Bacterial strains and plasmids
To perform isogenic comparisons, the reference E. coli strain CFT073  and the commensal E. coli strain HS were transformed with the kanamycin-resistant pBK-CMV empty vector (resulting in the strain referred to as E. coli mcr-) or a variant of pBK-CMV in which the mcr-1, mcr-3, -4, mcr-5, or mcr-10-encoding sequences were cloned downstream of the lac promoter (resulting in E. coli mcr+). To create rifampicin- or nalidixic acid-resistant bacteria, E. coli CFT073 was cultured overnight in Luria-Bertani (LB) medium and spread on LB agar plates containing rifampicin or nalidixic acid (50 μg/ml). One resistant clone was transformed using the pBK-CMV empty vector or pBK-CMV-mcr-1 vector. The presence of mcr-1 and its expression in the absence of a lac inducer resulted in colistin MIC values of 4 μg/mL, as usually observed in clinical isolates  and did not affect the growth of bacteria at 12 h (as well as up to at least 24 h) post-incubation in LB, Dulbecco’s modified Eagle medium (DMEM; Gibco, Waltham, MA, USA), or Roswell Park Memorial Institute 1640 (RPMI; Gibco) medium at 37°C (Supplementary Fig. 1). No inducer of the lac promoter was used in the experiments, and all experiments were performed with bacteria collected after 16 h of incubation at 37°C in LB medium.
The stability of pBK-CMV plasmid was assessed in E. coli mcr- and E. coli mcr+ by serial propagation for 10 days in LB broth medium at 37°C without kanamycin. The passages were performed every day by diluting the culture 1:1000 in 10 ml sterile LB medium. Serial dilutions were spread daily on LB agar plates with or without kanamycin and the number of CFUs was counted to assess the loss of plasmids. The data show that the loss of empty or mcr-1-encoding pBK-CMV is similar. Even after 10 days of intensive growth without antibiotic, ~25% of E. coli population conserved the plasmid (Supplementary Fig. 2). Throughout this work, CFUs were counted on plates supplemented with kanamycin (pBK-CMV antibiotic resistance) to target E. coli-containing pBK-CMV plasmids.
For experiments using bacterial supernatants, bacteria were centrifuged for 10 min at 5500 rpm, and the supernatant was collected and then sterilized using a 0.22-μm filter. In addition, pBR plasmid-encoding colicin E1, E2, and D as well as pLR1 plasmid-encoding colicin A were hosted in the laboratory E. coli strain C600 (kindly provided by Dr. Denis Duché, UMR7255 CNRS-Aix-Marseille Université).
Cloning of mcr genes
Genes mcr-1, mcr-3, mcr-4, mcr-5, and mcr-10 were amplified using the GoTaq DNA polymerase (Promega) and cloned in the pBK-CMV plasmid (mcr-3, mcr-4, mcr-5, and mcr-10) with the following primers: mcr1EcoRIFor 5’-GCGAATTCATGATGCAGCATACTTCTGTG-3’, mcr1XhoIRev 5’-GTTCTCGAGTCAGCGGATGAATGCGGTG-3’; mcr3BamHIFor 5’-CGGGATCCATGCCTTCCCTTATAAAAAT-3’, mcr3EcoRIRev 5’-CGGGGAATTCTTATTGAACATTACGACATTG-3’; mcr4BamHIFor 5’-GCGGATCCGTGATTTCTAGATTTAAGACG-3’, mcr4EcoRIRev 5’-GCAGAATTCCTAATACCTGCAAGGTGC-3’; mcr5BamHIFor 5’-GCGGATCCATGCGGTTGTCTGCATTTATCAC-3’, mcr5EcoRIRev 5’-GCGAATTCTCATTGTGGTTGTCCTTTTCTGC-3’; mcr10BamHIFor 5’- GCGGATCCATGCCCGTACTTTTCAGGATG-3’, mcr10EcoRIRev 5’-GCGAATTCCTATCCACGACATTCGCGGAAC-3’. Empty or mcr-containing pBK-CMV plasmids were electroporated into E. coli strains and selected using kanamycin (50 μg/ml). The mcr gene sequences were double-checked by sequencing (GATC, Konstanz, Germany).
