Multi-Locus Sequence Typing and Drug Resistance Analysis of Swine Origin Escherichia coli in Shandong of China and Its Potential Risk on Public Health

The extensive use of antibiotics has caused antimicrobial resistance and multidrug resistance in Escherichia coli and gradual expands it into a worldwide problem. The resistant E. coli could be transmitted to humans through animal products, thereby creating a problem for bacterial treatment in humans and resulting in a public health issue. This study aims to investigate the molecular typing and drug resistance of swine and human origin E. coli within the same prefecture-level cities of Shandong Province and the potential risk of E. coli on public health. The drug sensitivity results indicated that tetracycline (TE) (97.17%) is a major antibiotic with high drug resistance in 106 swine origin E. coli. There was a significant difference in the drug-resistant genotypes between the two sources, of which the blaTEM positive rate was the highest in the genera of β-lactams (99% in swines and 100% in humans). Among the 146 E. coli isolates, 98 (91.51% swine origin) and 31 (77.5% human origin) isolates were simultaneously resistant to three or more classes of antibiotics, respectively. The multi-locus sequence typing (MLST) results indicate that the 106 swine origin E. coli isolates are divided into 25 STs with ST1258, ST361, and ST10 being the dominant sequence analysis typing strains. There were 19 MLST genotypes in 40 strains of human E. coli from Tai'an, Shandong Province, with ST1193, ST73, ST648, ST131, ST10, and ST1668 being the dominant strains. Moreover, the cluster analysis showed that CCl0 and CC23 were the common clonal complexes (CCs) from the two sources. Our results provide a theoretical basis for guiding the rational use of antibiotics and preventing the spread of drug-resistant bacteria, and also provide epidemiological data for the risk analysis of foodborne bacteria and antimicrobial resistance in swine farms in Shandong Province.


INTRODUCTION
Escherichia coli is one of the most common bacteria in human and animal intestines, which can cause a variety of infectious diseases, such as peritonitis, cholecystitis, cystitis, and diarrhea. Meanwhile, it is also an important health indicator in food (1). In recent years, the unreasonable use of antibiotics, such as aminoglycoside, sulfonamide, and fluoroquinolone, has led to increasingly serious drug resistance of E. coli, particularly the emergence of multi-drug-resistant (MDR) strains and even superbugs, due to the production of extended-spectrum βlactamamide (ESBL), thereby bringing huge economic losses to Chinese animal husbandry and posing a serious threat to human health (2,3). All the main subtypes are the ESBLs producing E. coli isolated from diarrhea swinelets (4) in South Central Taiwan, slaughterhouse healthy swines (5) in central Portugal, farm healthy swines (6) in Denmark, and dairy farms (7) in Israel.
There are many ways for antibiotic residues to enter the environment. Some antibiotics like fluoroquinolones and tetracyclines (TEs) could not be completely metabolized in swines, consequently their residues may be detected in dust, feces, sewage, soil, surface water, and crops (8)(9)(10)(11)(12). These different antibiotic residue pools are ideal breeding grounds for resistance bacteria. The drug resistance of bacteria from edible animals in the process of breeding may be transmitted through the food chain, as well as exemplified by swine breeding, which is a part of human food chain. Unlimited proliferation of super bacteria and the highly frequent occurrence of MDR strains will bring new challenges to the existing medical and healthiness conditions in China (13).
Multi-locus sequence typing (MLST) is a method to accurately record the variations in the bacterial gene level by measuring the nucleotide sequences of four to eight housekeeping genes. MLST technology can conveniently transmit nucleic acid sequences through the internet to analyze the evolution and population biological characteristics of bacteria (14), thereby reflecting their epidemiology, pathogenicity, and evolution (15). Additionally, MLST can also be used to trace the source and spread of drugresistant strains. By using the MLST method and sequence analysis of one or two resistance genes, the E. coli-producing ESBL can be well-distinguished (16).
Previous studies mainly focused on the drug resistance of E. coli in chickens, swines, and other major food animals (17,18) while the relationship between the resistance of E. coli from swine and human sources was less studied. To provide a guidance for the rational use of antibiotics in clinical practice, we studied the drug sensitivity of E. coli strains in swine samples from the different areas of Shandong Province, China. In this study, the drug sensitivity results indicated that TE (97.17%) is a major antibiotic with high drug resistance in swine origin E. coli. So, we focused on the genetic evolutionary relationship between human-and swinederived TE-resistant E. coli and the potential risk of E. coli on clinical public. 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).

