Addressing the impact of phages on gut microbial ecology and functions using a synthetic bacterial community

The human gut is a complex ecosystem, harboring eukaryotic cells, bacteria and viruses. Alterations of the intestinal microbial communities are associated with an increasing number of human diseases. On the other hand, the gut microbiota protects the host against a variety of major human gastrointestinal pathogens. Bacteriophages (phages), viruses that infect bacteria, are important effectors and indicators of human health and disease by managing specific bacterial population structures and by interacting with the mucosal immune system. They are ubiquitous in nature and frequently ingested via food and drinking water. Moreover, bacteriophages are an attractive tool for microbiome engineering due to their specificity and the lack of known serious adverse effects on the host. However, most of our knowledge on phages is based on metagenomic studies and the functional role of virulent phages within the gastrointestinal microbiome remain poorly understood. To obtain functional insights on the effect of phages in the gastrointestinal microbiome and its function in health and disease, I established a model to investigate the interaction of bacteriophages and cognate host bacteria in the mammalian gut. Therefore, I isolated specific phages targeting members of a synthetic bacterial consortium, the Oligo-MM14, which consists of 14 well-characterized bacterial strains that form a stable community in gnotobiotic mice and provide colonization resistance against the human enteric pathogen Salmonella enterica serovar Typhimurium (S. Tm). First, I characterized the isolated phages in vitro with respect to plaque morphologies, genomic features, lysis behaviors and host ranges. Further, I developed methods for specific absolute quantification via qPCR for the single phages, which allowed me to track the abundance of phages and host bacteria in the OMM community in vitro and in vivo, revealing that the phages amplify at varying degrees while not disturbing the overall community composition. Furthermore, I showed, that phages lead to initial depletion of the target population in the mouse gut and thereafter coexist with the bacteria for up to a week after phage challenge. Moreover, the addition of phages targeting Escherichia coli and Enterococcus faecalis, two bacteria previously identified to mediate colonization resistance, led to a significant decrease of colonization resistance against S. Tm. This demonstrates that phages can affect microbial community functions. Infection susceptibility to S. Tm was markedly increased at an early time point after challenge with both phage cocktails but surprisingly, OMM14 mice were also susceptible to S. Tm infection 7 days after a single phage inoculation, when the targeted bacterial populations were back to pre-phage administration density. Since the abundance of the other bacteria in the gut is not affected by administration of the phage cocktails, this effect is specifically attributed to the impact of the phages on their bacterial hosts. This suggests, that phages targeting protective members of the microbiota may in general increase the risk for S. Tm infection. In summary, this work yields insights into phage-bacterial interactions in the gut and the effect of phages on fundamental microbiome functions, which will be important for evaluating the future use of phages for targeted microbiome manipulation

In vitro interaction network of a synthetic gut bacterial community

A key challenge in microbiome research is to predict the functionality of microbial communities based on community membership and (meta)-genomic data. As central microbiota functions are determined by bacterial community networks, it is important to gain insight into the principles that govern bacteria-bacteria interactions. Here, we focused on the growth and metabolic interactions of the Oligo-Mouse-Microbiota (OMM12) synthetic bacterial community, which is increasingly used as a model system in gut microbiome research. Using a bottom-up approach, we uncovered the directionality of strain-strain interactions in mono- and pairwise co-culture experiments as well as in community batch culture. Metabolic network reconstruction in combination with metabolomics analysis of bacterial culture supernatants provided insights into the metabolic potential and activity of the individual community members. Thereby, we could show that the OMM12 interaction network is shaped by both exploitative and interference competition in vitro in nutrient-rich culture media and demonstrate how community structure can be shifted by changing the nutritional environment. In particular, Enterococcus faecalis KB1 was identified as an important driver of community composition by affecting the abundance of several other consortium members in vitro. As a result, this study gives fundamental insight into key drivers and mechanistic basis of the OMM12 interaction network in vitro, which serves as a knowledge base for future mechanistic in vivo studies

Bacteriophages targeting protective commensals impair resistance against Salmonella Typhimurium infection in gnotobiotic mice.

