Antimicrobial resistance and population genomics of extensively drug resistant Escherichia coli in pig farms in mainland China: a national wide epidemiological and microbiological study

doi:10.21203/rs.3.rs-502741/v1

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Antimicrobial resistance and population genomics of extensively drug resistant Escherichia coli in pig farms in mainland China: a national wide epidemiological and microbiological study

https://doi.org/10.21203/rs.3.rs-502741/v1

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published 02 Mar, 2022

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Antimicrobial resistance (AMR) is one of the most urgent threats to the global public health, and the expanding use of antimicrobials in food animals is considered as a main reason for the worldwide rapid increasing of AMR. However, AMR in animals in many regions are poorly documented. China is the largest pig-rearing and pork consumption country in the world. In the present study, we identified AMR in pig farms from all provinces (including Tibet and Qinghai) of mainland China by investigation of a common indicator bacterium Escherichia coli from both pigs and the breeding environmental samples. A total of 2693 samples from pigs and environments in 67 pig farms in all 31 provinces of mainland China were collected between 1 October 2018 to 30 September 2019, and a total of 1871 E. coli strains were isolated. By testing the susceptibility of these 1871 E. coli isolates on 28 types of antibiotics that commonly used in both human and veterinary medicine, we found that resistance to tetracycline (96.26%), chloramphenicol (82.04%), moxifloxacin (81.56%), and trimethoprim/sulfamethoxazole (80.38%) were the broad phenotypes among these E. coli isolates from pig farms in China. A proportion of E. coli isolates were resistant to colistin (3.79%), carbapenems (imipenem [2.62%], meropenem [2.30%], ertapenem [2.46%]), and broad-spectrum-cephalosporins (ceftriaxone [29.56%], cefepime [14.00%]). More than 70% of the isolates displayed multidrug-resistant (MDR), and/or extensively drug-resistant (XDR) phenotypes, and MDR/XDR-E. coli was observed in pig farms in all provinces of mainland China. We also systematically revealed the distribution of O-serogroups, sequence types, resistance genes, virulence factors encoding genes, and putative plasmids of MDR/XDR-E. coli in pig farms from different provinces of China, and partially characterized the pathotypes of certain MDR/XDR-E. coli strains. In addition, the genetic transmission basis of the bla_NDM, mcr, ESBL-encoding, fluoroquinolone-resistance, and tetX genes were addressed in this study. Most importantly, we suggested a very high genetic propensity of the pig farm-sourced MDR/XDR-E. coli in spreading into humans. To the best of our knowledge, this is the first study on a national scale that the resistance phenotypes and population genomics of E. coli in pig farms in China are revealed. Our data presented herein will help understand the current profile of AMR in pigs and also provide reference for policy formulation of AMR control action in livestock in China.

Epidemiology

Applied & Industrial Microbiology

General Microbiology

Infectious Diseases

Escherichia coli

pig farms. antimicrobial resistance phenotypes

population genomics

China

Antimicrobial resistance (AMR) is one of the most urgent threats to global public health. Recently, the emergence and rapid increase of multidrug-resistant (MDR), extensively drug-resistant (XDR), and even pan-drug-resistant (PDR) bacteria, particularly those resist the last-resort drugs (carbapenems, colistin, and tigecycline) have catalyzed the serious concern that the world may get its first inkling of impending catastrophe of a return to the pre-antibiotic ^1–4. Several drug-resistant bacteria have received a worldwide great concern, of particular note is Escherichia coli ^1,5,6. This bacterial species is not only a leading cause of foodborne infections, but also represents a major reservoir of antimicrobial resistance genes (ARGs) due to its great capacity to accumulate ARGs, mostly through horizontal gene transfer ⁷. A recent study showed the total economic cost of AMR in Staphylococcus aureus, E. coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa reached $2.8 billion per year in the United States ⁸. In addition, E. coli has been found to play important roles in disseminations of bla _NDM−1, mcr, and/or tet(X3)/(X4); these ARGs mediates resistance to the last-resort drugs (carbapenems, colistin, and tigecycline) for gram-negative bacteria which may lead to non-antimicrobials available in both human and veterinary medicine ^3,4,9. In addition, extended-spectrum beta-lactamase (ESBL)-producing E. coli is also a major global health problem ^1,5.

