Epidemiological and genomic analysis revealed the significant role of flies in dissemination of carbapenem-resistant Enterobacteriaceae (CRE) in China.

While the role of flies as potential vectors for bacterial transmission is well recognized, the epidemiological features and genomic characteristics of associated antimicrobial-resistant strains remain underexplored. This study conducted a nationwide surveillance including 3689 flies and 838 isolates from sixteen human communities (HCs) and eight animal farms (AFs) across 21 provinces in China. Our findings revealed high carriage rates of carbapenem-resistant Enterobacteriaceae (CRE) in flies, with 27.0 % in AFs and 15.3 % in HCs. Among the 705 CRE strains identified, Providencia spp. (n = 356) and Escherichia coli (n = 231) were predominant, with 90.6 % attributed to the dissemination of the bla gene. Phylogenetic tree analysis highlighted frequent clonal transmission events, with over 18 and 13 clonal evolutionary clades in Providencia spp. and E. coli, respectively. Notably, spearman analysis linked the increase of CRE prevalence in AFs flies to environmental factors like precipitation (p < 0.028) and temperature (p < 0.037), while in HCs, it positively corelated with the total meat production (p < 0.027) in each province except for Hainan. Despite significant differences in ciprofloxacin resistance (p ≤ 0.001) among E. coli strains from HCs and AFs, core genome cluster analysis and Uniform Manifold Approximation and Projection (UMAP) approach indicated these strains exhibiting close relatedness to clinical strains. This comprehensive study underscores the critical role of flies in the escalation of CRE spread within the ecosystem in China. Flies found in both HCs and AFs contribute to the dissemination of CRE, highlighting the need for urgent interventions to address this pressing issue.