Genomics of an endemic cystic fibrosis Burkholderia multivorans strain reveals low within-patient evolution but high between-patient diversity

In many countries, Burkholderia multivorans is the most prevalent species within the Burkholderia cepacia complex (Bcc) found infecting the lungs of patients with cystic fibrosis (CF). Its positive identification is of immediate concern to the health of the patient as it is notoriously hard to eradicate using antibiotics and can cause necrosis of the lung tissues (cepacia syndrome). Infection control measures reduced the prevalence of B. cenocepacia in CF wards, but patients continue to acquire infections by B. multivorans from environmental sources. In most reported cases, the infecting strains are unique except in rare cases in which cross-infection is observed between patients. We report here an endemic strain of B. multivorans with sequence type ST-742 that has been infecting multiple patients, without evidence for cross-infection. We investigated the epidemiology and genomics of this ST-742 strain and show that it is microdiverse, as isolates between-patients exhibit numerous genomic differences, at scales that have not been observed previously when looking at evolutionary trajectories within-patients. Additionally, we found that the specific genomic background of a given strain may dictate the strategy of adaptation within the CF lung. Burkholderia multivorans is a member of the Burkholderia cepacia complex (Bcc), notorious for its pathogenicity in persons with cystic fibrosis. Epidemiological surveillance suggests that patients predominantly acquire B. multivorans from environmental sources, with rare cases of patient-to-patient transmission. Here we report on the genomic analysis of thirteen isolates from an endemic B. multivorans strain infecting four cystic fibrosis patients treated in different pediatric cystic fibrosis centers in Belgium, with no evidence of cross-infection. All isolates share an identical sequence type (ST-742) but whole genome analysis shows that they exhibit peculiar patterns of genomic diversity between patients. By combining short and long reads sequencing technologies, we highlight key differences in terms of small nucleotide polymorphisms indicative of low rates of adaptive evolution within patient, and well-defined, hundred Kbps-long segments of high enrichment in mutations between patients. In addition, we observed large structural genomic variations amongst the isolates which revealed different plasmid contents, active roles for transposase IS3 and IS5 in the deactivation of genes, and mobile prophage elements. Our study shows limited within-patient B. multivorans evolution and high between-patient strain diversity, indicating that an environmental microdiverse reservoir must be present for this endemic strain, in which active diversification is taking place. Furthermore, our analysis also reveals a set of 30 parallel adaptations across multiple patients, indicating that the specific genomic background of a given strain may dictate the route of adaptation within the cystic fibrosis lung

Enrichment, Sequencing, and Identification of DNA Bacteriophages from Fecal Samples

Research on individual viruses and phages, as well as viral populations (viromes), is greatly expanding. Phages and viromes are increasingly suspected to have numerous impacts on the ecosystem in which they reside by interacting directly or indirectly with the other organisms present in their environment. In particular, phage communities of the gut microbiota have been associated with a wide range of diseases. However, properly investigating intestinal viromes is still very challenging, both experimentally and analytically. This chapter proposes a simple and reproducible protocol to separate and enrich DNA phage particles from fecal samples, to sequence them, and finally obtain a basic but robust bioinformatic characterization and classification of the global bacteriophage community

Closed and high quality bacterial genomes of the Oligo Mouse Microbiota community

International audienceThe Oligo-Mouse-Microbiota (OMM 12) gnotobiotic murine model is an increasingly popular model for microbiota studies. However, following Illumina and PacBio sequencing, the genomes of the 12 strains could not be closed. Here, we used genomic chromosome conformation capture (Hi-C) data to reorganize, close and improve the quality of these 12 genomes

Viral Host Range database, an online tool for recording, analyzing and disseminating virus-host interactions

International audienceViruses are ubiquitous in the living world, and their ability to infect more than one host defines their host range. However, information about which virus infects which host, and about which host is infected by which virus, is not readily available. Results We developed a web-based tool called the Viral Host Range database to record, analyze and disseminate experimental host range data for viruses infecting archaea, bacteria and eukaryotes

Intestinal Bacteriophage Therapy: Looking for Optimal Efficacy

International audienceSeveral human intestinal microbiota studies suggest that bacteriophages, viruses infecting bacteria, play a role in gut homeostasis. Currently, bacteriophages are considered a tool to precisely engineer the intestinal microbiota, but they have also attracted considerable attention as a possible solution to fight against bacterial pathogens resistant to antibiotics. These two applications necessitate bacteriophages to reach and kill their bacterial target within the gut environment. Unfortunately, exploitable clinical data in this field are scarce. Here, we review the administration of bacteriophages to target intestinal bacteria in mammalian experimental models. While bacteriophage amplification in the gut was often confirmed, we found that in most studies, it had no significant impact on the load of the targeted bacteria. In particular, we observed that the outcome of bacteriophage treatments is linked to the behavior of the target bacteria toward each animal model. Treatment efficacy ranges from poor in asymptomatic intestinal carriage to high in intestinal disease. This broad range of efficacy underlines the difficulties to reach a consensus on the impact of bacteriophages in the gut and calls for deeper investigations of key parameters that influence the success of such interventions before launching clinical trials

