Luisa De Sordi

Varun Khanna

Laurent Debarbieux
email: laurent.debarbieux@pasteur.fr

The gut microbiota facilitates drifts in the genetic diversity and infectivity of bacterial viruses

The intestinal microbiota and human health are intimately linked but interactions between bacteria and bacteriophages in the context of the mammalian intestine remain largely unexplored. We used comparative population genomics to study a tripartite network consisting of a virulent bacteriophage, its bacterial host, and a phage-insensitive bacterial strain both in vitro and within the murine gut. The bacteriophage adapted to infect the insensitive strain when the three partners co-existed in the gut of conventional mice, but not dixenic mice or in planktonic cultures. The molecular changes associated with modifications in the bacteriophage host spectrum included single amino-acid substitutions and an unusual homologous intra genomic recombination event within the genome of the bacteriophage. An intermediate bacterial host isolated from the murine microbiota mediated bacteriophage adaptation. Our data indicate that by offering access to new hosts, the microbiota shifts the genetic diversity of bacteriophages thereby promoting long-term persistence of bacteriophage populations.

Introduction

Viruses are the most abundant biological entities in the intestinal microbiome, with a marked predominance of bacteriophages [START_REF] Minot | Rapid evolution of the human gut virome[END_REF][START_REF] Norman | Kingdom-agnostic metagenomics and the importance of complete characterization of enteric microbial communities[END_REF][START_REF] Virgin | The virome in mammalian physiology and disease[END_REF].

Culture-independent metagenomic approaches have been successful in characterising viral communities, but these methods are largely uninformative about their relationships with the matching bacterial hosts in the gut [START_REF] Breitbart | Metagenomic analyses of an uncultured viral community from human feces[END_REF][START_REF] Minot | The human gut virome: inter-individual variation and dynamic response to diet[END_REF][START_REF] Mirzaei | Menage a trois in the human gut: interactions between host, bacteria and phages[END_REF]. This is particularly true for virulent bacteriophages: their sequences, unlike those of temperate ones, cannot be deduced from bacterial genomes and identifying their vehicles of replication in the intestinal microbiome is often unachievable. For instance, the CrAssphage is the most widespread bacteriophage in the human population, but its host is still not known [START_REF] Dutilh | A highly abundant bacteriophage discovered in the unknown sequences of human faecal metagenomes[END_REF].

All humans carry a unique set of intestinal bacteriophages [START_REF] Faith | The long-term stability of the human gut microbiota[END_REF]. However, healthy individuals share a pool of conserved bacteriophages, which are different in type, number and abundance, from those found in patients suffering from Inflammatory Bowel Disease (IBD) [START_REF] Manrique | Healthy human gut phageome[END_REF][START_REF] Norman | Disease-specific alterations in the enteric virome in inflammatory bowel disease[END_REF]. More, significant bacteriophage changes in relative abundance, richness and diversity were recently reported to be associated with the development of human autoimmune disease [START_REF] Zhao | transplantation in Clostridium difficile infection is associated with treatment outcome[END_REF]. Indeed, there is increasing evidence that bacteriophages make a substantial contribution to the eubiosis of the human microbiome, although the mechanisms are yet to be defined [START_REF] De Paepe | Bacteriophages: an underestimated role in human and animal health?[END_REF][START_REF] Manrique | The Human Gut Phage Community and Its Implications for Health and Disease[END_REF]. In that perspective, it is no wonder that viral communities homogenise between donors and patients undergoing successful faecal microbiota transplant (FMT) [START_REF] Chehoud | Transfer of Viral Communities between Human Individuals during Fecal Microbiota Transplantation[END_REF]Zuo et al., 2017). However, it remains remarkable that filtered (bacteria-free) FMT provides curative results comparable to those of traditional FMT in resolving gut infections by Clostridium difficile [START_REF] Ott | Efficacy of Sterile Fecal Filtrate Transfer for Treating Patients With Clostridium difficile Infection[END_REF]. This implies that the viral community can re-establish itself by replicating on the new hosts available and thereby contribute to reshaping the bacterial population towards a healthy condition.

