Margaux Allain

Anne Claire Mahérault

Benoit Gachet

Caroline Martinez

Bénédicte Condamine

Mélanie Magnan

Isabelle Kempf

Erick Denamur

Luce Landraud
email: luce.landraud@aphp.fr

Dissemination of IncI plasmid encoding blaCTX-M-1 is not hampered by its fitness cost in the pig's gut

Keywords: CTX-M, Fitness cost, IncI plasmid, ESBL, RNA-Seq

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INTRODUCTION

Since the 2000s, a global epidemic of Extended Spectrum Beta-Lactamase (ESBL)producing Enterobacterales has emerged, linked to the dissemination of successful blaCTX-Mcarrying clones [START_REF] Murray | Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis[END_REF]. The CTX-M enzymes are mostly plasmid encoded and E. coli is the major host [START_REF] Rozwandowicz | Plasmids carrying antimicrobial resistance genes in Enterobacteriaceae[END_REF].

Among major multiresistant plasmids in Enterobacterales, IncF, IncI, IncC, IncN, and IncL plasmid families are the most commonly reported. The I-complex plasmids contain incompatibility groups I, K, B, and Z. IncI, or MOBP according to relaxase typing, is a group of low copy-number, narrow-host-range, conjugative plasmids, which vary in size from 50 to 250 kb [START_REF] Rozwandowicz | Plasmids carrying antimicrobial resistance genes in Enterobacteriaceae[END_REF][START_REF] Foley | Incompatibility Group I1 (IncI1) Plasmids: Their Genetics, Biology, and Public Health Relevance[END_REF]. Some indistinguishable IncI1 and IncIplasmids have been isolated from multiple, unrelated high-risk clones, identified in different periods and bacterial species, in distant geographical areas, defined as "epidemic" plasmids [START_REF] Guyomard-Rabenirina | Antimicrobial Resistance in Wildlife in Guadeloupe (French West Indies): Distribution of a Single blaCTX-M-1/IncI1/ST3 Plasmid Among Humans and Wild Animals[END_REF][START_REF] Petrin | Identification and characterization of a spreadable IncI1 plasmid harbouring a blaCTX-M-15 gene in an Italian human isolate of Salmonella serovar Napoli[END_REF][START_REF] Irrgang | Diversity of CTX-M-1-producing E. coli from German food samples and genetic diversity of the blaCTX-M-1 region on IncI1 ST3 plasmids[END_REF][START_REF] Carattoli | Resistance plasmid families in Enterobacteriaceae[END_REF]. IncI pST3 plasmid is the most commonly reported blaCTX-M-1-IncI1 plasmid in Europe, described in pathogenic human E. coli isolates, and in E. coli, and Salmonella spp. from food-producing animals [START_REF] Carattoli | Contemporary IncI1 plasmids involved in the transmission and spread of antimicrobial resistance in Enterobacteriaceae[END_REF]. Moreover, Lucas et al. recently showed that resistance to Extended-Spectrum Cephalosporins (ESC) in French pigs' E. coli isolates is mainly carried by highly similar blaCTX-M-1-IncI1/ST3 plasmids [START_REF] Lucas | Characterization of plasmids harboring blaCTX-M genes in Escherichia coli from French pigs[END_REF]. IncI plasmids have been recognized in clinically relevant bacteria from human, animal, and environmental sources, and as major vehicles for the dissemination of ESBL, notably CTX-M-15 and CTX-M-1 enzymes [START_REF] Rozwandowicz | Plasmids carrying antimicrobial resistance genes in Enterobacteriaceae[END_REF][START_REF] Carattoli | Contemporary IncI1 plasmids involved in the transmission and spread of antimicrobial resistance in Enterobacteriaceae[END_REF].

Previous studies using a series of experiments that included pigs inoculated with E. coli harboring blaCTX-M-1-encoding IncI plasmid showed the maintenance of the plasmid in its E. coli host, in the pig's gut. Moreover, the loss of the plasmid was a rare event in gut microbiota during these longitudinal studies of in vivo carriage in pigs [START_REF] Mourand | Rare Spontaneous Loss of Multiresistance Gene Carrying IncI/ST12 Plasmid in Escherichia coli in Pig Microbiota[END_REF]. In these models, the transfer of the blaCTX-M-1-encoding IncI plasmid between different E. coli strains occurred and underlines the dissemination capacity of the ESC resistance, independently of the exposure to antibiotic drugs [START_REF] Fleury | Impact of Ceftiofur Injection on Gut Microbiota and Escherichia coli Resistance in Pigs[END_REF][START_REF] Mourand | Escherichia coli Probiotic Strain ED1a in Pigs Has a Limited Impact on the Gut Carriage of Extended-Spectrum-β-Lactamase-Producing E. coli[END_REF]. Finally, understanding the factors of IncI1 plasmids success better is important, as they are major contributors to the dissemination of ESBL genes especially in the animal reservoir.

The arrival of a plasmid in a new bacterium produces a highly variable fitness cost depending on the plasmid-bacterium association [START_REF] Pietsch | Genome-Based Analyses of Fitness Effects and Compensatory Changes Associated with Acquisition of blaCMY-, blaCTX-M-, and blaOXA-48/VIM-1-Containing Plasmids in Escherichia coli[END_REF][START_REF] Wu | Fitness Advantage of mcr-1-Bearing IncI2 and IncX4 Plasmids in Vitro[END_REF][START_REF] Gama | Host dependent maintenance of a blaNDM-1-encoding plasmid in clinical Escherichia coli isolates[END_REF]. Plasmid persistence and dissemination in bacterial population, under conditions that do not select for plasmid-encoded genes, are influenced by the fitness effects associated with plasmid carriage [START_REF] San Millan | Fitness Costs of Plasmids: a Limit to Plasmid Transmission[END_REF][START_REF] Valle | Variability of plasmid fitness effects contributes to plasmid persistence in bacterial communities[END_REF]. Previous works have shown that compensatory adaptation frequently ameliorates the fitness of costly plasmids [START_REF] Mahérault | Advantage of the F2:A1:B-IncF Pandemic Plasmid over IncC Plasmids in In Vitro Acquisition and Evolution of bla CTX-M Gene-Bearing Plasmids in Escherichia coli[END_REF][START_REF] Harrison | Plasmid-mediated horizontal gene transfer is a coevolutionary process[END_REF][START_REF] Loftie-Eaton | Compensatory mutations improve general permissiveness to antibiotic resistance plasmids[END_REF]. This compensatory evolution may play a key role in the dynamics of the spread and evolution of antibiotic resistance plasmids [START_REF] Pietsch | Genome-Based Analyses of Fitness Effects and Compensatory Changes Associated with Acquisition of blaCMY-, blaCTX-M-, and blaOXA-48/VIM-1-Containing Plasmids in Escherichia coli[END_REF][START_REF] Loftie-Eaton | Evolutionary Paths That Expand Plasmid Host-Range: Implications for Spread of Antibiotic Resistance[END_REF][START_REF] Zwanzig | Mobile Compensatory Mutations Promote Plasmid Survival[END_REF][START_REF] Hall | Plasmid fitness costs are caused by specific genetic conflicts enabling resolution by compensatory mutation[END_REF]. However, most studies on the evolutionary dynamics of plasmid-bacterium associations have been performed in laboratory-based culture systems.

We took the unique opportunity of the previous studies performed in pigs for probiotic testing [START_REF] Mourand | Escherichia coli Probiotic Strain ED1a in Pigs Has a Limited Impact on the Gut Carriage of Extended-Spectrum-β-Lactamase-Producing E. coli[END_REF], to evaluate the fitness cost of IncI pST3 plasmid on bacterial isolates collected during an in vivo longitudinal study of carriage of a CTX-M-1 producing E. coli B1 commensal strain in pigs. We also investigated the impact of IncI pST3 plasmid on the bacterial host using whole-genome sequencing (WGS) and transcriptome sequencing and analysis to determine the effect of plasmid carriage on global gene expression.