The large and expanding use of antimicrobials in food animals is considered as a main reason for the worldwide rapid increasing of AMR ¹⁰. In the past two decades, meat production has plateaued in high-income countries but has largely grown in low- and middle-income countries particularly in Asia ¹¹. Under this background, antimicrobials are widely used in livestock either for growth promotion and/or for health maintain. Globally, 73% of all antimicrobials sold on the earth are used in animals raised for food ¹¹. The large and expanding use of antimicrobials can lead to the development of drug-resistant bacteria in the animal guts, and these resistant bacteria can spread to humans ¹. To date, AMR in animals in low- and middle-income countries are poorly documented, and this may be in part due to the absence of systematic surveillance systems and large epidemiological studies ¹¹.

China is the largest developing country in the world. Many provinces in China still belong to the low- and middle-income regions. China is also the number one country for antimicrobials producing, pig rearing and production, and pork consumption in the world ^12,13. Therefore, understanding the current profile of AMR in pig farms in China has a great significance for global AMR surveillance. However, pig producing in China is very complex with different sizes of pig farms and production models in various regions. To date, there is still lack of systemic data reflecting the condition of AMR in the pork production chain in China. Here, we identified AMR in pig farms in most regions in China by investigation of E. coli, which is a commonly used biomarker of AMR in pig farms ¹⁴. To the best of our knowledge, this is for the first time that the pig farms in all regions in mainland China were included for the epidemiological investigation. This study will contribute to understand the current profile of AMR in pigs and also provide reference for policy formulation of AMR control action in livestock in China.

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, stx_2eB, 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, bla_CTX−M−55 and bla_TEM−1B were most-frequently determined ESBL genes in E. coli isolates (bla_CTX−M−55 was carried in 209 sequenced isolates while bla_TEM−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 (bla_NDM−1, bla_NDM−5, bla_NDM−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, bla_NDM, 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 bla_NDM-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 bla_NDM-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 bla_NDM-, mcr-, and/or tetX4-carrying plasmids by Oxford Nanopore Sequencing (ONT). We obtained an 85.9-kb IncFII-type-bla_NDM−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 bla_NDM−1-carrying plasmid pXD33-05 was highly homologous to a bla_NDM−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 bla_NDM−1 as well as a 2.4‐kb one harboring a aminoglycoside resistance gene rmtB and a ESBL-encoding gene bla_TEM−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 bla_NDM-, 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).

In this study, we investigated AMR phenotypes of E. coli isolated from both pig samples and environmental samples in pig farms from all provincial regions in mainland China. As an important natural reservoir of ARGs and a commonly-used biomarker bacteria for monitoring AMR ^7,14, the resistance profile of E. coli from pig farms may reflect the AMR condition in these farms. To the best of our knowledge, this is the first time the AMR phenotypes of E. coli isolates from pig farms in all provinces of mainland China, including Tibet and Qinghai where there were little related data before ¹¹ being reported.

Our determination of resistance phenotypes of E. coli isolates from pig farms in different provinces in China on 28 types of antimicrobials commonly used in both human and veterinary medicine indicated a worrisome condition of AMR in pig farms (Figs. 1&2). MDR and even XDR were the common phenotypes of E. coli isolates recovered in this study, and MDR/XDR-E. coli isolates were widely determined in pig farms in different provinces in whole mainland China, including Tibet, Xinjiang, and Qinghai (Fig. 2). This worrisome condition is widely accepted as the result of overuse and abuse of antibiotics in pig industry in the country ^12,18. During the past decades, along with a rapid increase in economy, the production of meat, eggs, and milk in China has rapidly increased, especially for pork, the main source of animal protein for most Chinese people ^12,19. To meet increasing demand for pork, both the number and the size of pig farms have grown markedly, and a massive amount of antibiotics are used in the country to support its rapid increase in pig production ¹². According to available reports ^13,20,21, major classes of antibiotics extensively used in China’s pig farms include sulphonamides, tetracyclines, fluoroquinolones, macrolides, and β-lactams, and in particular, fluoroquinolones and β-lactams contributed more than half. Consistently, resistance to antibiotics belonging to those antibiotic classes were found to be the broad phenotypes for E. coli isolates from pig farms in China (Fig. 2).