Bacteriophage survival depends on the ability to perpetuate the antagonistic coevolution with bacterial hosts. This requires maximising the viral infection capacity towards each new population encountered [START_REF] Paterson | Antagonistic coevolution accelerates molecular evolution[END_REF]. When the ecological conditions are perturbed, this relationship can be destabilised with consequent increased extinction risk for the bacteriophage [START_REF] Wright | Ecological conditions determine extinction risk in co-evolving bacteria-phage populations[END_REF]. To limit this risk, viral genome plasticity generates genomic diversity to face the spatial and temporal variations typical of antagonistic interactions [START_REF] Buckling | Antagonistic coevolution between a bacterium and a bacteriophage[END_REF][START_REF] Vos | Local adaptation of bacteriophages to their bacterial hosts in soil[END_REF].

We hypothesised that the bacterial heterogeneity in the intestinal tract, would expand the genomic diversification of the coevolving viral population, promoting bacteriophage adaptation to different hosts. Using three experimental conditions (in vitro and in dixenic and conventional mice) we identified molecular events allowing one virulent bacteriophage to adapt to previously insensitive host bacteria, a phenomenon observed exclusively in the intestine.

We propose for bacteriophages to persist within this highly competitive environment they exploit the microbiota as a base to jump between different bacterial hosts.

Results

The intestinal environment promotes bacteriophage host-jump

Our experimental approach involved a tripartite network consisting of: (i) a novel virulent bacteriophage, P10, related to the myovirus Felix O1; (ii) its host, the pathogenic Escherichia coli strain LF82 and (iii) the commensal E. coli strain MG1655, to which P10 cannot bind, and is therefore non-infective (up to 10 10 pfu were tested) (Figure S1). The behaviour of these three partners was monitored for 24 days in parallel in three conditions: in vitro (planktonic cultures), in the gut of mice only colonised with strains LF82 and MG1655 (dixenic) and in the gut of conventional mice (Figure 1A and see Figure S2 for bacteriophage population densities). We screened for "host-jumps" or, in other words, a gain of function by P10; the function being the ability to infect the previously insensitive host, strain MG1655. No endogenous bacteriophage infecting either of the strains was isolated from mouse intestine from the control group.

Samples were analysed by plaque assay and sequencing of total bacteriophage populations (see below). Interestingly, bacteriophage P10 variants adapted to strain MG1655 (hereafter named ad_P10) and still infecting strain LF82 were isolated, but only from the gut of conventional mice (20% rate). No P10 variants able to jump hosts were obtained from dixenic mice or in vitro (Figure 1A and S2).

Adapted bacteriophages have extensively diversified host range

We investigated whether the ability of ad_P10 in infecting a previously insensitive host was associated with a more generalised effect on the bacteriophage host range. We isolated 16 ad_P10 bacteriophages and tested them on a collection of 105 Enterobacteriaceae strains (Figure 2 and Table S1). Almost half of the ad_P10 isolates had a reduced host range than the wild type strain (up to 50% reduction), and most others showed marginal difference; one exceptional ad_P10 variant had expanded its host range to 11 more strains than the wild-type P10 (Figure 2).

An unusual genome recombination event associated with host jump in virulent bacteriophages

To gain insight into the host jump mechanism and its variable effect on the host range of adapted bacteriophages, the genomes of two ad_P10 isolates were sequenced. In both genomes, a stretch of 216 or 280 nucleotides of the hypervariable region of the tail fiber gene gp91 underwent duplication, resulting in two interspersed repeats. The segment recombined within the hypervariable domain of the adjacent long tail fiber gene gp90, causing the deletion of either 237 or 273 nucleotides. This recombination was probably promoted by the presence of homologous flanking regions of 17 to 22 base pairs (Figure 3A). We named this event a "homologous intra genomic recombination" (hIGR) and investigated its frequency by sequencing the corresponding region in 20 randomly chosen ad_P10 isolates: it was present in 10 of the 20 isolates tested. Recombination is a major factor in genomic evolution, so we tested whether similar events had taken place in other bacteriophage genomes. A search in the database of all annotated bacteriophage tail fibers revealed that three other bacteriophages of the FelixO1-like family displayed the same tail fiber protein organisation as ad_P10: 60-to 70amino acids in the C-terminal hypervariable region were duplicated in the adjacent tail fiber-encoding gene sequence (Figure 3B). Similar hIGR events were also found, but at different positions, in the tail fiber genes of other bacteriophages (Figure 3B and Table S2).

