The study protocol and swine studies were approved by the Animal Care and Use Committee of Shandong Agricultural University, Tai'an, China. Human sample collection was carried out in accordance with the approved guidelines of the Ethics Committee of Tai'an City Central Hospital during routine checkups by medical professionals. All the subjects gave written informed consent in accordance with the Declaration of Helsinki.

As shown in Table 1, a total of 325 swine samples were collected from 5 fattening swine farms of 3 cities in Shandong Province from January to August 2018, with a sample and region distribution of the following: 230 from Tai'an City [100 in Daiyue District 1 (TDY1), 60 in Daiyue District 2 (TDY2), and 70 in Xin'tai District (TXT)], 50 in Laiwu District of Jinan City (JLW), and 45 from Jining City (JN). In the meantime, 40 non-repetitive TE-resistant E. coli strains were selected randomly from the 236 TE-resistant E. coli strains of clinical patient samples from 1 hospital in Tai'an from December 2017 to February 2018. The cases in this hospital came from Tai'an and surrounding prefecture-level cities, which are the same as the swine-sourced cases. In addition, these strains were identified using the Vitek-2 Compact Automatic Microbiology Analysis System (Biomérieux, Marcy-l'Étoile, France) in accordance with the standards of the American Society for Clinical and Laboratory Standardization Institute (CLSI) (19).

For the analyses of 146 isolated strains, the antimicrobial susceptibility testing was performed using the Kirby–Bauer disk (purchased from Thermo Fisher Scientific, Shanghai, China) diffusion method to test 29 commonly used antibiotics, including amikacin (AK), gentamicin (CN), imipenem (IPM), meropenem (MEM), cefazolin (KZ), ceftazidime (CAZ), cefotaxime (CTX), cefepime (FEP), aztreonam (ATM), ampicillin (AMP), ampicillin/sulbactam (SAM), piperacillin tazobactam (TZP), ciprofloxacin (CIP), levofloxacin (LEV), tetracycline (TE), chloramphenicol (C), piperacillin (PRL), cotrimoxazole (trimethoprim/sulfamethoxazole, SXT), moxifloxacin (MXF), colistin B (PB), and amoxicillin with clavulanic acid (AMC), methicillin (MET), cefuroxime (CXM), ceftriaxone (CRO), tobramycin (TOB), cefoxitin (FOX), cefoperazone/sulbactam (SCF), ertapenem (ETP), and tigecycline (TGC). The results of the minimal inhibitory concerting (MIC) of antibacterial drugs were interpreted according to the CLSI and were classified as sensitive, drug resistance, and intermediary, with the sensitive laboratory E. coli strain DH5α as a negative control (19). The MIC₉₀ values of antibiotics of 146 E. coli strains were shown in Supplementary Tables 1, 2. In this study, we defined the strains with resistance to three or more classes of antimicrobials (β-lactams as one class) as MDR strains and identified the synergistic effect among AMC, CTX, and FEP as the sign of ESBL (20).

For genotypic analyses of 146 isolated strains in this study, the bacterial DNA templates were prepared by the boiling method as follows: adding 10 μl of single bacteria into a 1.5-ml EP tube with the pre-added 300 μl of sterile water, heating at 105°C in a metal bath for 10 min, centrifugating at a speed of 12,000 r/min for 3 min, transferring the supernate into a new centrifuge tube as the bacterial nucleic acid template, and then storing at −20°C until use. PCR was performed as described previously (21–26) (Table 2) to detect the five genes for SXT resistance (qacEΔ1-sull for quaternary ammonium compounds (QACs-) sulfonamide-related gene and dfrA 1, dfrA 5, dfrA 12, and dfrA17 for four dihydrofolate reductase coding genes), eight TE-resistant genes (tetA, tetB, tetC, tetW, tetO, tetK, tetL, and tetM), four plasmid-mediated quinolone-resistant genes (PMQR) [qnrA, qnrB, qnrS, and aac (6′)-Ib-cr], three C-resistant genes (cat1, cmlA, and flor), and β-lactamase genes (bla_TEM, bla_SHV, bla_CTX−M, and bla_DHA). Due to the overlapping of 3′ end of QAC resistance gene qacEΔ1-sul1 with the first two codons of dihydropteroate synthase- (DHPS-) encoding gene sul1, only a pair of primers were designed in this study to complete the amplification of qacEΔ1-sul1 gene (21). The sequences of primers and annealing temperature used to test the presence of genes are described in Table 2. All PCR amplificons were sequenced by Sangon Biotech Co., Ltd., Shanghai, China, and the obtained DNA sequences were sequenced by BLAST with database at the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/genbank/).

