Antimicrobial resistance phenotypes and microbiological characteristics of E. coli isolates from pig farms in China. Between 1 October 2018 to 30 September 2019, a total of 2693 samples from pigs and breeding environments in 67 pig farms in all 31 provinces of mainland China were collected for E. coli isolation, and a total of 1871 E. coli strains including 1108 strains from pig samples (anal swabs and/or diarrheal feces) and 763 strains from environmental samples (swabs of drinking and/or fecal slurry, floors, drinking and/or food troughs) were finally obtained (Fig. 1A). Initially, we attempted to include more farms in each of the provinces, but the sudden outbreak of African Swine Fever (ASF) in late 2018 and the coronavirus disease (COVID-19) in late 2019 made it extremely difficult to get more samples from more farms. By testing the minimum inhibitory concentrations (MICs) of 28 types of antimicrobials belonging to aminoglycosides (amikacin [AMK], gentamicin [GEN], tobramycin [TOB]), carbapenems (imipenem [IPM], meropenem [MRP], ertapenem [ETP]), cephalosporins (cefazolin [CFZ], cefuroxime [CFX], cefoxitin [FOX], ceftazidime [CAZ], ceftriaxone [CRO], cefepime [CPM]), β-lactam combination agents (amoxicillin/clavulanate [AMC], ampicillin/sulbactam [AMS], piperacillin/tazobactam [PTZ]), monobactams (aztreonam [AZM]), phenicols (chloramphenicol [CHL]), tetracyclines (tetracycline [TET], minocycline [MIN], tigecycline [TGC]), fluoroquinolones (moxifloxacin [MXF], ciprofloxacin [CIP], levofloxacin [LVX], norfloxacin [NOR]), sulfonamides (trimethoprim/sulfamethoxazole [SXT]), fosfomycins (fosfomycin [FOS]), nitrofurantoins (nitrofurantoin [NIT]), and polymyxins (colistin [CL]) on all these 1871 isolates, the resistant phenotypes of E. coli strains in China were characterized (Figs. 1B & C). The results revealed that resistance to TET (percent resistant strains: 96.26%, n = 1801), CHL (82.04%, n = 1535), MXF (81.56%, n = 1526), and SXT (80.38%, n = 1504) were broad phenotypes of E. coli isolates from pig farms in China (Fig. 1C). In particularly, a number of E. coli isolates from pig farms were resistant to colistin (3.79%, n = 71), carbapenems (IPM [2.62%, n = 49], MRP [2.30%, n = 43], ETP [2.46%, n = 46]), and broad-spectrum-cephalosporins (CRO [29.56%, n = 553], CPM [14.00%, n = 262]) (Fig. 1C). Many isolates showed resistance to TGC (37.31%, n = 698), but most of them with MIC values ranging from 0.5 µg/ml to 1 µg/ml (92.98%, n = 649), only a very small proportion of them showed high-level of resistance (MIC ≥ 4 µg/ml; 0.72%, n = 5) (Figs. 1B & C). When comparing isolates from different samples of collection, resistance rates of isolates from pigs against SXT, GEN, CIP and NOR were significantly higher than those of the isolates from environmental samples, while isolates from environmental samples had significantly higher resistance rates against TGC, AMS, and AMC compared to isolates from pigs (Supplementary materials Figure S1).
The E. coli isolates from pig farms in different regions in China showed serious phenotypes of multidrug resistance and even extensively drug-resistance. More than 70% of the isolates were resistant to more than three of the twelve (sub-)classes (aminoglycosides, carbapenems, cephalosporins, β-lactam combination agents, monobactams, phenicols, tetracyclines, fluoroquinolones, sulfonamides, fosfomycins, nitrofurantoins, and polymyxins) of antibiotics tested (Figs. 2A & B). The mainland China consists of a total of 31 provinces, and MDR-E. coli isolates were determined in all 31 provinces, while XDR-E. coli isolates were determined in 29 provinces (except Sichuan and Tibet; Figs. 2C ~ E). In Sichuan and Tibet, 69.44% and 42.86% of the isolates from pig farms were resistant to more than three (sub-)classes of antibiotics, respectively. Notably, in Beijing and Ningxia, all isolates from pig farms displayed multidrug resistance (Figs. 2D & E). In particular, carbapenem-resistance isolates were found in 7 provinces but most of them were isolated from Henan province; isolates resistant to colistin were found in 12 provinces and most of them were also recovered from Henan province (Figs. 2B & C). TGC-resistant E. coli were found in 28 provinces including Tibet (Fig. 2D), but high-level TGC-resistant strains (MIC value ≥ 4 µg/ml) were only determined in Anhui, Hunan, Guizhou, Hebei and Hubei (Fig. 2C). E. coli strains resistant to broad-spectrum-cephalosporins (CRO and CPM) were isolated from pig farms in 30 and 24 provinces, respectively (Figs. 2C & D). Tibet was the only region where no strains from pig farms with the above particularly mentioned resistant phenotypes being detected (Figs. 2C & D).