Among the drug-resistant E. coli determined in pig farms in China, of particular note are the isolates with resistance phenotypes to carbapenems, CL, and TGC (Fig. 2). In particular, over 15% NDM-producing isolates carried CL resistance gene mcr-1. All of these antibiotics are proposed as the last-resort antibiotics for treating infections caused by MDR Gram-negative bacteria ^4,22. According to the recent official policy, the carbapenem antibiotics are not approved to be used in livestock in China, and it remains to be elucidated why E. coli isolates with resistance phenotypes to these antibiotics are recovered from pig farms. Notably, bacterial isolates with resistance phenotypes to carbapenems have also be recovered from poultry farms in China ²³. Although we cannot exclude the possibility that some pig farms might secretly use carbapenem antibiotics without receiving approval, a more preferrable possibility for the acquisition of these phenotypes is due to contaminated in-house environment ²³. The use of carbapenems in Chinese hospitals might cause the contamination of carbapenem-resistant bacteria or mobile carbapenem-resistant genes (e.g., bla_NDM) in environments (e.g., water, air, etc.). These resistant bacteria or ARGs may spread to livestock farms and in turn, lead to the emergence of carbapenem-resistant bacteria in animals. It was worth note that most of the carbapenem-resistant E. coli isolates recovered from pig farms in China in this study carried the NDM-producing gene bla_NDM (Fig. 4D). Continuous monitoring the persistence and spread of carbapenem-resistant bacteria in animals particularly food producing animals is essential, as these bacteria may represent high risks to human health.

The recovery of CL-resistant E. coli from pig farms might also due to the acquisition of CL-resistant mcr gene from the environment. Although the colistin withdrawal policy in 2017 and the decreasing use of colistin in agriculture have had a significant effect on reducing colistin resistance in both animals and humans in China ²⁴, plasmid-mediated CL resistance mcr genes might also persist in the environments of livestock farms ^23,25. The persistence of the mcr genes may contribute to the dissemination of CL-resistant bacteria. In agreement with this hypothesis, the plasmid-mediated colistin resistance genes mcr-1 and mcr-3 were widely determined in E. coli isolates with resistance phenotypes to CL (Fig. 4E). Notably, CL-resistant E. coli were found in pig farms in 11 provinces of China in this study, suggesting a still worrisome condition for the persistence and dissemination of CL resistance. More active actions should be taken to solve the problem and continuous monitoring is still necessary ²⁴. Although a large number of E. coli isolates from pig farms in China showed resistance to TGC (37.31%, n = 698), most of them displayed low-level of resistance (MIC values ranging from 0.5 µg/ml to 1 µg/ml; 92.98%, n = 649), and only five isolates displayed high-level of resistance (MIC ≥ 4 µg/ml; 0.72%) (Figs. 1B & C). However, the previously reported high-level TGC conferring gene tetX4 ³ was only found in high-level TGC resistant isolates. Instead, several tet genes were widely detected in low-level TGC resistant isolates (Figs. 1B & C). Phenotypes of low-level TGC resistance might be conferred by these tet genes such as tetA, tetB, tetC, and/or tetM ²⁶.

