Genetic Diversity, Host Adaptation, and Public Health Implications of Escherichia coli in Qinghai Plateau Animals

Genetic Diversity, Host Adaptation, and Public Health Implications of Escherichia coli in Qinghai Plateau Animals | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genetic Diversity, Host Adaptation, and Public Health Implications of Escherichia coli in Qinghai Plateau Animals Shengyi Han, Tian Shi, Shinan Zhang, Wenwen Zhou, Shuping Li, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6997435/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Escherichia coli is an important pathogen that causes diarrhea in humans and animals. In this study, we performed MLST typing, phylogenetic grouping, antimicrobial resistance phenotype, antibiotics resistance genes, and virulence genes of E. coli isolated from yaks and wild animals in the Qinghai Plateau in China. Methods We tested 88 and 36 E. coli strains from yaks and wild animals for antimicrobial susceptibility test, antibiotics resistance genes, virulence genes, phylogenetic groups and multi-locus sequence typing. Results Phylogenetic analysis revealed that the isolates were primarily classified into three categories: A (21/124, 16.9%), B1 (96/124, 77.4%), and B2 (7/124, 5.6%), with 37 distinct sequence types (STs), including the newly identified ST16626. PCR-based virulence gene screening detected 24 virulence genes, with ompA and fimC exhibiting universal prevalence (100%), followed by luxS (96.8%), etrA (79.8%), and flu (72.6%). Antibiotic resistance gene profiling identified five major categories: tetracyclines ( tetA [20.97%], tetB [29.03%], tetC [4.84%], tetD [4.84%] and tetM [2.42%]), sulfonamides ( sul1 [21.77%], sul2 [28.23%] and sul3 [9.68%]), quinolones ( qnrS [6.45%]), β-lactams ( blaTEM [88.71%], blaSHV [7.26%], CTX-M1 [2.42%], CTX-M8 [11.29%] and CTX-M9 [11.29%]), and aminoglycosides ( cat1 [67.74%], cat2 [54.03%], aadA2 [10.48%], aac(6')-Ib [9.68%] and aph(3')-vII [4.84%]). Antimicrobial susceptibility testing demonstrated resistance rates below 50% for all 12 tested drugs, with the highest prevalence observed for tetracycline (TCY, 41.94%), cefotaxime (CTX, 36.29%), ampicillin (AMP, 34.68%), and ciprofloxacin (CIP, 33.06%). Conclusions This study presents the first systematic elucidation of molecular epidemiological characteristics of E. coli in the Qinghai Plateau: Identification of 37 sequence types (including novel ST16626) revealed significant phylogenetic divergence between yak- and wildlife-derived strains and the recA gene deficiency phenomenon; Discovery of host-specific resistance-virulence gene profiles confirmed differential evolutionary adaptation; The findings provide molecular evidence for zoonotic disease risk early-warning and clarify the driving mechanism of livestock farming on antimicrobial resistance transmission. Escherichia coli antimicrobial resistance virulence MLST One-health Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Escherichia coli represents a ubiquitous microorganism that serves as a fundamental component of the intestinal microbiota in humans and animals [ 1 ]. While typically existing as a commensal organism in the gastrointestinal tract [ 2 ], E. coli can transition to a pathogenic state under conditions of host immune compromise or when colonizing extraintestinal sites [ 3 ]. This pathogenic transformation is mediated through two primary mechanisms: (1) acquisition of virulence determinants via mobile genetic elements, and (2) genetic mutations in essential bacterial components such as lipopolysaccharide transport systems [ 4 ]. The pathogenicity of virulent strains is directly associated with an array of virulence factors, including enterotoxins, virulence islands, adhesion molecules, outer membrane proteins, and iron acquisition systems, which collectively facilitate host colonization, invasion, and tissue damage [ 5 , 6 ]. Current classification recognizes seven distinct pathotypes of diarrheagenic E. coli: enteropathogenic (EPEC), enterohemorrhagic (EHEC), enterotoxigenic (ETEC), enteroinvasive (EIEC), enteroaggregative (EAEC), diffusely adherent (DAEC), and adherent-invasive (AIEC) [ 7 , 8 ]. Of particular public health significance, EHEC, EIEC and EAEC strains can be transmitted through the food chain, posing substantial One Health challenges at the human-animal-environment interface [ 7 ]. The clinical management of E. coli infections faces two major obstacles: (1) the remarkable diversity of pathogenic variants and their virulence factor combinations [ 9 – 11 ], and (2) the escalating crisis of antimicrobial resistance. Although antibiotics were initially effective in treating these infections [ 12 – 14 ], the emergence of resistant strains has been accelerated by the plasmid-mediated dissemination of resistance genes among bacterial populations [ 15 – 19 ]. Contemporary isolates frequently harbor class 1 integrons, extended-spectrum β-lactamases (ESBLs), AmpC β-lactamases, and plasmid-encoded quinolone resistance determinants (e.g., ermB , tetB , blaTEM ) [ 20 – 22 ], significantly complicating treatment regimens. Historically, ETEC has been the predominant causative agent of calf diarrhea since the first documented cases in the 19th century. In China, ETEC-associated outbreaks among cattle herds have been endemic since the 1980s, with particularly high morbidity and mortality rates in yaks from high-altitude regions. The epidemiological patterns of E. coli infections exhibit distinct geographical variations that correlate with regional socioeconomic development levels and hygiene standards. This investigation presents a comprehensive analysis of 124 E. coli isolates obtained from yaks and wildlife in the Qinghai-Tibet Plateau, employing multilocus sequence typing (MLST), phylogenetic analysis, antimicrobial susceptibility testing, and virulence/resistance gene profiling. The study specifically addresses three critical research questions: (1) the potential for cross-species transmission between domestic and wild animals, (2) host-specific associations with virulence factor distribution, and (3) the implications of horizontal gene transfer for One Health surveillance systems. Methods Strain collection location A total of 124 E. coli strains were isolated from Yaks (n = 88) and wildlife (n = 36) with diarrhea from 2022 to 2023 in The Tibetan Plateau region of China. The sampling area involved nine districts and counties in three prefectures in Qinghai Province (Fig. 1 ). Phylogenetic and MLST analysis Using the multiplex PCR method, and he primer information is listed in Table 1 . PCR detection of the three genes chuA , yjaA , and TspE4.C2 (Table 1 ) was conducted to determine the phylogenetic group typing of E. coli based on electrophoretic patterns. Primers were synthesized by Xi'an Tsingke Biological Co., Ltd. The PCR reaction system (25 µL) consisted of 1 µL each of three pairs of upstream and downstream primers, 3 µL of bacterial DNA template, 12.5 µL of 2×Taq Master Mix, and 3.5 µL of ddH 2 O. PCR amplification conditions were as follows: initial denaturation at 94℃ for 5 minutes; 30 cycles of denaturation at 94℃ for 30 seconds, annealing for 30 seconds, and extension at 72℃ for 30 seconds; final extension at 72℃ for 7 minutes. PCR products were analyzed by 1.5% agarose gel electrophoresis. Table 1 Primer Information for Systematic Evolution Grouping Primer Sequence(5༇→3༇) Length(bp) Annealing temperature ChuA GACGAACCAACGGTCAGGAT 279 55 TGCCGCCAGTACCAAAGACA YjaA TGAAGTGTCAGGAGACGCTG 211 55 ATGGAGAATGCGTTCCTCAC TspE4.C2 GAGTAATGTCGGGGCATTCA CGCGCCAACAAAGTATTACG 152 55 MLST of E. coli strains were identified by PCR, and the primer information is listed in Table 2 . Multiplex PCR was performed as previously described to detect the phylogenetic type of the strain. PCRs were performed in a final volume of 100 µL. PCR mix component was as follows: 4 µL of genomic DNA, 2 µL of each primer, 50 µL of 2×Spark Taq PCR master mix, 42 µL of Sterile Enzyme-Free Water. PCR amplification condition was as follows: initial denaturation at 95°C for 5 min followed by 30 cycles of 45 s at 95°C, 30 s at Tm and 1 min at 72°C, with a final extension of 10 min at 72°C. The target band is recycled, and the recovered product is sent to a sequencing company for sequencing. The sequencing results were uploaded to pubMLST, matched to each housekeeping gene, and ST typing was obtained after assembly. Table 2 MLST Typing Primers Information Primer Sequence(5༇→3༇) Length(bp) Annealing temperature(℃) adk ATTCTGCTTGGCGCTCCGG 583 54 CCGTCAACTTTCGCGTATTT fumC TCACAGGTCGCCAGCGCTTC 806 54 GTACGCAGCGAAAAAGATT gyrB TCGGCGACACGGATGACGGC 911 60 ATCAGGCCTTCACGCGCATC icd ATGGAAAGTAAAGTAGTTGTTCCGGCACA 878 54 GGACGCAGCAGGATCTGTT mdh ATGAAAGTCGCAGTCCTCGGCGCTGCTGGCGG 932 60 TTAACGAACTCCTGCCCCAGAGCGATATCTTTCTT purA CGCGCTGATGAAAGAGATGA 816 54 CATACGGTAAGCCACGCAGA recA CGCATTCGCTTTACCCTGACC 780 56 TCGTCGAAATCTACGGACCGGA Antimicrobial susceptibility testing Antimicrobial susceptibility was determined using the Kirby-Bauer disc agar diffusion method according to Clinical and Laboratory Standards Institute (CLSI) guidelines. A total of 11 antimicrobial agents (ampicillin, amikacin, ciprofloxacin, gentamicin, kanamycin, tetracycline, ofloxacin, norfloxacin, ceftriaxone, cefotaxime and sulfamethoxazole trimethoprim) were used to test the antimicrobial susceptibility of the isolated strains. Three replicates were set for each antibiotic test. The reference strain E. coli ATCC 25922 was used as the positive control. The control strain ATCC25922 and 124 test strains were separately inoculated onto LB agar medium and incubated overnight at 37℃. Single colonies were picked from the LB agar plates and transferred into regular LB broth, which was then incubated in a shaking incubator at 37℃ for 16–18 hours, with the bacterial suspension concentration adjusted using physiological saline. Inoculate 200 µL of the prepared bacterial suspension into an MH agar plate, and spread the suspension evenly over the surface of the MH agar medium with a sterile glass spreading rod. After the water on the plate has been completely absorbed, apply the antimicrobial susceptibility discs. Once the bacterial suspension is evenly spread, use sterilized forceps to place the antimicrobial susceptibility discs, ensuring that three discs are evenly distributed on each plate. During the application process, ensure that the discs firmly adhere to the medium's surface. Invert the plates and incubate at 37℃ for 16–18 hours, then observe the results. Measure the diameter of the inhibition zones with a caliper, in millimeters. Results were determined according to CLSI international standards for antimicrobial susceptibility testing. Some strains exhibited double rings, with measurements taken based on the outer ring. Analysis of putative virulence genes The virulence genes of E. coli strains were identified by PCR. A total of 35 tested virulence genes belongs to the following categories, including adhesion factor, hemolysin, toxin (resistant to heat-resistant enterotoxins, heat-sensitive enterotoxin), locus of enterocyte effacement, High Pathogenicity Island and some other virulence genes, primer information for all virulence genes can be found in Table 3 . Table 3 Primer information for virulence genes Primer Sequence Length Tm(℃) temperature K88 GATGAAAGACTCTGATTGCA GATTGCTACGTTCAGCGGAGCG 841 52 K99 CTGAAAAAAACACTGCTAGCTATT CATATAAGTGACTAAGAAGGATGC 543 52 F41 GATTGCTACGTTCAGCGGAGCG TCTGAGGTCATCCCAATTGTGG 628 52 987P GTTACTGCCAGTCTATGCCAAGTG TCGGTGTACCTGCTGAACGAATAG 463 52 fedA CATGATGGATCCATGAAAAGACTAGTGTTTATTTCTT CATGATGAATTCTTACTTGTAAGTAACCGCGTAAGCC 516 66 HPI AAGGATTCGCTGTTACCGGAC TCGTCGGGCAGCGTTTCTTCT 280 58 LEE ATATCCGTTTTAATGGCTATCT AATCTTCTGCGTACTGTGTTCA 425 58 STa GGGTTGGCAATTTTTATTTCTGTA ATTACAACAAAGTTCACAGCAGTA 183 56 STb ATGTAAATACCTACAACGGGTGAT TATTTGGGCGCCAAAGCATGCTCC 360 56 VT1 ATTCGCTGAATGTCATTCGCT ACGCTTCCCAGAATTGCATTA 664 56 VT2all ATTCGCTGAATGTCATTCGCT ACGCTTCCCAGAATTGCATTA 