Determination of putative pathogenic XDR- E. coli isolates. To understand the genomic characteristics of drug-resistant E. coli isolates from pig farms in different regions of China, we selected isolates (totally 515) with resistant phenotypes to either carbapenems (n = 49), CL (n = 71), TGC (MIC value ≥ 4 µg/ml; n = 5), or broad-spectrum-cephalosporins (n = 495) for next-generation sequencing (NGS). These isolates also displayed resistance to aminoglycosides (n = 334), phenicols (n = 476), tetracyclines (n = 510), fluoroquinolones (n = 473), sulfonamides (n = 452), and/or nitrofurantoins (n = 59) (Fig. 3A; Supplementary materials Table S1). In silico serotyping using the whole genome sequences identified 101 kinds of O-serogroups for the sequenced isolates, and O9a (n = 50), O101 (n = 46), and O8 (n = 39) were the predominant types (Fig. 3A; Supplementary materials Table S1). Notably, many O-serogroups which might have public health significance were also determined, including O101, O128ac, O11, O136, O28ac, O103, O149, O15, O45, O125ab, O9, O115, O159, O73, O25, O26, O29, O6, O8, O80, O143, O148, O153, O157, O166, O167, O78, O86, and O91 (Fig. 3A; Supplementary materials Table S1). Multilocus sequence typing (MLST) analyses revealed that these MDR-isolates belonged to 118 different sequence types (STs) and ST10 (n = 52), ST101 (n = 39), ST48 (n = 22) as well as ST5229 (n = 20) were the broadly determined STs (Fig. 3B). Interestingly, there was little or no correlation between the phylogenetic groups, STs, O-serotypes, and the place of isolation (Supplementary materials Table S1).
We next analyzed the virulence factors encoding genes (VFGs) carried by the MDR/XDR-E. coli isolates in this study. According to prediction, each of the isolates contained numerous VFGs (numbers of VFGs ranged from 78 to 284; Supplementary materials Table S2). Of particularly note were astA, eae, east1, ecpABCDER, efa1, eltAB, escCDFJNRSTUV, espABD, estIa, paa, pic, stb, stx2eB, tir, toxB (Fig. 3A; Supplementary materials Table S2). These genes encode important adherence factors and/or toxins, and E. coli isolates possessing these VFGs are assigned as pathogenic E. coli 15. In particular, these VFGs were determined in MDR/XDR-E. coli isolates with O-serotypes (O6, O8, O9, O11, O15, O25, O26, O28ac/O42, O29, O45, O73, O78, O80, O86, O91, O101, O103, O115, O125ab, O128ac, O136, O143, O148, O149, O153, O157, O159, O166, O167) that have public health significance (Fig. 3A).
Genomic associations with antimicrobial resistance phenotypes. We first searched for the presence of known acquired antimicrobial resistance genes (ARGs) in the genomic sequences. This approach led to the detection of 109 kinds of ARGs, including 28 aminoglycoside resistance genes, 35 β-lactam resistance genes, 7 phenicol resistance genes, five tetracycline resistance genes, ten quinolone resistance genes, as well as three sulfonamide resistance genes, seven macrolide resistance genes, two rifampicin resistance genes, seven trimethoprim resistance genes, two fosfomycin resistance genes, and four lincosamide resistance genes (Supplementary materials Table S3). In particular, blaCTX−M−55 and blaTEM−1B were most-frequently determined ESBL genes in E. coli isolates (blaCTX−M−55 was carried in 209 sequenced isolates while blaTEM−1B was carried in 181 ones) from pig farms in China (Fig. 4A); while qnrS1, oqxB, and oqxA were most-frequently determined quinolone resistance genes (presence in 233, 114, and 109 sequenced isolates, respectively; Fig. 4B). Among the tetracycline-resistant isolates, tet(A) and tet(M) were most-frequently determined resistance genes (presence in 453 and 170 sequenced isolates, respectively; Fig. 4C). Notably, three carbapenem resistance genes (blaNDM−1, blaNDM−5, blaNDM−7), two colistin resistance genes (mcr-1.1, mcr-3.1), and one tetracycline resistance genes (tetX4) were determined, and they conferred resistance to carbapenems, colistin, and tigecycline, respectively (Figs. 4C ~ E). Over 15% NDM-producing isolates carried colistin resistance gene mcr-1, while only one isolates carried both colistin resistance gene mcr-1 and high-level tetracycline resistance gene tetX4. We also observed there was a strong correlation between the presence of ARGs and the expected resistance phenotypes. Only carbapenem resistance isolates harboring the three carbapenem resistance genes, while only isolates with high level resistance to tigecycline (MIC value ≥ 4 µg/ml) containing tetX4; in addition, the mcr genes were only found in isolates with resistance phenotypes to colistin (Fig. 4).
