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May 21, 2015 - Effect of temperature on removal of antibiotic resistance genes by anaerobic digestion of activated sludge revealed by metagenomic approach.
Appl Microbiol Biotechnol (2015) 99:7771–7779 DOI 10.1007/s00253-015-6688-9

ENVIRONMENTAL BIOTECHNOLOGY

Effect of temperature on removal of antibiotic resistance genes by anaerobic digestion of activated sludge revealed by metagenomic approach Tong Zhang 1 & Ying Yang 1 & Amy Pruden 2

Received: 20 March 2015 / Revised: 7 May 2015 / Accepted: 8 May 2015 / Published online: 21 May 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract As antibiotic resistance continues to spread globally, there is growing interest in the potential to limit the spread of antibiotic resistance genes (ARGs) from wastewater sources. In particular, operational conditions during sludge digestion may serve to discourage selection of resistant bacteria, reduce horizontal transfer of ARGs, and aid in hydrolysis of DNA. This study applied metagenomic analysis to examine the removal efficiency of ARGs through thermophilic and mesophilic anaerobic digestion using bench-scale reactors. Although the relative abundance of various ARGs shifted from influent to effluent sludge, there was no measureable change in the abundance of total ARGs or their diversity in either the thermophilic or mesophilic treatment. Among the 35 major ARG subtypes detected in feed sludge, substantial reductions (removal efficiency >90 %) of 8 and 13 ARGs were achieved by thermophilic and mesophilic digestion, respectively. However, resistance genes of aadA, macB, and sul1 were enriched during the thermophilic anaerobic digestion, while resistance genes of erythromycin esterase type I, sul1, and tetM were enriched during the mesophilic anaerobic digestion. Efflux pump remained to be the major antibiotic resistance mechanism in sludge samples, but the portion of

Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6688-9) contains supplementary material, which is available to authorized users. * Tong Zhang [email protected] 1

Environmental Biotechnology Laboratory, Department of Civil Engineering, the University of Hong Kong, Pokfulam Road, Hong Kong, China

2

Department of Civil and Environmental Engineering, Virginia Tech, Blacksburg, VA 24061, USA

ARGs encoding resistance via target modification increased in the anaerobically digested sludge relative to the feed. Metagenomic analysis provided insight into the potential for anaerobic digestion to mitigate a broad array of ARGs. Keywords Antibiotic resistance genes . Thermophilic anaerobic digestion . Mesophilic anaerobic digestion . Metagenomic analysis

Introduction Antibiotic resistance is a growing health threat. In particular, increased morbidity and mortality that resulted from infections with multiantibiotic-resistant pathogens, such as extendedspectrum β-lactamase (ESBL)-producing Enterobacteriaceae, vancomycin-resistant Enterococcus (VRE), and methicillinresistant Staphylococcus aureus (MRSA), were highlighted as major concerns in a recent report from the US Centers for Disease Control and Prevention in 2013. Spread of antibiotic resistance among pathogens is a global problem, while development of new antibiotics is critical. Historically, it has been observed that resistance emerges soon after deployment of antibiotics (Levy 1982). This highlights the need for broad strategies to slow the rate at which resistance spreads, with growing interest in potential links between antibiotic resistance genes (ARGs) in the environment and the clinic (Kümmerer 2009a, b; Martinez 2008; Pruden et al. 2006; Wright 2010; Zhang et al. 2009b). In particular, proactive treatment of human and livestock wastestreams containing ARGs may help mitigate the spread of ARGs (Pruden et al. 2013). It is now evident that wastewater treatment plants (WWTPs) are a significant source of ARGs to the natural environment (LaPara et al. 2011; Storteboom et al. 2010). Municipal WWTPs receive wastewater containing antibiotic-resistant bacteria (ARB)

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and ARGs originating in the gastrointestinal tracts of humans, and a wide range of ARBs and ARGs have correspondingly been detected in activated sludge (AS) (Koczura et al. 2012; Yang et al. 2011, 2013; Zhang et al. 2009a). During wastewater treatment, some ARGs might attenuate, while others may proliferate as a result of complex microbial ecological processes. In addition to preferential survival and selection of ARBs, of particular concern is the potential for ARGs to spread among wastewater bacteria via horizontal gene transfer (Baquero et al. 2008; Summers 2006). ARGs originating from WWTPs have been observed to be transported to and persist in receiving water bodies influenced by effluent (LaPara et al. 2011; Storteboom et al. 2010; Xu et al. 2014) and soil subject to land application of residual biosolids (Munir et al. 2011). Thus, WWTPs have promise as important nodes for controlling the spread of ARGs via the natural environment (Pruden et al. 2013). It has been estimated that the loading rate of ARGs to the environment from biosolids is about 1000 times that of aqueous effluent (Munir et al. 2011). According to a report from North East Biosolids and Residuals Association (NEBRA), about 7,180,000 t of sludge was generated in the USA in 2004. Numerous methods can be used to treat the excess sludge before disposal or land application, such as anaerobic digestion, aerobic digestion, chemical stabilization, etc. Among them, anaerobic digestion is widely applied since it can economically and efficiently reduce the sludge volume (Novak et al. 2007), generate biogas (Kwietniewska and Tys 2014), and remove pathogens (Sahlstrom 2003). Removal of ARGs could be an added benefit, but little is known about the effect of sludge digestion conditions on ARGs. A few studies have investigated the fate of ARGs during anaerobic digestion and suggested that temperature is a critical variable for ARG removal (Diehl and LaPara 2010; Ghosh et al. 2009; Ma et al. 2011). Generally, prior studies indicate that different ARGs respond differently under mesophilic or thermophilic conditions, with thermophilic digestion generally outperforming mesophilic digestion. However, prior studies only provided information on a handful of ARGs due to limitations of the quantitative polymerase chain reaction (q-PCR) methodology. The objective of this study was to determine the effect of temperature (mesophilic versus thermophilic) on the removal of ARGs by anaerobic sludge digestion. Metagenomic analysis based on high-throughput DNA sequencing was applied in order to determine the effect on a broad array of ARGs.