RESULTS

Characterization of ancestral and in vivo evolved UB12 strains. According to the wholegenome sequencing results, E. coli UB12-RC recipient strain belongs to O100:H40 serotype, B1 phylogroup, and ST2520, according to the Warwick multilocus sequence typing (MLST) scheme. This strain harbors a mutation in rpoB after selection in Mueller-Hinton medium (MH) containing 250 mg/liter rifampicin [START_REF] Mourand | Rare Spontaneous Loss of Multiresistance Gene Carrying IncI/ST12 Plasmid in Escherichia coli in Pig Microbiota[END_REF], and contains two plasmids: one P1-like phage-plasmid (IncY, 98119 bp) [START_REF] Pfeifer | Bacteria have numerous distinctive groups of phage-plasmids with conserved phage and variable plasmid gene repertoires[END_REF], and one small mobilizable plasmid (Col156 plasmid, 5223 bp) (Table 1). In addition to the IncY and Col156 resident plasmids, the UB12-TC transconjugant harbors a blaCTX-M-1-IncI plasmid (108699 bp), and one colicinogenic plasmid, belonging to the ColE1 group (ColRNAI plasmid, 6724 bp), which disseminated from the M63-DN donor strain. The blaCTX-M-1-IncI plasmid is of plasmid multilocus sequence type 3 (pST3) of the IncI1 family and presents 99% identity and 72 % coverage to R64 plasmid, a prototype of the IncI1 group (ascension N° AP005147) [START_REF] Carattoli | Contemporary IncI1 plasmids involved in the transmission and spread of antimicrobial resistance in Enterobacteriaceae[END_REF]. Moreover, the core genome of IncI ST3 plasmid presents with other blaCTX-M-1-IncI1/ST3 plasmid previously described 100% identity and 99% coverage (ascension n° KJ484635) [START_REF] Wang | Nucleotide sequences of 16 transmissible plasmids identified in nine multidrug-resistant Escherichia coli isolates expressing an ESBL phenotype isolated from food-producing animals and healthy humans[END_REF]. The blaCTX-M-1 gene is associated with an ISEcp1 mobile element found at the 5' end of the gene, and the ISEcp1-blaCTX-M-1 transposition unit is inserted in the shufflon region of the plasmid. Other resistance genes carried in the blaCTX-M-1-IncI/ST3 plasmid are dfrA17, sul2, and aadA5 (Table 1).

Three cefotaxime-susceptible and rifampicin-resistant isolates, collected at days 7, 18, and 20, during longitudinal carriage of the UB12-TC strain in the pigs, have spontaneously lost the IncI plasmid in vivo and harbor Col156, IncY, and ColRNAI plasmids (referenced as UB12-TCpIncI) (Table 1).

To identify mutations and rearrangements that occur during the carriage of UB12-TC strains in the pig's gut, we used the open-source Breseq pipeline [START_REF] Deatherage | Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq[END_REF] and the ancestral UB12-TC strain as reference genome. In the first step, we excluded all single nucleotide polymorphisms (SNP), insertions or deletions detected comparing mapping reads of the UB12-TC ancestral strain to the assembly of itself. In the second step, we determined genetic differences occurring between the UB12-RC recipient strain, seven UB12-TC isolates, collected in stools between day 7 and 45 after oral inoculation of pigs, i.e. in vivo evolved (UB12-TC ive ), and the cefotaxime-susceptible and rifampicin-resistant isolates (UB12-TCpIncI) (Table 2). All transconjugants showed one intergenic point mutation, upstream of the nadA gene, except two (Table 2). In chromosomal DNA, we detected seven other events between the UB12-RC recipient strain, the UB12-TC strain and six UB12-TC ive transconjugants: one intergenic point mutation between the ygeH and ygeG genes, one synonymous point mutation in the kdsB gene, and five nonsynonymous point mutations in the ecpC, ydeE, ttdB, thiH genes, and in the hlyE pseudogene (Table 2). In total, one to three point mutations per strain were detected. In plasmid DNA, a common rearrangement was detected in the shufflon region of the IncI plasmid in three transconjugants. None of other plasmids showed any modification.

No fitness cost decrease of UB12 strains during digestive carriage in pigs. To evaluate the IncI plasmid cost on the bacterial fitness, we compared the maximum growth rates (MGRs) of the E. coli M63-DN donor strain, the UB12-RC recipient strain and the ancestral UB12-TC transconjugant. The MGR of M63-DN donor strain (3.55 div h-1) was significantly higher than the MGR of UB12-RC strain (3.38 div h-1) (P=0.02). The difference between the MGR of UB12-RC strain and the MGR of UB12-TC transconjugant (3.28 div h-1) was nonsignificant (P=0.09) (Fig 1).

To evaluate the adaptation of IncI plasmid in the E. coli UB12 strain, we compared the MGRs of the UB12-TC strain, the M63-DN donor, and ten clones (UB12-TC ive and UB12-TCpIncI) isolated from the stools of the pigs, between day 7 and day 45, during an in vivo longitudinal study of carriage of the UB12-TC transconjugant [START_REF] Fleury | Impact of Ceftiofur Injection on Gut Microbiota and Escherichia coli Resistance in Pigs[END_REF]. Among these clones, three UB12-TC transconjugants had spontaneously lost the IncI plasmid (UB12-TCpIncI). As no significant fitness cost variation was detected between the MGRs of the evolved UB12-TC ive transconjugants on the one hand, and between the MGRs of the UB12-TCpIncI transconjugants on the other hand, further statistical analyses were performed after pooling the MGRs of the clones from each group. The difference between the MGRs of UB12-TC transconjugants (3.28 div h-1) and the MGRs of UB12-TC ive evolved transconjugants (3.23 div h-1) was nonsignificant (P=0.08), as the difference between the MGRs of UB12-RC clones and the MGRs of the UB12-TC ive transconjugants (P=0.07). Interestingly, the differences between the MGRs of UB12-TCpIncI (3.40 div h-1) and the MGRs of UB12-TC or UB12-TC ive clones were significant (P=0.004 and P=1.61 10-7 respectively) (Fig 1).

No fitness cost decrease during in vitro experiment evolution of UB12-TC. To assess if the absence of decrease in in vivo fitness cost could be reproduced in vitro in a constant welldefined environment, we conducted an in vitro evolution experiment in LB during 30 days (300 generations in total) and evaluated the impact of the IncI plasmid on the bacterial host after evolution. We measured the MGRs of five independent ancestral transconjugants (UB12-TC), and of five independent clones for each lineage after in vitro evolution (UB12-TC ite ), compared to the MGRs of five plasmid-free ancestral and evolved control strains (UB12-RC and UB12-RC ite ). First, no significant fitness cost variation was detected between the MGRs of the evolved clones belonging to the same lineage. Further statistical analyses were performed after pooling the MGRs of the clones from each lineage. The differences between the MGRs of UB12-TC (3.49 div h-1) and UB12-TC ite (3.35 div h-1) clones, and the MGRs of plasmid-free control strains UB12-RC and UB12-RC ite (3.49 div h-1 and 3.42 div h-1, respectively) were nonsignificant (Fig 2). Although no significant difference was found between ancestral and evolved strains, the MGRs of UB12-TC decreased after the evolution experiment (3.49 div h-1 versus 3.35 div h-1 for UB12-TC and UB12-TC ite , respectively). Of note, there was a trend (P= 0.054) for increased cost fitness in evolved strains ( 1). Eight NI (72%) harbor more than two plasmids, while two NI (NI-3 and NI-11) harbor the IncI plasmid alone (Table 1).

The core genomic divergence, estimated by the number of core genome SNPs between strains, is variable and dependent on the sequence type and phylogroup of strains, from 18513 to 77175 (between E. coli B1 ST2520 UB12-TC strain, and respectively, E. coli B1 ST2540 NI-4 clone and E. coli B2 ST95 NI-9 clone) (Fig 3A, Table S1).

