Differential impacts of early, extended-spectrum, and last-resort β-lactams on manure-derived microbiomes and resistomes in an in vitro model

Understanding the environmental dissemination of antimicrobial resistance (AMR) is crucial for mitigating its spread and reducing the clinical burden of resistant infections. In a previous study, we demonstrated that copiotrophic enrichment in a medium that stimulates the growth of gut-associated bacteria can reveal clinically associated bacterial populations and antibiotic resistance genes (ARGs) that are not detected through direct environmental sampling. In the present study, we applied this enrichment approach to poultry manure to examine how β-lactamase-resistant antibiotics (cefotaxime and meropenem) shape the composition and resistance profiles of copiotrophic, gut-like microbial community relative to an earlier β-lactam (ampicillin) and to antibiotic-free controls. Our results show that extended-spectrum and last-resort β-lactams induced more pronounced shifts in microbial diversity, resistome, and mobilome profiles compared to ampicillin. When focusing on specific taxa, such as clinically relevant enteric bacteria and both susceptible and β-lactam-resistant coliforms, differential responses were observed correlating to the antimicrobial range of action. Moreover, cefotaxime and meropenem exerted different effects on the relative abundance of enteric bacteria and plasmids harboring multiple ARGs. Together, these findings underline how the diversity of pathogen and AMR indicators in gut microbiomes may be shaped by different selective pressures imposed by β-lactamase-resistant antibiotics. IMPORTANCE This study explores how different generations of β-lactam antibiotics shape microbial communities and antibiotic resistance in a gut-like in vitro system. Using a metabolically driven synthetic community derived from manure, we found that advanced β-lactams such as cefotaxime and meropenem led to stronger and more specific shifts in both community composition and resistance gene profiles compared to ampicillin. These results emphasize that the ecological impact of antibiotics depends not only on whether bacteria are inhibited but also on the spectrum of activity of each compound. In addition, the system we used provides a simple and controllable way to study interactions among antibiotics, microbial communities, and the resistome. Overall, this work offers a clearer ecological view of how clinically important antibiotics may influence microbiomes and the spread of antibiotic resistance genes.