Population genomic analyses indicates that the patterns of mutation differ between in vitro and in vivo populations

We used population genomics analyses to compare P10 populations evolving in vitro, in dixenic mice and in conventional mice. The number of single-nucleotide polymorphisms (SNPs) accumulated over 24 days (17 to 28 SNPs, at frequency higher than 1%; Table S3) did not differ significantly (p=0.67) and did not depend on the presence of strain MG1655 in the system (p=0.1) or on the host-jump event (p=0.93) (Mann-Whitney test). This suggests that the adaptation was not a consequence of a higher mutation rate in the intestinal environment than in laboratory cultures.

We found that gene gp37, which is homologous to rIIA (one of the r genes for lysis inhibition in T4 [START_REF] Paddison | The roles of the bacteriophage T4 r genes in lysis inhibition and fine-structure genetics: a new perspective[END_REF]) was a hotspot for mutations in all populations, implicating this gene in general viral fitness that was not investigated further (Figure 1B-C). Some genomic regions were differentially mutated in vitro versus in vivo. In liquid cultures, P10 persistently infected only one host, strain LF82, and none of the SNPs in these populations were highly frequent (< 20%), suggesting that they had a limited role in viral fitness. The genes most frequently affected in this condition included those encoding enzymes involved in nucleic acid metabolism, possibly fine-tuning the infection mechanism (Figure 1B-C and Table S3).

By contrast, numerous mutations were only found in bacteriophages replicating in the intestinal tract. Many were present at high frequency and were located in the genomic region predicted to encode for structural proteins. Most of the high-frequency SNPs, like the hIGR described above, were in the two tail fiber genes (gp90 and gp91).

A single point mutation in a tail fiber gene is sufficient for a host jump Tail fiber gene gp91 was only modified in ad_P10 populations (those that showed a host-jump) with a particular SNP (A G at position 55079) at a frequency of 100% (confirmed by PCR sequencing of 20 individual ad_P10 bacteriophages; Figure 1B-C and Table S3). It was absent from non-adapted populations from all three conditions tested. This variant results in a singleamino acid change at position 284 (Y284H). We tested its involvement in host specificity and constructed a recombinant P10 bacteriophage (rP10) carrying this mutation (Y284H): this single SNP was sufficient to confer the ability to infect strain MG1655 (Figure S3). However, this happened at the expense of bacteriophage replication in its original host, strain LF82 (decrease of nearly 100-fold) without conferring highly efficient replication in strain MG1655 (Figure S3). However, consecutive replication in this new host rapidly led to adaptation such that rP10 replication in strain MG1655 was ten times higher than P10 in strain LF82 (Figure S3). As few as four replication passages in vitro were sufficient for this massive gain of infectivity on the new host. Genomic sequencing showed that this improvement was accompanied by additional mutations in the genome of rP10 that likely contributed to increasing phage replication efficiency on the new host. We counted 7 additional SNPs, of which 4 were non-synonymous and one deletion (1bp) (Table S4). Two of the non-synonymous mutations were located within each of the tail fiber genes, one in the DNA polymerase gene and the others in genes of unknown function (Table S4).