Inactivation of Mcr-1 catalytic site
To inactivate the Mcr-1 enzyme produced from pBK-CMV, the threonine at position 285 that is involved in the binding of the active Zn atoms in the Mcr-1 catalytic site  was replaced by an alanine by site-directed mutagenesis using the InFusion HD Cloning Kit (Takara, Kusatsu, Japan), the Platinum SuperFi II DNA Polymerase (Invitrogen, Waltham, MA, USA), and T285AFor 5’-ATACGCCGCCGATGTGCCGCACGATGTG-3’, and T285ARev 5’-ACATCGGCGGCGTATTCTGTGCCGTG-3’ primers. The mcr gene sequence was double-checked by double-strand sequencing (GATC, Konstanz, Germany).
Bacteria were grown overnight at 37°C on LB containing kanamycin (50 μg/ml). They were washed with 10-mM phosphate buffer (pH 7.2), suspended in 100 μl to obtain an optical density of 0.5 MacFarland, and then incubated for 2 h with antimicrobial peptides (1 μg for hBD1 and hBD2, 0.2 μg for hBD3 and LL37, 2 μg for HD-5, and 4 μg for HD-6). The bacteria were then spread on LB agar plates and incubated overnight at 37°C, and the CFUs were counted. All peptides used were obtained from the Peptide Institute, Osaka, Japan.
Bacteriocin resistance assays
The antibacterial activity of bacteriocins against E. coli mcr-1+ and E. coli mcr-1- was investigated in agar medium by an antibiogram-like approach as previously described . Briefly, a bacterial suspension of 0.5 MacFarland was prepared from an overnight agar plate culture and diluted in sterile broth to obtain a final inoculum of approximately 105 CFU/ml. After inoculation of Mueller-Hinton (MH) agar culture medium with this suspension, 10 μl of colicin-producing E. coli was spotted on the MH agar plate. After overnight incubation at 37°C, the plates were examined for colicin sensitivity, which was observed as clear zones of lysis in the overlaid strains. The inhibitory activity of colicins was then quantified in LB broth, as previously reported . Briefly, colicin-producing E. coli and E. coli mcr-1+ or E. coli mcr-1- at a 100:1 ratio were co-cultivated in 10 ml of LB broth at 37°C in a shaking incubator. After 24 h of incubation, serial dilutions of cocultures were spread on blood sheep agar plates (bioMérieux, France), and the CFUs of E. coli CFT073 mcr-1+ and E. coli CFT073 mcr-—which have a hemolysin phenotype (unlike E. coli C600)—were counted.
LPS was purified as previously described . Briefly, bacteria were grown overnight at 37°C in 50 ml of LB containing kanamycin (50 μg/ml). They were collected by centrifugation, washed twice with phosphate-buffered saline (PBS, Gibco) containing 0.15 mM CaCl2 and 0.5 mM MgCl2, and then disrupted by sonication. To eliminate the remaining nucleic acids and proteins, lysates were treated with 200 μg/ml proteinase K (1 h at 65°C with gentle mixing) and then with 40 μg/ml DNase and 80 μg/ml RNase (37°C, in the presence of 1 μl/ml 20% MgSO4 and 4 μl/ml chloroform overnight with gentle mixing). Finally, an equal volume of hot (68°C) 90% phenol was added to the mixtures, followed by vigorous shaking at 68°C for 15 min. Suspensions were then cooled on ice and centrifuged at 8500 rpm for 15 min. Aqueous phases were pooled, and phenol phases were re-extracted with 10 ml of distilled water at 68°C. Pooled aqueous phases were extensively dialyzed against distilled water at 4°C, and the purified LPS product was finally lyophilized. For experiments, LPS was dissolved in PBS.
The human intestinal epithelial HT-29 (ATCC® HTB-38™) and the human monocyte THP-1 (ATCC® TIB-202™) cell lines were maintained in an atmosphere containing 5% CO2 at 37°C in the culture media recommended by ATCC. The human mucus-producing intestinal epithelial HT29-16E cell line  were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal calf serum (Lonza), 1% l-glutamine (Life Technologies), 200 U penicillin, 50 mg of streptomycin, and 0.25 mg of amphotericin B per litre. In addition, THP-1 monocytes were differentiated into macrophages by treatment with 20 ng/ml phorbol myristate acetate (PMA) for 18 h.
Cells were infected with a multiplicity of infection (MOI) of 100 (HT-29) or 10 (THP-1) for the indicated time. To determine bacterial adhesion, cells were extensively washed with sterile PBS, and serial dilutions were spread on LB agar plates. After one night at 37°C, the number of CFUs was counted.
