Genomic Insight into the Mobility of Antibiotic Resistance Genes in Multidrug-Resistant Escherichia coli Isolated from Dewatered Sludge Cakes

Background/Objectives: Municipal wastewater treatment plants (WWTPs) act as reservoirs for antibiotic-resistant bacteria, which pose a threat to global public health. In this study, we used whole-genome sequencing (WGS) to characterize antibiotic resistance genes (ARGs) and their association with mobile genetic elements (MGEs) in five multidrug-resistant (MDR) Escherichia coli isolates from dewatered sludge cake samples collected from a municipal WWTP in Cheongju, Republic of Korea. Methods: Susceptibility to nine antibiotics was evaluated via disk diffusion assay. Among the isolates exhibiting multidrug resistance (MDR) to three or more antibiotic classes, five isolates were randomly selected for whole-genome sequencing using the Illumina NovaSeqX platform. Additionally, we compared the genomic structures of five WWTP isolates with 35 environmental E. coli isolates from South Korea deposited in the NCBI pathogen database. ARGs and MGEs, including plasmids, integrons, and insertion sequences (ISs), were detected in the genome assemblies. Results: ARGs were differentially distributed between chromosomal and plasmid-derived contigs. Efflux pump-related genes were predominantly located on the chromosome across all isolates, whereas several beta-lactamase genes (e.g., blaTEM-30 and blaTEM-33), fluoroquinolone, and tetracycline resistance genes were localized on putative plasmid contigs. Furthermore, we characterized specific MGEs associated with these ARGs, including a class 1 integron gene cassette (dfrA17–aadA5–qacEΔ1–sul1) and an IS-mediated module (mph(A)–mrx–IS6100). Core-genome multilocus sequence typing (cgMLST) revealed that these MDR isolates represented diverse genetic lineages rather than a single clonal cluster. Conclusions: The results from this study highlight the necessity of enhanced post-treatment management of wastewater byproducts and WGS-based surveillance to mitigate the environmental spread of MDR bacteria.