Variable cost of IncI plasmid in transconjugants E. coli NI from the pig's gut. To evaluate the impact of the IncI pST3 plasmid on new bacterial host, we proposed to compare the MGRs of the UB12-TC strain, the M63-DN donor, and eleven cefotaxime-resistant and rifampicinsusceptible NI from the stools of the pigs, between day 4 and day 22. These isolates all harbored the IncI pST3plasmid that have disseminated between different E. coli in the pigs' microbiota after inoculation (Table 1). The MGRs of four NI were not significantly different from that of the donor strain M63-DN, while seven NI showed an MGR significantly lower than those of the M63-DN strain (P between 2.7 10 -5 and 0.015) (Fig 3B).

In order to determine the relative abundance of the newly formed transconjugants in the pig's gut, we compared the titer of cefotaxime-resistant and rifampicin-susceptible E. coli clones to all cefotaxime-resistant E. coli population, on the days during which each NI was collected. We observed a variable relative abundance, ranging from 10% to 30% for NI-1, NI- Transcriptomic analysis of UB12 strains. We compared the transcriptome of the ancestral UB12-TC transconjugant with the UB12-RC recipient strain to determine the impact of plasmid acquisition on host gene expression. We also compared the transcriptome of two UB12-TC ive transconjugants, isolated between day 28 and 30 in the pig's gut, and of one UB12-TCpIncI transconjugant with the UB12-RC recipient and the ancestral UB12-TC strains to determine the impact of the IncI pST3plasmid maintenance on the bacterial host in the pig's gut. We combined the results from differential gene expression analysis of two independent biological replicates for each clone, except for UB12-TC.

We observed a low significant transcriptional response of the acquisition of plasmids into UB12-TC, with ten significantly (> 2-log change) differentially expressed genes (adjusted P value (Padj) < 0.05) between UB12-RC and UB12-TC strains. Six genes were down-regulated and four genes were up-regulated in UB12-TC compared to UB12-RC. Functions affected by plasmids acquisition included genes involved in mobility and chemotaxis (down-regulation of flgB gene; up-regulation of motA and cheA genes), in stress response (down-regulation of cyuP and ydcI genes; up-regulation of gnsA gene) or genes encoding for phage proteins (downregulation of three chromosomal genes; up-regulation of one IncY plasmid-gene) (Table 3).

No significant change in chromosomal and plasmid gene expression was observed in the UB12-TC ive clones, compared to UB12-TC before carriage in the pig's gut.

However, we detected a transcriptional response between the UB12-RC and UB12-TC ive strains, with a total number of significantly (> 2-log change) differentially expressed genes (Padj < 0.05) ranging from 37 to 39. Among these differentially expressed genes observed between UB12-RC and both UB12-TC ive clones, most of down-regulated chromosomal genes encoded for phage proteins (12 genes). Other down-regulated genes were fimA gene, encoding for type 1 fimbriae major subunit, and two genes involved in metabolism (lpp and garP genes, significantly detected down-regulated only for one of UB12-TC ive clones) (Table 3). In one or both of UB12-TC ive clones, we detected up-regulated genes involved in various stress response, such as ariR (oxidative stress), bhsA (multiple stress resistance), yhcN (hydrogene peroxide stress), cspA (temperature response), and soxS (superoxide stress mastor regulator), and genes encoding for transporters in various metabolic pathways (srlA, for sorbitol; narK or napD, for nitrate/nitrite; and malEFK, for maltose). Other functional classes of genes detected as significantly up-regulated were those involved in biofilm (crfC and pdeH genes), in mobility (motAB and fliC genes), and in chemotaxis (cheB and tap genes) (Table 3).

All genes involved in mobility and chemotaxis, differentially expressed between UB12-TC ive and UB12-RC strains, were also up-regulated in the UB12-TCpIncI clone. Moreover, other genes included in the chemotaxis system (tar, cheZYR, and tgr genes) and the flagella systems (fliS, flgN, flgKL, and flgM genes) were up-regulated between UB12-TCpIncI and UB12-RC strains. Interestingly, fliA, the other major regulator of the flagella systems, appeared significantly differentially expressed (down-regulation) between UB12-TC and UB12-TCpIncI clones.

Using the replicase repA gene for comparison, we showed that the transcription of IncI plasmid genes was not different between UB12-TC and UB12-TC ive clones (Table S2). We observed that most of the IncI plasmid genes (111 genes, 89.5%) were expressed at low level or not expressed compared to repA transcription. Only 13 of predicted CDS (10%) were transcribed at level between 4-and 35-fold higher than repA. The most expressed plasmidic gene was annotated as a conserved protein of unknown function, located just upstream of oriT and the nikA gene (encoding for a Ribbon-helix-helix relaxosome protein), which was transcribed at level 5-fold higher than repA. Two other loci in plasmid core regions were transcribed at level between 5-and 16-fold higher than repA, the excAB genes and an ORF located just upstream, and the parBM genes. Two accessory regions of the IncI plasmid encoding resistance genes were also transcribed at high level, the ISEcp-1-blaCTX-M-1 cassette and three genes (glmM, sul2, and drfA) located on an ISCR2 mobile genetic element (Table S2).

We observed that the IncY plasmid genes were also expressed at low level. Only 10% (11 genes) of the coding sequences were transcribed at higher level than repA, and all annotated as conserved proteins of unknown function (Table S2).

Swimming motility of strains. To confirm the transcriptomic data on genes involved in mobility, we tested the swimming ability of all the strains used in our study in semi-solid LB agar tubes. We observed that the swimming ability decreased in UB12-TC strains compared to UB12-RC and M63-DN strains. While the ancestral UB12-TC strain presented a comparable swimming motility phenotype to all UB12-TC ive clones, UB12-TCpIncI clones presented an increase in the ability to swim in semi-solid LB agar tubes. Of note, we observed various phenotypes, from non-motile to motile, for all eleven NI pig's clones, which all harbored IncI plasmid (Table 1).

DISCUSSION

Multiresistance plasmids are widely distributed across bacteria and one of the main drivers of antibiotic resistance spread. The success of a plasmid-borne resistance is constrained by the fitness cost that the multiresistance plasmid imposes on the bacterial cell. It is not clear whether epidemic blaCTX-M-1-IncI1 plasmids, one of the most common plasmid type in ESBL producers of animal origin [START_REF] Rozwandowicz | Plasmids carrying antimicrobial resistance genes in Enterobacteriaceae[END_REF][START_REF] Carattoli | Contemporary IncI1 plasmids involved in the transmission and spread of antimicrobial resistance in Enterobacteriaceae[END_REF], conferred a fitness cost on the host bacterium. Previous studies have shown that the blaCTX-M-1-IncI1 plasmid imposes no or negligible fitness costs on its E. coli host in vitro [START_REF] Johnson | In Vivo Transmission of an IncA/C Plasmid in Escherichia coli Depends on Tetracycline Concentration, and Acquisition of the Plasmid Results in a Variable Cost of Fitness[END_REF] and persists without antimicrobial usage [START_REF] Fischer | The IncI1 plasmid carrying the blaCTX-M-1 gene persists in in vitro culture of a Escherichia coli strain from broilers[END_REF]. On the other hand, using in vitro competition experiments, Freire Martín et al. showed that the IncI1 plasmid-cured isolate outcompeted the original plasmid-carrying isolate, or plasmid-complemented strains, suggesting a fitness burden was imposed by carriage of this plasmid [START_REF] Freire Martín | Curing vector for IncI1 plasmids and its use to provide evidence for a metabolic burden of IncI1 CTX-M-1 plasmid pIFM3791 on Klebsiella pneumoniae[END_REF]. These studies were performed using in vitro models and/or laboratory-adapted strains with limited clinical relevance. Here, we investigated the cost of a blaCTX-M-1-IncI1/ST3 epidemic plasmid in a commensal E. coli animal strain, before and after oral inoculation of pigs. In our model, blaCTX-M-1-IncI1/ST3 plasmid imposed no measurable burden on the recipient strain after conjugation and we observed no modification of the bacterial growth rate of the blaCTX-M-1-IncI1 plasmidcarrying strain after the longitudinal carriage in the pig's gut. However, a significant difference of bacterial growth rate was found between the blaCTX-M-1-IncI1/ST3 plasmid-carrying strain and the transconjugants that had spontaneously lost the IncI1 plasmid during in vivo carriage, suggesting a small fitness burden exists. The loss of IncI plasmid is a rare event [START_REF] Mourand | Rare Spontaneous Loss of Multiresistance Gene Carrying IncI/ST12 Plasmid in Escherichia coli in Pig Microbiota[END_REF], as expected because of the presence of addiction systems on these large conjugative multiresistance plasmids. As a whole, our observations could suggest that blaCTX-M-1-IncI1 epidemic plasmids do not induce a sufficient fitness cost on its host bacterium to drive their extinction in gut microbiota, in the absence of antibiotics.