We also detected the point mutations associated with AMR in the E. coli isolates from pig farms. Point mutations were determined in gyrA, gyrB, parC, parE, pmrA, pmrB, folP, ampC, rpoB, 23SrRNA, 16S_rrsB, 16S_rrsC, and 16S_rrsH (Supplementary materials Table S4). In addition to RNA mutations observed in 23SrRNA, 16S_rrsB, 16S_rrsC, and 16S_rrsH, point mutations in parC were determined in most of the pig farm E. coli isolates in China (n = 514) while mutations in ropB were determined in small numbers of E. coli isolates (n = 22) (Fig. 5A). Point mutations caused 31 types of amino acid changes in GyrA, and “S83L” (n = 301) as well as “D87N” (n = 203) were the most common mutations (Fig. 5B). In GyrB, point mutations caused 21 types of amino acid changes, and “A618T” (n = 18), “E219K” (n = 18), “H652R” (n = 17), and “S492N” (n = 15) were the most common mutations (Fig. 5C). Point mutations caused 39 types of amino acid changes in ParC, and “E62K” (n = 514) as well as “S80I” (n = 237) were the most common mutations (Fig. 5D). In ParE, point mutations caused 29 types of amino acid changes, and “S458A” (n = 53) was the most common mutation (Fig. 5E). In PmrA, point mutations caused 11 types of amino acid changes, and “G144S” (n = 30) was the most common mutation (Fig. 5F). Point mutations caused 22 types of amino acid changes in PmrB, and “D283G” (n = 247), “Y358N” (n = 224) as well as “H2R” (n = 109) were the most common mutations (Fig. 5G). In FolP, point mutations caused 14 types of amino acid changes, and “F4Y” (n = 20) was the common change (Fig. 5H). In AmpC, point mutations caused 23 types of amino acid changes, and deletion of arginine at site 24 (R24*; n = 18) was the common change (Fig. 5I). In RpoB, point mutations caused 19 types of amino acid changes (Fig. 5J)
Genetic basis of the ESBL, fluoroquinolone-resistance, blaNDM, mcr, and tetX genes transmission. To understand the genetic basis of the ARG transmission, we next analyzed the presence of ARG-associated plasmids. This approach led to the detection of 53 groups of plasmid replicons (Supplementary materials Table S5). We found a ColRNAI type plasmid was presented in most of the E. coli isolates (n = 272) from pig farms in China, followed by IncFIB (n = 266), and IncX1 (n = 236) (Fig. 6A). We determined a total of 53 groups of plasmids in ESBL-genes carrying isolates (n = 495), quinolone-resistance-genes carrying isolates (n = 473), and all tet-carrying isolates (n = 510) from pig farms in China, respectively; and each of the isolates contained 0 to 14 of the reference plasmids (Figs. 4A ~ C; Supplementary materials Table S5). Plasmids including ColRNAI, IncFIB, and IncX1 were observed in most of the ESBL-genes carrying isolates [ColRNAI (260/495), IncFIB (257/495), IncX1 (228/495)], quinolone-resistance-genes carrying isolates [ColRNAI (248/473), IncFIB (257/473), IncX1 (228/473)], as well as the tet-carrying isolates [ColRNAI (270/510), IncFIB (265/510), IncX1 (235/510)] from pig farms in China. However, plasmids including Col(MP18), Col3M, IncB/O/K/Z, and IncU were rarely determined in these isolates. Short-read mapping of all blaNDM-carrying isolates from pig farms in China (n = 45) determined 34 groups of plasmids, and each of the isolates carried 2 to 14 of the reference plasmids (Fig. 4D; Supplementary materials Table S5). Most blaNDM-carrying isolates carried IncFII(pHN7A8) (27/45), ColRNAI (25/45), IncX3 (24/45), Col(MG828) (21/45), and Col156 (20/45). However, some plasmids such as Col(BS512), Col3M, IncFIB(Mar), IncFIB(pHCM2), IncFII(pCoo), and IncP1 were found only rarely. A total of 41 groups of plasmids were determined in the mcr-carrying isolates (n = 69), and each of the isolates carried 3 to 14 of the reference plasmids (Fig. 4E; Supplementary materials Table S5). Most mcr-carrying isolates carried ColRNAI (50/69), IncX1 (39/69), RepA (36/69), IncHI2 (36/69), and IncHI2A (36/69); while several ones including Col(KPHS6), Col(Ye4449), IncFII(pCoo), and IncP1 were rarely found.