484 56 LTⅠ TAGAGACCGGTATTACAGAAATCTGA TCATCCCGAATTCTGTTATATATGTC 282 56 SLT2 GGTTATGCCTCAGTCATTATTAA GAATGAAGAAGATGTTTATAGCGG 281 56 SLT2e GAATGAAGAAGATGTTTATAGCGG TTTTATGGAACGTAGGTAYYACC 454 56 F17 GCAGAAAATTCAATTTATCCTTGG CTGATAAGCGATGGTGTAATTAAC 537 57 F18 GTGAAAAGACTAGTGTTTATTTC CTTGTAAGTAACCGCGTAAGC 510 55 ompA ACGCTGTTTCACGTTGTCA AACCCGTATGTTGGCTTTG 753 55 ompT TCATCCCGGAAGCCTCCCTCACTACTAT TAGCGTTTGCTGCACTGGGCTTCTGATAC 496 55 fimC GTTCATGGCAATGGTGGTT AGTTCCGGCATTCAACTCT 514 60 fimA GCTCTGGCTGATACTACACC TTATTGATACTGAACCTTGA 495 58 espA TATCAGGCACAAAGCGATCTGTC TATCTCCGGTTATTTACCAAGGG 432 55 aer TACCGGATTGTAATATGCAGACCGT AATATCTTCCTCCAGTCCGGAGAAG 602 55 flu CTGGTATGGAATCACTTACGGG GAGAATGCTCCCAGGCGGTTTAT 965 57 luxS ATGCCGTTGTTAGATAGC CTAGATGTGCAGTTCCTGC 516 55 hlyA GCATCATCAAGCGTACGTTCC AATGAGCCAAGCTGGTTAAGCT 534 60 hlyE CTCAATCGGCATCCACAT GTTTCCCTTCAACAACCC 456 52 hlyF TGGCCACAGTCGTTTAGGGTGCTTACC GGCGGTTTAGGCATTCCGATACTCAG 450 58 eaeA CTGAACGGCGATTACGCGAA CCAGACGATACGATCCAG 798 52 papC GGGCGTGATAACGATTC ATTTGCCAGCGGACTAC 234 47 sfaD GCAACAGCAACGCTGGTTGCATCAT AGAGAGAGCCACTCTTATACGGACA 410 62 feaG GAATCTGTCCGAGAATATCA GTTGGTACAGGTCTTAATGC 499 55 etrA CTTCTTCCTAACGAAACTATCATTA TGACATATCAACTTTCTCTTACGC 913 50 rfc ATCCATCAGGAGGGGACTGGA AACCATACCAACCAATGCGAG 788 63 sepA TAAAACCCGCCGCCTGAGTA TGCCGGTGAACAGGAGGTTT 611 62 fasA GTAACTCCACCGTTTGGTATC AAGTTACTGCCAGTCTATGC 409 58 PCRs were performed in a final volume of 50 µl. PCR mix component was as follows: 2 µL of genomic DNA, 1 µL of each primer, 25 µL of 2×Spark Taq Pcr master mix, 21 µL of Sterile Enzyme-Free Water. PCR amplification condition was as follows: initial denaturation at 95°C for 5 min followed by 30 cycles of 45 s at 95°C, 30 s at Tm and 1 min at 72°C, with a final extension of 10 min at 72°C. Analysis of antibiotic-resistance genes Antibiotic-resistance genes of E. coli strains were identified by PCR. A total of 30 tested virulence genes belongs to five broad categories, including tetracyclines( tetA 、 tetB 、 tetC 、 tetD 、 tetG 、 tetM 、 tetX ), sulfonamides( sul1 、 sul2 、 sul3 ), quinolones༈ qnrA 、 qnrB 、 qnrS 、 qepA ༉, β-lactams༈ blaDHA 、 blaTEM 、 blaCMY-2 、 blaSHV 、 CTX-M1 、 CTX-M2 、 CTX-M8 、 CTX-M9 、 CTX-M25 ༉, aminoglycosides༈ cat1 、 cat2 、 aac(3')-IV 、 aadA2 、 rmtB 、 aac(6')-Ib 、 aph(s')-vII ༉. Selected drug resistance gene primer information is provided in Table 4 . Table 4 Resistance gene primer information Primer Sequence Length Tm(℃) Temperature CTX-M1 ATGGTTAAAAAATCACTGCGTCAGTTC TCACAAACCGTTGGTGACGATTTTAGCCGC 876 52 CTX-M2 ATGATGACGCAGAGCATTCGCCGCTCA TCAGAAACCGTGGGTTACGATTTTCGC 876 55 CTX-M8 ACTTCAGCCACACGGATTCA CGAGTACGTCACGACGACTT 878 55 CTX-M9 ATGGTGACAAAGAGAGTGCA CCCTTCGGCGATGATTCTC 870 55 CTX-M25 CACACGAATTGAATGTTCAG TCACTCCACATGGTGAGT 924 50 tetA CACTATGGCATTCTGCTGGC CATAGATCGCCGTGAAGAGG 948 60 tetB GCCCAGTGCTGTTGTTGTC AAGACCAAGACCCGCTAATG 553 60 tetC TCCTGCTCGCTTCGCTACT TGGTCGTCATCTACCTGC 730 58 tetD AAACCATTACGGCATTCTGC GACCGGATACACCATCCATC 787 56 tetG CGGTCTTATGGGTGCTCTA CCTTGCTTGTTACTGAC 721 58 tetM TTATCAACGGTTTATCAGG CGTATATATGCAAGACG 397 55 tetX CAATAATTGGTGGTGGACCC TTCTTACCTTGGACATCCCG 468 56 sul1 TCAGACGTCGTGGATGTCG CGAAGAACCGCACAATCTCG 393 57 sul2 CCTGTTTCGTCCGACACAGA GAAGCGCAGCCGCAATTCAT 435 59 sul3 AGATGTGATTGATTTGGGAGC TAGTTGTTTCTGGATTAGAGCCT 443 53 qnrA TCAGCAAGAGGATTTCTCA GGCAGCACTATTACTCCCA 627 55 qnrB ACGATGCCTGGTAGTTGTCC ACGACATTCGTCAACTGCAA 469 55 qnrS ACGACATTCGTCAACTGCAA TAAATTGGCACCCTGTAGGC 299 56 qepA GCAGGTCCAGCAGCGGGTAG CTTCCTGCC CGAGTATCGTG 299 56 blaDHA AACTTTCACAGGTGTGCTGGGT CCGTACGCATACTGGCTTTGC 387 63 blaTEM ATAAAATTCTTGAAGACGAAA GACAGTTACCAATGCTTAATC 1080 52 blaSHV CACTCAAGGATGTATTGTG TTAGCGTTGCCAGTGCTCG 885 63 BlaCMY-2 ATGATGAAAAAATCGTTATGC TTGCAGCTTTTCAAGAATGCG 1143 57 cat1 CTTGTCGCCTTGCGTATAAT ATCCCAATGGCATCGTAAAG 508 53 cat2 AACGGCATGATGAACCTGAA ATCCCAATGGCATCGTAAAG 547 53 rmtB ACATCAACGATGCCCTCAC AAGTTCTGTTCCGATGGTC 472 53 aadA2 GGTGCTAAGCGTCATTGAGC GCTTCAAGGTTTCCCTCAGC 470 57 aac(6’)-Ib TTGCGATGCTCTATGAGTGGCTA CTCGAATGCCTGGCGTGTTT 482 56 aph (3’)-Ⅶ TCCACAGGATGGCAAGATCC TTCAACGGGAAACGTCTTGC 690 55 aac(3’)-Ⅳ GGCCACTTGGACTGATCGAG GCGGATGCAGGAAGATCAAC 409 58 PCRs were performed in a final volume of 50 µL. PCR mix component was as follows; 2 µL of genomic DNA, 1 µL of each primer, 25 µL of 2×Spark Taq Pcr master mix, 21 µL of Sterile Enzyme-Free Water. PCR amplification condition was as follows: initial denaturation at 95°C for 5 min followed by 30 cycles of 45 s at 95°C, 30 s at Tm and 1 min at 72°C, with a final extension of 10 min at 72°C. Results phylogenetic and MLST analysis A total of 124 isolates were confirmed as E. coli via biochemical and 16s RNA. The majority of the E. coli isolates belonged to phylogroup B1 (77.42%, n = 96), with the other isolates belonging to phylogroup A (16.94%, n = 21) and B2 (5.65%, n = 7). However, no isolates were assigned to phylogenetic group D (Table S1 ). The phylogroups B1 were the most common in the Yak and wildlife samples. MLST analysis revealed the presence of highly diverse sequence types (STs, N = 37 unique STs) among E. coli isolates subjected to sequencing (Table S2 ). Among yak-derived E. coli isolates, 37 different STs were identified, including ST2522, ST58, ST56, ST101, ST40, ST155, ST156, ST10, ST1252, ST3884, ST21, ST2166, ST2035, ST1665, ST1101, ST446, ST342, ST73, ST43, ST1249, ST1642, ST10601, ST85, ST345, ST16080, ST1079, ST6178, ST2973, ST75, ST109, ST1426, ST2179, ST223, ST602, ST2253, and a novel ST16626 (Fig. 2 ). The phylogenetic tree reveals that yak-derived E. coli strains and wild animal-derived E. coli strains are distinctly separated into two branches, with several yak-derived E. coli strains also observed within the wild animal branch. Through common mapping of their source regions, phylogenetic groups, and MLST, it is evident that there are differences among E. coli strains from different origins (Fig. 3 ). Antimicrobial susceptibility testing Antimicrobial susceptibility testing revealed that 124 E. coli isolates exhibited resistance rates of 41.5% to TCY, 34.7% to AMP, 16.9% to AMK, 25.0% to GEN, 16.1% to OFX, 15.3% to NOR, 17.7% to CRO, 36.3% to CTX, 25.8% to SXT, 24.2% to KAN, and 33.1% to CIP against 11 antibiotics, with all resistance rates below 50.0% (Table S3 ). The comparison of antibiotic resistance rates between yak-derived and wildlife-derived isolates revealed that the resistance rates of yak-derived isolates to various antibiotics were consistently higher than those of wildlife-derived isolates, as shown in Fig. 4 . This phenomenon may be due to the frequent use of antibiotics in the farming process of yaks. Analysis of putative virulence genes A total of 124 strains were tested for 35 virulence genes, and all 124 strains were found to carry virulence genes, including sfaD , eaeA , F17 , espA , aer , fimA , ompT , h lyF , flu , ompA , fimC , luvs , etrA , PAPC , hlyA , HPI , LEE , VT1 , SLT2 , 987P , F41 , sepA , hlyE , and STb . The detection rates of each virulence gene are shown in Fig. 5 and Table S4 . The analysis reveals that yak-derived E. coli strains exhibit both differences and similarities in the carriage of virulence genes compared to wildlife-derived E. coli strains. The differences lie in the detection of five virulence genes— 987P , F41 , sepA , hlyE , and STb —exclusively in wildlife E. coli strains, as well as hlyA , espA , and eaeA also being detected only in wildlife E. coli strains. The similarities include the detection of all other virulence genes except the aforementioned eight, with high detection rates for etrA , luxS , fimC , and ompA shown in Fig. 5 . The analysis of the number of virulence genes carried by each strain revealed that 124 E. coli strains exhibited 88 different virulence gene profiles, with each strain carrying more than three virulence genes. The most common virulence gene profile was " F17 + flu + ompA + fimC + fimA + luxs + hlyA + etrA ," which accounted for 6.45% of all virulence gene profiles. Analysis of antibiotic-resistance genes The results of drug resistance gene detection are shown in Fig. 7 , indicating positive detections for β-lactams, tetracyclines, sulfonamides, aminoglycosides, and quinolones. Among β-lactam genes, bla-TEM , bla-SHV , CTX-M1 , CTX-M8 , and CTX-M9 were detected at rates of 88.7%, 7.3%, 2.4%, 11.3%, and 11.3%, respectively. For tetracyclines, tetA , tetB , tetC , tetD , and tetM were detected at rates of 20.9%, 29.0%, 4.8%, 4.8%, and 2.4%, respectively. Sulfonamide genes sul1 , sul2 , and sul3 were detected at rates of 21.8%, 28.2%, and 9.7%, respectively. The quinolone qnrS was detected at a rate of 6.5%. For aminoglycosides, cat1 , cat2 , aadA2 , aac(6')-Ib , and aph(3')-vII were detected at rates of 67.7%, 54.0%, 10.5%, 9.7%, and 4.8%, respectively. A comparative analysis of the detection rates of resistance genes in yak and wildlife isolates revealed that the eight resistance genes bla-SHV , CTX-M1 , tetC , tetD , sul3 , cat2 , aadA2 , and aac(6')-Ib were exclusively detected in yak isolates, with no detection observed in wildlife, as shown in Fig. 8 and Table S5 . Discussion This comprehensive study characterized 124 E. coli isolates from yaks and wildlife in Qinghai Plateau through phylogenetic analysis, MLST typing, antimicrobial susceptibility testing, and virulence/resistance gene profiling. Phylogenetic grouping showed predominance of phylogroup B1 (77.42%), followed by A (16.94%) and B2 (5.65%), with no D group isolates. MLST revealed 37 distinct sequence types, including novel ST16626, with phylogenetic separation between yak and wildlife strains though some overlap existed. Notably, 45 strains (36.3%) lacked recA , preventing ST typing - a significant finding given recA crucial roles in DNA repair and SOS response [ 23 – 25 ], and its emerging potential as a therapeutic target to combat heteroresistance [ 26 – 28 ]. Antimicrobial testing demonstrated resistance rates below 50% for all tested antibiotics, with highest resistance to tetracycline (41.94%), cefotaxime (36.29%), and ampicillin (34.68%). Yak-derived isolates showed consistently higher resistance than wildlife strains, likely reflecting agricultural antibiotic use. All strains carried virulence genes (35 tested), with 88 unique virulence profiles identified. Key differences emerged in virulence gene carriage: 987P , F41 , sepA , hlyE , STb , hlyA , espA and eaeA were exclusive to wildlife strains, while etrA , luxS , fimC and ompA showed high prevalence across both groups. Resistance gene analysis detected β-lactam ( bla-TEM 88.7%), tetracycline ( tetB 29.0%), sulfonamide ( sul2 28.2%), quinolone ( qnrS 6.5%), and aminoglycoside ( cat1 67.7%) resistance genes, with eight genes (including bla-SHV and CTX-M1 ) unique to yak isolates. The study highlights E. coli complex biology - as both commensal and pathogen through virulence factor acquisition, and as a reservoir of diverse resistance mechanisms including ESBL production and biofilm formation regulated by luxS / AI-2 quorum sensing [ 29 ]. These findings demonstrate: (1) significant strain diversity with host adaptation, (2) evidence of cross-species transmission potential, and (3) emerging resistance patterns in this unique high-altitude ecosystem, underscoring the need for One Health surveillance to address this public health challenge. The recA -deficient strains and host-specific virulence/resistance patterns represent particularly noteworthy findings requiring further investigation. Conclusions This first comprehensive characterization of E. coli at the Qinghai yak-wildlife interface reveals significant strain diversity with host-specific adaptations, provides evidence of potential cross-species transmission, and documents moderate but concerning antimicrobial resistance patterns, underscoring the imperative for integrated One Health surveillance to monitor emerging threats in high-altitude ecosystems. Abbreviations E. coli: Escherichia coli MLST: Multilocus sequence typing PCR: Polymerase Chain Reaction ‌LB Broth: Luria-Bertani Broth MH agar: Mueller-Hinton Agar CLSI: Clinical and Laboratory Standards Institute AMP: ampicillin AMK: amikacin CIP: ciprofloxacin GEN: gentamicin KAN: kanamycin TCY: tetracycline OFX: ofloxacin NOR: norfloxacin CRO: ceftriaxone CTX: cefotaxime SXT: sulfamethoxazole trimethoprim Declarations Ethics approval and consent to participate Laboratory Animal Management Committee of Qinghai Academy of Animal Husbandry and Veterinary Sciences ruled that no formal ethics approval was required to conduct this research. Before conducting the research, informed consent was obtained from all the owners of the yak farms included in this study. Consent for publication Not applicable. Availability of data and materials All data are available in the main text or the supplementary materials. Competing interests The authors declare no competing interests. Funding This study was supported by the Qinghai Province Science and Technology Achievement Transformation and Key R&D Project (2024ZY014). Author Contributions Conceptualization S.H and T.S.; methodology, T.S.; software, S.Z., and S.H; formal analysis, R.G.; investigation S. L, W. Z and L.L; writing—original draft preparation, S.H; writing—review and editing, S.H and T.S; project administration, S.L.; funding acquisition S.L. All authors have read and agreed to the published version of the manuscript. Acknowledgements Not applicable. References Jesser KJ, Levy K. Updates on defining and detecting diarrheagenic Escherichia coli pathotypes. Curr Opin Infect Dis. 2020;33(5):372–80. Raetz CR, Whitfield C. Lipopolysaccharide endotoxins. Annu Rev Biochem. 