Antimicrobial resistance and population genomics of multidrug-resistant Escherichia coli in pig farms in mainland China

Abstract

The expanding use of antimicrobials in livestock is an important contributor to the worldwide rapid increase in antimicrobial resistance (AMR). However, large-scale studies on AMR in livestock remain scarce. Here, we report findings from surveillance of E. coli AMR in pig farms in China in 2018-2019. We isolated E. coli in 1,871 samples from pigs and their breeding environments, and found AMR in E. coli in all provinces in mainland China. We detected multidrug-resistance in 91% isolates and found resistance to last-resort drugs including colistin, carbapenems and tigecycline. We also identified a heterogeneous group of O-serogroups and sequence types among the multidrug-resistant isolates. These isolates harbored multiple resistance genes, virulence factor-encoding genes, and putative plasmids. Our data will help to understand the current AMR profiles of pigs and provide a reference for AMR control policy formulation for livestock in China.

Fig. 1. Isolation and antimicrobial resistance characteristics of E. coli isolates from pig farms in different provinces of China.

a Numbers of E. coli isolates recovered from different types of samples collected from Chinese pig farms. Numbers in brackets refer to the numbers of E. coli isolates recovered from these samples; b Numbers of E. coli isolates resistant to different antibiotic classes tested; c Heatmap showing the percentage of farm-originating E. coli isolates resistant to each of the antibiotics tested; d Percentages of farm-originating E. coli isolates resistant to different antibiotic classes in different provinces of mainland China. AMK amikacin, GEN gentamicin, TOB tobramycin, IPM imipenem, MRP meropenem, ETP ertapenem, CFZ cefazolin, CFX cefuroxime, FOX cefoxitin, CAZ ceftazidime, CRO ceftriaxone, CPM cefepime, AMC amoxicillin/clavulanate, AMS ampicillin/sulbactam, PTZ piperacillin/tazobactam, AZM aztreonam, CHL chloramphenicol, TET tetracycline, MIN minocycline, TGC tigecycline, MXF moxifloxacin, CIP ciprofloxacin, LVX levofloxacin, NOR norfloxacin, SXT trimethoprim/sulfamethoxazole, FOS fosfomycin, NIT nitrofurantoin, CL colistin.

Fig. 2. Distribution and sequence types of multidrug-resistant E. coli isolates from Chinese pig farms.

a A map of China showing the percentages of multidrug-resistant E. coli isolates from pig farms in different provinces of China (“x” refers to percent multidrug-resistant isolates); different colors represent percentages; small circles in green, purple and blue show the distribution of carbapenem-resistant, colistin-resistant, and tigecycline-resistant E. coli isolates, respectively; numbers in small circles represent the numbers of carbapenem-resistant, colistin-resistant, and tigecycline-resistant E. coli isolates, respectively, in different provinces; ESBL-producing E. coli isolates are also indicated. b Analysis of the minimum spanning tree of the 515 sequenced E. coli isolates based on the sequence type. TGC tigecycline, ESBL extended-spectrum beta-lactamase, MLST multilocus sequence typing, ST sequence type.

Fig. 3. Distribution of antimicrobial resistance phenotypes, antimicrobial resistance genes and plasmid replicons.

The phylogenetic tree was generated based on the concatenated multilocus sequence typing (MLST) alleles using fastMLST v0.0.15, followed by multiple sequence alignment using MAFFT v7.407 and phylogenetic inference using FastTree. The tree was visualized using the Interactive Tree Of Life (iTOL v.5). Raw data for the MLST-based tree and its annotation codes are provided in Supplementary Data 11.

Fig. 4. Genetic characteristics of the bla_NDM-carrying, mcr-carrying, and/or tetX4-carrying plasmids identified in this study.

a Circle map showing the structure of the bla_NDM-carrying plasmid pXD33-05; b Circle map showing the structure of the mcr-carrying plasmid pXD33-06; c Circle map showing the structure of the tetX4-carrying plasmid pHB50-tetX4; arrows in purple, green, gray, orange, and blue refer to antimicrobial resistance genes, mobilization-associated genes (including insertion elements), hypothetical protein-encoding genes, functional protein-encoding genes, and type IV pilus formation-related genes, respectively; d Circle map showing sequence alignments of pXD33-05 and those plasmids with a backbone shared with other Enterobacteriaceae bacteria; circles from inside to outside indicate the GC content of pXD33-05 (circle 1), the GC skew of pXD33-05 (circle 2), pXD33-05 (circle 3), the E. coli plasmid pHNEC55 (GenBank accession no. KT879914; circle 4), the E. coli plasmid pHNHNC02 (GenBank accession no. MG197497; circle 5), and the K. pneumoniae plasmid pKPC-L388 (GenBank accession no. CP029225; circle 6); the plasmid backbone regions are highlighted in light green, while the multidrug resistance regions are highlighted in light red; nucleotide identities are shown with different colors below the maps; e Circle map showing the sequence alignments of pXD33-06 and those plasmids with a backbone shared with other Enterobacteriaceae bacteria; circles from inside to outside indicate the GC content of pXD33-06 (circle 1), the GC skew of pXD33-06 (circle 2), pXD33-06 (circle 3), the E. coli plasmid pWI2-mcr (GenBank accession no. LT838201; circle 4), the Salmonella plasmid pSH15G2169 (GenBank accession no. MH522417; circle 5), and the K. pneumoniae plasmid 16BU137_mcr-1.1 (GenBank accession no. MT316509; circle 6); nucleotide identities are shown with different colors below the map.

Fig. 5. Phylogenetic trees of 515 multidrug-resistant E. coli isolates from pig farms together with 287 publicly available draft genomes of human commensal E. coli across China.

a Circular tree showing the phylogenetic relationship of the 802 E. coli isolates. The tree was generated based on the core genome SNPs using the snippy-multi program implemented in Snippy v 4.4.0 (https://github.com/tseemann/snippy), and was rooted on the E. fergusonii sequence. The snippy based tree was inferred using FastTree2, and was visualized using the Interactive Tree Of Life (iTOL v.5). Raw data for the Snippy tree and its annotation codes are provided in Supplementary Data 12; b Venn diagram showing the shared and unique sequence types between pig farm-originating E. coli and human commensal E. coli.