Miseq technology

Genomic DNAs (gDNAs) of all bacterial strains sequenced were extracted in two steps: a mechanical treatment was first performed using acid washed glass beads (G4649-500g) (Sigma-Aldrich, Lyon, France) and a FastPrep BIO 101 instrument (Qbiogene, Strasbourg, France) at maximum speed (6.5 m/s) for 90 s. Then, after a 2–2.5 h lysozyme incubation at 37 °C, DNA was extracted on the EZ1 biorobot with the EZ1 DNA Tissue kit (Qiagen). The elution volume was 50 µL. Each gDNA was quantified by a Qubit assay with the high sensitivity kit (Life technologies, Carlsbad, CA, USA).
The strategy used for sequencing genomic DNAs varied from one bacterial strain to another. Four strategies were used: (i) one run with the Paired-End strategy using the MiSeq technology (Illumina, San Diego, CA, USA); (ii) two successive runs with the Paired-End Strategy using the MiSeq technology (Illumina); (iii) one run with the Paired-End Strategy using the MiSeq technology (Illumina) + one MinION sequencing run (Oxford Nanopore Technologies Ltd., Oxford, UK); (iv) one run with the Mate-Pair Strategy (Illumina) + 1 MinION sequencing run (Oxford Nanopore Technologies Ltd.). Only one of these four sequencing strategy was used for each of the studied strains (Table S1).

Fresh extruded stools were collected before EAE treatment and immediately positioned in carbon dioxide ice for the gut microbial analysis. Feces DNA was extracted using QuickGene DNA tissue kit from Kurabo Company (Neyagawa, Japan) and sent for PCR amplification and sequencing of the V3 and V4 region of bacterial 16S rRNA genes using the Illumina MiSeq technology at BGI Co. (Shenzhen, China).
For alpha diversity analysis, we rarified the OTU to several metrics, including curves of OTU rank, rarefaction and Shannon, and calculated indexes of Shannon, Chao1, Simpson, and ACE. For beta diversity analysis, principal component analysis (PCA) was performed using QIIME (23 (link)). The LDA effect size (LEfSe) analysis was performed for the quantitative analysis of biomarkers among each group (24 (link)). Briefly, LEfSe analysis, LDA threshold of >4, used the non-parametric factorial Kruskal-Wallis (KW) sum-rank test and then used the (unpaired) Wilcoxon rank-sum test to identify the most differently abundant taxa.

Plasmid pHN6DS2, as a representative IncN1-IncHI2/ST3 plasmid from ST93 E. coli isolate, was selected to extract from the transconjugant using QIAGEN^® Plasmid Midi Kit (Qiagen, Hilden, Germany) and sequenced by Illumina Miseq technology (Illumina, San Diego, CA, United States). Sequence reads were assembled into contigs with SOAPdenovo version 2.04. Nine contigs of pHN6DS2 were assembled into the complete plasmid sequence with PCR amplification and Sanger sequencing (Supplementary Table S3) using related mcr-1-carrying plasmids as references by BLAST³. Analysis and annotation of plasmid pHN6DS2 were performed using the RAST server (Aziz et al., 2008 (link)), ISfinder⁴, ResFinder⁵, RAC⁶, BLAST⁷, and the Gene Construction Kit 4.5 (Textco BioSoftware, Inc., Raleigh, NC, United States). The remaining transconjugants or original isolates containing ∼244.4 kb mcr-1-bearing plasmid were examined for pHN6DS2-like plasmids by PCR and sequencing (Supplementary Table S4).