Phylogenetic Analysis of blaCTX-M and blaTEM Genes in E. coli from Hospital Wastewater.

Molecular surveillance of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in environmental reservoirs is essential for understanding antimicrobial resistance (AMR) transmission within the One Health framework. This study aimed to characterize the genetic diversity and phylogenetic relationships of blaCTX-M and blaTEM genes in ESBL-producing E. coli isolated from hospital wastewater in Manila and Quezon City, Philippines. Seventeen isolates carrying blaCTX-M-1, blaCTX-M-9, and blaTEM-1 genes, confirmed by multiplex PCR, were subjected to DNA sequencing and phylogenetic analysis alongside global reference strains. Phylogenetic analysis of the blaCTX-M gene sequences revealed two distinct clusters: six blaCTX-M-15 variants clustered within the CTX-M-1 clade, showing close relatedness to strains from Thailand, Iran, and neighboring Southeast Asian clinical and environmental samples, while two blaCTX-M-27 clustered within the CTX-M-9 group, closely related to isolates from India, Australia, and Spain. All blaTEM-1 gene sequences aligned with globally disseminated TEM-1 references. The co-occurrence of multiple ESBL gene variants in individual effluent samples underscores active horizontal gene transfer facilitated by mobile genetic elements in wastewater environments. These findings reveal substantial genetic diversity of ESBL determinants and demonstrate the convergence of clinical and environmental AMR reservoirs through hospital effluents. Incorporating genetic surveillance of hospital wastewater into national AMR action plans can enhance detection of emerging resistance variants, inform risk assessment, and guide targeted interventions to mitigate environmental dissemination. Future work should integrate whole-genome sequencing to elucidate plasmid dynamics and resistance gene mobilization mechanisms, advancing One Health strategies to curb the AMR threat.