Pyroligneous acids (PAs) amendments could reduce soil antibiotic resistance genes (ARGs) pollution, but their impacts on horizontal transformation of extracellular ARGs (eARGs) remain unclear. Here, a wood residues derived PA was selected to investigate its effect on ARG dissemination via transformation using a soil microcosm experiment and an in vitro transformation system. The PA application effectively decreased the abundances of representative ARGs and mobile genetic elements, demonstrating that the weakened horizontal gene transfer alleviated ARG pollution in the soil. PA showed an amount-dependent inhibition on the transformation as well as the three distilled fractions and chemical components, proving that their important roles in inhibiting eARG transformation. The relatively low-amount (1 mu L mL-1) of PA suppressed the transformation mainly by destroying the plasmid pBR322 structure, while the high-amount (10-100 mu L mL-1) of PA inhibited the transformation due to the inactivation of recipient Escherichia coli DH5 alpha by inducing oxidative stress and destroying cell membrane, and damages of plasmid by reducing eARGs abundance and broking the base deoxyribose, and phosphate skeletons. These findings expand the understanding of PA amendments mitigating ARG pollution in agricultural soils via inhibiting horizontal gene transformation, and also provide a practical strategy to remediate soil ARG pollution.

Antibiotic resistance (AR) has been extensively studied in natural habitats and clinical applications. AR is mainly reported with the use and misuse of antibiotics; however, little is known about its presence in antibiotic-free remote supraglacial lake environments. This study evaluated bacterial strains isolated from supraglacial lake debris and meltwater in Dook Pal Glacier, northern Pakistan, for antibiotic-resistant genes (ARGs) and metaltolerant genes (MTGs) using conventional PCR. Several distinct ARGs were reported in the bacterial strains isolated from lake debris (92.5%) and meltwater (100%). In lake debris, 57.5% of isolates harbored the blaTEM gene, whereas 58.3% of isolates in meltwater possessed blaTEM and qnrA each. Among the ARGs, qnrA was dominant in debris isolates (19%), whereas in meltwater isolates, qnrA (15.2%) and blaTEM (15.2%) were dominant. ARGs were widely distributed among the bacterial isolates and different bacteria shared similar types of ARGs. Relatively greater number of ARGs were reported in Gram-negative bacterial strains. In addition, 92.5% of bacterial isolates from lake debris and 83.3% of isolates from meltwater harbored MTGs. Gene copA was dominant in meltwater isolates (50%), whereas czcA was greater in debris bacterial isolates (45%). Among the MTGs, czcA (18.75%) was dominant in debris strains, whereas copA (26.0%) was greater in meltwater isolates. This presents the co-occurrence and co-selection of MTGs and ARGs in a freshly appeared supraglacial lake. The same ARGs and MTGs were present in different bacteria, exhibiting horizontal gene transfer (HGT). Both positive and negative correlations were determined between ARGs and MTGs. The research provides insights into the existence of MTGs and ARGs in bacterial strains isolated from remote supraglacial lake environments, signifying the need for a more detailed study of bacteria harboring ARGs and MTGs in supraglacial lakes.