By performing whole genome sequencing, the population genomics of MDR/XDR-E. coli in pig farms in China was revealed. During the analyses, we characterized several O-serogroups that are frequently associated with E. coli pathotypes including Enterotoxigenic E. coli (ETEC), Enteropathogenic E. coli (EPEC), Enterohaemorrhagic E. coli (EHEC), Enteroaggregative E. coli (EAEC), Enteroinvasive E. coli (EIEC), and Diffusely adherent E. coli (DAEC) ^27,28 (Fig. 3). Importantly, several marker VFGs of E. coli pathotypes (e.g., astA, eae, east1, stb, stx_2eB, toxB, etc.^27,28) were also determined in E. coli isolates belonging to these O-serogroups (Fig. 3). The wide presence of these MDR/XDR-E. coli in pig farms represents a great health risks and should receive more attentions. Determination of ARGs identified mobile genes conferring resistance to the tested antibiotic classes in E. coli isolates in pig farms in China (Fig. 3; Supplementary materials Table S3). In addition, a large number of macrolide-resistance genes were also determined. These findings suggest that the extensive use of sulphonamides, tetracyclines, fluoroquinolones, macrolides, and β-lactams in China’s pig farms facilitates the acquisition of related resistance genes in E. coli in farms. Along with the detection of multiple ARGs, we also detected many putative plasmids in E. coli isolates from pig farms in China (Supplementary materials Table S4). Several groups of these plasmids (e.g., IncA/C, IncB/O, IncFIB, IncX1, IncFIIA, IncX2, IncY, etc.) are capable of carrying transfer, MDR, and virulence functions in broad-host-range ²⁹. The presence of those plasmids may accelerate the dissemination of ARGs and even VFGs as well. We also determined several groups of plasmids that were rarely associated with the spread of specific ARGs. For example, the IncX3 plasmid has been reported to be account for majority of bla_NDM carriage in livestock farms ^23,30. However, we determined an IncFII-type-bla_NDM-carrying plasmid (Fig. 6B), and this type of plasmid was observed in all E. coli isolates co-producing NDM and MCR (Supplementary materials Figure S2). In addition, the mcr-carrying plasmid determined E. coli isolates co-producing NDM and MCR in this study was also different from the plasmid mediating the dissemination of mcr in China reported recently ⁴ (Fig. 6C). The presence of these plasmids makes the dissemination of bla_NDM−1 and/or mcr more heterogeneous. It was worth note that the elements mediating the spread of high-level TGC resistance gene tetX4 in E. coli isolates from pig farms in this study shared highly homologous to those reported previously ^3,17,31 (Fig. 6D), suggesting the dissemination of tetX4 might be not as heterogeneous as bla_NDM and mcr. However, continuous monitoring should be taken in the future. Most notably, the MDR/XDR-E. coli isolates from pig farms displayed a very close relatedness to the E. coli strains from humans in China (Fig. 7). Most pig farm-origin E. coli isolates in this study differed by only less than 1000 SNPs to the human-originated E. coli strains, and many differed by only less than 10 SNPs (Fig. 7; Supplementary materials Table S7). These findings suggest a very high genetic propensity of farm sourced MDR/XDR-E. coli in spreading into humans in China.

Although this work has a limitation that we could not include more pig farms in different provinces in China for sample collection due to the outbreak of ASF and COVID-19, our sample collection still covers pig farms in all provinces of China. On a national scale for the first time, we characterized the resistance phenotypes as well as population genomics of E. coli in pig farms in China. Our results revealed a worrisome condition of AMR in pig farms in China and there is still a long way for China to take actions to reduce AMR in livestock. Fortunately, Chinese government has taken a series of active actions to solve the urgent AMR conditions in animal husbandry. A noteworthy action is the Ministry of Agriculture and Rural Affairs (MARA) has issued a policy to ban the addition of antibiotics for promoting animal growth in feed from July 1, 2020 (MARA Announcement No.194, 07-10-2019). In this study, we also systematically revealed the distribution of O-serogroups, sequence types, ARGs, VFGs, as well as putative plasmids of MDR/XDR-E. coli in pig farms in different provinces of China. These data will provide comprehensive insights to help understand AMR in pig farms, and may also be beneficial for the government to make policies for reducing AMR in pig industry in the country. Notably, we also determined many MDR/XDR-E. coli with potential pathogenicity to humans and most importantly, we found there was a very high genetic propensity of pig farm-sourced MDR/XDR-E. coli in spreading into humans. The persistence and dissemination of these isolates represent important health risks and should receive more attentions.

