Identifying Extended-spectrum β-lactamase (ESBL) producing E. coli Hotspots in Integrated Livestock Farming using Whole Genome Sequencing

• ESBL- E. coli was found in 35.1% of samples, especially in poultry fertilizer, broiler stools, and livestock feed. • The dominant strain was ST155, with ST175 and ST360 linked to human ExPEC infections. • Similar bacteria in layer and duck feed suggest a shared contamination source. • Common resistance genes included blaCTX-M15, blaCTX-M55, blaTEM33, blaOXA, and blaCMY, with more gene variation in poultry than ruminants. • IncI and IncF plasmids, found in multiple sources, carried virulence genes that help bacteria survive and cause disease. • The ESBL E. coli isolate from a farm worker was 99% similar to a hospital patient’s, suggesting the farm worker could develop similar symptoms like urinary infections, diarrhea, or serious blood infections. In 2019, antibiotic resistance posed a significant health threat, leading to 1.27 million deaths worldwide, and the risk expected to persist into the future. Livestock farming is a hotspot for antimicrobial resistance owing to the consumption of antimicrobials and environmental contamination of the surroundings. However, few studies have thoroughly sampled Extended Spectrum β-Lactamase producing E. coli (ESBL E. coli) throughout the entire process within a single farm, from the initial feed spots to the final fertilizer spots. This study used comprehensive sampling, interviews, antibiotic resistance profiling, and whole-genome sequencing to identify ESBL E. coli hotspots in an integrated teaching farm in Indonesia. All of 134 samples were obtained of livestock and human stool, feed concentrates, water, and livestock fertilizer. E. coli and ESBL E. coli were isolated and characterized using selective media, indole assays, and double-disk synergy testing. DNA from 18 isolates was extracted, sequenced, and analyzed. The investigation discovered ESBL E. coli in 35.1% of the samples (47/134). ESBL E. coli concentrations were highest in poultry fertilizer (7.4 log 10 CFU/g), broiler stool (6.7 log 10 CFU/g), and broiler feed. Multiple β-lactam antibiotic resistance genes and extraintestinal pathogenic E. coli virulence genes were identified through genomic analysis, indicating a considerable threat of antibiotic resistance and pathogenicity in this strain. The close genetic relationship between the isolates suggests cross-contamination within the farm environment. Specific hotspots for ESBL E. coli were identified, including broiler stools, poultry fertilizers, and duck stools, based on ESBL E. coli concentration and phylogenetic tree analyses. This study provides new evidence that ESBL E. coli extends beyond livestock stools to feed concentrates and water, while hotspot mapping uncovers hidden hotspots across the farm. The findings advance One Health by guiding stronger biosecurity, waste management, and antimicrobial resistance surveillance in livestock systems.