2002;71:635–700. Pakbin B, Brück WM, Rossen JWA. Virulence factors of Enteric Pathogenic Escherichia coli : A Review. Int J Mol Sci. 2021;22(18):9922. Tenaillon O, Skurnik D, Picard B, Denamur E. The population genetics of commensal Escherichia coli . Nat Rev Microbiol. 2010;8:207–17. Clements A, Young JC, Constantinou N, Frankel G. Infection strategies of enteric pathogenic Escherichia coli . Gut Microbes. 2012;3:71–87. Pakbin B, Akhondzadeh Basti A, Khanjari A, Azimi L, Karimi A. Differentiation of stx1A gene for detection of Escherichia coli serotype O157: H7 and Shigella dysenteriae type 1 in food samples using high resolution melting curve analysis. Food Sci Nutr. 2020;8:3665–72. Yang S-C, Lin C-H, Aljuffali IA, Fang J-Y. Current pathogenic Escherichia coli foodborne outbreak cases and therapy development. Arch Microbiol. 2017;199:811–25. Alegbeleye OO, Sant’Ana AS. Pathogen subtyping tools for risk assessment and management of produce-borne outbreaks. Curr Opin Food Sci. 2020;32:83–9. Kaito C, Yoshikai H, Wakamatsu A, et al. Non-pathogenic Escherichia coli acquires virulence by mutating a growth-essential LPS transporter[J]. PLoS Pathog. 2020;16(4):e1008469. Kaper, Nataro HL, Mobley. Pathogenic Escherichia coli [J]. Nat Rev Microbiol. 2004;2(2):123–40. Allocati. Escherichia coli in Europe: an overview[J]. Int J Environ Res Public Health. 2013;10(12):6235–54. Jang. Environmental Escherichia coli : ecology and public health implications-a review[J]. J Appl Microbiol. 2017;123(3):570–81. Leekitcharoenphon P, Johansson MHK, Munk P, et al. Genomic evolution of antimicrobial resistance in Escherichia coli [J]. Sci Rep. 2021;11(1):15108. Awad WS. Molecular characterization of pathogenic Escherichia coli isolated from diarrheic and in-contact cattle and buffalo calves[J]. Trop Anim Health Prod. 2020;52(6):3173–85. Capps, Identification. Shiga toxin subtypes and prevalence of minor serogroups of Shiga toxin-producing Escherichia coli in feedlot cattle feces[J]. Sci Rep. 2021;11(1):8601. Croxen. Recent advances in understanding enteric pathogenic Escherichia coli [J]. Clin Microbiol Rev. 2013;26(4):822–80. Gomes. Diarrheagenic Escherichia coli [J]. Brazilian J Microbiol. 2016;47:3–30. Gaastra W, Svennerholm AM. Colonization factors of human enterotoxigenic Escherichia coli (ETEC) [J]. Trends Microbiol. 1996;4(11):444–52. Wolf MK. Occurrence, distribution, and associations of O and H serogroups, colonization factor antigens, and toxins of enterotoxigenic Escherichia coli [J]. Clin Microbiol Rev. 1997;10(4):569–84. Francis DH. Enterotoxigenic Escherichia coli infection in pigs and its diagnosis[J]. J Swine Health Prod. 2002;10:171–5. Yang S-C, Lin C-H, Aljuffali IA, Fang J-Y. Current pathogenic Escherichia coli foodborne outbreak cases and therapy development. Arch Microbiol. 2017;199:811–25. Alegbeleye OO, Sant’Ana AS. Pathogen subtyping tools for risk assessment and management of produce-borne outbreaks. Curr Opin Food Sci. 2020;32:83–9. Kowalczykowski SC. Biochemical and biological function of Escherichia coli RecA protein: behavior of mutant RecA proteins. Biochimie. 1991;73(2–3):289–304. Rehrauer WM, Lavery PE, Palmer EL, Singh RN, Kowalczykowski SC. Interaction of Escherichia coli RecA protein with LexA repressor. I. LexA repressor cleavage is competitive with binding of a secondary DNA molecule. J Biol Chem. 1996;271(39):23865–73. Moreau PL. Role of Escherichia coli RecA protein in SOS induction and post-replication repair. Biochimie. 1985;67(3–4):353–6. Diaz-Diaz S, Yerbes P, Recacha E, et al. RecA inactivation as a strategy to reverse the heteroresistance phenomenon in clinical isolates of Escherichia coli . Int J Antimicrob Agents. 2023;61(2):106721. Recacha E, Machuca J, Díaz-Díaz S, et al. Suppression of the SOS response modifies spatiotemporal evolution, post-antibiotic effect, bacterial fitness and biofilm formation in quinolone-resistant Escherichia coli . J Antimicrob Chemother. 2019;74(1):66–73. Machuca J, Recacha E, Gallego-Mesa B, et al. Effect of RecA inactivation on quinolone susceptibility and the evolution of resistance in clinical isolates of Escherichia coli . J Antimicrob Chemother. 2021;76(2):338–44. Alshammari M, Ahmad A, AlKhulaifi M, Al Farraj D, Alsudir S, Alarawi M, et al. Reduction of biofilm formation of Escherichia coli by targeting quorum sensing and adhesion genes using the CRISPR/Cas9-HDR approach, and its clinical application on urinary catheter. J Infect Public Health. 2023;16:1174–83. Additional Declarations No competing interests reported. Supplementary Files TableS1.xlsx TableS2.xlsx TableS3.xlsx TableS4.xlsx TableS5.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6997435","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":498112268,"identity":"6eab35ce-7fd2-47b0-8628-7f34240c1079","order_by":0,"name":"Shengyi Han","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Shengyi","middleName":"","lastName":"Han","suffix":""},{"id":498112269,"identity":"e810a5bb-b5dc-4e07-a4c1-2434f9fa1bb5","order_by":1,"name":"Tian Shi","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Tian","middleName":"","lastName":"Shi","suffix":""},{"id":498112270,"identity":"1755ccc3-e1cf-42e7-8454-759b6884a481","order_by":2,"name":"Shinan Zhang","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Shinan","middleName":"","lastName":"Zhang","suffix":""},{"id":498112271,"identity":"631e41c3-c842-4a5b-a098-0ef07a24d7d9","order_by":3,"name":"Wenwen Zhou","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Wenwen","middleName":"","lastName":"Zhou","suffix":""},{"id":498112272,"identity":"cf2d4c0d-73a3-49d8-8788-bbcbb37ba60a","order_by":4,"name":"Shuping Li","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Shuping","middleName":"","lastName":"Li","suffix":""},{"id":498112273,"identity":"be200282-ae46-4901-b44e-e8b38d308322","order_by":5,"name":"Guoyuan Hu","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Guoyuan","middleName":"","lastName":"Hu","suffix":""},{"id":498112274,"identity":"5acf0004-182c-492d-81b7-fdfe5bc4722f","order_by":6,"name":"Lingxia Li","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Lingxia","middleName":"","lastName":"Li","suffix":""},{"id":498112275,"identity":"d6a3a145-411d-487b-ab1e-5b3fc5898c3b","order_by":7,"name":"Shengqing Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuUlEQVRIiWNgGAWjYBACPgYGxgcJBjZybOztB4jTwsbAwGzwoSLNmI/nTALRWtgkZ5w5nDhPwsGASC38hzcb87alpbdJMCQw/KjYRoQWibTCx7xtNrlt0o0HGHvO3CZGC48xyJbcNpkDCcyMbcRo4T9jJs3bdjidTSLBgEgtDDlmIO8nkKBFIq0YFMiGbcBAPkiUX/j5D28ERaW8fHv7wQc/KojQAgSI6DhAlHoULaNgFIyCUTAKsAIAZGA41YpxwSQAAAAASUVORK5CYII=","orcid":"","institution":"Qinghai University","correspondingAuthor":true,"prefix":"","firstName":"Shengqing","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-06-28 11:23:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6997435/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6997435/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88882092,"identity":"d0d08d6f-864c-46f8-8614-7739a6d8547c","added_by":"auto","created_at":"2025-08-12 11:19:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":342397,"visible":true,"origin":"","legend":"\u003cp\u003eGeographical distribution of strain collection sites across Qinghai Province, China, including: Qumalei County, Yushu County, Zhiduo County, and Zaduo County in Yushu Prefecture; Haiyan County in Haibei Prefecture; and Maqin County in Golog Prefecture.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6997435/v1/851930efe37e2d1eae26c41c.png"},{"id":88883040,"identity":"90113dbe-9873-4faf-8454-8685c97f6731","added_by":"auto","created_at":"2025-08-12 11:27:42","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1111136,"visible":true,"origin":"","legend":"\u003cp\u003eMinimum evolution tree based on multilocus sequence typing (MLST) analysis. The phylogenetic relationships among different sequence types (STs) are shown, with red numbers indicating absolute genetic distances between STs.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6997435/v1/01813a6c7a6075ec53c1083f.jpg"},{"id":88882095,"identity":"dec737bf-b4cd-48ef-a319-70ee023d7360","added_by":"auto","created_at":"2025-08-12 11:19:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":898377,"visible":true,"origin":"","legend":"\u003cp\u003eMLST phylogenetic tree of 124 isolates, displaying their sequence types (STs), geographical origins, isolation sources, and phylogenetic clade classifications.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-6997435/v1/fe2061adecdf6b62446477a6.png"},{"id":88881647,"identity":"3c845292-b891-49f1-8682-4cc1e456d972","added_by":"auto","created_at":"2025-08-12 11:11:43","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1438802,"visible":true,"origin":"","legend":"\u003cp\u003eAntimicrobial resistance profiles of isolated strains. (A) Resistance rates of 124 isolates to 11 antimicrobial agents. (B) Comparative resistance rates of isolates from different sources against the antimicrobial agents.\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6997435/v1/fb4ae3067b7e9871a5e4b5b9.jpg"},{"id":88881640,"identity":"2218596e-b3f5-40ce-9964-cd92db139985","added_by":"auto","created_at":"2025-08-12 11:11:43","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1584291,"visible":true,"origin":"","legend":"\u003cp\u003ePrevalence of virulence genes. (A) Detection rates of virulence genes across all \u003cem\u003eE. coli \u003c/em\u003eisolates. (B) Comparative detection rates of virulence genes in yak-derived versus wildlife park-derived strains.\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6997435/v1/0fd3b3a42ff06db8ddffffe4.jpg"},{"id":88883046,"identity":"fe2a3010-d463-4a22-8a5c-e1c3fa6e8d30","added_by":"auto","created_at":"2025-08-12 11:27:43","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2888279,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap analysis of 124 isolates showing their sequence types (STs), virulence factor (VF) profiles, geographical regions, isolation sources, and phylogenetic classifications. Red indicates presence and white indicates absence of virulence genes.\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6997435/v1/3befcc532efa077f8cf3c54a.jpg"},{"id":88882098,"identity":"90b441b4-2250-4991-abb2-4eb6c7df2181","added_by":"auto","created_at":"2025-08-12 11:19:43","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1615002,"visible":true,"origin":"","legend":"\u003cp\u003ePrevalence of resistance genes. (A) Detection rates of antimicrobial resistance genes (ARGs) in all E. coli isolates. (B) Comparative detection rates of ARGs in yak-derived versus wildlife park-derived strains.