Development of ciprofloxacin resistance coincides with non-cognate plasmid loss during biological wastewater treatment.

Antibiotic pressure in wastewater and sludge is a critical driver shaping the environmental resistome and facilitating the spread of resistance. To assess this influence, activated sludge was exposed to ciprofloxacin (CIP) for 30 days. CIP markedly altered the resistome by increasing the absolute abundances of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) while reducing plasmids. In a parallel 60-day evolution experiment using an activated-sludge-derived E. coli under 2 μg/L CIP, evolved clones were classified into distinct groups based on ciprofloxacin resistance level, growth rate, and plasmid copy number. Group 3, consisting of plasmid-free clones with high growth rates and high-level CIP resistance, represented the fittest population and was predominantly associated with the gyrA-S83L mutation. Their rapid cell division likely outpaced plasmid duplication and segregation, leading to plasmid loss. In contrast, Group 2 comprised plasmid-bearing clones with elevated plasmid copy numbers but impaired growth and intermediate resistance, and was predominantly associated with the gyrA-D87G mutation. Group 1 retained traits most similar to the ancestor. Transcriptomics revealed different gene regulatory strategies employed by Groups 2 and 3 in response to CIP. Analysis of wastewater-derived plasmids and E. coli genomes further supported that CIP-driven chromosomal adaptations are associated with variations in plasmid prevalence and plasmid-borne resistomes. Collectively, these findings demonstrate that interactions between chromosomal resistance mechanisms and plasmid retention under antibiotic pressure are associated with differences in resistome structure in wastewater treatment systems, and highlight the need to account for these processes when managing antibiotic resistance in activated sludge.