Metagenomic profiling and transfer dynamics of antibiotic resistance determinants in a full-scale granular sludge wastewater treatment plant

Abstract

In the One Health context, wastewater treatment plants (WWTPs) are central to safeguard water resources. Nonetheless, many questions remain about their effectiveness to prevent the dissemination of antimicrobial resistance (AMR). Most surveillance studies monitor the levels and removal of selected antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in intracellular DNA (iDNA) extracted from WWTP influents and effluents. The role of extracellular free DNA (exDNA) in wastewater is mostly overlooked. In this study, we analyzed the transfer of ARGs and MGEs in a full-scale Nereda® reactor removing nutrients with aerobic granular sludge. We tracked the composition and fate of the iDNA and exDNA pools of influent, sludge, and effluent samples. Metagenomics was used to profile the microbiome, resistome, and mobilome signatures of iDNA and exDNA extracts. Selected ARGs and MGEs were analyzed by qPCR. From 2,840 ARGs identified, the genes arr-3 (2%), tetC (1.6%), sul1 (1.5%), oqxB (1.2%), and aph(3")-Ib (1.2%) were the most abundant among all sampling points and bioaggregates. Pseudomonas, Acinetobacter, Aeromonas, Acidovorax, Rhodoferax, and Streptomyces populations were the main hosts of ARGs in the sludge. In the effluent, 478 resistance determinants were detected, of which 89% from exDNA potentially released by cell lysis during aeration in the reactor. MGEs and multiple ARGs were co-localized on the same extracellular genetic contigs. These can pose a risk for AMR dissemination by transformation into microorganisms of receiving water bodies. Total intracellular ARGs decreased 3-42% as a result of wastewater treatment. However, the ermB and sul1 genes increased by 2 and 1 log gene copies mL-1, respectively, in exDNA from influent to effluent. The exDNA fractions need to be considered in AMR surveillance, risk assessment, and mitigation.

Catch me if you can: Capturing extracellular DNA transformation in mixed cultures via Hi-C sequencing

“…Recently, we highlighted that the extracellular free DNA (exDNA) in wastewater is a rich pool of MGEs (65%; 5-9 µg exDNA L −1 ) [3] that can co-localize ARGs [4]. Quantifying DNA uptake from the environment by natural transformation is of key interest [5] since it can pose a risk for the development of ARB in wastewater environments and their discharge into nature and aquatic reservoirs.…”

“…Gram-positive ones have a thick peptidoglycan layer that absorbs antibiotics and detergents easier, leading to faster cell death and slower development of resistance [63]. We recently showed that most microorganisms co-localizing MGEs and ARGs in a full-scale WWTP were Gram-negative [4]. A diverse microbial community like activated sludge is composed of many potential hosts that encompass a diversity of mechanisms to maintain and transfer plasmids, including donor-mediated conjugation once the plasmid is transformed [64].…”

“…Gram-positive ones have a thick peptidoglycan layer that absorbs antibiotics and detergents easier, leading to faster cell death and slower development of resistance [63]. We recently showed that most microorganisms co-localizing MGEs and ARGs in a full-scale WWTP were Gram-negative [4].…”

Antibiotic resistance response to sulfamethoxazole from the intracellular and extracellular DNA fractions of activated sludge

“…These compartments must be comprehensively taken into account when evaluating AR, following the One-Health approach (Miłobedzka et al, 2022). Wastewater treatment plants (WWTPs) should act as barriers to limit the spread of both, antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), to the environment (Bürgmann et al, 2018; Calderón-Franco et al, 2022; Pallares-Vega et al, 2019). WWTP effluents, and wastewater catchment areas, are currently considered as one of the major release points of ARB and ARGs to the environment (Rizzo et al, 2013).…”

“…In contrast, a higher concentration (around 20%) of exDNA than iDNA has been detected in sediments in natural environments with nutrient scarcity (Mao et al, 2014). An increase (up to 2 log 10 gene copies mL -1 ) of some ARGs ( ermB and sul1 ) in the exDNA pool has been detected across a WWTP process, eventually discharged in the effluent (Calderón-Franco et al, 2022). The exDNA pool can therefore not be underestimated in AR surveillance in wastewater systems.…”