Temporal dynamics of antibiotic resistant bacteria and antibiotic resistance genes in activated sludge upon exposure to starvation

The activated sludge represents a huge reservoir for antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). Owing to the wastewater fluctuation, annual maintenance and storage requirement, the activated sludge in wastewater treatment plants (WWTPs) may suffer from substrate deficiency (i.e., starvation). Whereas the starvation has been confirmed to regulate the antibiotic resistance in numerous pure bacteria, its impacts on the antibiotic resis-tance in activated sludge remain unclear. Here, the dynamics of sulfonamide and tetracycline ARB and corresponding ARGs in three forms including intracellular ARGs (iARGs), adsorbed extracellular ARGs (aeARGs) and free extracellu-lar ARGs (feARGs) in activated sludge upon exposure to starvation were investigated. The results showed that, among the different electron donors (i.e., carbon, nitrogen and phosphate), carbon starvation could effectively reduce the absolute abundance of ARB and aeARGs by up to 1.68 lgs and 2.62 lgs, respectively, and released a small amount of feARGs in wastewater with the maximum value of 1.1 x 105 copies/mL due to the high degree of sludge cell lysis and DNA adsorption/degradation. For the different acceptor conditions (that is, alternating anaerobic-aerobic, anaerobic, anoxic and aerobic), the anaerobic-aerobic starvation obviously mitigated the absolute abundance of ARB, aeARGs and iARGs by 0.71 lgs, 3.41 lgs and 1.35 lgs, respectively, via the substantial sludge cell lysis and DNA degradation. These findings demonstrated the response patterns and mechanisms of bacterial resistance in activated sludge to starvation stress, and thus provide clues to control the risk of antibiotic resistance in WWTPs by the starvation strategy.