After ESBL-producer E. coli inoculation, blaCTX-M-1-IncI1/ST3 plasmid disseminated rapidly to natural isolates in the pig's gut, highlighting the usually very efficient conjugation system of IncI plasmids [START_REF] Carattoli | Contemporary IncI1 plasmids involved in the transmission and spread of antimicrobial resistance in Enterobacteriaceae[END_REF][START_REF] Knudsen | Transfer of a blaCTX-M-1-carrying plasmid between different Escherichia coli strains within the human gut explored by whole genome sequencing analyses[END_REF]. As reference the bacterial growth rate of the blaCTX-M-1-IncI donor strain, the comparative growth rate of the eleven newly formed transconjugants in the pig's gut appeared extremely variable, related to variable fitness of these IncI plasmid-carrying new spontaneous transconjugants. Although it is not possible to determine the competitiveness of the host cells before IncI plasmid acquisition due to the experimental design of our study, this suggests that the fitness cost could not act as a strong barrier to transmission of the blaCTX-M-1-IncI1 epidemic plasmid, among natural populations of E. coli in a relevant environmental niche such as the gut. Interestingly, the relative abundance of some of the newly formed transconjugants was greater than 50%, suggesting that these clones represented dominant ESCresistant E. coli strains in the pig's gut at this time.

Previous studies have reported in vivo transfers of multidrug-resistant plasmids despite competition from fecal flora, and a high risk of rapid increase of transconjugants becoming the dominant population under antibiotic treatment [START_REF] Knudsen | Transfer of a blaCTX-M-1-carrying plasmid between different Escherichia coli strains within the human gut explored by whole genome sequencing analyses[END_REF][START_REF] Hadziabdic | In Vivo Transfer and Microevolution of Avian Native IncA/C 2 bla NDM-1 -Carrying Plasmid pRH-1238 during a Broiler Chicken Infection Study[END_REF][START_REF] Duval-Iflah | R-plasmic transfer from Serratia liquefaciens to Escherichia coli in vitro and in vivo in the digestive tract of gnotobiotic mice associated with human fecal flora[END_REF]. Some studies have shown that antibiotics exposure increased conjugative transfer of blaCTX-M-1-IncI plasmids [START_REF] Liu | Antibiotic-Induced, Increased Conjugative Transfer Is Common to Diverse Naturally Occurring ESBL Plasmids in Escherichia coli[END_REF] and it has been suggested that the transfer rate of the IncI plasmid may be underestimated in in vitro study models [START_REF] Fischer | Competition between Escherichia coli Populations with and without Plasmids Carrying a Gene Encoding Extended-Spectrum Beta-Lactamase in the Broiler Chicken Gut[END_REF]. The high predominance and worldwide spread of some IncI1 plasmids, especially among animal ESBL-producer E. coli, could be explained by a low fitness cost imposed on the new recipient strain, combined with a high conjugation efficiency and potentiated by a high exposure of animals to antibiotics in agricultural settings.

A complex plasmid-chromosome cross-talk is involved in plasmid domestication into their host cells and often appears to be dependent of the plasmid-host pairing studied. Among these plasmid-host adaptation effects associated with a potential fitness benefit, only changes in chromosomal gene expression have been observed in some plasmid-host evolutions [START_REF] Buckner | Clinically Relevant Plasmid-Host Interactions Indicate that Transcriptional and Not Genomic Modifications Ameliorate Fitness Costs of Klebsiella pneumoniae Carbapenemase-Carrying Plasmids[END_REF].

These modifications in the expression of chromosomal genes for the bacterium adaptation to the acquisition of the plasmid appeared highly variable both in the degree of alteration and in the range of cellular functions that are affected [START_REF] Buckner | Clinically Relevant Plasmid-Host Interactions Indicate that Transcriptional and Not Genomic Modifications Ameliorate Fitness Costs of Klebsiella pneumoniae Carbapenemase-Carrying Plasmids[END_REF][START_REF] Harr | Gene expression analysis indicates extensive genotypespecific crosstalk between the conjugative F-plasmid and the E. coli chromosome[END_REF][START_REF] Dunn | Limited and Strain-Specific Transcriptional and Growth Responses to Acquisition of a Multidrug Resistance Plasmid in Genetically Diverse Escherichia coli Lineages[END_REF][START_REF] Jousset | Transcriptional Landscape of a blaKPC-2 Plasmid and Response to Imipenem Exposure in Escherichia coli TOP10[END_REF][START_REF] Lang | Transcriptome Mapping of pAR060302, a bla CMY-2 -Positive Broad-Host-Range IncA/C Plasmid[END_REF][START_REF] Long | Phenotypical profile and global transcriptomic profile of Hypervirulent Klebsiella pneumoniae due to carbapenemase-encoding plasmid acquisition[END_REF]. However, chromosome-encoded functions most commonly targeted by plasmid carriage include respiration, signaling, energy production, various metabolism pathways, and virulence factors, especially adhesion-related functions involved in biofilm formation and mobility [START_REF] Billane | Why do plasmids manipulate the expression of bacterial phenotypes?[END_REF][START_REF] Vial | Plasmid-chromosome cross-talks[END_REF]. To analyse the impact of the blaCTX-M-1-IncI1/ST3 plasmid on the commensal E. coli host, we performed WGS and RNAsequencing of the ancestral strains on the one hand, and UB12 isolates collected from pigs' feces on the other hand. In our study, a very small number of SNPs in chromosomal and plasmid coding sequences were detected in the UB12 isolates, without clear convergence. In addition, RNA-sequencing revealed that only the expression of a small set of chromosomal genes was altered in the host cell after acquisition of the IncI ST3 plasmid, involving chromosomal genes encoding prophages proteins and genes found in different processes such as metabolism, stress response, and cell mobility. We showed that the transcriptional expression of chemotaxis and flagellar genes change between IncI plasmid-carrying and plasmid-free UB12 isolates collected after oral inoculation of pigs, in accordance with the modification in the swimming capacities of these clones. None of mutations observed in our data were associated with change in gene expression, suggesting that we observed a physiological response. It is interesting to note that chromosomal genes implicated in cell mobility are also involved in the bacterial response to environmental changes especially in the highly dynamic and compartmentalized gut environment [START_REF] Gauger | Role of Motility and the flhDC Operon in Escherichia coli MG1655 Colonization of the Mouse Intestine[END_REF]. In our model, plasmid-host adaptation could be the result of both the specific plasmid-host pairing studied and adaptation to environmental changes in the pig's gut. Overall, we observed that the blaCTX-M-1-IncI1/ST3 plasmid has only a limited effect on the host cell, targeting cellular functions classically known to be affected in the host bacterium after plasmid acquisition.