Genotype Detection of Antibiotic Resistance
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 (qacE 1-sull for quaternary ammonium compounds (QACs-) sulfonamiderelated 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 plasmidmediated 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 qacE 1-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 qacE 1-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/).

MLST Analysis
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  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).

Isolation and Identification of Swine Origin E. coli Strains
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%).

Antibiotic Resistance Spectrum of E. coli
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).

Multiple Drug Resistance of E. coli
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).

MLST-Based Genotyping
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) Table 4   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 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.

DISCUSSION
Antibiotics-based treatment of colibacillosis is frequently used in the production of swines. Due to the pressure in the long term, the irregular use and abuse of antibiotics usually lead to the resistance of E. coli to antibiotics, even multiple drug resistance. A 100% isolation rate of E. coli reported by Guan et al. (28) suggested that the prevalence of swine colibacillosis is serious. The reported isolation rates of pathogenic E. coli from swines separately were 100% in Zhoukou area (29), 88.5% in Shanxi Province (30), and 68.4% in Jiangsu Province (31). In this study, the isolation rate of E. coli from swines was 32.62% (106/325), which is relatively low and closer to the value (36.2%) in Henan Province (32). The detection and analysis of resistance of E. coli isolates from swine revealed that the resistance rate of isolates to TE, C, AMP, PRL, and SXT was higher than 80.19%, while that of the other 16 antibiotics was <38.68%.
Tetracyclines, aminoglycosides, and β-lactams are the first antibiotic choices for the clinical prevention and treatment of E. coli. Due to the abuse or unreasonable use of antibiotics for many years, E. coli drug-resistant genes appear constantly. Based on the MIC value detection of antibiotic types, we further detected the drug-resistant genes of corresponding antibiotics and combined the results of the two methods to interpretate the final result. The bla TEM gene was mostly positive for βlactams, with 99% for swine E. coli. The tetC gene in TE genotype was the highest, with 95.28%. The positive rate of qacE 1-sulI gene with 93.4% was the highest in SXT. aac(6 ′ )-Ib gene was the highest in quinolones, with 93.4%. The positive rate of cmlA gene with 97.2% was the highest in Cs. It has been found that β-lactams are the main genotypes of swine E. coli in China, while bla SHV is the main genotype in France, Germany, and Taiwan (33). Wang et al. (34) detected 64.0% of the tetA resistance genes from swines in Jilin Province. The results of Kuo et al. (35) showed that the main resistance gene was florR (82.90%). Especially, Zhang et al. (36) pointed out that ESBL E. coli causes a high recurrence rate in patients with urinary tract infection, furthermore, the recurrence is related to the genotype bla CTX−M and bla TEM of this pathogen. In 2016, it was reported that ESBL E. coli in a hospital of India had the most bla SHV types, followed by bla TEM and bla CTX−M types (37). Our study indicated that bla TEM genotype is the main genotype of E. coli from both the sources, while bla SHV genotype is almost not detected, as contrary to the abovementioned studies. The detection rate of TE resistance gene tetC with 95.28% indicated that tetC is a common resistance gene in E. coli in Shandong Province. Obviously, the existence of tetC resistance gene is closely related to the long-term widespread use of TE drugs in treatment, prevention, and other aspects (38). The presence of many complex types of drug resistance genes in Shandong should lay a foundation for the clinical prevention and treatment of the disease, which receive enough attention.
In recent years, the detection rate of drug-resistant genes has reached a new high level, and most are multi-genotype drugresistant strains. Zhu et al. (39) conducted a test of the drug sensitivity of 50 strains of E. coli isolated and preserved in the 1970s, and the results showed the strains being antibioticsensitive to varying degrees and the presence of multiple drugresistant strains, and this trend was on the rise. Sellera et al. (40) found that the rapid spread of MDR strains caused the public choice of alternative antibiotics used in the control of bacteria. The continuous emergence of drug resistance in E. coli and the cross of drug-resistant genes result in the difficulty in the disease prevention and treatment (41). Our finding was consistent with the previous reports. The results of antibiotics of 106 strains of E. coli showed that 98 strains were resistant to 3 or over 3 types of 21 kinds of antibiotics, and multiple drug resistance reached 92.45%. The strains of 7 (15 strains)/5 (15 strains) drug-resistant antibacterial spectra involved in 7/6 ST types with a distribution in all the 5 detection regions (Supplementary Table 4). The two kinds of drug-resistant antibacterial spectra were 28.30%. These results indicated the severity of the multi-drug resistance rate of E. coli in Shandong Province.
