Systematic evaluation of the impact of standard storage conditions on plasmid conjugation behavior in wastewater samples

Filter mating experiment is widely used to study the conjugation behavior of plasmids and associated antibiotic resistance in environmental settings, however, the influence and biases brought by sample storage conditions (temperature and duration) were not yet systematically elaborated. This study systematically investigated the influence of standard storage conditions (4 °C, −20 °C, −80 °C) on plasmid conjugation behavior in influent (Inf) and activated sludge (AS) samples from sewage treatment plants (STP). The findings revealed a significant reduction in conjugation efficiency under all the tested storage conditions except for 1-week storage at 4 °C. Notably, storing at −80 °C maintained conjugation activities in activated sludge more effectively compared to −20 °C. However, the preservation performance was less effective for influent samples, which consist mainly of anaerobe-dominant communities. Systematic loss of IncH-type plasmids was observed in influent samples stored at 4 °C and −20 °C. Correspondingly, the plasmid-carrying resistome genotypes detected in the influent samples showed a clear downward trend with the increase in storage duration when stored at 4 °C and −20 °C. A relatively uniform composition in terms of incompatibility type and resistome profile was observed across activated sludge samples, regardless of the varied storage conditions. This study highlights the critical impact of storage conditions on plasmid conjugation behavior and resistome composition, offering valuable insights for optimal sample handling in resistome research.