Gut microbial communities protect the host against a variety of major human gastrointestinal pathogens. Bacteriophages (phages) are ubiquitous in nature and frequently ingested via food and drinking water. Moreover, they are an attractive tool for microbiome engineering due to the lack of known serious adverse effects on the host. However, the functional role of phages within the gastrointestinal microbiome remain poorly understood. Here, we investigated the effects of microbiota-directed phages on infection with the human enteric pathogen Salmonella enterica serovar Typhimurium (S. Tm), using a gnotobiotic mouse model (OMM14) for colonization resistance (CR). We show, that phage cocktails targeting Escherichia coli and Enterococcus faecalis acted in a strain-specific manner. They transiently reduced the population density of their respective target before establishing coexistence for up to 9 days. Infection susceptibility to S. Tm was markedly increased at an early time point after challenge with both phage cocktails. Surprisingly, OMM14 mice were also susceptible 7 days after a single phage inoculation, when the targeted bacterial populations were back to pre-phage administration density. Concluding, our work shows that phages that dynamically modulate the density of protective members of the gut microbiota can provide opportunities for invasion of bacterial pathogens, in particular at early time points after phage application. This suggests, that phages targeting protective members of the microbiota may increase the risk for Salmonella infection

Chromosome folding and prophage activation reveal specific genomic architecture for intestinal bacteria

International audienceBackground Bacteria and their viruses, bacteriophages, are the most abundant entities of the gut microbiota, a complex community of microorganisms associated with human health and disease. In this ecosystem, the interactions between these two key components are still largely unknown. In particular, the impact of the gut environment on bacteria and their associated prophages is yet to be deciphered. Results To gain insight into the activity of lysogenic bacteriophages within the context of their host genomes, we performed proximity ligation-based sequencing (Hi-C) in both in vitro and in vivo conditions on the 12 bacterial strains of the OMM 12 synthetic bacterial community stably associated within mice gut (gnotobiotic mouse line OMM 12 ). High-resolution contact maps of the chromosome 3D organization of the bacterial genomes revealed a wide diversity of architectures, differences between environments, and an overall stability over time in the gut of mice. The DNA contacts pointed at 3D signatures of prophages leading to 16 of them being predicted as functional. We also identified circularization signals and observed different 3D patterns between in vitro and in vivo conditions. Concurrent virome analysis showed that 11 of these prophages produced viral particles and that OMM 12 mice do not carry other intestinal viruses. Conclusions The precise identification by Hi-C of functional and active prophages within bacterial communities will unlock the study of interactions between bacteriophages and bacteria across conditions (healthy vs disease)

In vitro interaction network of a synthetic gut bacterial community.

A key challenge in microbiome research is to predict the functionality of microbial communities based on community membership and (meta)-genomic data. As central microbiota functions are determined by bacterial community networks, it is important to gain insight into the principles that govern bacteria-bacteria interactions. Here, we focused on the growth and metabolic interactions of the Oligo-Mouse-Microbiota (OMM12) synthetic bacterial community, which is increasingly used as a model system in gut microbiome research. Using a bottom-up approach, we uncovered the directionality of strain-strain interactions in mono- and pairwise co-culture experiments as well as in community batch culture. Metabolic network reconstruction in combination with metabolomics analysis of bacterial culture supernatants provided insights into the metabolic potential and activity of the individual community members. Thereby, we could show that the OMM12 interaction network is shaped by both exploitative and interference competition in vitro in nutrient-rich culture media and demonstrate how community structure can be shifted by changing the nutritional environment. In particular, Enterococcus faecalis KB1 was identified as an important driver of community composition by affecting the abundance of several other consortium members in vitro. As a result, this study gives fundamental insight into key drivers and mechanistic basis of the OMM12 interaction network in vitro, which serves as a knowledge base for future mechanistic in vivo studies