For the MLST analysis of the isolated E. coli strains, the internal fragments of seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA) were amplified by PCR from bacterial DNA (the primers are indicated in Supplementary Table 3), and the resultant sequences were imported into the E. coli MLST database website (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli/documents/primers Coli_html) to determine sequence types (STs) and clonal complexes (CCs) (Supplementary Tables 4, 5). The clustering of the different ST types of E. coli was carried out using the eBURST v3.0 software, which could reveal the existence of the same origin strains among distinct strains of various geographical sources as described previously (27).

As shown in Table 1, a total of 106 E. coli strains were isolated from 325 fattening swine farm samples with a contamination rate of 32.62%. Of these samples, the contamination rate by JLW and JN samples was the highest (40%) followed by TDY2 samples (35%), TXT samples (31%), and TDY1 samples (25%).

As indicated in Figure 1A, 21 antibiotics were tested and 13 kinds displayed high resistance to E. coli strains in swines as TE was the highest one (97.17%), followed by C (93.4%), AMP (89.62%), PRL (85.85%), and SXT (80.19%). The rates of the resistance to E. coli strains in other swines were lower than KZ (38.68%), SAM (25.47%), MXF (23.58%), CIP (22.64%), LEV (21.7%), CN (3.77%), CTX (2.83%), and ATM (1.87%). Three kinds of antibiotics such as PB (40.56%), TZP (0.94%), and AMC (0.94%) were the intermediate, while five kinds of antibiotics such as FEP, AK, IPM, MEM, and CAZ were sensitive (Figure 1).

In addition to TE (100%), the other 22 antibiotics with high resistance to human-derived E. coli strains were AMP (90%), followed by MET (82.5%), SAM (75%), CXM (60%), KZ (57.5%), LEV (55%), CIP (55%), CTX (52.5%), CRO (52.5%), FEP (52.5%), ATM (45%), TOB (40%), CN (37.5%), CAZ (20%), FOX (15%), TZP (7.5%), and SCF (5%). In addition, the other five species (i.e., ETP, AK, MEM, TGC, and IPM) were sensitive (Figure 1B). The results of antibiotic susceptibility rates of the two sources of E. coli were summarized in Table 3.

Swine-sourced drug resistance spectrum could be divided into 35 spectral types, 15 stains with 7 drug resistance/KZ+AMP+PRL+PB+SXT+C+TE and 15 stains with 5 drug resistance/AMP+PRL+SXT+C+TE being the most popular drug-resistant ones (Supplementary Table 4). The strains of seven drug resistance antibacterial spectra involved in seven ST types, which were distributed in all the five detection regions, while five drug resistances involved six ST types, which were also distributed in all the five detection areas (Supplementary Table 4). The two kinds of drug resistance antibacterial spectra were 28.30%. The other strains had more than three drug resistance types, except of No. 3 strain (from JLW and TE), No. 45 strain (from JN and C), and No. 103/94 strain (from TDY2) (-) only with one drug resistance (Supplementary Table 4).

The drug-resistant spectrum of the human source could be divided into 34 spectral types, with KZ+CTX+FEP+AMP+CIP+MET+CM+CAZ+TOB+SAM+ATM+LEV+TE being the most popular drug-resistant one (Supplementary Table 5).

Among the 106 E. coli strains isolated from swines, 98 strains displayed MDR (i.e., resistance to three or more classes of antibiotics at the same time) accounting for 92.45% of the total isolates. Among them, the four classes of antibiotic-resistant (4R) strains were the most common ones, accounting for 32.08% (34/106), followed by strains resistant to the five classes of antibiotics (5R) accounting for 29.25% (31/106), resistant to the six classes of antibiotics (6R) occupying 20.75% (22/106), and the seven classes of antibiotics (7R) with 0.94% (1/106), respectively (Figure 2).

Among the randomly selected 40 TE-resistant human E. coli strains, 31 strains exhibited multi-drug resistance, accounting for 77.5% of the total isolates. The strains resistant to 4R and 5R were the most common ones, each accounting for 27.5% (11/40), followed by the strains resistant to 6R, occupying 7.5% (3/40) (Figure 2).

