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.

耐多药（Multidrug-resistant, MDR）粪肠球菌（Enterococcus faecium）是乳制品中备受关注的条件致病菌。其耐药基因与毒力基因可通过食物链传播，对公共卫生构成威胁。宁夏回族自治区是中国主要的乳制品产区。本研究针对2019至2024年间宁夏地区牛乳来源的耐多药粪肠球菌，开展耐药性与基因组特征分析，为风险评估及乳品安全管控提供依据。
2019至2024年间，研究团队在银川、银南、银北地区共采集1314份牛乳样本。通过平板筛选、质谱鉴定、微量肉汤稀释法及溶血试验鉴定耐多药粪肠球菌。依托全基因组测序，开展平均核苷酸同源性（average nucleotide identity, ANI）、单核苷酸多态性（single-nucleotide polymorphism, SNP）、多位点序列分型（multilocus sequence typing, MLST）、泛基因组及直系同源基因簇（clusters of orthologous groups, COG）分析，重点聚焦抗菌药物耐药基因（antimicrobial resistance genes, ARGs）、毒力因子（virulence factors, VFs）与可移动遗传元件（mobile genetic elements, MGEs）。
采用多响应置换过程（multi-response permutation procedure, MRPP）、分子方差分析（analysis of molecular variance, AMOVA）、多元置换分散度分析（permutational analysis of multivariate dispersions, Permdisp）、主坐标分析（principal coordinates analysis, PCoA）及随机森林模型，对基因群落进行比较并筛选驱动基因。同时对抗菌药物耐药基因、最低抑菌浓度（minimum inhibitory concentration, MIC）与毒力因子开展相关性分析。
本研究共分离得到91株粪肠球菌。不同年份与地区的分离率存在显著差异。溶血表型以β溶血为主。对氟苯尼考、头孢噻呋及氯霉素的耐药率超过60%，而对万古霉素与利奈唑胺的耐药率逐年上升。系统发育聚类分析显示菌株分为2个亚型、3个进化支与10种序列型（sequence types, STs）。泛基因组中以云基因为主导，多数云基因与碳水化合物转运及代谢相关。
共鉴定得到265个抗菌药物耐药基因，其中vanF与optrA的检出率超过95%。多数菌株携带质粒与转座子。在检出的185个毒力因子中，与黏附及定植相关的基因最为常见。耐药基因（optrA、cfrA、vanF）之间存在强相关性，部分基因与MIC90及毒力因子存在关联。抗菌药物耐药基因群落随年份存在差异，PmrF为主要驱动基因。
2019至2024年间，宁夏地区牛乳来源的粪肠球菌分离率因年份与地区而异。抗生素耐药性（尤其是耐多药表型）随时间推移呈上升趋势，菌株携带多样化的抗菌药物耐药基因、毒力因子与可移动遗传元件。较高的遗传多样性以及随年份变化的抗菌药物耐药基因与毒力因子结构，反映了当地的养殖模式特征。总体而言，加强对牛乳来源粪肠球菌的监测，有助于提升乳品安全性与公共卫生水平。