\u003c/p\u003e","description":"","filename":"Fig.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6997435/v1/659eae0d0fbe5efcddf2b4e8.jpg"},{"id":88881644,"identity":"7394606b-0ef3-4f28-82e0-5ce91c762084","added_by":"auto","created_at":"2025-08-12 11:11:43","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2736293,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap analysis of 124 isolates showing their sequence types (STs), antimicrobial resistance gene (ARG) profiles, geographical regions, isolation sources, and phylogenetic classifications. Red indicates presence and white indicates absence of resistance genes.\u003c/p\u003e","description":"","filename":"Fig.8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6997435/v1/82aaaf837cde8db1af942593.jpg"},{"id":90700719,"identity":"818da414-bb70-43b4-9e8f-4b20d9c543fd","added_by":"auto","created_at":"2025-09-05 23:31:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13150775,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6997435/v1/ddac7515-88f8-4067-935a-083c68b70b43.pdf"},{"id":88882094,"identity":"43b194ac-00e4-46aa-97bc-6a006fb62bfc","added_by":"auto","created_at":"2025-08-12 11:19:42","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14445,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6997435/v1/57eb343558ea0ffa3268bce2.xlsx"},{"id":88881636,"identity":"99c4a0b3-34b0-4d32-8e83-bb0de16d54df","added_by":"auto","created_at":"2025-08-12 11:11:43","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17590,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6997435/v1/b4caa391af91068331186a27.xlsx"},{"id":88881633,"identity":"77b56162-5f1c-48f4-9fe4-1ac907a493a7","added_by":"auto","created_at":"2025-08-12 11:11:42","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":17791,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6997435/v1/5d8842271008501c8a668258.xlsx"},{"id":88882096,"identity":"b6385f35-8d78-4fd7-8954-c1531a3025c6","added_by":"auto","created_at":"2025-08-12 11:19:43","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":19512,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6997435/v1/e33a450da41bf561260f1551.xlsx"},{"id":88883041,"identity":"5f749bb6-d888-4ef7-88be-d7b4dc3b0629","added_by":"auto","created_at":"2025-08-12 11:27:43","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":17090,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6997435/v1/fdd410bb7f8b352b766ebac1.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genetic Diversity, Host Adaptation, and Public Health Implications of Escherichia coli in Qinghai Plateau Animals","fulltext":[{"header":"Background","content":"\u003cp\u003eEscherichia coli represents a ubiquitous microorganism that serves as a fundamental component of the intestinal microbiota in humans and animals [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. While typically existing as a commensal organism in the gastrointestinal tract [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], E. coli can transition to a pathogenic state under conditions of host immune compromise or when colonizing extraintestinal sites [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This pathogenic transformation is mediated through two primary mechanisms: (1) acquisition of virulence determinants via mobile genetic elements, and (2) genetic mutations in essential bacterial components such as lipopolysaccharide transport systems [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The pathogenicity of virulent strains is directly associated with an array of virulence factors, including enterotoxins, virulence islands, adhesion molecules, outer membrane proteins, and iron acquisition systems, which collectively facilitate host colonization, invasion, and tissue damage [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCurrent classification recognizes seven distinct pathotypes of diarrheagenic E. coli: enteropathogenic (EPEC), enterohemorrhagic (EHEC), enterotoxigenic (ETEC), enteroinvasive (EIEC), enteroaggregative (EAEC), diffusely adherent (DAEC), and adherent-invasive (AIEC) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Of particular public health significance, EHEC, EIEC and EAEC strains can be transmitted through the food chain, posing substantial One Health challenges at the human-animal-environment interface [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe clinical management of E. coli infections faces two major obstacles: (1) the remarkable diversity of pathogenic variants and their virulence factor combinations [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e–\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and (2) the escalating crisis of antimicrobial resistance. Although antibiotics were initially effective in treating these infections [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e–\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], the emergence of resistant strains has been accelerated by the plasmid-mediated dissemination of resistance genes among bacterial populations [\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e–\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Contemporary isolates frequently harbor class 1 integrons, extended-spectrum β-lactamases (ESBLs), AmpC β-lactamases, and plasmid-encoded quinolone resistance determinants (e.g., \u003cem\u003eermB\u003c/em\u003e, \u003cem\u003etetB\u003c/em\u003e, \u003cem\u003eblaTEM\u003c/em\u003e) [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e–\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], significantly complicating treatment regimens.\u003c/p\u003e\u003cp\u003eHistorically, ETEC has been the predominant causative agent of calf diarrhea since the first documented cases in the 19th century. In China, ETEC-associated outbreaks among cattle herds have been endemic since the 1980s, with particularly high morbidity and mortality rates in yaks from high-altitude regions. The epidemiological patterns of E. coli infections exhibit distinct geographical variations that correlate with regional socioeconomic development levels and hygiene standards.\u003c/p\u003e\u003cp\u003eThis investigation presents a comprehensive analysis of 124 E. coli isolates obtained from yaks and wildlife in the Qinghai-Tibet Plateau, employing multilocus sequence typing (MLST), phylogenetic analysis, antimicrobial susceptibility testing, and virulence/resistance gene profiling. The study specifically addresses three critical research questions: (1) the potential for cross-species transmission between domestic and wild animals, (2) host-specific associations with virulence factor distribution, and (3) the implications of horizontal gene transfer for One Health surveillance systems.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eStrain collection location\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 124 \u003cem\u003eE. coli\u003c/em\u003e strains were isolated from Yaks (n = 88) and wildlife (n = 36) with diarrhea from 2022 to 2023 in The Tibetan Plateau region of China. The sampling area involved nine districts and counties in three prefectures in Qinghai Province (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhylogenetic and MLST analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUsing the multiplex PCR method, and he primer information is listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. PCR detection of the three genes \u003cem\u003echuA\u003c/em\u003e, \u003cem\u003eyjaA\u003c/em\u003e, and \u003cem\u003eTspE4.C2\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) was conducted to determine the phylogenetic group typing of \u003cem\u003eE. coli\u003c/em\u003e based on electrophoretic patterns. Primers were synthesized by Xi'an Tsingke Biological Co., Ltd. The PCR reaction system (25 µL) consisted of 1 µL each of three pairs of upstream and downstream primers, 3 µL of bacterial DNA template, 12.5 µL of 2×Taq Master Mix, and 3.5 µL of ddH\u003csub\u003e2\u003c/sub\u003eO. PCR amplification conditions were as follows: initial denaturation at 94℃ for 5 minutes; 30 cycles of denaturation at 94℃ for 30 seconds, annealing for 30 seconds, and extension at 72℃ for 30 seconds; final extension at 72℃ for 7 minutes. PCR products were analyzed by 1.5% agarose gel electrophoresis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimer Information for Systematic Evolution Grouping\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePrimer\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSequence(5༇→3༇)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLength(bp)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAnnealing temperature\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eChuA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGACGAACCAACGGTCAGGAT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e279\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTGCCGCCAGTACCAAAGACA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eYjaA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTGAAGTGTCAGGAGACGCTG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e211\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eATGGAGAATGCGTTCCTCAC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eTspE4.C2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGAGTAATGTCGGGGCATTCA\u003c/p\u003e\u003cp\u003eCGCGCCAACAAAGTATTACG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e152\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMLST of \u003cem\u003eE. coli\u003c/em\u003e strains were identified by PCR, and the primer information is listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Multiplex PCR was performed as previously described to detect the phylogenetic type of the strain. PCRs were performed in a final volume of 100 µL. PCR mix component was as follows: 4 µL of genomic DNA, 2 µL of each primer, 50 µL of 2×Spark Taq PCR master mix, 42 µL of Sterile Enzyme-Free Water. PCR amplification condition was as follows: initial denaturation at 95°C for 5 min followed by 30 cycles of 45 s at 95°C, 30 s at Tm and 1 min at 72°C, with a final extension of 10 min at 72°C. The target band is recycled, and the recovered product is sent to a sequencing company for sequencing. The sequencing results were uploaded to pubMLST, matched to each housekeeping gene, and ST typing was obtained after assembly.