We observed that most of the blaCTX-M-1-IncI1/ST3 plasmid genes were expressed at low level or not expressed in host cell. Our observation indicates that the IncI plasmid transcription is highly regulated in this model, which is consistent with findings from other studies reporting transcriptional profiles of other large conjugative plasmids [START_REF] Lang | Transcriptome Mapping of pAR060302, a bla CMY-2 -Positive Broad-Host-Range IncA/C Plasmid[END_REF]. Recent studies have reported the involvement of plasmid genes encoding for nucleoid-associated proteins (NAPs) homologs, such as H-NS-like proteins, in transcriptional regulation networks between plasmids and host chromosome [START_REF] Vial | Plasmid-chromosome cross-talks[END_REF][START_REF] Doyle | An H-NS-like Stealth Protein Aids Horizontal DNA Transmission in Bacteria[END_REF][START_REF] Takeda | Distribution of Genes Encoding Nucleoid-Associated Protein Homologs in Plasmids[END_REF][START_REF] Dorman | H-NS-like nucleoid-associated proteins, mobile genetic elements and horizontal gene transfer in bacteria[END_REF]. Plasmids encoding H-NS-like "stealth" protein reduce their fitness cost by silencing transcriptional activities of their own genes, as well as some chromosomal genes, and therefore minimize the impact of plasmid introduction in the bacterium. Large conjugative plasmids frequently carry NAP gene homologs but, using in silico analysis, similar genes were not yet identified in plasmids of the IncI incompatibility group [START_REF] Shintani | Nucleoid-associated proteins encoded on plasmids: Occurrence and mode of function[END_REF]. However, a large number of IncI backbone genes are yet annotated as hypothetical proteins. Future studies could be interesting to characterize new regulators still unknown among these genes, and to better understand the cross-talk between IncI plasmids and the host chromosome.

Our study has further limitations. First, we performed the experiments in a unique commensal E. coli host strain and the genetic background of strains plays an important role in plasmid-host adaptation. Second, due to the design of our study, we used in vitro plating of the bacteria and therefore we could not directly observe the E. coli populations of interest in the pig's gut. This approach might have introduced a bias as a consequence of short adaptation of the IncI plasmid in the new recipient strain. Nevertheless, our study is the first to provide direct evidence of in vivo plasmid transfer in the pig's gut and to confirm that blaCTX-M-1-IncI1/ST3 plasmid fitness cost on its bacterial hosts is negligible in vivo, in a relevant environmental niche such as the gut.

MATERIALS AND METHODS

Bacterial strains and plasmids. For this study, a total of 24 strains were selected from a pig strain collection of ANSES laboratory (Ploufragan, France). The E. coli transconjugant UB12-TC was obtained by conjugation between a randomly chosen pan-susceptible pig commensal E. coli isolate, made resistant to 250 mg/liter rifampicin (recipient strain UB12-RC, B1, O100:H40) and an ESBL-resistant E. coli pig isolate (donor strain M63-DN, B1, O147:H7).

Each strain belongs to ST2520 and ST2521, respectively, according to the Warwick MLST scheme.

Thus, E. coli UB12-TC is resistant to rifampicin and harbors a IncI1 pST3 plasmid encoding blaCTX-M-1, sul2, dfrA17, and aadA5 genes. Seven cefotaxime-and rifampicin-resistant isolates (named UB12-TC ive ), three cefotaxime-susceptible and rifampicin-resistant isolates (named UB12-TCpIncI), and eleven cefotaxime-resistant and rifampicin-susceptible isolates (named NI-1 to NI-11) were chosen among all strains collected during a series of experiments conducte to evaluate the carriage of ESBL-producing E coli in the pig's gut [START_REF] Fleury | Impact of Ceftiofur Injection on Gut Microbiota and Escherichia coli Resistance in Pigs[END_REF][START_REF] Mourand | Escherichia coli Probiotic Strain ED1a in Pigs Has a Limited Impact on the Gut Carriage of Extended-Spectrum-β-Lactamase-Producing E. coli[END_REF]. These isolates have been collected from day 4 to day 45 after pig inoculation.

Animals and experimental design. As previously described in Mourand et al. [START_REF] Mourand | Rare Spontaneous Loss of Multiresistance Gene Carrying IncI/ST12 Plasmid in Escherichia coli in Pig Microbiota[END_REF][START_REF] Mourand | Escherichia coli Probiotic Strain ED1a in Pigs Has a Limited Impact on the Gut Carriage of Extended-Spectrum-β-Lactamase-Producing E. coli[END_REF], the experiments were conducted in the ANSES, Ploufragan, France, animal facilities. The trials were conducted with 6-to 8-week-old specific pathogen-free and initially ESC-resistant E. colifree Large White piglets. The animals were randomized before the experiment. They did not receive any antibiotic treatment prior to the trial and were given the same non-supplemented feed. Each room contained eight pigs placed in two pens of four animals each. For both trials, UB12-TC was inoculated on the first day (day 0): each pig was orally given a 10-ml suspension prepared from strain cultivated on MH agar containing cefotaxime (2 mg/liter). Individual fecal samples were collected from animals on day 0 before inoculation, and between day 1 and 45.

The fecal samples were stored at -70°C until analysis.

The numbers of cefotaxime-resistant E. coli (including UB12-TC and NI) in the individual fecal samples were estimated by spreading 100 µl of 10-fold dilutions on MacConkey agar plates (containing respectively 2 mg/liter cefotaxime), in triplicate. The titers of total E. coli population were determined by plating serial dilutions on MacConkey agar plates without antibiotic. After incubation at 37°C, the colonies obtained on supplemented or nonsupplemented MacConkey agar plates were enumerated, and the titers were calculated for each pig per day. The relative abundance of cefotaxime-and rifampicin-susceptible (NI clones) was calculated based on the ability of 2 to 5 isolates obtained from each pig on each day on cefotaxime-supplemented media to grow on rifampicin-supplemented media, and expressed as a percentage.

Antimicrobial susceptibility testing. Antimicrobial susceptibility testing by the disk diffusion method (ampicillin, ticarcillin, piperacillin, amoxicillin-clavulanic acid, ticarcillin-clavulanic acid, piperacillin-tazobactam, cefotaxime, ceftazidime, aztreonam, cefoxitin, cefepime, ertapenem and imipenem, association of sulphonamides + trimethoprim, tetracycline, streptomycin, nalidixic acid, ciprofloxacin, and rifampicin) was performed according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines (http://www.eucast.org).

Motility assay. Donor strain M63-DN, recipient strain UB12-RC, ancestral UB12-TC, all UB12-TC ive and UB12-TCpIncI clones were grown overnight in LB medium, washed, suspended in isotonic water to a concentration of 1 X10 2 cells ml -1 and used to inoculate semisolid LB agar tubes containing 0.35% agar, incubated for 48 h at 37°C. Experiments were repeated 3 times. For data analysis, three categories were considered (motile, partially motile, and nonmotile).

In vitro evolution experiments. Five randomly selected colonies of UB12-RC and UB12-TC were inoculated into 5 ml of LB and grown under constant shaking at 200 rpm. After overnight incubation at 37°C, 5 l of each culture was inoculated into five tubes containing 5 ml of fresh LB. At this step, the 10 cultures were considered the ancestors of each lineage (day 0 of the evolution assay). Next, the 10 cultures were propagated by daily serial transfer, with a 1/1,000 dilution (5 l of culture grown overnight diluted into 5 ml of fresh LB without antibiotic), for 30 days (corresponding to 300 generations in total) and incubated overnight at 37°C under constant stirring at 200 rpm. Finally, after 30 days, 10 independent lineages were obtained (denoted "ite"). At day 0 and every 5 or 6 days of the evolution assay, the culture was isolated on LB agar for purity, and antibiotic susceptibility was tested by disk diffusion. A culture sample from each lineage was collected and stored at -80°C with glycerol for future analysis.

