Differential size-dependent response patterns and antibiotic resistance development mechanism in anammox consortia

Antibiotic resistance is a global threat to human and animal health. Anaerobic ammonia oxidation (anammox) is an efficient and innovative wastewater treatment technology, which can be served as a promising approach to teat antibiotic wastewater. This study systematically investigated effects of sulfamethazine on the performance, microbial community dynamics and the resistome in anammox systems inoculated with different-sized granular sludge. The activity and performance of small (< 0.5 mm) anammox granules were more susceptible to sulfamethazine stress than those of medium (0.5-1.0 mm) and large (1.0-2.0 mm) granules. Sulfamethazine addition greatly increased the diversity and abundance of mobile genetic elements (MGEs) and antibiotic resistance genes (ARGs). Based on the metagenomic analysis, the horizontal transfer of ARGs in the anammox system was upregulated through bacterial oxidative stress, pili synthesis and type IV secretion system. In addition, two strains of sulfamethazine-resistant bacteria (Pseudomonas asiatica sp. nov. and Pseudomonas shirazica sp. nov.) were isolated from the anammox system. Their whole genome sequencing results showed that the most abundant plasmid was pkF7158B, which mediated the horizontal transfer of two main multidrug resistance genes (cpxR and mexB). This work provides a holistic insight into microbial heterogeneity of different-sized anammox granular sludge and their evolution and resistance development mechanism.