore yield PAS. We also use this model to study the consequences of PAS on the emergence of antibiotic resistance. A significant percentage of hyper-mutagenic filamentous bacteria are effectively killed by phages due to their increased susceptibility to infection. As a result, the addition of even a very low number of bacteriophages produced a strong reduction of the mutagenesis rate of the entire bacterial population. We confirm this prediction experimentally using reporters for bacterial DNA repair. Our work highlights the multiple benefits associated with the combination of sublethal doses of antibiotics with bacteriophages.

One of the greatest current challenges in structural biology is to study protein dynamics over a wide range of timescales in complex environments, such as the cell. Among magnetic resonances suitable for this approach, electron paramagnetic resonance spectroscopy coupled to site‐directed spin labeling (SDSL‐EPR) has emerged as a promising tool to study protein local dynamics and conformational ensembles. In this work, we exploit the sensitivity of nitroxide labels to report protein local dynamics at room temperature. We demonstrate that such studies can be performed while preserving both the integrity of the cells and the activity of the protein under investigation. Using this approach, we studied the structural dynamics of the chaperone NarJ in its natural host, Escherichia coli. We established that spin‐labeled NarJ is active inside the cell. We showed that the cellular medium affects NarJ structural dynamics in a site‐specific way, while the structural flexibility of the protein is maintained. Finally, we present and discuss data on the time‐resolved dynamics of NarJ in cellular context.

Salmonella enterica, a Gram-negative zoonotic bacterium, is mainly a food-borne pathogen and the main cause of diarrhea in humans worldwide. The main reservoirs are found in poultry farms, but they are also found in wild birds. The development of antibiotic resistance in S. enterica species raises concerns about the future of efficient therapies against this pathogen and revives the interest in bacteriophages as a useful therapy against bacterial infections. Here, we aimed to decipher and functionally annotate 10 new Salmonella phage genomes isolated in Spain in the light of phage therapy. We designed a bioinformatic pipeline using available building blocks to de novo assemble genomes and perform syntaxic annotation. We then used genome-wide analyses for taxonomic annotation enabled by vContact2 and VICTOR. We were also particularly interested in improving functional annotation using remote homologies detection and comparisons with the recently published phage-specific PHROG protein database. Finally, we searched for useful functions for phage therapy, such as systems encoded by the phage to circumvent cellular defenses with a particular focus on anti-CRISPR proteins. We, thus, were able to genetically characterize nine virulent phages and one temperate phage and identify putative functions relevant to the formulation of phage cocktails for Salmonella biocontrol.