Sample collection, identification, and antimicrobial susceptibility testing

Between 1 October 2018 to 30 September 2019, a surveillance project on AMR in E. coli from pig farms in China was set. In each of the 31 provinces in mainland China, 2 ~ 3 different pig farms were randomly selected to collect swabs from fresh feces and rectal swabs of pigs (with approximately 40 samples per farm), as well as swabs of drinking and fecal slurry, floors, troughs (for each point at least three samples per farm were collected). All samples were shipped with dry ice immediately for E. coli isolation. Swabs were incubated in Luria Bertani (LB) broth (Sigma-Aldrich, MO, USA) at 37°C, 180 rpm, for overnight. Afterwards, swab cultures were streaked on MacConkey agars and were incubated at 37°C for 16 hours. E. coli isolates were confirmed by PCR amplification of the 16S rRNA gene with primers (F: 5’-GAAGCTTGCTTCTTTGCT-3’, R: 5’-GAGCCCGGGGATTTCACAT-3’) documented previously ³². PCR assays amplifying the presence of seven house-keeping genes of E. coli (adk, fumC, gyrB, icd, mdh, purA, and recA) ³³ were set for double confirmation.

The antimicrobial susceptibility of E. coli isolates was determined by testing the MIC values of different kinds of antibiotics on the bacterium, following the Clinical & Laboratory Standards Institute (CLSI, United States) recommended microbroth dilution protocol (CLSI M100, 28th Edition). A total of 28 types of antibiotics (AMK, GEN, TOB, IPM, MRP, ETP, CFZ, CFX, FOX, CAZ, CRO, CPM, AMC, AMS, PTZ, AZM, CHL, TET, MIN, TGC, MXF, CIP, LVX, NOR, SXT, FOS, NIT, CL) were included for the tests. Results were interpreted using the CLSI breakpoints (CLSI M100, 28th Edition). If CLSI breakpoint was not available, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoint (v 8.1) was alternatively used for the interpretation. Each antibiotic was tested with three duplicates. E. coli ATCC 25922 was used as quality control.

Whole genome sequencing and data availability

All E. coli strains with resistant phenotypes to one of the carbapenems tested, broad-spectrum-cephalosporins (ceftriaxone and cefepime), tigecycline, or colistin, were selected for NGS. Bacterial genomic DNA was extracted by using a commercial DNA Kit (TIANGEN, Beijing, China). The quality and concentration of the bacterial genomic DNA was evaluated via electrophoresis on a 1% agarose gel as well as NanoDrop2000 (Thermo Scientific, Waltham, MA, USA) and Qubit 4 Fluorometer (Thermo Scientific, Waltham, USA). Libraries were constructed based on the qualified DNA by using a NEBNext Ultra™ II DNA Library Prep Kit (New England BioLabs, Ipswich, USA), and were sequenced on a NovaSeq 6000 platform using the pair-end 150-bp sequencing protocol (Novogene, Beijing, China). Raw reads with low quality were removed as described previously ³⁴. High-quality reads were de novo assembled via SPAdesv3.9.0 to generate genome contigs.

The complete genome sequences of bla_NDM− 1-, mcr-1-, and/or tetX4-carrying plasmids were generated by ONT sequencing in combination with the Illumina technology. Plasmid DNA was extracted using the phenol-chloroform protocol combined with Phase Lock Gel tubes (Qiagen GmbH) and was detected by the agarose gel electrophoresis as well as quantified by Qubit® 2.0 (Thermo Scientific, Waltham, USA). Libraries for ONT and Illumina sequencing were prepared using an SQK-LSK109 kit and a NEBNext® Ultra™ DNA Library Prep kit, respectively. Prepared DNA libraries were sequenced using Nanopore PromethION platform and Illumina NovaSeq PE150 at Novogene Co. LTD (Tianjin, China), respectively. ONT and Illumina short reads were finally assembled and combined using the Unicycler v0.4.4 software with default parameters.

Whole genome sequences (WGSs) of E. coli isolates have been deposited into GenBank (BioProject accession no. PRJNA688628). GenBank accession numbers are given in Supplementary materials Table S1.