Individual fitness assays. Fitness, defined here as the Maximum Growth Rate (MGR), was determined from growth curves using a high-precision technique developed in-house as described previously [START_REF] Mahérault | Advantage of the F2:A1:B-IncF Pandemic Plasmid over IncC Plasmids in In Vitro Acquisition and Evolution of bla CTX-M Gene-Bearing Plasmids in Escherichia coli[END_REF]. For each strain, the fitness assay was repeated six times. The measurements were collected using a Python script developed in our laboratory, and the MGR was obtained by the lm function in R (http://www.R-project.org/).

Whole-genome sequencing. WGS was performed using Illumina technology. Briefly, DNA was prepared for paired-end sequencing on the Illumina MiniSeq (Integragen, MA, USA) for all strains. DNA samples were extracted using the genomic DNA NucleoMag tissue kit from Macherey-Nagel (Hoerdt, France). DNA libraries were prepared and indexed, and tagmentation was processed using the Nextera DNA Flex library Prep kit (Illumina, USA). The sample concentrations were normalized in equimolar concentration at 1 nM. The pooled libraries were sequenced to a read length of 2 by 150 bp with Illumina MiniSeq High output reagent Kit. The genomes were sequenced at an average depth of 50X.

DNA sequence analysis and UB12-TC genome comparison. Illumina genome sequence reads were assessed for quality using FastQC v0.11.8 and subsequently trimmed with a cutoff phred score of 30 using TrimGalore v0.4.5. All genomes were de novo assembled using SPAdes v.3.13.1 software and analysed with an in-house bioinformatics pipeline (https://github.com/iame-researchCenter/Petanc) adapted from Bourrel et al. [START_REF] Bourrel | Colistin resistance in Parisian inpatient faecal Escherichia coli as the result of two distinct evolutionary pathways[END_REF]. Briefly, genome typing was performed including MLST determination, phylogrouping, and serotyping.

The pipeline was also used to get information about plasmid replicons, resistance and virulence genes, the prediction of a plasmid sequence or a chromosome sequence for each contig using PlaScope [START_REF] Royer | PlaScope: a targeted approach to assess the plasmidome from genome assemblies at the species level[END_REF].

For each sample, sequenced reads were mapped to a reference sequence to analyse the mutations and rearrangements. For reference plasmid sequences, we used the plasmid sequences extracted from GenBank (http://www.ncbi.nlm.nih.gov/GenBank/) (ColRNAI, accession n° J01566; IncY, accession n° MH42254; Col156, accession n° EU090225 and IncI, accession n° KJ484635) and de novo assembled contigs as predicted to plasmid genome of UB12-TC strain with PlaScope, included in our pipeline. For reference chromosomal genome, first, we calculated the mash distance between UB12-TC genome sequence and E. coli genomes from RefSeq database using Mash v.2.0 and we selected the closest E. coli strain assembly ASM190094v1. Then, all predicted chromosomal contigs of the UB12-TC genome were rearranged as scaffold with RAGOUT v2.3 (https://github.com/fenderglass/Ragout) [START_REF] Kolmogorov | Ragout--a reference-assisted assembly tool for bacterial genomes[END_REF] and the reference genome (E. coli assembly ASM190094v1). All scaffolds of the UB12-TC strain obtained were annotated on the MicroScope platform [START_REF] Médigue | MicroScope-an integrated resource for community expertise of gene functions and comparative analysis of microbial genomic and metabolic data[END_REF]. Identification of the point mutations and rearrangements between UB12-TC and UB12-TC ive were performed using the open computational pipeline Breseq v0.27.1 [START_REF] Deatherage | Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq[END_REF]. All SNPs identified in the ancestor clones that were sequenced were subtracted from the sequences of the evolved clones.

Transcriptome sequencing. RNA sequencing was performed on the recipient strain UB12-RC, the ancestral transconjugant UB12-TC, two evolved UB12-TC transconjugants (UB12-TC ive , detected in pig' stools at day 28 and 31) and one UB12-TC transconjugant that had lost the plasmid IncI (UB12-TCpIncI, detected in pig' stools at day 20).

For RNA extraction, a total of 10 cultures representing two biological replicates per strain were independently grown. For each strain, 6 l of an overnight culture was added in to 6 ml of fresh LB and was incubated at 37˚C under constant stirring at 200 rpm until reaching an OD600 of approximately 0.6. 3 ml of each culture were taken, representing two technical replicates, and mixed with RNA protect (Qiagen, Courtaboeuf, France) according to the manufacturer's recommendations. Cells were immediately pelleted by a 10 min centrifugation at 5,000 g. RNA was prepared using the Quick-RNA Fungal/Bacterial MiniPrep kit (Ozyme, ZymoResearch, USA). DNA was digested using Turbo DNAse (Thermofisher, UK) based on the manufacturer's protocol. Verification of complete removal of any contaminating DNA was performed by PCR amplification of recA housekeeping gene. The final RNA solution was quantified with the Qubit TM RNA High Sensitivity assay kit (Thermofisher Scientific, UK) and RNA quality was evaluated using the Agilent 2100 Bioanalyzer RNA 6000Nano Kit (Agilent Technologies, Santa Clara, CA). The RNA was immediately stored at -80°C.

For each replicate, 8 l of RNA was treated to remove 16S and 23S rRNAs and cDNA libraries were prepared using the Zymo-Seq RiboFree® Total RNA Library Kit (Ozyme, ZymoResearch, USA). The cDNA quantity was determined with the Qubit TM dsDNA Broad Range assay kit. Paired-end sequencing of the pooled libraries was performed with an Illumina MiniSeq Mid Output reagent kit and an Illumina NextSeq 500/550 High Output Kit v2.5 (Illumina, USA), configured to 2 X 150-bp cycles, yielding approximately 8 and 400 million reads, respectively. Transcriptome analysis. To assure high sequence quality, the Illumina cDNA reads in FASTQ format were trimmed to remove all sequencing adapters using TrimGalore v0.4.5., according to the manufacturer's recommendations. Then, cDNA reads were trimmed again, using TrimGalore v.0.4.5., with a cutoff phred of 30, and the reads shorter than 70 bp were discarded.

A final quality check was done using FastQC v0.11.8. Reads were then mapped to the UB12-TC reference genome sequence (see above) using BWA alignment software and BWA-MEM algorithm v.0.7.17. (52) Mapping statistics were verified using BAMStat v.1.25. and SAMtools idxstat v.1.9. [START_REF] Li | The Sequence Alignment/Map format and SAMtools[END_REF]. Reads counts were performed using featureCounts v.2.0.3.

(http://subread.sourceforge.net) [START_REF] Liao | featureCounts: an efficient general purpose program for assigning sequence reads to genomic features[END_REF]. An R package, DESseq2 v.1.34. was used to normalize and to identify differentially expressed genes between each sample (http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html) [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[END_REF]. A cutoff of q value of < 0.05 and a fold change of > 2 were used to measure statistical significance.

Statistics. For statistical data analysis from the fitness assays, we used an analysis of variance (ANOVA) and unpaired Student's test using R software, with P values of < 0.05 being considered significant.

  Fig 2). Description of the newly transconjugants E. coli isolates (NI) with IncI plasmid. As the recipient host background could interfere in the spread of multidrug resistance plasmids, we analyzed the genetic diversity of eleven cefotaxime-resistant and rifampicin-susceptible newly formed transconjugants (natural isolates, NI) harboring IncI. The phylogroups of NI are diverse (one A, B2, and D, two C, three B1, and G) (Fig 3A). Based on plasmid-families replicon, the plasmidome of NI appears varied with multiple combinations of plasmid types. Most of the eleven isolates (eight NI) carry Col-like plasmids, alone or co-carried with IncF plasmids. One isolate (NI-1) carries an IncY plasmid, sharing 98% identity and 71% coverage to the IncY plasmid of UB12-TC (Table

2 ,

 2 NI-3, and NI-6, from 40% to 50% for NI-4, NI-9, NI-10, and NI-11, and more than 65% for NI-5, NI-7, and NI-8.

Fig 1 :

 1 Fig 1 : Boxplot representation of the maximum growth rate (MGR) of each pool lineage: MGR of the donor strain (M63-DN), the recipient strain (UB12-RC), the ancestral transconjugant (UB12-TC, IncI, pST3), seven in vivo evolved transconjugants (UB12-TC ive ), and three evolved transconjugants which had lost in vivo the IncI plasmid (UB12-TCpIncI). UB12-TC ive and UB12-TCpIncI were isolated in stools between day 7 and day 45 during longitudinal study of in vivo carriage in pigs. For each box, the central mark indicates the median, and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. Each point corresponds to one measurement per clone, which were repeated six times per strains. Asterisks indicate significant differences (* = P < 0.05, ** = P < 0.01 and *** = P < 0.001). NS = not significant.

