Horizontal transfer of intracellular and extracellular ARGs in sludge compost under sulfamethoxazole stress

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Aerobic composting has been considered a safe way to recycle sludge, providing a green recycling system that conserves resources. Sludge from sewage treatment plants has been identified as a significant reservoir for ARGs. However, it remains unclear whether the residual antibiotics in sludge affect the horizontal transfer mechanism of intracellular and extracellular antibiotic resistance genes (iARGs, eARGs) in compost. Here, intracellular, extracellular and total genomic DNA was extracted during sulfamethoxazole (SMX) selective pressure sludge composting. After composting, most of the sul in the sludge was removed, the removal rate of iARGs and eARGs was around 80 % and 90 %. The presence of antibiotic stress in sludge would promote the horizontal transfer of ARGs. Metagenomic sequencing confirmed that the sul gene cluster was carried by multiple mobile genetic elements, such as the Tn3 transposon and intl1, which promoted the horizontal transfer of iARGs. SMX selective pressure increased the expression of genes responsible for SOS response (29.52 %), type IV secretion systems (28.09 %) and type IV pili (40.45 %). Combined with the KEGG annotation analysis, SMX facilitated intake to eARGs by hosts, such as Pseudomonas, via type IV pili. Overall, this is the first study to verify that the horizontal transfer of iARGs and eARGs is promoted by the presence of antibiotic residues in sludge during composting.