HT29-16E cells were seeded on sterile glass cover slips. After 21 days of culture, the cells were infected at MOI of 100 for 30 min. After several washes, cells were fixed, incubated at room temperature in the hybridization buffer (20 mM Tris-HCl (pH 7.8), 1.25 M NaCl, 0.01% SDS, Denhardt’s solution 1X), and then incubated for 3 h with the DNA probe (5 ng/μL; 5’-GCA AAG GTA TTA ACT TTA CTC TTC TCC-Cy2-3’). Cells were washed with hot hybridization buffer (48°C) and stained for 10 min using DAPI (1 μg/mL) and WGA (1 μg/mL). Cells were washed 3 times and observed using a Zeiss LSM 800 confocal microscope. Bacteria were counted using image analysis software Imaris.
Enzyme-linked immunosorbent assays (ELISAs)
The amounts of KC, IL-1β, IL-6, IL-8, and TNF-α secreted in the cell culture supernatants or present in mouse tissues and lipocalin-2 present in mouse faeces were determined by ELISA (R&D Systems) in accordance with the manufacturer’s instructions.
Mouse sensitivity to E. coli mcr-1+ or E. coli mcr-1- and their derivate LPS
Six- to 8-week-old C57/BL6 male mice (Charles River, Ecully, France) received an intraperitoneal administration of 5 mg/ml LPS or 109 CFUs of bacteria in 0.2 ml of PBS.
E. coli mcr-1+ or E. coli mcr-1- infection using the oligo-mouse-microbiota 12 (OMM12) model
OMM12 (C57Bl/6J) mice are gnotobiotic mice that harbor a defined consortium of 12 bacterial strains isolated from the murine gut and are devoid of E. coli strains (see Table S1) . These mice are also designated stable defined moderately diverse microbiota mice (sDMDMm2) . OMM12 mouse breeding pairs were kindly provided by Dr. Basic and Prof. Bleich (Hannover Medical School, Institute for Laboratory Animal Science, Hannover, Germany) and bred under germ-free conditions in a flexible isolator at the INRAe mouse facility (UMR454 MEDIS, Theix, France). Mouse colonies were regularly checked for potential contaminants using qPCR and culture-based approaches.
OMM12 mouse experiments were performed using an individually ventilated and positively pressurized cage system (IsoCage P—bioexclusion system, Tecniplast, France). Each experiment was performed using either 8- or 18-week-old littermates subjected to 12:12 light/dark cycles with access to food and water ad libitum. Briefly, OMM12 mice were transferred into the IsoCage P system 2 days prior to the beginning of the experiment to allow for the gut microbiota to stabilize. Further, the mice were orally infected with 103 bacteria/mouse resuspended in 50 μl of PBS and then sacrificed 10 days post-infection by cervical dislocation.
E. coli mcr-1+ and E. coli mcr-1- infection using a conventional mouse model
Experiments were performed using wild-type or Camp knock-out six- to 8-week-old C57/BL6 male mice (Charles River and The Jackson Laboratory, respectively) housed in a specific pathogen-free animal facility at the University of Clermont Auvergne, France. Mice were fed standard chow ad libitum throughout the experiments, had free access to sterile water, and were subjected to 12:12 light/dark cycles. In addition, they received oral gavage with a 200-μl suspension containing 108 CFUs of E. coli mcr-1+ or E. coli mcr-1- and then sacrificed 3 days post-infection by cervical dislocation.
For the competition experiments, mice received streptomycin in drinking water (2.5 g/L) for 48 h. The antibiotic was then discontinued, and 24 h later, the mice received 108 CFUs of E. coli mcr-1+ (rifampicin resistant strain) and 108 CFUs of E. coli mcr-1- (nalidixic acid-resistant strain) in 200 μl of PBS. At the indicated time, serial dilutions of feces were spread on LB agar plates containing kanamycin (50 μg/ml) and either rifampicin (50 μg/ml) or nalidixic acid (50 μg/ml) in order to count E. coli mcr-1+ and E. coli mcr-1- CFUs, respectively.
Quantification of E. coli mcr-1+ and E. coli mcr-1- in mice feces
Fecal samples were collected at the indicated times, diluted in sterile PBS, and then plated on LB agar containing kanamycin (50 μg/ml) and either rifampicin (50 μg/ml) or nalidixic acid (50 μg/ml) to quantify the number of E. coli mcr-1+ and E. coli mcr-1-. Bacterial identification was performed using chromogenic medium or mass spectrometry, and the presence of mcr-1 in isolates was confirmed by PCR. No such resistant or mcr-1-positive bacteria were found in uninfected mice. Intestinal tissues were longitudinally opened, washed in 3 ml of sterile PBS, and then either homogenized using a Tissue Master 125 Homogenizer (Omni International) to quantify E. coli tissue-associated loads or directly incubated in sterile DMEM containing antibiotics to quantify the secreted cytokines.
