An observational study on the spatiotemporal evolution and genomic characteristics of multidrug-resistant Enterococcus faecium isolated from dairy in Ningxia, China (2019-2024)

Multidrug-resistant (MDR) Enterococcus faecium is an opportunistic pathogen of concern in dairy products. Its resistance and virulence genes can spread through the food chain, posing risks to public health. The Ningxia Hui Autonomous Region is a major dairy-producing area in China. This study assessed drug resistance and genomic characteristics of MDR-E. faecium isolated from milk in Ningxia between 2019 and 2024, providing a basis for risk evaluation and dairy safety control. From 2019 to 2024, 1,314 milk samples were collected in Yinchuan, Yinnan, and Yinbei. MDR-E. faecium was identified using plate screening, mass spectrometry, broth microdilution, and hemolysis detection. Whole-genome sequencing enabled average nucleotide identity (ANI), single-nucleotide polymorphism (SNP), multilocus sequence typing (MLST), pan-genome, and clusters of orthologous groups (COG) analyses, focusing on antimicrobial resistance genes (ARGs), virulence factors (VFs), and mobile genetic elements (MGEs). Multi-response permutation procedure (MRPP), analysis of molecular variance (AMOVA), permutational analysis of multivariate dispersions (Permdisp), principal coordinates analysis (PCoA), and a random forest model were applied to compare gene communities and identify driver genes. Correlation analysis was conducted between ARGs, minimum inhibitory concentration (MIC), and VFs. Ninety-one E. faecium strains were isolated. Isolation rates differed significantly across years and regions. β-hemolysis dominated the hemolytic phenotype. Resistance to florfenicol, ceftiofur, and chloramphenicol exceeded 60%, while resistance to vancomycin and linezolid rose annually. Phylogenetic clustering revealed two subtypes, three clades, and 10 sequence types (STs). Cloud genes predominated in the pan-genome, with many linked to carbohydrate transport and metabolism. A total of 265 ARGs were identified; vanF and optrA showed detection above 95%. Most strains carried plasmids and transposons. Among the 185 detected VFs, adhesion and colonization-related genes were most frequent. Strong correlations occurred among resistance genes (optrA, cfrA, vanF), and some genes were associated with MIC90 and VFs. ARG communities differed by year, with PmrF as the main driver. From 2019 to 2024, dairy-derived E. faecium in Ningxia varied in isolation rate by year and region. Antibiotic resistance, particularly MDR, increased over time, and strains carried diverse ARGs, VFs, and MGEs. High genetic diversity and year-dependent ARG and VF structures reflected local breeding patterns. Overall, strengthened monitoring of dairy-derived E. faecium can improve dairy safety and public health.