Calderon-Franco, D. | bioRxiv Science

bioRxiv2020

Antibiotic resistance genes and mobile genetic elements removal from treated wastewater by sewage-sludge biochar and iron-oxide coated sand

Disinfection of treated wastewater in wastewater treatment plants (WWTPs) is used to minimize emission of coliforms, pathogens, and antibiotic resistant bacteria (ARB) in the environment. However, the fate of free-floating extracellular DNA (eDNA) that do carry antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) is overlooked. Water technologies are central to urban and industrial ecology for sanitation and resource recovery. Biochar produced by pyrolysis of sewage sludge and iron-oxide-coated sands recovered as by-product of drinking water treatment were tested as adsorbents to remove ARGs and MGEs from WWTP effluent. DNA adsorption properties and materials applicability were studied in batch and up-flow column systems at bench scale. Breakthrough curves were measured with ultrapure water and treated wastewater at initial DNA concentrations of 0.1-0.5 mg mL-1 and flow rates of 0.1-0.5 mL min-1. Batch tests with treated wastewater indicated that the adsorption profiles of biochar and iron-oxide coated sand followed a Freundlich isotherm, suggesting a multilayer adsorption of nucleic acids. Sewage-sludge biochar exhibited higher DNA adsorption capacity (1 mg g-1) and longer saturation breakthrough times (4 to 10 times) than iron-oxide coated sand (0.2 mg g-1). The removal of a set of representative ARGs and MGEs was measured by qPCR comparing the inlet and outlet of the plug-flow column fed with treated wastewater. ARGs and MGEs present as free-floating eDNA were adsorbed by sewage-sludge biochar at 85% and iron-oxide coated sand at 54%. From the environmental DNA consisting of the free-floating extracellular DNA plus the intracellular DNA of the cells present in the effluent water, 97% (sewage-sludge biochar) and 66% (iron-oxide coated sand) of the tested genes present were removed. Sewage-sludge biochar displayed interesting properties to minimize the spread of antimicrobial resistances to the aquatic environment while strengthening the role of WWTPs as resource recovery factories. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/302018v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@bdc78borg.highwire.dtl.DTLVardef@177a537org.highwire.dtl.DTLVardef@1ffefdeorg.highwire.dtl.DTLVardef@1dd1457_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LISewage-sludge biochar and iron oxide coated sands were tested to adsorb DNA and cells. C_LIO_LIBiochar removed 97% of genes tested from environmental DNA of unfiltered effluent. C_LIO_LI85% of ARGs and MGEs of free-floating extracellular DNA were retained by biochar. C_LIO_LIBiochar is a WWTP by-product that can be re-used for public health sanitation. C_LI

bioengineering↗

bioRxiv2020

A novel method to isolate free-floating extracellular DNA from wastewater for quantitation and metagenomic profiling of mobile genetic elements and antibiotic resistance genes

Antibiotic resistant genes (ARGs) and mobile genetic elements (MGEs) can be found in the free-floating extracellular DNA (eDNA) fraction of microbial systems. These xenogenic components can generate bacterial cells resistant to one or more antibiotics by natural transformation. Because of low concentration in wastewater, the obtaining of a high quality and a high yield of eDNA extract is challenging. We developed a method using chromatography to isolate eDNA without causing cell lysis (often unchecked) from complex wastewater matrices. The chromatographic step involved a diethylaminoethyl-cellulose-monolithic column to capture the eDNA found in cell-free filtered wastewater samples (e.g. influent wastewater, activated sludge and treated effluent wastewaster). Free-floating eDNA yields from 1 L of influent, activated sludge and treated effluent water reached 12.5 {+/-} 1.9 g, 12.3 {+/-} 1 g and 5.6 {+/-} 2.9 g of raw eDNA and 9.0 {+/-} 0.7 g, 5.6 {+/-} 0.46 g and 2.6 {+/-} 1.3 g of purified eDNA, respectively. In order to check the suitability of free-floating eDNA extracts for molecular analysis, qPCR and metagenomics were performed. eDNA extracts from treated effluent water were analyzed by qPCR to quantify a selected panel of ARGs and MGEs. Microbiome, resistome, and mobilome profiles from activated sludge free-floating eDNA were measured by metagenomic sequencing. Between iDNA and eDNA fractions, qPCR showed differences of 0.94, 1.11, 1.92 and 1.32 log10 gene copies mL-1 for sulfonamides resistant genes (sul1 and sul2), {beta}-lactamase resistance gene blaCTXM, and the class 1 integron-integrase (intI1) MGE, respectively. These differences highlighted the crucial need for an isolation method to discern both iDNA and eDNA to understand ARGs persistence and quantity in complex cultures. The eDNA yields obtained from 1 L of activated sludge (3.6 g of total suspended solids L-1) samples were substantially higher than the amount of DNA template needed for high-throughput sequencing (>1 g) in service facilities. Subsystems classification showed that the eDNA metagenome was mainly composed by MGEs (65.1%). The 35.9% rest related to traditional functional genetic signatures. It was the first time the resistome from the eDNA fraction was analyzed showing lower number of primary aligned reads when compared to the iDNA and a predominance of aminoglycosides and {beta}-lactamams. Metagenome results showed that eDNA can not be discarded as a pool of ARGs and MGEs for horizontal gene transfer. This novel isolation method was powerful to elucidate the molecular compositions of free-floating eDNA fractions in complex environmental samples such as wastewater environments at different microbial densities. Data obtained using this extraction method will foster xenogenic and microbial risk assessments across urban and natural water systems. This will support water authorities in the delineation of measures to adopt at wastewater treatment plants to remove them and safeguard environmental and public health. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/072397v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1905305org.highwire.dtl.DTLVardef@3a774borg.highwire.dtl.DTLVardef@6e1262org.highwire.dtl.DTLVardef@790c42_HPS_FORMAT_FIGEXP M_FIG Picture created with BioRender C_FIG

molecular biology↗