Bacterial diversity within the microbiota favours viral host-jump

We investigated why host-jumps were only observed for bacteriophages replicating in the gut of conventional mice. We hypothesised that this event is mediated by the infection of one or more intermediate bacterial hosts within the intestinal microbiota leading to the observed modifications of the bacteriophage genetic diversity. We isolated a gut bacterial strain that was infected by ad_P10 at high efficacy of plating (EOP) of 1 from faeces of conventional mice not exposed to any bacterial inoculum (see methods). By contrast, wild-type P10 was only poorly able to form plaques (EOP=10 -2 ) on this strain. This supports the idea that the P10 host-jump is concomitant with an increased lytic efficacy against an alternative host in the same environment. Sequence analysis identified this bacterium as a murine E. coli strain that we named Mouse E. coli (MEc) 1. We studied the role of this putative intermediate host in the host-jump of P10 resulting in ad_P10 by using co-cultures in vitro: we mixed P10 with strains MEc1 or MG1655, with or without the presence of strain LF82, or with a mix of the three E. coli strains. After seven days of incubation, the efficacy with which the wild-type P10 population infected MEc1 increased 10-fold whenever this strain was present (Figure 4A). If strain MG1655 was also present, the host jump was observed, although it was delayed to day 24 when the preferential host of P10, LF82, was in the system (Figure 4A-B). By contrast, no host-jump was observed when P10 was mixed in vitro in the presence of LF82 and MEc1, alone or in combination, nor, as expected, in the presence of MG1655 alone (Figure 4A-B). This indicates that host-jumps become prevalent when there is a strong pressure conferring advantage on the new infecting population, such as in conditions where the density of the preferential hosts is 180 low or null.

181

Discussion

To persist in the environment, obligate parasites, such as viruses, rely on the availability of suitable hosts. Genetic mutations during viral replication can generate viral variants that can acquire the ability to infect new hosts [START_REF] Poirier | Virus population dynamics during infection[END_REF]. This is illustrated by several human pandemics caused by viruses originally incapable of infecting human cells (for example HIV, flu virus H1N1, and Ebola virus). Our work demonstrates that the abundance of potential hosts favours shifts in viral infectivity: this suggests that the diversity of intestinal microbiota enables the persistence of bacteriophages by promoting diversification due to the availability of divergent infection paths.

Numerous studies have described the behaviour of bacteria and bacteriophages in silico and in vitro, defining population dynamics, emergence of bacterial resistance and bacteriophage adaptation [START_REF] Betts | Contrasted coevolutionary dynamics between a bacterial pathogen and its bacteriophages[END_REF][START_REF] Kerr | Local migration promotes competitive restraint in a host-pathogen 'tragedy of the commons[END_REF][START_REF] Morgan | The effect of migration on local adaptation in a coevolving host-parasite system[END_REF][START_REF] Weitz | Phage-bacteria infection networks[END_REF][START_REF] Wichman | Adaptive molecular evolution for 13,000 phage generations: a possible arms race[END_REF]. However, how these phenomena apply to the homeostasis of human viral communities remains largely unknown [START_REF] Scanlan | Bacteria-Bacteriophage Coevolution in the Human Gut: Implications for Microbial Diversity and Functionality[END_REF].

The host jump that we observed in the intestinal tract had a large effect on the host spectrum of the P10 population. In the murine intestinal tract, this resulted in a host expansion from one to two or three (possibly more) bacterial strains coevolving with P10. However, the infectivity towards a set of strains that has not coevolved with the bacteriophage, was very variable and often restricted, compared to the wild-type P10. There are at least two possible explanations for these observations: either the host-jump emerged several times in different bacteriophage variants, or a single event was followed by additional mutations affecting their host range phenotype.

Environmental studies have shown that the structure of bacteriophage-host infection networks are typically nested, providing a stable community at the site of sampling [START_REF] Beckett | Coevolutionary diversification creates nested-modular structure in phage-bacteria interaction networks[END_REF][START_REF] Flores | Statistical structure of host-phage interactions[END_REF][START_REF] Koskella | Bacteria-phage coevolution as a driver of ecological and evolutionary processes in microbial communities[END_REF]. Our results confirm that this pattern is not limited to bacterial hosts isolated from the same environment; it also applies to the ability to infect other potential host strains, reflecting a more generalised, space-and timeindependent pattern of behaviour.

Host-jumps were concomitant with the accumulation of mutations in the P10 genome. These mutations largely differed from those found in vitro and were mostly in the tail fiber genes.