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMLST Typing Primers Information\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePrimer\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSequence(5༇→3༇)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLength(bp)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAnnealing temperature(℃)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eadk\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eATTCTGCTTGGCGCTCCGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e583\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e54\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCCGTCAACTTTCGCGTATTT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003efumC\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTCACAGGTCGCCAGCGCTTC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e806\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e54\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGTACGCAGCGAAAAAGATT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003egyrB\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTCGGCGACACGGATGACGGC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e911\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eATCAGGCCTTCACGCGCATC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eicd\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eATGGAAAGTAAAGTAGTTGTTCCGGCACA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e878\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e54\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGGACGCAGCAGGATCTGTT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003emdh\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eATGAAAGTCGCAGTCCTCGGCGCTGCTGGCGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e932\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTTAACGAACTCCTGCCCCAGAGCGATATCTTTCTT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003epurA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCGCGCTGATGAAAGAGATGA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e816\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e54\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCATACGGTAAGCCACGCAGA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003erecA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCGCATTCGCTTTACCCTGACC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e780\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTCGTCGAAATCTACGGACCGGA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntimicrobial susceptibility testing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAntimicrobial susceptibility was determined using the Kirby-Bauer disc agar diffusion method according to Clinical and Laboratory Standards Institute (CLSI) guidelines. A total of 11 antimicrobial agents (ampicillin, amikacin, ciprofloxacin, gentamicin, kanamycin, tetracycline, ofloxacin, norfloxacin, ceftriaxone, cefotaxime and sulfamethoxazole trimethoprim) were used to test the antimicrobial susceptibility of the isolated strains. Three replicates were set for each antibiotic test. The reference strain \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 was used as the positive control.\u003c/p\u003e\u003cp\u003eThe control strain ATCC25922 and 124 test strains were separately inoculated onto LB agar medium and incubated overnight at 37℃. Single colonies were picked from the LB agar plates and transferred into regular LB broth, which was then incubated in a shaking incubator at 37℃ for 16–18 hours, with the bacterial suspension concentration adjusted using physiological saline. Inoculate 200 µL of the prepared bacterial suspension into an MH agar plate, and spread the suspension evenly over the surface of the MH agar medium with a sterile glass spreading rod. After the water on the plate has been completely absorbed, apply the antimicrobial susceptibility discs. Once the bacterial suspension is evenly spread, use sterilized forceps to place the antimicrobial susceptibility discs, ensuring that three discs are evenly distributed on each plate. During the application process, ensure that the discs firmly adhere to the medium's surface. Invert the plates and incubate at 37℃ for 16–18 hours, then observe the results. Measure the diameter of the inhibition zones with a caliper, in millimeters. Results were determined according to CLSI international standards for antimicrobial susceptibility testing. Some strains exhibited double rings, with measurements taken based on the outer ring.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of putative virulence genes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe virulence genes of \u003cem\u003eE. coli\u003c/em\u003e strains were identified by PCR. A total of 35 tested virulence genes belongs to the following categories, including adhesion factor, hemolysin, toxin (resistant to heat-resistant enterotoxins, heat-sensitive enterotoxin), locus of enterocyte effacement, High Pathogenicity Island and some other virulence genes, primer information for all virulence genes can be found in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimer information for virulence genes\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003ePrimer\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSequence\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLength\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTm(℃)\u003c/p\u003e\u003cp\u003etemperature\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eK88\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGATGAAAGACTCTGATTGCA\u003c/p\u003e\u003cp\u003eGATTGCTACGTTCAGCGGAGCG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e841\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eK99\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCTGAAAAAAACACTGCTAGCTATT\u003c/p\u003e\u003cp\u003eCATATAAGTGACTAAGAAGGATGC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e543\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eF41\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGATTGCTACGTTCAGCGGAGCG\u003c/p\u003e\u003cp\u003eTCTGAGGTCATCCCAATTGTGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e628\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003e987P\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGTTACTGCCAGTCTATGCCAAGTG\u003c/p\u003e\u003cp\u003eTCGGTGTACCTGCTGAACGAATAG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e463\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003efedA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCATGATGGATCCATGAAAAGACTAGTGTTTATTTCTT\u003c/p\u003e\u003cp\u003eCATGATGAATTCTTACTTGTAAGTAACCGCGTAAGCC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e516\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eHPI\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAAGGATTCGCTGTTACCGGAC\u003c/p\u003e\u003cp\u003eTCGTCGGGCAGCGTTTCTTCT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e280\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eLEE\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATATCCGTTTTAATGGCTATCT\u003c/p\u003e\u003cp\u003eAATCTTCTGCGTACTGTGTTCA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e425\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eSTa\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGGGTTGGCAATTTTTATTTCTGTA\u003c/p\u003e\u003cp\u003eATTACAACAAAGTTCACAGCAGTA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e183\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eSTb\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATGTAAATACCTACAACGGGTGAT\u003c/p\u003e\u003cp\u003eTATTTGGGCGCCAAAGCATGCTCC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e360\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eVT1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATTCGCTGAATGTCATTCGCT\u003c/p\u003e\u003cp\u003eACGCTTCCCAGAATTGCATTA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e664\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eVT2all\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATTCGCTGAATGTCATTCGCT\u003c/p\u003e\u003cp\u003eACGCTTCCCAGAATTGCATTA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e484\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eLTⅠ\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTAGAGACCGGTATTACAGAAATCTGA\u003c/p\u003e\u003cp\u003eTCATCCCGAATTCTGTTATATATGTC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e282\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eSLT2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGGTTATGCCTCAGTCATTATTAA\u003c/p\u003e\u003cp\u003eGAATGAAGAAGATGTTTATAGCGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e281\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eSLT2e\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGAATGAAGAAGATGTTTATAGCGG\u003c/p\u003e\u003cp\u003eTTTTATGGAACGTAGGTAYYACC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e454\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eF17\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGCAGAAAATTCAATTTATCCTTGG\u003c/p\u003e\u003cp\u003eCTGATAAGCGATGGTGTAATTAAC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e537\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eF18\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGTGAAAAGACTAGTGTTTATTTC\u003c/p\u003e\u003cp\u003eCTTGTAAGTAACCGCGTAAGC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e510\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eompA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eACGCTGTTTCACGTTGTCA\u003c/p\u003e\u003cp\u003eAACCCGTATGTTGGCTTTG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e753\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eompT\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eTCATCCCGGAAGCCTCCCTCACTACTAT\u003c/p\u003e\u003cp\u003eTAGCGTTTGCTGCACTGGGCTTCTGATAC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e496\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003efimC\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eGTTCATGGCAATGGTGGTT\u003c/p\u003e\u003cp\u003eAGTTCCGGCATTCAACTCT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e514\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003efimA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eGCTCTGGCTGATACTACACC\u003c/p\u003e\u003cp\u003eTTATTGATACTGAACCTTGA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e495\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eespA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eTATCAGGCACAAAGCGATCTGTC\u003c/p\u003e\u003cp\u003eTATCTCCGGTTATTTACCAAGGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e432\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eaer\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eTACCGGATTGTAATATGCAGACCGT\u003c/p\u003e\u003cp\u003eAATATCTTCCTCCAGTCCGGAGAAG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e602\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eflu\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCTGGTATGGAATCACTTACGGG\u003c/p\u003e\u003cp\u003eGAGAATGCTCCCAGGCGGTTTAT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e965\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e57\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eluxS\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATGCCGTTGTTAGATAGC\u003c/p\u003e\u003cp\u003eCTAGATGTGCAGTTCCTGC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e516\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003ehlyA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGCATCATCAAGCGTACGTTCC\u003c/p\u003e\u003cp\u003eAATGAGCCAAGCTGGTTAAGCT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e534\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003ehlyE\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCTCAATCGGCATCCACAT\u003c/p\u003e\u003cp\u003eGTTTCCCTTCAACAACCC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e456\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e52\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003ehlyF\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGGCCACAGTCGTTTAGGGTGCTTACC\u003c/p\u003e\u003cp\u003eGGCGGTTTAGGCATTCCGATACTCAG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e450\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eeaeA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCTGAACGGCGATTACGCGAA\u003c/p\u003e\u003cp\u003eCCAGACGATACGATCCAG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e798\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e52\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003epapC\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGGGCGTGATAACGATTC\u003c/p\u003e\u003cp\u003eATTTGCCAGCGGACTAC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e234\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e47\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003esfaD\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGCAACAGCAACGCTGGTTGCATCAT\u003c/p\u003e\u003cp\u003eAGAGAGAGCCACTCTTATACGGACA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e410\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e62\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003efeaG\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGAATCTGTCCGAGAATATCA\u003c/p\u003e\u003cp\u003eGTTGGTACAGGTCTTAATGC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e499\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eetrA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCTTCTTCCTAACGAAACTATCATTA\u003c/p\u003e\u003cp\u003eTGACATATCAACTTTCTCTTACGC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e913\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003erfc\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATCCATCAGGAGGGGACTGGA\u003c/p\u003e\u003cp\u003eAACCATACCAACCAATGCGAG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e788\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e63\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003esepA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTAAAACCCGCCGCCTGAGTA\u003c/p\u003e\u003cp\u003eTGCCGGTGAACAGGAGGTTT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e611\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e62\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003efasA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGTAACTCCACCGTTTGGTATC\u003c/p\u003e\u003cp\u003eAAGTTACTGCCAGTCTATGC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e409\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePCRs were performed in a final volume of 50 µl. PCR mix component was as follows: 2 µL of genomic DNA, 1 µL of each primer, 25 µL of 2×Spark Taq Pcr master mix, 21 µL of Sterile Enzyme-Free Water. PCR amplification condition was as follows: initial denaturation at 95°C for 5 min followed by 30 cycles of 45 s at 95°C, 30 s at Tm and 1 min at 72°C, with a final extension of 10 min at 72°C.