Hi-C untangles the temporal dynamics of the children's gut resistome and mobilome, highlighting the role of transposable elements

Many metagenomic studies lack the ability to measure the temporal dynamics of the intestinal resistome (the collection of antibiotic resistance genes [ARGs]) and mobilome (the collection of all mobile genetic elements that enable their transfer) and link the genetic features to specificspecies in the gut. We applied Hi-C sequencing and shotgun metagenomics to study fecal matter from children (n = 15) living in semi-rural communities of Quito, Ecuador. We sampled at three differentperiods, with a 4- to 6-month interval between each sample collection. To understand the dynamics of ARGs from differentgenetic perspectives, we focused on identifying classes of mobile ARGs that are classifiedas high risk to human health. We selected those ARGs that appeared at least twice across sampling periods in the same child and focused the longitudinal analysis on the subset of children (n = 6) where these high-risk ARGs were consistently detected. The study demonstrated the temporal dynamics of these mobile ARGs from the taxonomic, plasmid, and transposable element perspectives, including insertion sequences and transposons. Our findingsreveal that while plasmid composition fluctuatesover time, transposons play a crucial role in the stability and dissemination of ARGs. Specifically,aph(3″)-Ib and aph(6)-Id genes were consistently mobilized by transposons across multiple multidrug-resistant Escherichia coli strains. These results highlight the importance of transposons in shaping the gut resistome and suggest that tracking regionally significanttransposons could improve our understanding of ARG transmission in small geographic areas. IMPORTANCE Antibiotic resistance (ABR) is a growing global challenge, and particularly high-risk antibiotic resistance genes (ARGs) are a threat to public health. While plasmids are often considered the cornerstone of the spread of ARGs, our study emphasizes the critical role of transposons in the persistence and mobility of ARGs within the gut microbiota. By integrating Hi-C sequencing and shotgun metagenomics, we show that transposons mediate the transfer and persistence of ARGs across differentEscherichia coli lineages, while plasmid composition changes over time. Recognizing the impact of transposons on resistome dynamics can help refinestrategies to mitigate ABR transmission, particularly in regions where the impact of resistance is most significant,such as low- and middle-income countries. Our findingsprovide new insights into the mechanisms driving the persistence of ABR in the human gut, which are essential for developing more effectivepublic health interventions and incorporating transposable elements into surveillance efforts.