Fig 2 :

 2 Fig 2 : Boxplot representation of the maximum growth rate (MGR) of each pool lineage: MGR of five ancestral UB12-RC and UB12-TC control strains and evolved UB12-RC and UB12-TC strains at day 30. For each box, the central mark indicates the median, and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. Each point corresponds to one measurement per clone, which were repeated six times per transconjugant and control. NS = not significant.

Fig 3 :

 3 Fig 3 : (A), Phylogenetic tree of the 11 newly formed transconjugants detected among pig E.coli commensal isolates (NI clones, E. coli, IncI, pST3), the M63-DN donor strain, and the UB12-TC transconjugant strain (in bold) are presented. The tree is rooted on an Escherichia clade (E1492cladeI,[START_REF] Denamur | The population genetics of pathogenic Escherichia coli[END_REF]). The phylogenetic tree was constructed with IQ-TREE v1.6.9 from the core genome genes using Roary v1.007002. From left to right are presented the Sequence Type (according to the Warwick University scheme), the O and H serotypes, the fimH allele, and the fitness cost of NI clones compared to the donor strain (M63-DN) (NA= not applicable, NS= not significant, * = P < 0.05, ** = P < 0.01 and *** = P < 0.001). Each branch of the same phylogenetic group are colored (yellow, D; red, B2; orange, G; blue, A; light green, C; dark green, B1). Information on O group, H type, and fimH allele are given in Table1. (B), Boxplot representation of the maximum growth rate (MGR) of each strain : donor strain (M63-DN), UB12-TC ancestral transconjugant and NI clones, isolated in stools between day 4 and day 22 during longitudinal study of in vivo carriage in pigs. For each box, the central mark indicates the median, and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. Each point corresponds to one measurement per clone, which were repeated six times per strains. Asterisks in red indicate significant differences (* = P < 0.05, ** = P < 0.01

  protein Phage related gene -3,2043637 0,09624996 -3,83463554 6,8963E-05 -3,97393099 2,0185E-05 -0,70851469 0,9998186

Table :

 : List of differentially expressed genes from UB12-TC clones compared to UB12-RC recipient strain.Boxes in color indicate genes that were statistically significantly different compared to UB12-RC (positive value = upregulated; negative value = down-regulated) with padj <0.05 (dark gray) and with padj <0.1 (light gray) ; log2FC indicates the log2(fold-change), padj indicates the adjusted p-value ; a Gene Ontology terms according the BioCys Genome Database collection and eggNOG-mapper v2