Material and methods Operation of bench-scale anaerobic digesters Bench-scale anaerobic digesters were set up using 1-L fiveneck flasks with a working volume of 900 mL. One

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thermophilic anaerobic digester and one mesophilic anaerobic digester were maintained at 55±2 °C and 35±2 °C, respectively, through circulating hot water around the flask. Both thermophilic and mesophilic anaerobic digesters were initiated with 600 mL of anaerobic digested sludge (ADS) and 300-mL AS collected from Shatin WWTP (ST WWTP) in Hong Kong. The bench-scale anaerobic digesters were fed with concentrated AS collected from the aeration tank in ST WWTP on October 2011. The collected AS was concentrated by gravity sedimentation and centrifugation to a volatile suspended solid (VSS) level of 15,086 mg/L and stored at −20 °C as the source of feed sludge through the duration of the experiment. Both digesters were mixed by magnetic stirrers. To maintain the sludge retention time (SRT) of 15 days, 180 mL of mixture from each digester was replaced by the same volume of feed sludge every 3 days. pH was controlled above 7.0 by adding 1-M sodium carbonate solution via an automatic pH controller (pH-201, MSITECH, Singapore). Biogas was collected in Tedlar bags (Supleco, Sigma-Aldrich). The volume of the generated biogas was measured using a glass syringe. The composition of the biogas was determined using a gas chromatograph (GC) (Hewlett Packard 5890II, USA) with a thermal conductivity detector (TCD). Argon was used as the carrier gas at a flow rate of 30 mL/min. Injector, detector, and column temperatures were kept at 57, 180, and 50 °C, respectively. VSS was measured according to standard methods (Eaton and Franson 2005). Sample collection, DNA extraction, and high-throughput sequencing A sample of digested sludge was collected from each digester after the volume of the generated biogas and total solid reduction rates were stable (78 days from the operation of digesters) before the addition of feed sludge. The ADS and AS samples used for DNA analysis were immediately fixed with 100 % ethanol at a ratio of 1:1 (v/v) after collection and stored at −20 °C before DNA extraction. DNA extraction was performed using a FastDNA® Spin Kit for Soil (MP Biomedicals, Santa Ana, CA, USA) from approximately 200 mg of sludge pellet collected by centrifugation. DNA concentration and purity were determined by microspectrophotometry (NanoDrop ND-1000, NanoDrop Technologies, Wilmington, DE, USA). DNA extractions were performed in triplicate for each sample type, and the resulting extracts were composited for sequencing to average out bias in sampling and extraction. High-throughput sequencing was conducted at the Beijing Genomics Institute (BGI, Shenzhen) using Illumina Hiseq 2000 with the sequencing strategy of index PE101+8+101 cycle (Paired-End sequencing, 101-bp reads and 8-bp index sequence). About 5 μg of DNA of the feed sludge, thermophilic ADS, and mesophilic ADS was used for library construction

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and DNA sequencing. Approximately 2.4 Gb of data was generated for each sample. Metagenomic data were deposited in MG-RAST with the accession number of 4601903.3 (feed sludge), 4601904.3 (mesophilically digested sludge), and 4601905.3 (thermophilically digested sludge). Bioinformatics analysis In order to compare the abundance and diversity of ARG-like sequences in different sludge samples, sequencing data were first trimmed to the same sequencing depth for each sample type, i.e., 2,389,000 reads for each dataset. The trimmed datasets of feed sludge, thermophilically digested sludge, and mesophilically digested sludge were named as F-AS, TADS, and M-ADS, respectively. ARG-like sequences were identified by aligning the sequences against the optimized antibiotic resistance gene database (ARDB) using BLASTX with the E value cutoff of 1e−5 (Yang et al. 2013). The searched sequences from BLAST results were further identified using the cutoff of sequence identity of 90 % and hit length longer than 25 amino acids (aa) (Kristiansson et al. 2011; Zhang et al. 2011). The identified ARG-like sequences were classified using the structured database of ARDB and the customized Python script as reported previously (Yang et al. 2013). Statistical analysis Since the abundance of ARG sequences in metagenomic datasets is very low, the levels of ARG-like sequences in the present study were described using the unit of Bppm.^ The unit of Bppm^ (one read in one million reads) was defined as the portion of ARG-like sequences in Btotal metagenome sequences.^ The composition of ARG sequences was described using the unit of B%,^ which represents the portion of a type or a subtype of ARG-like sequences in Btotal ARG-like sequences.^ The units of Bppm^ and B%^ were also used in previous studies to describe the abundance and distribution of ARGs in environmental samples (Chen et al. 2013; