Genomic epidemiology of blaCTX−M−65-positive clinical Enterobacteriaceae reveals clonal diversity and shared plasmid backbones

CTX-M alleles show heterogeneous host and plasmid associations, but the clinical and mobile-genetic contexts of blaCTX−M−65 remain incompletely characterized. A total of 373 third-generation cephalosporin-resistant Enterobacteriaceae isolates were collected and subsequently subjected to antimicrobial susceptibility testing, PCR-based blaCTX−M−65 confirmation, whole-genome sequencing and comparative genomic analysis. 20 blaCTX−M−65-positive isolates (20/373, 5.4%) were identified from the total of 373 isolates, comprising Escherichia coli (n = 14), Salmonella Typhimurium (n = 5) and Klebsiella pneumoniae (n = 1). These isolates exhibited multidrug resistance to several antibiotics including cefuroxime (100%) and levofloxacin (70%). Phylogenetic analysis identified a closely related ST155 E. coli pair (9 core-genome SNPs across different hospital departments) and ST34 S. Typhimurium cluster (< 20 core-genome SNPs within pediatrics). A K. pneumoniae ST11 isolate co-harboring blaKPC−2 and blaCTX−M−65 was identified. Analysis of 1421 global blaCTX−M−65-positive plasmids showed that IncHI2, IncFIA/IB and IncFII/R plasmids were prevalent. Representative plasmids from this study shared high backbone similarity with public plasmids reported from China and South Korea and ISEcp1, IS26 and IS903 were commonly identified within blaCTX−M−65 -associated genetic contexts. blaCTX−M−65 occurred across diverse bacterial hosts and related plasmid backbones, demonstrating the need for continued genomic surveillance. λ blaCTX−M−65 was detected in 5.4% of third-generation cephalosporin-resistant clinical Enterobacteriaceae isolates. λ A closely related ST155 E. coli pair and an ST34 S. Typhimurium cluster were identified. λ Host-specific analyses identified frequent IncHI2- and IncF-associated plasmid backbones. λ ISEcp1, IS26 and IS903 were common mobile elements surrounding blaCTX−M−65.