About 26 genotypes of 5R commonly used in a clinic were studied, and the results revealed that the detection rate of Cs-resistant flor gene (100%) was the highest one in E. coli of swine origin, followed by bla_TEM gene (99%) of β-lactamases and cmlA gene of C (97.17%), tetW genes (96.22%) and tetC gene (95.28%) of TEs, and the qacEΔ1-sulI gene of SXT (93.4%) and quinolones aac (6′)-Ib gene (93.4%) (Figure 3). Meanwhile, the detection rate of bla_TEM gene (100%) of β-lactamases was the highest in human E. coli, followed by tetC gene of TEs (97.5%), 90% of drfA 17 gene of SXT, and 90% of tetA gene of TE (Figure 3).

There are 25 MLST genotypes in the 106 E. coli strains, with ST1258, ST361, and ST10 being the dominant strains. ST1258 was the most popular strain type among all the tested E. coli strains, which were detected in each swine farm. A total of 10 strains of ST361 were detected in 4 regions [JLW (1), TXT (2), TDY1 (3), and TDY2 (4)] (Supplementary Table 4). There are 19 MLST genotypes in 40 strains of human E. coli from Shandong Province, with ST1193, ST73, ST648, ST131, ST10, and ST1668 being the dominant strains. ST1193 covered five strains (12.5%), followed by ST73, ST648, and ST131 each containing four strains (each accounting for 10.00%), ST10 and ST1668 each containing three strains (each occupying 7.50%), ST457, ST393, ST69, and ST617 each containing two strains (each occupying 5%), while each of the other nine genotypes contained one strain (Supplementary Table 5).

The cluster analysis of swine E. coli isolates showed that the 25 different ST types can be classified into 3 CCs, namely CC10 [ST10 (7) and ST48 (1)], CC155 [ST58 (3)], and CC23 [ST410 (2)], and the other 21 ST types including no CCs. CC10 contains seven strains, including ST10 [JN (1), TDY1 (5), and TDY2 (1)] and ST48 [JN (1)]. The three ST58 swine E. coli strains derived from TDY2 belonged to the group CC155, while the other two ST410 swine E. coli strains derived from TDY2 belonged to the group CC23 (Supplementary Table 4).

There were 10 CCs in 40 human E. coli strains such as CC14 (ST1193), CC73 (ST73), CC648 (ST648), CC131 (ST131), CC10 (ST10, ST617, ST6896, ST5296, and ST710), CC31 (ST393), CC69 (ST69), CC38 (ST2003), CC95 (ST2619), and CC23 (ST88). CC10 contains multiple ST types, such as sputum ST10 (2), urine ST10 (1), sputum ST617 (1), and blood ST617 (1), sputum ST6896 (1), blood ST5296 (1), and urine ST710 (1), respectively. Two ST393 from the urine and blood each belonging to CC31. One strain of ST 88 detected from the sputum belongs to CC23 (Supplementary Table 5). Moreover, the results of the cluster analysis showed that CCl0 and CC23 with different ST types were the common CCs between the two sources of E. coli. CC10 was the most important one, including six ST types [swine E. coli [ST10 (7) and ST48 (1)], while human CC10 contained more ST types, such as [ST10 (3), ST617 (2), ST6896 (1)], ST5296 (1), and ST710 (1)], accounting for 0.11% (16/146).

Finally, a strain evolution diagram was constructed using the eBURST v3.0 software in accordance with the MLST analysis and identification of the 106 strains of E. coli from swines and 40 strains from human sources, as shown in Figure 4. Figure 4A indicated that ST10 is the common ancestor of each ST type of E. coli from swines, and 11 single-locus variants (SLVs) are closely related to ST10. ST542 was identified as the SLV of ST4429 and connected. On the other hand, three derived SLVs of ST48 (ST3529, ST58, and ST5420) were identified and linked. This process continued to expand outward, and ST3529 further expanded two SLVs (ST2628 and ST5851). Similarly, ST4417 was identified as the SLV of ST5420 and linked. Three derived SLVs (ST3685, ST767, and ST906) of ST58 were identified and linked. Additionally, ST3685 further extended ST410 outward, identified ST5694 as the SLV of ST906 and linked the identified SLV, and further extended ST1258 outward. Figure 4B demonstrated that ST10 was the common ancestor of each ST type of human E. coli, and eight SLVs were closely related to it. Then, it allocated each SLV of the eight SLVs, marked ST2674 as the SLV of ST710, and linked the marked SLV. Furthermore, two derived SLVs (ST88 and ST5296) of ST2674 were identified and linked. Similarly, three derived SLVs (ST2003, ST7176, and ST69) of ST393 were identified and linked. Additionally, the two derived SLVs (ST1193 and ST73) of ST1668 were identified and linked, and the two derived SLVs (ST131 and ST2619) of ST73 were identified and linked.