Quantitative (q)PCR of bacterial 16S rRNA genes
Fecal DNA extraction was performed as described in Herp et al. (2019), with the only difference being that DNA was diluted to a final concentration of 10 ng/μl in dH2O . The DNA concentration was determined using a Qubit 3 fluorometer and its corresponding kit (Qubit® dsDNA HS Assay kit (0.2–100 ng), Thermo Fisher Scientific). Absolute 16S rRNA gene quantification was performed as described in Brugiroux et al. (2016), with modifications . In addition, QPCR assays were run and analysed on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). All qPCRs were performed using 60°C as the optimal annealing temperature, except for the qPCR detection of the I48 strain, which was performed using 54°C to optimize amplification efficiency. Detection of E. coli was assessed using the optimal OMM12 qPCR program (95°C for 10 min, followed by 45 cycles of 95°C for 15 s and 60°C for 1 min), with E. coli forward (5’-CATGCCGCGTGTATGAAGAA-3’) and reverse (5’-CGGGTAACGTCAATGAGCAAA-3’) primers and the corresponding probe (Taqman FAM-labeled probe 5’-TATTAACTTTACTCCCTTCCTCCCCGCTGAA-3’) published by Huijsdens et al. .
Sequencing of the bacterial 16S rRNA genes
Fecal samples were immediately frozen in liquid nitrogen after defecation and stored at −80°C. Within 1 month, DNA was extracted from the fecal samples according to the International Human Microbiome Standards protocol, as previously described . A negative control devoid of DNA as well as bacterial and DNA-positive controls were also processed throughout the entire experiment (ZymoBIOMICS®, Zymo Research). The genomic DNA was amplified with fusion primers targeting the variable V3 and V4 regions of the 16S rRNA gene with indexing barcodes, as previously described . All samples were pooled for 2 x 300 bp paired-end sequencing on the Illumina MiSeq platform (Illumina), in accordance with the manufacturer’s specifications, at the Clermont-Ferrand University Hospital, France. The reads were deposited in the European Nucleotide Archive (project ID: PRJEB33293).
Microbiota composition analysis
Paired-end read assembly, quality and length filtering, OTU picking (100% sequence identity threshold), and chimera removal were performed with UPARSE . After quality-filtering and trimming, an average of 26,175 sequences were acquired for each sample. Allele-specific variants were assigned to taxonomy by QIIME 2 (https://qiime2.org/) with the SILVA database (version 132, https://www.arb-silva.de/). Sequence counts were normalized to their sample size and then multiplied by the size of the smaller sample (n = 5,500). Sequences that were not observed more than three times in at least 15% of the samples (n = 4) were discarded.
The statistical analyses were performed in R (https://www.r-project.org/), with vegan (https://CRAN.R-project.org/package=vegan) and phyloseq packages . The Kruskal-Wallis test was used to estimate alpha diversity differences among groups, and pairwise comparison was performed with the Wilcoxon test with a correction of p values in accordance with the FDR procedure. Beta diversity was assessed from Jensen-Shannon and generalized UniFrac indices, which were reported after non-metric multidimensional scaling (NMDS). Adonis (PERMANOVA) and PermDISP2 tests were used to assess significant differences among groups. Significant differences in taxon abundances between groups were detected with the DEseq2 approach and correction of p values according to the FDR procedure.
Pairwise comparative modelling (PCM)
PCM was used to predict mcr-1 analogues in the intestinal microbiota, as previously described . PCM is based on using homology modeling to increase the specificity of the functional prediction of proteins, particularly when they are distantly related to potential homologs. Taxonomy was assigned, as previously described , by combining results obtained from a BLASTN against the National Centre for Biotechnology Information (NCBI) genome database (minimal 70% identity and 80% coverage), a BLASTN against the IMOMI in-house database (minimal 85% identity and 90% coverage), and the taxonomy of the metagenomic unit whenever applicable.
Animal protocols were in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the University of Clermont Auvergne and were approved by the French Ministry of National Education, Higher Education, and Research (APAFIS#16354, #22507, #22770).
Values are expressed as means ± SEMs. Statistical tests were performed with GraphPad Prism version 6.07 software, using a two-tailed Student’s t test or a Mann-Whitney U test depending on normality determined using the D’Agostino-Pearson omnibus normality test. P values less than 0.05 were considered statistically significant.