Multidrug-resistant Escherichia coli causing diarrhea in yak calves on the Qinghai-Tibet Plateau: phenotypic characterization, whole-genome sequencing, and pathogenicity analysis

Vet World Vol.19 March-2026 Article - 5

Research Article

Veterinary World, 19(3): 948-963

https://doi.org/10.14202/vetworld.2026.948-963

Qian Chen^1,2, Di Wu^1,2, Zhen Yang^1,2, Chang Sun^1,2, Shulin Tang^1,2, Changjiang Chen³, Bin Wei³, Qing Liu³, Pengxia Bai⁴, Hongjuan Zhang^1,2, Shengyi Wang^1,2, and Baocheng Hao^1,2

1. Key Laboratory of New Animal Drug Project, Gansu Province, China.

2. Key Laboratory of Veterinary Pharmaceutical Development, Ministry of Agriculture and Rural Affairs, Lanzhou Institute of Husbandry and Pharmaceutical Sciences of Chinese Academy of Agriculture Sciences, Lanzhou 730050, China.

3. Animal Husbandry and Veterinary Station of Huangyuan County, Xining, 812100, China.

4. Qinghai College of Animal Husbandry and Veterinary Technology, Xining, 812100, China.

Background and Aim: Calf diarrhea represents a major threat to yak (Bos grunniens) husbandry on the Qinghai-Tibet Plateau, where extreme environmental conditions (high altitude, low oxygen, cold temperatures) and irregular antibiotic use may accelerate the emergence of multidrug-resistant (MDR) bacterial pathogens. This study aimed to isolate and identify the predominant bacterial agents responsible for diarrhea in yak calves, determine their antimicrobial resistance profiles, and investigate the genomic features and pathogenicity of the most resistant strain to provide evidence-based guidance for prevention and control.

Materials and Methods: Rectal swabs were collected from 12 naturally diarrheic yak calves across four geographically distinct farms in Huangyuan County, Qinghai Province, during the peak season (June–July). Bacterial isolates were obtained through enrichment in Luria-Bertani broth followed by plating on Luria-Bertani agar, and identified by Gram staining, 16S rRNA gene amplification (primers 27F/1492R), and Sanger sequencing with BLAST comparison (>99.5% identity). Antimicrobial susceptibility was assessed using the Kirby-Bauer disk diffusion method with 17 antibiotics representing eight classes, interpreted according to Clinical and Laboratory Standards Institute VET08-Ed4 breakpoints. The most MDR isolate (HYCQ01) underwent whole-genome sequencing (WGS) on the Oxford Nanopore Technologies MinION platform. Genome assembly quality was evaluated with BUSCO v5.4.7; virulence factors and antibiotic resistance genes were annotated against the Virulence Factor Database and Comprehensive Antibiotic Resistance Database, respectively (BLASTP, e-value ≤ 1e-5, identity ≥ 40%, length ≥ 50 bp). Pathogenicity was tested in 20 male C57BL/6 mice (7–8 weeks, 20 ± 2 g) via intraperitoneal injection of 1.0 × 10⁸ colony-forming units/mL bacterial suspension (100 μl/kg); survival was monitored, and organ histopathology (heart, jejunum, kidneys, liver, lungs, spleen) was examined after hematoxylin-eosin staining. All animal procedures were approved by the Experimental Animal Ethics Committee (No. 2024-030). Data were analyzed using GraphPad Prism 10.1.2 with one-way analysis of variance.

Results: Eight Escherichia coli strains were isolated from the 12 samples and confirmed by 16S rRNA sequencing. All isolates displayed MDR phenotypes, showing 100% resistance to penicillin G and clindamycin, 87.5% to sulfafurazole, and 75.0% to erythromycin. WGS of HYCQ01 revealed 32 resistance classes and 152 resistance genes, consistent with its phenotype (including β-lactamases, macrolide-lincosamide resistance determinants, and tetracycline efflux pumps). Virulence genes included Type III secretion system components, alginate biofilm regulators, iron acquisition systems (pvdE), and hemolysins (rck). Phylogenetically, HYCQ01 clustered near enterotoxigenic E. coli O139:H28 but exhibited a hybrid profile combining animal-associated colonization factors (F17, CFA/I) with atypical extraintestinal traits. In the mouse model, HYCQ01 induced 100% mortality within 27 h post-challenge (p < 0.0001) and caused severe histopathological damage in spleen and jejunum, indicating strong systemic invasiveness.

Conclusion: MDR E. coli, exemplified by the hybrid strain HYCQ01, predominates as a causative agent of yak calf diarrhea on the Qinghai-Tibet Plateau, shaped by local ecological pressures and horizontal gene transfer. These results highlight the urgent need for region-specific antimicrobial resistance surveillance, rational antibiotic stewardship, and exploration of non-antibiotic alternatives (probiotics, plant-derived antimicrobials) within a One Health framework. WGS data are deposited at NCBI under BioProject PRJNA1289237.

Keywords: antimicrobial resistance, diarrhea, Escherichia coli, multidrug-resistant, pathogenicity, Qinghai-Tibet Plateau, whole-genome sequencing, yak calves.

How to cite this article: Chen Q, Wu D, Yang Z, Sun C, Tang S, Chen C, Wei B, Liu Q, Bai P, Zhang H, Wang S, Hao B. Multidrug-resistant Escherichia coli causing diarrhea in yak calves on the Qinghai-Tibet Plateau: phenotypic characterization, whole-genome sequencing, and pathogenicity analysis. Vet. World. 2026;19(2):948-963.

Received: 24-11-2025 Accepted: 04-02-2026 Published online: 12-03-2026

Corresponding author: Shengyi Wang and Baocheng Hao E-mail: wangshengyi@caas.cn and haobaocheng@caas.cn

DOI: 10.14202/vetworld.2026.948-963

Copyright: Chen, et al. This article is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.