Freeze–Thaw Cycles Greatly Affect the Conjugative Transfer of Antibiotic-Resistant Genes: Effects of the Secretion of Extracellular Polymeric Substances

Abstract

Freeze–thaw (FT) cycle, a ubiquitous and important physical process in natural environments, may influence the horizontal gene transfer (HGT) process of antibiotic-resistant genes (ARGs), yet its impact and underlying mechanisms remain unclear. This study investigated the impact and mechanisms of FT cycles on ARG conjugative transfer among intra- and interspecies under different solution ionic strength conditions. The results showed that despite ionic strengths and species pairs, different FT treatment cycles induced diverse influences on ARG conjugation frequency with inhibition effect of 1 FT cycle (decreased by 3.5–76-fold) and facilitation effect of 2 and 3 FT cycles (promoted by 2–27-fold). The reduced conjugation frequency after 1 FT treatment cycle is mainly attributed to energy deficiency caused by the starvation process during FT treatment. The enhanced cell–cell adhesion induced by the increased hydrophobicity and secretion of bacterial extracellular polymeric substances (EPS) facilitated the formation of conjugative pilus, primarily promoting the ARG conjugative transfer process after 2 and 3 FT treatment cycles. The results of our study clearly show that FT cycles (intensified by the ongoing climate change) exhibit profound influence on the dissemination of ARGs, which helps understand and predict ARG risks in different natural environments.