Fate of Antibiotic Resistance Genes During Anaerobic Digestion of Wastewater Solids

Bacterial resistance to antibiotics has become a worldwide health problem, resulting in untreatable infections and escalating healthcare costs. Wastewater treatment plants are a critical point of control between anthropogenic sources of pathogens, antibiotic resistant bacteria (ARBs), antibiotic resistance genes (ARGs), and the environment through discharge of treated effluent and land application of biosolids. Recent studies observing an apparent resuscitation of pathogens and pathogen indicators and the widening realization of the importance of addressing environmental reservoirs of ARGs all lead toward the need for improved understanding of ARG fate and pathogen inactivation kinetics and mechanisms in sludge stabilization technologies.

This research has investigated the fate of two pathogens, methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli, and various ARGs under pasteurization, anaerobic digestion, biosolids storage, and land application conditions. Pathogen die-off occurs at a rate specific to each pathogen and matrix in ambient and mesophilic temperature environments. Viable but nonculturable (VBNC) states are initiated by thermal treatments, such as thermophilic digestion and possibly pasteurization, and allow the persistence of pathogen cells and any ARGs contained therein through treatment and into the receiving environment where resuscitation or transformation could occur.

Raw sludge ARG content does affect digester effluent quality, although the predominant mechanisms of ARG persistence may be different in mesophilic versus thermophilic digestion. In both thermophilic and mesophilic digestion, a correlation was observed between raw sludge and digester ARGs associated with Class 1 integrons, possibly as a result of horizontal gene transfer. ARB survival was shown to contribute to ARG content in mesophilic digestion, but not thermophilic digestion. Thermophilic digestion may achieve a higher ARG reduction because of reduced microbial diversity compared to mesophilic digestion. However, it is evident that horizontal gene transfer still does occur, particularly with highly mobile integrons, so that complete reduction of all ARGs would not be possible with thermophilic digestion alone.

Surprisingly, the experiments that introduced various concentrations of antibiotic sulfamethoxazole and antimicrobial nanosilver did not induce enhanced rates of horizontal gene transfer.

Finally, ARG concentrations in biosolids increased during cold temperature storage suggesting that there is a stress induction of horizontal gene transfer of integron-associated ARGs.