We generated the complete genome sequences of the blaNDM-, mcr-, and/or tetX4-carrying plasmids by Oxford Nanopore Sequencing (ONT). We obtained an 85.9-kb IncFII-type-blaNDM−1-carrying plasmid (designated pXD33-05) and a 33.3-kb IncX4-type-mcr-1-carrying plasmid (designated pXD33-06) from an isolate XD33 co-producing NDM and MCR (GenBank accession no. JAENDM000000000). Interestingly, sequence alignments revealed that a pXD33-05-like plasmid and a pXD33-06-like plasmid were also presented in the other isolates co-producing NDM and MCR (Supplementary materials Figure S2). Sequence alignment also revealed that blaNDM−1-carrying plasmid pXD33-05 was highly homologous to a blaNDM−1-carrying plasmid pHNEC55 (GenBank accession no. KT879914) (Fig. 6B). The average nucleotide identity (ANI) between the backbones of pXD33-05 and pHNEC55 was higher than 99%. However, the MDR elements between the two plasmids were different. Structurally, the MDR elements of pXD33-05 consisted of two ARG cassettes, including a 7.6-kb cassette harboring a bleomycin resistance gene, a aminoglycoside resistance gene aph(3')-VI and blaNDM−1 as well as a 2.4‐kb one harboring a aminoglycoside resistance gene rmtB and a ESBL-encoding gene blaTEM−1B (Fig. 6B). The 7.6‐kb cassette was flanked by an IS6 and an IS3 elements, while the 2.4-kb cassette was flanked by a Tn3 and an IS6 elements. Sequence comparisons showed that the mcr-1-carrying plasmid pXD33-06 was highly homologous to plasmid pWI2-mcr (GenBank accession no. LT838201) (Fig. 6C). However, it displayed little homology to the high-impact mcr-bearing plasmid pHNSHP45 (GenBank accession no. KP347127) reported in China 4 (Fig. 6C). In addition to mcr-1, no other ARGs were found on pXD33-06 (Fig. 6C). We also analyzed the genetic environments of tetX4 which mediates resistance to high level tigecycline in E. coli isolates from pig farms in China. ONT sequencing on two high-level-TGC resistant isolates HB50 and SY36 revealed that tetX4 was carried by an IncX1 plasmid in both isolates, which was highly homologous to a previously reported E. coli tetX4-harboring plasmid pYY76-1-2 (GenBank accession no. CP040929) 16 (Fig. 6D). In all determined tetX4-carrying elements, tetX4 was adjacent to an ISCR2 element (Fig. 6D). Plasmid conjugation experiments revealed that most of the blaNDM-, mcr-, and/or tetX4-carrying plasmids were conjugative and conferred phenotypes of carbapenem-, colistin-, and high-level tigecycline (MIC value ≥ 4 µg/ml) resistance to the bacterial receipts, respectively (Supplementary materials Table S6).
High genetic propensity of farm sourced XDR- E. coli in spreading into humans. To determine the genetic propensity of the MDR/XDR-E. coli isolates to spread into the human sector, the genetic relatedness of the 515 MDR/XDR-E. coli isolates from pig farms in China to 287 publicly available draft genomes of human commensal E. coli (Bioproject no. PRJNA4001047) were investigated 17. The 802 E. coli isolates were phylogenetically divided into three lineages (Fig. 7A), and the 515 MDR/XDR-E. coli isolates from pig farms displayed a close relatedness to the 287 human E. coli strains (Fig. 7B). A large proportion of pig-farm originated MDR/XDR-E. coli isolates showed high genetic similarity (443/515, differed by only less than 1000 SNPs) to the human originated E. coli strains in China (Fig. 7; Supplementary materials Table S7). Of particularly concern is that 44.27% (228/515) of the MDR/XDR-E. coli isolates from pig farms differed by only less than 100 SNPs (as small as 3 SNPs) from the human E. coli isolated in China (Fig. 7; Supplementary materials Table S7).