Regarding a few studies on ESBLs E. coli from animal and food sources in China, in recent years, growing reports from foreign investigators showed the potentiality of detecting the same drug-resistant genes present in animals also in people who are in close contact with animals (42)(43)(44). MLST can reflect the evolutionary biology of bacteria. There is also a certain correlation between the drug-resistant genes of E. coli and ST types. Importantly, E. coli from human and animal sources belonging to STs ST10, ST131, and ST648, has become multipotency and MDR bacteria, and ST10 (CTX-M-1) has been previously found in livestock, poultry meat, and healthy humans (45). Wang et al. (46) pointed out that ST10, ST218, ST3037, ST744, ST6929, and ST48 all belong to CC10, and the ST10 group is considered to be the most prevalent ST group of ESBLproducing E. coli from humans now. According to the related reports, CCl0 is a common clone group of strains in livestock (47). In line with this, ST10 was also found in ST type of E. coli from both sources in this study. Among them, the two ST types (ST10 and ST48) of JN belong to CC10 group. Similarly, the detection of CCl0 in a large-scale fattening swine farm, together with a local hospital case as a supplementary example, suggests the possible occurrence of pathogen transmission from animal to human in this area. Dissanayake et al. show that ST10, ST23, ST95, ST117, and ST131 are the main popular ST types of EXPEC (48), Among them, ST10 is the most common type in human, poultry, and swine source EXPEC at home and abroad (49,50). In this study, the presence of ST1258 was detected in JLW (6), TXT (11), JN (9), TDY1 (8), and TDY2 (8). As the most common type of ST strains from swines, ST1258 has not been found in the related literature of E. coli, while it has been detected in Bacillus cereus (51), Acinetobacter dijkshoorniae (52), and Streptococcus pneumoniae (53). There is an intimation that attention should be paid to the epidemic of the type ST1258. In conclusion, the results showed that the ST types of E. coli collected in this experiment were complex and diverse, showing a genetic diversity.
Furthermore, there is still a need for verifying which CCl0 and CC23 aggregation may pose the transmission risk to humans through the food chain or not. Zhou et al. (54) found that the ST type of swine ExPEC was classified into ST410, ST88, ST612, ST2505, and ST2371, and was closely related to ST23, belonging to CC23. ST23 is commonly found in human hemolytic uremia syndrome (Expec) (O157:H7) and avian ExPEC (55,56). In this study, the ST type of the tested bacteria belonging to CC23 was attributed to ST410 [TDY2 (2)] from swine and ST88 (1) from Tai'an human, which was consistent with the abovementioned conclusion. Although ST648 is not recognized as the main prevalent ST type, it is also widely distributed in swine, human, and poultry sources ExPEC (57). There no ST648 was found in swines, but ST648 was found in humans [urine (2) and blood (2)]. To some extent, porcine E. coli has the same genetic background as human and avian ExPEC. There was a certain correlation between the ST types of swine E. coli, its antibiotic spectrum, and resistance genotypes.

CONCLUSION
This study shows that the antibiotic resistance of E. coli is a serious issue as represented by several fattening swine farms in Shandong Province. The detection rate of clinical multi-drug resistance is high, and the main types of Cs and β-lactamase are mainly flor and bla TEM gene. The ST1258 is a novel popular genotype in some swine herds in Shandong Province. The cluster analysis showed that CCl0 and CC23 were the common CCs from the two sources. Our results provide a theoretical basis for guiding the rational use of antibiotics and preventing the spread of drug-resistant bacteria, and also provide epidemiological data for the risk analysis of foodborne bacteria and antimicrobial resistance in swine farms in Shandong Province.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Ethics Committee of Tai'an City Central Hospital. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Animal Care and use Committee of Shandong Agricultural University, Tai'an, China.

AUTHOR CONTRIBUTIONS
FW, MJ, and SZ participated in the study design. WW and LY conducted the study and drafted the manuscript. WH and FZ collected the important background information. All the authors read and approved the final manuscript.

FUNDING
This work was supported by the Natural Science Foundation of Shandong (ZR2020MC181 and ZR2016HL44) and the Fund of Shandong Agricultural Major Application Technology Innovation (SD2019XM009).