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of antibiotic-resistance genes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAntibiotic-resistance genes of \u003cem\u003eE. coli\u003c/em\u003e strains were identified by PCR. A total of 30 tested virulence genes belongs to five broad categories, including tetracyclines(\u003cem\u003etetA\u003c/em\u003e、\u003cem\u003etetB\u003c/em\u003e、\u003cem\u003etetC\u003c/em\u003e、\u003cem\u003etetD\u003c/em\u003e、\u003cem\u003etetG\u003c/em\u003e、\u003cem\u003etetM\u003c/em\u003e、\u003cem\u003etetX\u003c/em\u003e), sulfonamides(\u003cem\u003esul1\u003c/em\u003e、\u003cem\u003esul2\u003c/em\u003e、\u003cem\u003esul3\u003c/em\u003e), quinolones༈\u003cem\u003eqnrA\u003c/em\u003e、\u003cem\u003eqnrB\u003c/em\u003e、\u003cem\u003eqnrS\u003c/em\u003e、\u003cem\u003eqepA\u003c/em\u003e༉, β-lactams༈\u003cem\u003eblaDHA\u003c/em\u003e、\u003cem\u003eblaTEM\u003c/em\u003e、\u003cem\u003eblaCMY-2\u003c/em\u003e、\u003cem\u003eblaSHV\u003c/em\u003e、\u003cem\u003eCTX-M1\u003c/em\u003e、\u003cem\u003eCTX-M2\u003c/em\u003e、\u003cem\u003eCTX-M8\u003c/em\u003e、\u003cem\u003eCTX-M9\u003c/em\u003e、\u003cem\u003eCTX-M25\u003c/em\u003e༉, aminoglycosides༈\u003cem\u003ecat1\u003c/em\u003e、\u003cem\u003ecat2\u003c/em\u003e、\u003cem\u003eaac(3')-IV\u003c/em\u003e、\u003cem\u003eaadA2\u003c/em\u003e、\u003cem\u003ermtB\u003c/em\u003e、\u003cem\u003eaac(6')-Ib\u003c/em\u003e、\u003cem\u003eaph(s')-vII\u003c/em\u003e༉. Selected drug resistance gene primer information is provided in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eResistance gene primer information\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePrimer\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSequence\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLength\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTm(℃)\u003c/p\u003e\u003cp\u003eTemperature\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCTX-M1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eATGGTTAAAAAATCACTGCGTCAGTTC\u003c/p\u003e\u003cp\u003eTCACAAACCGTTGGTGACGATTTTAGCCGC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e876\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e52\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCTX-M2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eATGATGACGCAGAGCATTCGCCGCTCA\u003c/p\u003e\u003cp\u003eTCAGAAACCGTGGGTTACGATTTTCGC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e876\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCTX-M8\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eACTTCAGCCACACGGATTCA\u003c/p\u003e\u003cp\u003eCGAGTACGTCACGACGACTT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e878\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCTX-M9\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eATGGTGACAAAGAGAGTGCA\u003c/p\u003e\u003cp\u003eCCCTTCGGCGATGATTCTC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e870\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCTX-M25\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCACACGAATTGAATGTTCAG\u003c/p\u003e\u003cp\u003eTCACTCCACATGGTGAGT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e924\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003etetA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCACTATGGCATTCTGCTGGC\u003c/p\u003e\u003cp\u003eCATAGATCGCCGTGAAGAGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e948\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003etetB\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGCCCAGTGCTGTTGTTGTC\u003c/p\u003e\u003cp\u003eAAGACCAAGACCCGCTAATG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e553\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003etetC\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTCCTGCTCGCTTCGCTACT\u003c/p\u003e\u003cp\u003eTGGTCGTCATCTACCTGC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e730\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003etetD\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAAACCATTACGGCATTCTGC\u003c/p\u003e\u003cp\u003eGACCGGATACACCATCCATC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e787\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003etetG\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCGGTCTTATGGGTGCTCTA\u003c/p\u003e\u003cp\u003eCCTTGCTTGTTACTGAC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e721\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003etetM\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTTATCAACGGTTTATCAGG\u003c/p\u003e\u003cp\u003eCGTATATATGCAAGACG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e397\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003etetX\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCAATAATTGGTGGTGGACCC\u003c/p\u003e\u003cp\u003eTTCTTACCTTGGACATCCCG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e468\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003esul1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTCAGACGTCGTGGATGTCG\u003c/p\u003e\u003cp\u003eCGAAGAACCGCACAATCTCG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e393\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e57\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003esul2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCCTGTTTCGTCCGACACAGA\u003c/p\u003e\u003cp\u003eGAAGCGCAGCCGCAATTCAT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e435\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e59\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003esul3\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAGATGTGATTGATTTGGGAGC\u003c/p\u003e\u003cp\u003eTAGTTGTTTCTGGATTAGAGCCT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e443\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e53\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eqnrA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTCAGCAAGAGGATTTCTCA\u003c/p\u003e\u003cp\u003eGGCAGCACTATTACTCCCA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e627\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eqnrB\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eACGATGCCTGGTAGTTGTCC\u003c/p\u003e\u003cp\u003eACGACATTCGTCAACTGCAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e469\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eqnrS\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eACGACATTCGTCAACTGCAA\u003c/p\u003e\u003cp\u003eTAAATTGGCACCCTGTAGGC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e299\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eqepA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGCAGGTCCAGCAGCGGGTAG\u003c/p\u003e\u003cp\u003eCTTCCTGCC CGAGTATCGTG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e299\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eblaDHA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAACTTTCACAGGTGTGCTGGGT\u003c/p\u003e\u003cp\u003eCCGTACGCATACTGGCTTTGC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e387\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e63\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eblaTEM\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eATAAAATTCTTGAAGACGAAA\u003c/p\u003e\u003cp\u003eGACAGTTACCAATGCTTAATC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1080\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e52\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eblaSHV\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCACTCAAGGATGTATTGTG\u003c/p\u003e\u003cp\u003eTTAGCGTTGCCAGTGCTCG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e885\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e63\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBlaCMY-2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eATGATGAAAAAATCGTTATGC\u003c/p\u003e\u003cp\u003eTTGCAGCTTTTCAAGAATGCG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1143\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e57\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ecat1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCTTGTCGCCTTGCGTATAAT\u003c/p\u003e\u003cp\u003eATCCCAATGGCATCGTAAAG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e508\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e53\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ecat2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAACGGCATGATGAACCTGAA\u003c/p\u003e\u003cp\u003eATCCCAATGGCATCGTAAAG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e547\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e53\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ermtB\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eACATCAACGATGCCCTCAC\u003c/p\u003e\u003cp\u003eAAGTTCTGTTCCGATGGTC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e472\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e53\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eaadA2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGGTGCTAAGCGTCATTGAGC\u003c/p\u003e\u003cp\u003eGCTTCAAGGTTTCCCTCAGC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e470\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e57\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eaac(6’)-Ib\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTTGCGATGCTCTATGAGTGGCTA\u003c/p\u003e\u003cp\u003eCTCGAATGCCTGGCGTGTTT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e482\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eaph (3’)-Ⅶ\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTCCACAGGATGGCAAGATCC\u003c/p\u003e\u003cp\u003eTTCAACGGGAAACGTCTTGC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e690\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eaac(3’)-Ⅳ\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGGCCACTTGGACTGATCGAG\u003c/p\u003e\u003cp\u003eGCGGATGCAGGAAGATCAAC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e409\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePCRs were performed in a final volume of 50 µL. PCR mix component was as follows; 2 µL of genomic DNA, 1 µL of each primer, 25 µL of 2×Spark Taq Pcr master mix, 21 µL of Sterile Enzyme-Free Water. PCR amplification condition was as follows: initial denaturation at 95°C for 5 min followed by 30 cycles of 45 s at 95°C, 30 s at Tm and 1 min at 72°C, with a final extension of 10 min at 72°C.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003ephylogenetic and MLST analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 124 isolates were confirmed as \u003cem\u003eE. coli\u003c/em\u003e via biochemical and 16s RNA. The majority of the \u003cem\u003eE. coli\u003c/em\u003e isolates belonged to phylogroup B1 (77.42%, n\u0026thinsp;=\u0026thinsp;96), with the other isolates belonging to phylogroup A (16.94%, n\u0026thinsp;=\u0026thinsp;21) and B2 (5.65%, n\u0026thinsp;=\u0026thinsp;7). However, no isolates were assigned to phylogenetic group D (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The phylogroups B1 were the most common in the Yak and wildlife samples.