Amongst those, we report a previously undescribed hIGR and provide evidence for similar hIGRs in other bacteriophages, with implications for viral fitness and possibly adaptation to different hosts. Modular exchange between different bacteriophages is known to drive genomic evolution by homologous, illegitimate or site-specific recombination [START_REF] Brussow | Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion[END_REF][START_REF] Ignacio-Espinoza | Phylogenomics of T4 cyanophages: lateral gene transfer in the 'core' and origins of host genes[END_REF][START_REF] Yap | Recombination hotspots in bacteriophage T4 are dependent on replication origins[END_REF]. Here we provide a demonstration of intra-genomic recombination in bacteriophages not harbouring genes homologous to known recombinases, such as all FelixO1-like bacteriophages. Possibly, these bacteriophages exploit the host recombinase machinery, for example that associated with integrated prophages (LF82 harbours four of them [START_REF] Miquel | Complete genome sequence of Crohn's disease-associated adherent-invasive E. coli strain LF82[END_REF] ), to mediate the evolution of their own genomes which contain "ready-to-use" recombination sites. Such recombination would be a novelty in virulent bacteriophages and further investigation is required to determine whether it can be found elsewhere than in tail fiber genes.

In all adapted bacteriophages, host-jump was associated to accumulation of point mutations.

Although many localized in the tail fiber genes, one particular SNP was sufficient in itself to drive host-jump. However, it did not confer a selective infectious advantage over the wild-type bacteriophage population which continues to replicate on the available original host (optimal fitness). This provides an explanation of why it was not selected and amplified in planktonic cultures or in dixenic mice in the presence of only two bacterial strains. This is consistent with the results of previous experimental and theoretical coevolutionary studies [START_REF] Ashby | Effects of epistasis on infectivity range during host-parasite coevolution[END_REF].

In antagonistic coevolution, the arms-race dynamics between viruses and hosts lead to increased viral infectivity via a constant supply of genomic mutations. However this comes at a cost in terms of fitness, preventing widely infective variants from fixing [START_REF] Buckling | Antagonistic coevolution between a bacterium and a bacteriophage[END_REF][START_REF] Gandon | Host-parasite coevolution and patterns of adaptation across time and space[END_REF][START_REF] Poullain | The evolution of specificity in evolving and coevolving antagonistic interactions between a bacteria and its phage[END_REF] and favouring fluctuating selection on alternative host genotypes [START_REF] Gomez | Bacteria-phage antagonistic coevolution in soil[END_REF][START_REF] Hall | Host-parasite coevolutionary arms races give way to fluctuating selection[END_REF]. Our study widen this concept to infection of different host strains (host-jump) generating viral diversity directly proportional to host diversity.

The kinetics of events leading to host jump is not known. The single SNP we report might have appeared first, followed by other adaptive mutations, as we observed with the specific recombinant variant we generated, or it could have been the other way around. These events are likely to be affected by the uneven spatial distribution of bacteriophage hosts in the intestine. Indeed, their accessibility differs between sites and their density and diversity can vary providing different opportunities for bacteriophage spatial adaptation. Here, the available hosts can drive fluctuating selection of differentially infectious bacteriophage variants resulting from the accumulation of genomic mutations. A schematic representation of fitness landscape showing viral subpopulations and the available hosts is presented in Figure 4C. The extreme endpoints are the extinction of the least fit and least competitive variants, and the expansion, and probably improved persistence, of those acquiring the ability to infect previously insensitive hosts. Although we did not found significant differences in the accumulation of SNPs when comparing our three experimental conditions, our results should not suggest that the surrounding environment has no impact on genomic evolution. Accumulation of deleterious mutation leads to inevitable extinction, but this happens at a more rapid pace in vivo (in the gut), being this an "open" system where the least fit can be eliminated, compared to in vitro closed conditions (in flasks). Therefore, mutations found in populations isolated from the intestinal tract are more likely to only reflect advantageous genomic variants.

We propose that these interactions play key roles in maintaining bacteriophage and microbial diversities in the intestinal tract. In this context, disruption of such bacteriophage-bacteria interactions may play crucial role in causing dysbiosis associated to antibiotic treatments or inflammatory disease. Our work shows that the microbiota is a neglected source of evolutionary pressure: it promotes drifts in viral genomic diversity and host ranges by facilitating host jumping and providing an opportunity to persist in this ecological niche. show a map of SNPs present at a frequency higher than 5% in P10 populations after 24 days in vitro (IV), in conventional mice (CM) an in dixenic mice (DM), in the presence of one strain (1S), LF82, or two strains (2S), LF82 and MG1655. Two adapted (ad_) populations were sequenced separately, only one is shown here for clarity. These showed the highest frequency of mutations, mainly gathered in the tail fiber genes gp90 and gp91. For a full list of SNPs and positions in all populations refer to table S3. Relevant genes names are indicated along with functional clusters (NA, nucleic acids) whose positions have been deduced from genes annotation. Ad_P10 (n=16) were tested for infectivity (light blue) versus 105 Enterobacteriaceae (see Table S1) and compared to the ancestral P10 bacteriophage (purple). The shared acquired infectivity towards strain MG1655 has a very variable effect on the global host range. Only positive results are shown for clarity. S2).