Bioinformatical analysis

ResFinder 4.1 ³⁵ was used to determine putative acquired antimicrobial resistance genes (ARGs) and point mutations associated with AMR. PlasmidFinder 2.1 ³⁶ was used to determine putative plasmids carried by the sequenced strains. In silico serogroups and sequence types (ST) of the sequenced strains were determined by SerotypeFinder 2.0 ³⁷ and multilocus Sequence Typing (MLST) 2.0 ³⁸, respectively. Sequence alignments were performed by using the MAFFT software version 7.471 ³⁹. RAST Sever was used for sequence annotations ⁴⁰. Average nucleotide identities between two genome sequences were calculated by ANI calculator ⁴¹. A comparative genome analysis was performed and visualized using the BRIG package ⁴² and/or the EasyFig package ⁴³. Phylogenetic trees based on whole-genome single nucleotide polymorphisms (WG-SNPs) were generated using Parsnp (version 1.2) software ⁴⁴ and were visualized using Interactive Tree Of Life (iTOL v.5) ⁴⁵. The draft genomes of 287 human commensal E. coli (PRJNA400107) were downloaded from NCBI and included for phylogenetical analysis in this study ¹⁷.

Plasmid conjugation experiments

Plasmid conjugation experiments between carbapenem-resistant E. coli, colistin-resistant E. coli, and/or tigecycline-resistant E. coli (donors) and rifampin-resistant E. coli C600 (recipient) were performed as described previously ²⁵. Briefly, bacterial donor and recipient strains at mid-log phase (OD₆₀₀ = 0.5 ~ 0.6) were mixed at a ratio of 1:3 (v/v). Bacterial mixture was spotted on nitrocellulose membranes that were pre-plated on LB agars. An incubation at 37°C for 12 h was given to each of the plates, and bacteria on the membrane were washed using LB broth followed by being shaken at 37°C for 4 h. Transconjugants were selected on LB agar plates with rifampin (1000 mg/L) plus imipenem (20 mg/L) [to screen carbapenem-resistant transconjugants], or rifampin (1000 mg/L) plus colistin (2 mg/L) [to screen colistin-resistant transconjugants], or rifampin (1000 mg/L) plus tigecycline (4 mg/L) [to screen tigecycline-resistant transconjugants]. Antimicrobial susceptibility of the transconjugants was determined using broth microdilution method as mentioned above.

Supporting Information

All authors declare no competing interests.

Acknowledgements

The authors sincerely acknowledge our colleagues for sample collection. Our work was supported by the National Key R&D Program of China (grant numbers: 2017YFC1600101 and 2017YFC1600103), the earmarked fund for China Agriculture Research System (grant number: CARS-35), the Natural Science Foundation of Hubei Province (grant number: 2020CFB525), China Postdoctoral Science Foundation (grant number: 2018M640719), Walmart Foundation (Project number: 61626817) and Walmart Food Safety Collaboration Center. The funders had no role in the study design, data collection, data analysis, data interpretation, or the manuscript writing.

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There is NO Competing Interest.

TableS1.xlsx
Table S1. General features of sequenced XDR-E. coli isolates from pig farms in China.
TableS2.xlsx
Table S2. Virulence factor-encoding genes (VFGs) carried by the sequenced XDR-E. coli isolates from pig farms in China.
TableS3.xlsx
Table S3. Antimicrobial resistance genes (ARGs) carried by the sequenced XDR-E. coli isolates from pig farms in China.
TableS4.xlsx
Table S4. Point mutations associated with antimicrobial resistance determined in the sequenced XDR-E. coli isolates from pig farms in China.
TableS5.xlsx
Table S5. Plasmid replicons determined in the sequenced XDR-E. coli isolates from pig farms in China.
TableS6.xlsx
Table S6. Phenotypic characteristics of the transconjugants of the carbapenem-resistant, colistin-resistant, and/or high-level-tigecycline-resistant E. coli.
TableS7.xlsx
Table S7. Numbers of single nucleotide polymorphisms (SNPs) determined between the pig farm originated XDR-E. coli isolates and the human commensal E. coli across China.
FigureS1.tif
Figure S1. Resistance rate of E. coli isolates from pigs and/or environmental samples to each of the antibiotics tested in the present study. 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.
FigureS2.tif
Figure S2. Comparison of the 85.9-kb IncFII-type-blaNDM-1-carrying plasmid (pXD33-05; panel A) and the 33.3-kb IncX4-type-mcr-1-carrying plasmid (pXD33-06; panel B) with the genomes of NDM and MCR co-producing E. coli isolates from pig farms in China.
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Antimicrobial resistance and population genomics of extensively drug resistant Escherichia coli in pig farms in mainland China: a national wide epidemiological and microbiological study

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