\u003c/p\u003e\u003cp\u003eMLST analysis revealed the presence of highly diverse sequence types (STs, N\u0026thinsp;=\u0026thinsp;37 unique STs) among \u003cem\u003eE. coli\u003c/em\u003e isolates subjected to sequencing (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Among yak-derived \u003cem\u003eE. coli\u003c/em\u003e isolates, 37 different STs were identified, including ST2522, ST58, ST56, ST101, ST40, ST155, ST156, ST10, ST1252, ST3884, ST21, ST2166, ST2035, ST1665, ST1101, ST446, ST342, ST73, ST43, ST1249, ST1642, ST10601, ST85, ST345, ST16080, ST1079, ST6178, ST2973, ST75, ST109, ST1426, ST2179, ST223, ST602, ST2253, and a novel ST16626 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The phylogenetic tree reveals that yak-derived \u003cem\u003eE. coli\u003c/em\u003e strains and wild animal-derived \u003cem\u003eE. coli\u003c/em\u003e strains are distinctly separated into two branches, with several yak-derived \u003cem\u003eE. coli\u003c/em\u003e strains also observed within the wild animal branch. Through common mapping of their source regions, phylogenetic groups, and MLST, it is evident that there are differences among \u003cem\u003eE. coli\u003c/em\u003e strains from different origins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntimicrobial susceptibility testing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAntimicrobial susceptibility testing revealed that 124 \u003cem\u003eE. coli\u003c/em\u003e isolates exhibited resistance rates of 41.5% to TCY, 34.7% to AMP, 16.9% to AMK, 25.0% to GEN, 16.1% to OFX, 15.3% to NOR, 17.7% to CRO, 36.3% to CTX, 25.8% to SXT, 24.2% to KAN, and 33.1% to CIP against 11 antibiotics, with all resistance rates below 50.0% (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). The comparison of antibiotic resistance rates between yak-derived and wildlife-derived isolates revealed that the resistance rates of yak-derived isolates to various antibiotics were consistently higher than those of wildlife-derived isolates, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. This phenomenon may be due to the frequent use of antibiotics in the farming process of yaks.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of putative virulence genes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 124 strains were tested for 35 virulence genes, and all 124 strains were found to carry virulence genes, including \u003cem\u003esfaD\u003c/em\u003e, \u003cem\u003eeaeA\u003c/em\u003e, \u003cem\u003eF17\u003c/em\u003e, \u003cem\u003eespA\u003c/em\u003e, \u003cem\u003eaer\u003c/em\u003e, \u003cem\u003efimA\u003c/em\u003e, \u003cem\u003eompT\u003c/em\u003e, h\u003cem\u003elyF\u003c/em\u003e, \u003cem\u003eflu\u003c/em\u003e, \u003cem\u003eompA\u003c/em\u003e, \u003cem\u003efimC\u003c/em\u003e, \u003cem\u003eluvs\u003c/em\u003e, \u003cem\u003eetrA\u003c/em\u003e, \u003cem\u003ePAPC\u003c/em\u003e, \u003cem\u003ehlyA\u003c/em\u003e, \u003cem\u003eHPI\u003c/em\u003e, \u003cem\u003eLEE\u003c/em\u003e, \u003cem\u003eVT1\u003c/em\u003e, \u003cem\u003eSLT2\u003c/em\u003e, \u003cem\u003e987P\u003c/em\u003e, \u003cem\u003eF41\u003c/em\u003e, \u003cem\u003esepA\u003c/em\u003e, \u003cem\u003ehlyE\u003c/em\u003e, and \u003cem\u003eSTb\u003c/em\u003e. The detection rates of each virulence gene are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e. The analysis reveals that yak-derived \u003cem\u003eE. coli\u003c/em\u003e strains exhibit both differences and similarities in the carriage of virulence genes compared to wildlife-derived \u003cem\u003eE. coli\u003c/em\u003e strains. The differences lie in the detection of five virulence genes\u0026mdash;\u003cem\u003e987P\u003c/em\u003e, \u003cem\u003eF41\u003c/em\u003e, \u003cem\u003esepA\u003c/em\u003e, \u003cem\u003ehlyE\u003c/em\u003e, and \u003cem\u003eSTb\u003c/em\u003e\u0026mdash;exclusively in wildlife \u003cem\u003eE. coli\u003c/em\u003e strains, as well as \u003cem\u003ehlyA\u003c/em\u003e, \u003cem\u003eespA\u003c/em\u003e, and \u003cem\u003eeaeA\u003c/em\u003e also being detected only in wildlife \u003cem\u003eE. coli\u003c/em\u003e strains. The similarities include the detection of all other virulence genes except the aforementioned eight, with high detection rates for \u003cem\u003eetrA\u003c/em\u003e, \u003cem\u003eluxS\u003c/em\u003e, \u003cem\u003efimC\u003c/em\u003e, and \u003cem\u003eompA\u003c/em\u003e shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The analysis of the number of virulence genes carried by each strain revealed that 124 \u003cem\u003eE. coli\u003c/em\u003e strains exhibited 88 different virulence gene profiles, with each strain carrying more than three virulence genes. The most common virulence gene profile was \"\u003cem\u003eF17\u0026thinsp;+\u0026thinsp;flu\u0026thinsp;+\u0026thinsp;ompA\u0026thinsp;+\u0026thinsp;fimC\u0026thinsp;+\u0026thinsp;fimA\u0026thinsp;+\u0026thinsp;luxs\u0026thinsp;+\u0026thinsp;hlyA\u0026thinsp;+\u0026thinsp;etrA\u003c/em\u003e,\" which accounted for 6.45% of all virulence gene profiles.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of antibiotic-resistance genes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe results of drug resistance gene detection are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, indicating positive detections for β-lactams, tetracyclines, sulfonamides, aminoglycosides, and quinolones. Among β-lactam genes, \u003cem\u003ebla-TEM\u003c/em\u003e, \u003cem\u003ebla-SHV\u003c/em\u003e, \u003cem\u003eCTX-M1\u003c/em\u003e, \u003cem\u003eCTX-M8\u003c/em\u003e, and \u003cem\u003eCTX-M9\u003c/em\u003e were detected at rates of 88.7%, 7.3%, 2.4%, 11.3%, and 11.3%, respectively. For tetracyclines, \u003cem\u003etetA\u003c/em\u003e, \u003cem\u003etetB\u003c/em\u003e, \u003cem\u003etetC\u003c/em\u003e, \u003cem\u003etetD\u003c/em\u003e, and \u003cem\u003etetM\u003c/em\u003e were detected at rates of 20.9%, 29.0%, 4.8%, 4.8%, and 2.4%, respectively. Sulfonamide genes \u003cem\u003esul1\u003c/em\u003e, \u003cem\u003esul2\u003c/em\u003e, and \u003cem\u003esul3\u003c/em\u003e were detected at rates of 21.8%, 28.2%, and 9.7%, respectively. The quinolone \u003cem\u003eqnrS\u003c/em\u003e was detected at a rate of 6.5%. For aminoglycosides, \u003cem\u003ecat1\u003c/em\u003e, \u003cem\u003ecat2\u003c/em\u003e, \u003cem\u003eaadA2\u003c/em\u003e, \u003cem\u003eaac(6')-Ib\u003c/em\u003e, and \u003cem\u003eaph(3')-vII\u003c/em\u003e were detected at rates of 67.7%, 54.0%, 10.5%, 9.7%, and 4.8%, respectively. A comparative analysis of the detection rates of resistance genes in yak and wildlife isolates revealed that the eight resistance genes \u003cem\u003ebla-SHV\u003c/em\u003e, \u003cem\u003eCTX-M1\u003c/em\u003e, \u003cem\u003etetC\u003c/em\u003e, \u003cem\u003etetD\u003c/em\u003e, \u003cem\u003esul3\u003c/em\u003e, \u003cem\u003ecat2\u003c/em\u003e, \u003cem\u003eaadA2\u003c/em\u003e, and \u003cem\u003eaac(6')-Ib\u003c/em\u003e were exclusively detected in yak isolates, with no detection observed in wildlife, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e and Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis comprehensive study characterized 124 \u003cem\u003eE. coli\u003c/em\u003e isolates from yaks and wildlife in Qinghai Plateau through phylogenetic analysis, MLST typing, antimicrobial susceptibility testing, and virulence/resistance gene profiling. Phylogenetic grouping showed predominance of phylogroup B1 (77.42%), followed by A (16.94%) and B2 (5.65%), with no D group isolates. MLST revealed 37 distinct sequence types, including novel ST16626, with phylogenetic separation between yak and wildlife strains though some overlap existed. Notably, 45 strains (36.3%) lacked \u003cem\u003erecA\u003c/em\u003e, preventing ST typing - a significant finding given \u003cem\u003erecA\u003c/em\u003e crucial roles in DNA repair and SOS response [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and its emerging potential as a therapeutic target to combat heteroresistance [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Antimicrobial testing demonstrated resistance rates below 50% for all tested antibiotics, with highest resistance to tetracycline (41.94%), cefotaxime (36.29%), and ampicillin (34.68%). Yak-derived isolates showed consistently higher resistance than wildlife strains, likely reflecting agricultural antibiotic use. All strains carried virulence genes (35 tested), with 88 unique virulence profiles identified. Key differences emerged in virulence gene carriage: \u003cem\u003e987P\u003c/em\u003e, \u003cem\u003eF41\u003c/em\u003e, \u003cem\u003esepA\u003c/em\u003e, \u003cem\u003ehlyE\u003c/em\u003e, \u003cem\u003eSTb\u003c/em\u003e, \u003cem\u003ehlyA\u003c/em\u003e, \u003cem\u003eespA\u003c/em\u003e and \u003cem\u003eeaeA\u003c/em\u003e were exclusive to wildlife strains, while \u003cem\u003eetrA\u003c/em\u003e, \u003cem\u003eluxS\u003c/em\u003e, \u003cem\u003efimC\u003c/em\u003e and \u003cem\u003eompA\u003c/em\u003e showed high prevalence across both groups. Resistance gene analysis detected β-lactam (\u003cem\u003ebla-TEM\u003c/em\u003e 88.7%), tetracycline (\u003cem\u003etetB\u003c/em\u003e 29.0%), sulfonamide (\u003cem\u003esul2\u003c/em\u003e 28.2%), quinolone (\u003cem\u003eqnrS\u003c/em\u003e 6.5%), and aminoglycoside (\u003cem\u003ecat1\u003c/em\u003e 67.7%) resistance genes, with eight genes (including \u003cem\u003ebla-SHV\u003c/em\u003e and \u003cem\u003eCTX-M1\u003c/em\u003e) unique to yak isolates. The study highlights \u003cem\u003eE. coli\u003c/em\u003e complex biology - as both commensal and pathogen through virulence factor acquisition, and as a reservoir of diverse resistance mechanisms including ESBL production and biofilm formation regulated by \u003cem\u003eluxS\u003c/em\u003e/\u003cem\u003eAI-2\u003c/em\u003e quorum sensing [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. These findings demonstrate: (1) significant strain diversity with host adaptation, (2) evidence of cross-species transmission potential, and (3) emerging resistance patterns in this unique high-altitude ecosystem, underscoring the need for One Health surveillance to address this public health challenge. The \u003cem\u003erecA\u003c/em\u003e-deficient strains and host-specific virulence/resistance patterns represent particularly noteworthy findings requiring further investigation.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis first comprehensive characterization of \u003cem\u003eE. coli\u003c/em\u003e at the Qinghai yak-wildlife interface reveals significant strain diversity with host-specific adaptations, provides evidence of potential cross-species transmission, and documents moderate but concerning antimicrobial resistance patterns, underscoring the imperative for integrated One Health surveillance to monitor emerging threats in high-altitude ecosystems.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cem\u003eE. coli: Escherichia coli\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMLST: Multilocus sequence typing\u003c/p\u003e\n\u003cp\u003ePCR: Polymerase Chain Reaction\u003c/p\u003e\n\u003cp\u003e\u0026zwnj;LB Broth: Luria-Bertani Broth\u003c/p\u003e\n\u003cp\u003eMH agar: Mueller-Hinton Agar\u003c/p\u003e\n\u003cp\u003eCLSI: Clinical and Laboratory Standards Institute\u003c/p\u003e\n\u003cp\u003eAMP: ampicillin\u003c/p\u003e\n\u003cp\u003eAMK: amikacin\u003c/p\u003e\n\u003cp\u003eCIP: ciprofloxacin\u003c/p\u003e\n\u003cp\u003eGEN: gentamicin\u003c/p\u003e\n\u003cp\u003eKAN: kanamycin\u003c/p\u003e\n\u003cp\u003eTCY: tetracycline\u003c/p\u003e\n\u003cp\u003eOFX: ofloxacin\u003c/p\u003e\n\u003cp\u003eNOR: norfloxacin\u003c/p\u003e\n\u003cp\u003eCRO: ceftriaxone\u003c/p\u003e\n\u003cp\u003eCTX: cefotaxime\u003c/p\u003e\n\u003cp\u003eSXT: sulfamethoxazole trimethoprim\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cstrong\u003e\u003cbr\u003e\u003c/strong\u003eLaboratory Animal Management Committee of Qinghai Academy of Animal Husbandry and Veterinary Sciences ruled that no formal ethics approval was required to conduct this research. Before conducting the research, informed consent was obtained from all the owners of the yak farms included in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are available in the main text or the supplementary materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Qinghai Province Science and Technology Achievement Transformation and Key R\u0026amp;D Project (2024ZY014).