METHOD DETAILS

Experimental conditions of coevolution

Bacteriophage P10, strains LF82 and MG1655 were mixed in planktonic cultures (in vitro setting) or introduced in mice (in vivo settings) as indicated in Figure 1A and as detailed below.

In vitro

Approximately 10 8 cfu of each bacterial strains grown to stationary phase were inoculated both individually and combined, in 1ml of LB supplemented with streptomycin (50 μg/μL) incubated statically at 37°C. Three days later 2 x 10 6 pfu of P10 were added to the mix. Every seven days bacterial and bacteriophage titers and infectivity towards both initial bacterial strains were obtained from 100 μL of the sample and cultures were refreshed starting from a 10-fold dilution in fresh medium. Three technical replicates of three independent experiments were performed.

In vivo

Ten groups of 1 to 5 mice for each in vivo model (dixenic and conventional mice) were force-fed strains LF82 and MG1655 (approximately 10 8 cfu of each strain per mouse, grown to stationary phase), followed, three days later, by a single bacteriophage administration (2 x 10 6 pfu per mouse). In conventional mice, streptomycin (5 g/L) was added to drinking water from three days before bacterial administration when it was lowered to 0.5 g/L and renewed weekly.

Absence of detectable endogenous E. coli and bacteriophages infecting LF82 or MG1655 was confirmed prior to bacteriophage administration by plaque assay. A control group consisted in 5 mice caged together and receiving only streptomycin. Whenever a mouse displayed an adaptation event, those sharing the same cage also carried such bacteriophage variants. Due to likeliness of coprophagy in these animals, each cage was considered to have hosted a single adaptation event.

Bacteriophage P10 was also independently mixed with strain LF82 or MG1655 in vitro and in conventional mice during 24 days and no plaques were ever recovered on lawns of MG1655.

Bacteria and bacteriophage quantification

Bacterial and bacteriophage samples from liquid cultures or freshly collected fecal samples were weighed and homogenized in PBS, serially diluted in PBS and 4 µL of each dilution were spotted onto LB or Drigalski agar supplemented with streptomycin and kanamycin or ampicillin for the quantification of strains LF82 or MG1655, respectively. Bacteriophage titers were synteny and for detection of recombination. No other bacteriophage than P10 was ever purified and sequenced from amplification on strains LF82 or MG1655.

For researching hIGR events in other bacteriophage genomes, we downloaded the annotation of all bacteriophages available on PhAnToMe (http://www.phantome.org/) (September 2016) and looked for homologous regions in all annotated tail fiber sequences within each bacteriophage genome using megablast blastn v2.2.18 (with option -F F). Genome mutations were identified using the BRESEQ Variant Report -v0.26 pipeline [START_REF] Barrick | Genome evolution and adaptation in a long-term experiment with Escherichia coli[END_REF] and the annotation of identified variants was processed using SnpEff v4.1 [START_REF] Cingolani | A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3[END_REF].

Bacteriophage recombination

An adenine to guanidine substitution at nucleotide position 55079 of P10 genome was introduced in the gp91 gene by site-directed mutagenesis using Polymerase Chain Reaction (PCR). The mutated gene was cloned as an EcoRI-XbaI fragment in plasmid pUC18 [START_REF] Norrander | Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis[END_REF] and transformed into LF82. P10 was added to a culture of LF82, carrying such plasmid (or an empty plasmid as control), in early logarithmic growth phase, at an M.O.I. of 0.1, cogrowing with strain MG1655 in equal proportions to enrich the recombinant population. A lysate recovered after 4 hrs incubation at 37°C was plated on a lawn of either strain LF82 or MG1655 to recover ad_P10 plaques. Presence of the mutation was confirmed by PCR in 20 plaques isolated on MG1655 and following three rounds of purification. No plaques on MG1655 were observed for the control group.