The intestinal virome: lessons from animal models

International audienceMucosal surfaces in contact with the environment host specific microbiota. The intestinal tract harbours the most abundant and diverse bacterial and viral populations interacting with each other as well as with the host. Viruses of the microbiota are important components of this ecosystem, as shown by viral alterations associated with various pathologies. However, practical and ethical constraints limit functional studies of the virome in humans, making animal models invaluable experimental tools to understand its impact on intestinal physiology. In this review, we present the recent advances in the study of virome in animal models. We focus on the strategies used to characterise viral changes in disease models and approaches to modulate the microbiota using viruses. In reviewing the interplay between viruses, bacteria, and the animal host, we highlight the potential and limitations of these models in elucidating the role of the virome in determining human health and disease

Prophylactic Administration of a Bacteriophage Cocktail Is Safe and Effective in Reducing Salmonella enterica Serovar Typhimurium Burden in Vivo

International audienceNontyphoidal Salmonella bacteria are the causative agent of salmonellosis, which accounts for the majority of foodborne illness of bacterial etiology in humans. Here, we demonstrate the safety and efficacy of the prophylactic administration of a bacteriophage preparation termed FOP (foodborne outbreak pill), which contains lytic phages targeting Salmonella (SalmoFresh phage cocktail), Shiga toxin-producing Escherichia coli (STEC), and Listeria monocytogenes, for lowering Salmonella burdens in OMM12 gnotobiotic mice. Prophylactic administration of FOP significantly reduced the levels of Salmonella in feces and in intestinal sections compared to the levels in controls. Moreover, the overall symptoms of the disease were also considerably lessened. Dose-dependent administration of FOP showed that phage amplification reached similarly high levels in less than 48 h independent of dose. In addition, 16S rRNA gene analysis showed that FOP did not alter the intestinal microbiota of healthy OMM12 mice and reduced microbiota perturbations induced by Salmonella. FOP maintained its full potency against Salmonella in comparison to that of SalmoFresh, its Salmonella-targeting component phages alone. Altogether, the data support that preventive administration of FOP may offer a safe and effective approach for reducing the risk of foodborne infections caused by Salmonella and, potentially, other foodborne bacteria (namely, STEC and L. monocytogenes) targeted by the FOP preparation

The Spatial Heterogeneity of the Gut Limits Predation and Fosters Coexistence of Bacteria and Bacteriophages

International audienceThe ecological dynamics underlying the coexistence between antagonistic populations of bacteria and their viruses, bacteriophages (phages), in the mammalian gut microbiota remain poorly understood. We challenged a murine synthetic bacterial community with phages to study the factors allowing phages-bacteria coexistence. Coexistence was not dependent on the development of phage-resistant clones nor on the ability of phages to extend their host range. Instead, our data suggest that phage-inaccessible sites in the mucosa serve as a spatial refuge for bacteria. From there, bacteria disseminate in the gut lumen where they are predated by luminal phages fostering the presence of intestinal phage populations. The heterogeneous biogeography of microbes contributes to the long-term coexistence of phages with phage-susceptible bacteria. This observation could explain the persistence of intestinal phages in humans as well as the low efficiency of oral phage therapy against enteric pathogens in animal models and clinical trials

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)

The gut environment regulates bacterial gene expression which modulates susceptibility to bacteriophage infection

International audienceAbundance and diversity of bacteria and their viral predators, bacteriophages (phages), in the digestive tract are associated with human health. Particularly intriguing is the long-term coexistence of these two antagonistic populations. We performed genome-wide RNA sequencing on a human enteroaggregative Escherichia coli isolate to identify genes differentially expressed between in vitro conditions and in murine intestines. We experimentally demonstrated that four of these differentially expressed genes modified the interactions between E. coli and three virulent phages by either increasing or decreasing its susceptibility/resistance pattern and also by interfering with biofilm formation. Therefore, the regulation of bacterial genes expression during the colonization of the digestive tract influences the coexistence of phages and bacteria, highlighting the intricacy of tripartite relationships between phages, bacteria, and the animal host in intestinal homeostasis