	Genome Reference	Locus Reference	Gene	Product	GO Terms a	log2FC	Padj	log2FC	Padj	log2FC	Padj	log2FC	Padj
	ECOL4764K		cheA	chemotaxis protein CheA	Chemotaxis (NT)	2,44783641 0,00237824 1,6618073 0,02643089 1,61793383 0,05792439 3,39694919 3,1361E-17
	ECOL4764K		tar	methyl-accepting chemotaxis protein Tar Chemotaxis (NT)	0,35904909 0,99975668 0,90038027 0,56711099 1,08193808 0,12425088 2,52614687 1,8061E-09
	ECOL4764K		cheR	chemotaxis protein methyltransferase	Chemotaxis (J)	1,31467434 0,99975668 0,53731584 0,99726782 0,74834092 0,94241864 1,94461227 2,5904E-05
	ECOL4764K		cheB	protein-glutamate methylesterase/protein glutamine deamidase	Chemotaxis (NT)	1,61017833 0,86086269 1,95395442 0,00038597 1,90873908 0,02550016 3,37827951 4,9075E-13
	ECOL4764K		cheY	chemotaxis protein CheY	Chemotaxis (KT)	-0,38604911 0,99975668 0,84638707 0,72815763 0,84992797 0,41766744 1,44825921 0,00533935
	ECOL4764K		cheZ	chemotaxis protein CheZ	Chemotaxis (J)	0,14320588 0,99975668 0,47044846 0,99726782 0,53222483 0,99982751 1,46782071 0,01350632
	ECOL4764K		trg	methyl-accepting chemotaxis protein Trg Chemotaxis (NT)	0,22621629 0,99975668 0,47635971 0,99726782 0,34401042 0,99982751 1,32683919 0,01396423
	ECOL4764K		tap	methyl-accepting chemotaxis protein Tap Chemotaxis (NT)	1,64986384 0,27943748 1,79254785 0,00465004 1,92087014 0,00359162 3,55572749 1,2378E-20
	ECOL4764K		yeeN	putative transcriptional regulator YeeN	Function Unknown (K)	1,56735099 0,18151996 1,87188402 0,08396312 1,94557903 0,01514422 1,26820165 0,9998186
	ECOL4764K			protein of unknown function	Function Unknown (S)	1,18936804 0,64643806 0,95973201 0,3662016 1,05386371 0,04779514 0,54491486 0,9998186
	ECOL4764K			conserved protein of unknown function	Function Unknown (S)	1,06079641 0,72325589 1,91186226 0,12351174 1,61571376 0,03232358 0,69774954 0,9998186
	ECOL4764K		yeeX	DUF496 domain-containing protein YeeX Function Unknown (S)	1,48446556 0,12053696 1,32006713 0,00832163 1,55265854 0,00204096 1,26712195 0,42012273
	ECOL4764K			SIR2_2 domain-containing protein	Function unknown (S)	1,78235876 0,05216601 2,05694257 0,00025802 2,08265483 3,475E-05 1,69739614 0,9998186
	ECOL4764K		yjcZ	uncharacterized protein YjcZ	Function unknown (S)	1,01765908 0,99975668 0,54035335 0,99726782 0,85744316 0,39623688 2,44202815 0,00029627
	ECOL4764K			YjbI protein	Function unknown (S)	-0,9133541 0,99975668 2,28376821 0,0383436 2,27538051 0,05809278 0,78170087 0,9998186
	ECOL4764K		ybdZ	enterobactin biosynthesis protein YbdZ	Function unknown (S)	-2,29213602 0,99975668 3,13394637 0,02211293 1,40956129 0,22322039	0,911427 0,9998186
	ECOL4764K		srlA	sorbitol-specific PTS enzyme IIC2 component	Metabolism (G)	0,45129968 0,99975668 1,62365045 0,99726782 1,2762467 0,03441003 1,14754227	0,811379
	ECOL4764K		napD	NapA signal peptide-binding chaperone NapD	Metabolism (P)	0,8201188 0,99975668 2,29289548 0,05600281 2,87501484 0,00812277 2,25275005 0,06791626
	ECOL4764K		narK	nitrate:nitrite antiporter NarK	Metabolism (P)	-0,38390016 0,99975668 1,05699721 0,9733515 2,61825819 0,04321861 1,92704218 0,78516139
	ECOL4764K		lamB	maltose outer membrane channel/phage lambda receptor protein	Metabolism (M)	-0,33199188 0,99975668 3,02915833 0,03103617 2,85400101 0,01182163 1,67912886 0,9998186
	ECOL4764K		malK	maltose ABC transporter ATP binding subunit	Metabolism (P)	-0,91271454 0,99975668 2,91750617 0,00246239 2,87200557 0,0036916 1,26124927 0,01393791
	ECOL4764K		malE	maltose ABC transporter periplasmic binding protein	Metabolism (P)	-0,07773947 0,99975668 2,29063854 0,05361344 2,32236222 0,03578512 1,12427349 0,0616188
	ECOL4764K		malF	maltose ABC transporter membrane subunit MalF	Metabolism (G)	0,13807123 0,99975668 2,87308868 0,00955001 2,83601098 0,01086782 1,03170565 0,41672289
	ECOL4764K		gnsA	putative phosphatidylethanolamine synthesis regulator GnsA	Metabolism (S)	1,9499088 0,01334079 1,62445128 0,56803068 1,48140702 0,44180191 0,75222366 0,9998186
	ECOL4764K		fliS	flagellar biosynthesis protein FliS	Mobility (N)	-0,3836078 0,99975668 1,10600922 0,47705249 1,05056155 0,31125954 1,59581178 0,00035853
	ECOL4764K		fliC	flagellar filament structural protein	Mobility (N)	1,27948099 0,3604938 1,36332621 0,00715182 1,47471013 0,00223327 2,60139882 6,3047E-11
	ECOL4764K		motA	motility protein A	Mobility (N)	2,39223387 0,01243818 2,45116842 2,1365E-06 2,67928714 5,6538E-11 4,82804021 5,9129E-22
	ECOL4764K		motB	motility protein B	Mobility (N)	1,88765993 0,12857638 1,62232638 0,00689759 1,54818363 0,0235335 3,54866687	6,12E-19
	ECOL4764K		flgL	flagellar hook-filament junction protein 2 Mobility (N)	0,27038769 0,99975668 0,61263279 0,99726782	0,572498 0,99982751 1,70499166 1,8969E-05
	ECOL4764K		flgK	flagellar hook-filament junction protein 1 Mobility (N)	0,10274037 0,99975668 0,36238444 0,99726782 0,50076412 0,99982751 2,0173971 0,02322431
	ECOL4764K		flgM	anti-sigma factor for FliA (sigma(28))	Mobility (N)	0,34000603 0,99975668 0,69973937 0,99726782 0,5804435 0,99982751 1,97572269 0,00049616
	ECOL4764K		flgN	flagellar biosynthesis protein FlgN	Mobility (N)	-0,77326134 0,99975668 0,78099782 0,99726782 0,9081652 0,8880041 1,78599881 0,01350632
	ECOL4764K		crfC	clamp-binding sister replication fork colocalization protein	Mobility/Biofilm (S)	1,7097451 0,07723678 1,95494295 0,00099113 2,21853205 0,00020283 4,27686078 4,6418E-07
	ECOL4764K		pdeH	c-di-GMP phosphodiesterase PdeH	Signal transduction/ Biofilm (T) 1,46385624 0,99975668 1,66923588 0,00445625 1,71644685 0,00093775 3,34376659 9,1986E-09
	ECOL4764K		aer	aerotaxis sensor receptor, flavoprotein	Signal transduction (T)	0,7750516 0,99975668 0,55372989 0,99726782 0,57298795 0,99982751 2,01637057 7,1499E-08
	ECOL4764K		ariR	regulator of acid resistance, influenced by indole	Stress response (K)	-1,49354048 0,99975668 2,80049268 0,04360618 1,93625417 0,0816273 0,09458701 0,9998186
	ECOL4764K		soxS	DNA-binding transcriptional dual regulator SoxS	Stress response (K)	0,87424041 0,99975668 2,21650721 0,00259711 2,98162412 1,9613E-08 0,28148909 0,9998186
	ECOL4764K		bhsA	DUF1471 domain-containing multiple stress resistance outer membrane protein	Stress response (M)	1,59776978 0,2905904 2,42131465 0,00118746 2,25488823 2,7044E-06 0,96289078 0,9998186
	ECOL4764K		yhcN	DUF1471 domain-containing stress-induced protein YhcN	Stress response (S)	1,87275468 0,07206742 3,39817662 0,00145485 2,98627883 0,00046437 1,27996133 0,9998186
	ECOL4764K		cspA	cold shock protein CspA	Transcription (K)	1,12139443 0,61924355 1,5684485 0,00214343 2,0167162 8,7657E-06 1,01535241 0,9998186
	ECOL4764K		infA	translation initiation factor IF-1	Translation (J)	1,31763897 0,30382261 1,39096931 0,02693564 1,16110079 0,16528867 0,66463366 0,9998186
	p4764_IncY.171940636		Phage protein	Phage related gene	3,14455603 0,00149673 2,18084744 0,00086307 2,52625482 0,00024066 0,9660949 0,9998186
	ECOL4764K		ghoT	toxin of the GhoTS toxin-antitoxin system Virulence (S)	0,83692078 0,99975668 1,12522596 0,57856784 1,84114319 0,01535673 1,14237385 0,9998186
	ECOL4764K			AAA family ATPase		1,18850346 0,64643806 1,3958336 0,06825492 1,4229527 0,03606433 0,93340617 0,9998186
	ECOL4764K		ydcI	putative DNA-binding transcriptional repressor YdcI	Transcription (K)	-2,69473373 0,01855179 -1,07572986 0,99726782 -1,21206268 0,57141967 -0,93255822 0,9998186
	ECOL4764K		lpp	murein lipoprotein	Stress response (M)	-1,7994587 0,26242052 -3,3785418 0,00014215 -2,13192355 0,27055292 -2,82669822 0,00395717
	ECOL4764K		fimA	type 1 fimbriae major subunit	Adherence, virulence (NU) -0,17459055 0,99975668 -1,37091961 0,02693564 -1,26245507 0,04858227 -1,17411151 0,05009408
	ECOL4764K			conserved protein of unknown function	Function unknown (S)	-2,65112498 0,99975668 -3,08426947	0,030812 -2,78481638 0,03064935 -1,09159107 0,9998186
	ECOL4764K			conserved protein of unknown function	Function unknown (S)	-0,88397112 0,99975668 -1,59301313 0,0223801 -1,35985068 0,05618409 -1,08447934 0,9998186
	ECOL4764K		cyuP	putative D/L-serine transporter	Metabolism (E )	-2,46467882 0,00398487 -0,45755875 0,99726782 -0,01010979 0,99982751 0,09586933 0,9998186
	ECOL4764K		garP	galactarate/glucarate/glycerate transporter GarP	Metabolism (P)

ACKNOWLEDGMENTS

We thank Jean Marc Guigo (Institut Pasteur) for useful discussions and Olivier Clermont, Kevin La and Gwenaelle Mourand (ANSES) for technical help. This work was partially supported by a grant from the Fondation pour la Recherche Médicale to E.D. (grant number DEQ20161136698), ANSES (French Agency for Food, Environmental, and Occupational Health and Safety), France-Agrimer (grant 2012-0388), and by the Conseil Départemental des Côtes d'Armor. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Data availability. All reads have been deposited at the European Nucleotide Archive (project accession number PRJEB8070, ID samples from ERS14303872 to ERS14303895)

Ethics. The experiments were performed in accordance with French animal welfare regulations, and the protocol was approved by the ANSES/ENVA/UPEC ethical committee (ComEth authorizations 12-032 and 15-050 [no. APAFIS: 2015061813246553]). The experiments were conducted in the ANSES, Ploufragan, France, animal facilities.

Table 1: Characterization of donor E. coli strain, evolved and ancestral E. coli UB12 clones, and new in vivo transconjugants used in this study. a, detailed in [START_REF] Lucas | Characterization of plasmids harboring blaCTX-M genes in Escherichia coli from French pigs[END_REF][START_REF] Fleury | Impact of Ceftiofur Injection on Gut Microbiota and Escherichia coli Resistance in Pigs[END_REF]; b, according to the Warwick multilocus sequence typing (MLST) scheme; c, PlasmidFinder, IncI plasmid is in bold ; d , (+) total spreading within medium, (+/-) partial spreading, and (-) not spreading