Bacteriophage fitness test

The infection efficiency of the recombinant P10 bacteriophage (rP10) carrying the Y284H mutation in the Gp91 protein compared to the wild type P10 bacteriophage was measured as follows. First, rP10 was replicated on the MG1655 strain during four cycles of four hours at multiplicity of infection (M.O.I.) = 0.001. The resulting bacteriophage lysate was named rP10 Experiments were independently replicated three times and means were compared by unpaired, one-tailed, t-test.

MEc1 isolation and sequencing

Frozen feacal samples from streptomycin-treated control mice (not exposed to P10 or LF82 or MG1655) were serially diluted and plated aerobically onto Drigalski plates to select for possible Enterobacteriaceae. Isolated positive colonies were replicated in liquid cultures to approximate OD600=0.2, and 6 µL of culture were spotted onto agar plates and exposed to 1 x 10 5 pfu of P10 in a 4µL double spot assay [START_REF] Saussereau | Effectiveness of bacteriophages in the sputum of cystic fibrosis patients[END_REF]. MEc1 was chosen as representative of 50 bacteriophage-sensitive colonies out of 60. Bacteriophage-bacteria co-culture was performed as detailed above in the experimental conditions of coevolution. Genomic sequencing (study

FIGURE LEGENDS Figure 1 .

 LEGENDS1 FIGURE LEGENDS

(

  C) Mutation Y284H in gene gp91 (position 55079; highlighted in orange) is the only genomic modification common to all ad_P10 clones. The figure shows a genomic map of bacteriophage P10 with enlargement of the terminal region of the Gp91 protein. The alignment of sequences from P10 and 20 isolated ad_P10 (numbered 1 to 20) is deduced by PCR amplification of the genomic region encompassing all amino-acid substitutions (highlighted in pink and yellow) found within this protein.

Figure 2 .

 2 Figure 2. Broad host range variations are associated to bacteriophage adaptation.

Figure 3 .

 3 Figure 3. Homologous intra genomic recombination (hIGR) events are widespread in

Figure 4

 4 Figure 4 Intermediate hosts from the microbiota alter bacteriophage infectivity.

  Figure 1

[

  MG]. Then, P10, rP10 and rP10 [MG] were added to exponentially growing cultures at OD600=0.2 of either strains LF82 + MG1655, LF82 + MEc1 or MEc1 + MG1655 mixed in equal proportions and at M.O.I. = 0.01. A negative control included the same amount of bacteriophages mixed to growing medium only. Co-cultures were incubated at 37°C statically during 4 hrs and bacteriophages were titered on each of the bacterial strains. Data were normalized as coefficient of multiplication compared to the initial bacteriophage inoculum.

Acknowledgments:

This work was supported by the DigestScience Foundation (Lille, France). We thank Guillaume Achaz, Mart Krupovic, Sylvain Gandon, Eduardo Rocha and Marco Vignuzzi for the critical reading of an early version of this manuscript. We also thank the Centre de Gnotobiologie and the sequencing platform of Institut Pasteur for the use of their facilities and managing help. We thank the teams of Christel Neut (Communauté d'Universités et d'Etablissements Lille Nord de France, Inserm U995, Lille, France) and Nicolas Barnich (Université Clermont Auvergne, Clermont-Ferrand, France) for sharing their E. coli collections. We are grateful to Matthieu Galtier, Marine Henry and Damien Maura for setting up the co-colonisation protocol and to Chantal Archambeau and Estelle Atse for media and buffers preparation.

DATA AND SOFTWARE AVAILABILITY

The genomic sequences of bacteriophage P10, ad_P10 and rP10 as well as bacteriophage populations have been deposited in the ENA under study accession number PRJEB18073.

The genomic sequence of strain MEc1 has been deposited in the ENA under study accession number PRJEB21810.

Author Contributions: Conceptualization, L.D.S. and L.D.; Methodology, L.D.S., V.K., L.D.; Investigation, L.D.S.; Formal Analysis, L.D.S. and V.K.; Writing, L.D.S., V.K. and L.D.; Funding Acquisition, L.D.