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization S.H and T.S.; methodology, T.S.; software, S.Z., and S.H; formal analysis, R.G.; investigation S. L, W. Z and L.L; writing\u0026mdash;original draft preparation, S.H; writing\u0026mdash;review and editing, S.H and T.S; project administration, S.L.; funding acquisition S.L. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJesser KJ, Levy K. Updates on defining and detecting diarrheagenic \u003cem\u003eEscherichia coli\u003c/em\u003e pathotypes. Curr Opin Infect Dis. 2020;33(5):372\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRaetz CR, Whitfield C. Lipopolysaccharide endotoxins. Annu Rev Biochem. 2002;71:635\u0026ndash;700.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePakbin B, Br\u0026uuml;ck WM, Rossen JWA. Virulence factors of Enteric Pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e: A Review. Int J Mol Sci. 2021;22(18):9922.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTenaillon O, Skurnik D, Picard B, Denamur E. The population genetics of commensal \u003cem\u003eEscherichia coli\u003c/em\u003e. Nat Rev Microbiol. 2010;8:207\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eClements A, Young JC, Constantinou N, Frankel G. Infection strategies of enteric pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e. Gut Microbes. 2012;3:71\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePakbin B, Akhondzadeh Basti A, Khanjari A, Azimi L, Karimi A. Differentiation of stx1A gene for detection of \u003cem\u003eEscherichia coli\u003c/em\u003e serotype O157: H7 and \u003cem\u003eShigella dysenteriae\u003c/em\u003e type 1 in food samples using high resolution melting curve analysis. Food Sci Nutr. 2020;8:3665\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang S-C, Lin C-H, Aljuffali IA, Fang J-Y. Current pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e foodborne outbreak cases and therapy development. Arch Microbiol. 2017;199:811\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlegbeleye OO, Sant\u0026rsquo;Ana AS. Pathogen subtyping tools for risk assessment and management of produce-borne outbreaks. Curr Opin Food Sci. 2020;32:83\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKaito C, Yoshikai H, Wakamatsu A, et al. Non-pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e acquires virulence by mutating a growth-essential LPS transporter[J]. PLoS Pathog. 2020;16(4):e1008469.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKaper, Nataro HL, Mobley. Pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e[J]. Nat Rev Microbiol. 2004;2(2):123\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAllocati. \u003cem\u003eEscherichia coli\u003c/em\u003e in Europe: an overview[J]. Int J Environ Res Public Health. 2013;10(12):6235\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJang. Environmental \u003cem\u003eEscherichia coli\u003c/em\u003e: ecology and public health implications-a review[J]. J Appl Microbiol. 2017;123(3):570\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLeekitcharoenphon P, Johansson MHK, Munk P, et al. Genomic evolution of antimicrobial resistance in \u003cem\u003eEscherichia coli\u003c/em\u003e[J]. Sci Rep. 2021;11(1):15108.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAwad WS. Molecular characterization of pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e isolated from diarrheic and in-contact cattle and buffalo calves[J]. Trop Anim Health Prod. 2020;52(6):3173\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCapps, Identification. Shiga toxin subtypes and prevalence of minor serogroups of Shiga toxin-producing \u003cem\u003eEscherichia coli\u003c/em\u003e in feedlot cattle feces[J]. Sci Rep. 2021;11(1):8601.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCroxen. Recent advances in understanding enteric pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e[J]. Clin Microbiol Rev. 2013;26(4):822\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGomes. Diarrheagenic \u003cem\u003eEscherichia coli\u003c/em\u003e[J]. Brazilian J Microbiol. 2016;47:3\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGaastra W, Svennerholm AM. Colonization factors of human enterotoxigenic \u003cem\u003eEscherichia coli\u003c/em\u003e (ETEC) [J]. Trends Microbiol. 1996;4(11):444\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWolf MK. Occurrence, distribution, and associations of O and H serogroups, colonization factor antigens, and toxins of enterotoxigenic \u003cem\u003eEscherichia coli\u003c/em\u003e[J]. Clin Microbiol Rev. 1997;10(4):569\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFrancis DH. Enterotoxigenic \u003cem\u003eEscherichia coli\u003c/em\u003e infection in pigs and its diagnosis[J]. J Swine Health Prod. 2002;10:171\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang S-C, Lin C-H, Aljuffali IA, Fang J-Y. Current pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e foodborne outbreak cases and therapy development. Arch Microbiol. 2017;199:811\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlegbeleye OO, Sant\u0026rsquo;Ana AS. Pathogen subtyping tools for risk assessment and management of produce-borne outbreaks. Curr Opin Food Sci. 2020;32:83\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKowalczykowski SC. Biochemical and biological function of \u003cem\u003eEscherichia coli\u003c/em\u003e RecA protein: behavior of mutant RecA proteins. Biochimie. 1991;73(2\u0026ndash;3):289\u0026ndash;304.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRehrauer WM, Lavery PE, Palmer EL, Singh RN, Kowalczykowski SC. Interaction of \u003cem\u003eEscherichia coli\u003c/em\u003e RecA protein with LexA repressor. I. LexA repressor cleavage is competitive with binding of a secondary DNA molecule. J Biol Chem. 1996;271(39):23865\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoreau PL. Role of \u003cem\u003eEscherichia coli\u003c/em\u003e RecA protein in SOS induction and post-replication repair. Biochimie. 1985;67(3\u0026ndash;4):353\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDiaz-Diaz S, Yerbes P, Recacha E, et al. RecA inactivation as a strategy to reverse the heteroresistance phenomenon in clinical isolates of \u003cem\u003eEscherichia coli\u003c/em\u003e. Int J Antimicrob Agents. 2023;61(2):106721.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRecacha E, Machuca J, D\u0026iacute;az-D\u0026iacute;az S, et al. Suppression of the SOS response modifies spatiotemporal evolution, post-antibiotic effect, bacterial fitness and biofilm formation in quinolone-resistant \u003cem\u003eEscherichia coli\u003c/em\u003e. J Antimicrob Chemother. 2019;74(1):66\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMachuca J, Recacha E, Gallego-Mesa B, et al. Effect of RecA inactivation on quinolone susceptibility and the evolution of resistance in clinical isolates of \u003cem\u003eEscherichia coli\u003c/em\u003e. J Antimicrob Chemother. 2021;76(2):338\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlshammari M, Ahmad A, AlKhulaifi M, Al Farraj D, Alsudir S, Alarawi M, et al. Reduction of biofilm formation of \u003cem\u003eEscherichia coli\u003c/em\u003e by targeting quorum sensing and adhesion genes using the CRISPR/Cas9-HDR approach, and its clinical application on urinary catheter. J Infect Public Health. 2023;16:1174\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Escherichia coli, antimicrobial resistance, virulence, MLST, One-health","lastPublishedDoi":"10.21203/rs.3.rs-6997435/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6997435/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e is an important pathogen that causes diarrhea in humans and animals. In this study, we performed MLST typing, phylogenetic grouping, antimicrobial resistance phenotype, antibiotics resistance genes, and virulence genes of \u003cem\u003eE. coli\u003c/em\u003e isolated from yaks and wild animals in the Qinghai Plateau in China.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe tested 88 and 36 \u003cem\u003eE. coli\u003c/em\u003e strains from yaks and wild animals for antimicrobial susceptibility test, antibiotics resistance genes, virulence genes, phylogenetic groups and multi-locus sequence typing.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003ePhylogenetic analysis revealed that the isolates were primarily classified into three categories: A (21/124, 16.9%), B1 (96/124, 77.4%), and B2 (7/124, 5.6%), with 37 distinct sequence types (STs), including the newly identified ST16626. PCR-based virulence gene screening detected 24 virulence genes, with \u003cem\u003eompA\u003c/em\u003e and \u003cem\u003efimC\u003c/em\u003e exhibiting universal prevalence (100%), followed by \u003cem\u003eluxS\u003c/em\u003e (96.8%), \u003cem\u003eetrA\u003c/em\u003e (79.8%), and \u003cem\u003eflu\u003c/em\u003e (72.6%). Antibiotic resistance gene profiling identified five major categories: tetracyclines (\u003cem\u003etetA\u003c/em\u003e [20.97%], \u003cem\u003etetB\u003c/em\u003e [29.03%], \u003cem\u003etetC\u003c/em\u003e [4.84%], \u003cem\u003etetD\u003c/em\u003e [4.84%] and \u003cem\u003etetM\u003c/em\u003e [2.42%]), sulfonamides (\u003cem\u003esul1\u003c/em\u003e [21.77%], \u003cem\u003esul2\u003c/em\u003e [28.23%] and \u003cem\u003esul3\u003c/em\u003e [9.68%]), quinolones (\u003cem\u003eqnrS\u003c/em\u003e [6.45%]), β-lactams (\u003cem\u003eblaTEM\u003c/em\u003e [88.71%], \u003cem\u003eblaSHV\u003c/em\u003e [7.26%], \u003cem\u003eCTX-M1\u003c/em\u003e[2.42%], \u003cem\u003eCTX-M8\u003c/em\u003e [11.29%] and \u003cem\u003eCTX-M9\u003c/em\u003e [11.29%]), and aminoglycosides (\u003cem\u003ecat1\u003c/em\u003e [67.74%], \u003cem\u003ecat2\u003c/em\u003e [54.03%], \u003cem\u003eaadA2\u003c/em\u003e [10.48%], \u003cem\u003eaac(6')-Ib\u003c/em\u003e [9.68%] and \u003cem\u003eaph(3')-vII\u003c/em\u003e [4.84%]). Antimicrobial susceptibility testing demonstrated resistance rates below 50% for all 12 tested drugs, with the highest prevalence observed for tetracycline (TCY, 41.94%), cefotaxime (CTX, 36.29%), ampicillin (AMP, 34.68%), and ciprofloxacin (CIP, 33.06%).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThis study presents the first systematic elucidation of molecular epidemiological characteristics of \u003cem\u003eE. coli\u003c/em\u003e in the Qinghai Plateau: Identification of 37 sequence types (including novel ST16626) revealed significant phylogenetic divergence between yak- and wildlife-derived strains and the recA gene deficiency phenomenon; Discovery of host-specific resistance-virulence gene profiles confirmed differential evolutionary adaptation; The findings provide molecular evidence for zoonotic disease risk early-warning and clarify the driving mechanism of livestock farming on antimicrobial resistance transmission.\u003c/p\u003e","manuscriptTitle":"Genetic Diversity, Host Adaptation, and Public Health Implications of Escherichia coli in Qinghai Plateau Animals","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-12 11:11:38","doi":"10.21203/rs.3.rs-6997435/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ab84a310-40ab-4040-9d2c-2406e533a0c4","owner":[],"postedDate":"August 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-05T23:23:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-12 11:11:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6997435","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6997435","identity":"rs-6997435","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}