STAR METHODS

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Laurent Debarbieux (laurent.debarbieux@pasteur.fr)

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Microorganisms

Bacteriophage P10 (its full name is LF82_P10 -study accession number PRJEB18073) was isolated and purified from wastewater, with an enrichment technique [START_REF] Debarbieux | Bacteriophages can treat and prevent Pseudomonas aeruginosa lung infections[END_REF] using E. coli strain LF82. Adsorption of P10 to strains LF82 and MG1655 was measured by adding 2 x 10 5 pfu to exponentially growing cultures (OD600=0.3) and redrawing aliquots every 60 seconds during 10 minutes to titer the unabsorbed bacteriophage [START_REF] Hyman | Practical methods for determining phage growth parameters[END_REF]. To distinguish both E. coli strains we introduced two different antibiotic and fluorescent markers. Strain LF82S [START_REF] Galtier | Bacteriophages targeting adherent invasive Escherichia coli strains as a promising new treatment for Crohn's disease[END_REF] was disrupted at the ampC locus by insertion of a DsRed fluorescence marker gene along with a kanamycin cassette by lambda Red mediated homologous recombination to produce strain LF82 DsRed, named LF82 in this manuscript. Ampicillin resistance and GFP marker genes were P1 transduced from E. coli MG1655 λattampgfp [START_REF] Da Re | Tight modulation of Escherichia coli bacterial biofilm formation through controlled expression of adhesion factors[END_REF] into strain E. coli MG1655 resistant to streptomycin [START_REF] Da Re | Identification of commensal Escherichia coli genes involved in biofilm resistance to pathogen colonization[END_REF] producing strain MG1655 GFP (named MG1655 in this manuscript). The techniques used have been described in detail elsewhere [START_REF] Datsenko | One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[END_REF][START_REF] Thomason | E. coli genome manipulation by P1 transduction[END_REF].

Strains were routinely cultured in lysogeny broth (LB), or on LB agar or Drigalski agar (Bio-Rad, determined by spotting the same dilutions onto LB agar plates covered with a bacterial lawn of the host strain and supplemented with the appropriate antibiotics. The plates were incubated for 18 hrs at 37°C. The detection limit was 5 x 10 2 cfu or pfu per millilitre of culture or gram of faeces.

Host range of adapted bacteriophages Ad_P10 (n=16) were tested for infectivity, as described above, against different E. coli strains (n=105) listed in Table S1 and compared to the profile of wild type P10.

Analysis of genomic sequences

Sequencing of bacteriophages and bacteriophage populations (study accession number PRJEB18073) was performed using Illumina sequencing technology (Illumina Inc., San Diego, CA). Bacteriophage DNA was extracted from a sterile bacteriophage solution, using DNase and RNase pretreatments followed by a phenol-chloroform extraction, modified from Pickard [START_REF] Pickard | Preparation of bacteriophage lysates and pure DNA[END_REF]. For bacteriophage sequence analysis, the quality of Illumina reads was visualised by FastQC v0.10.1 Brabraham Bioinformatics (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Bacteriophage assembly was performed using a workflow implemented in Galaxy-Institut Pasteur using clc_assembler v4.4.2 and clc_mapper v4.4.2 (CLC Bio, Qiagen). P10 annotation was performed by the RAST v2.0 server [START_REF] Aziz | The RAST Server: rapid annotations using subsystems technology[END_REF] followed by manual curation. For synteny analysis, we aligned all the contigs of phage genomes using blastn v2.2.18 [START_REF] Altschul | Basic local alignment search tool[END_REF] (with option -F F) against the sequence of P10. Artemis Comparison Tool [START_REF] Carver | ACT: the Artemis Comparison Tool[END_REF] was used to visualize the accession number PRJEB21810) was performed using Illumina technology (Illumina Inc., San Diego, CA) and analyzed as above.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical analysis

Statistical analyses (Mann Whitney test and Student t-test) were conducted with Prism 5 software (GraphPad). p< 0.05 was considered statistically significant.

Sample size was determined by the "resource equation method" [START_REF] Mead | The Design of Experiments: Statistical Principles for Practical Applications[END_REF].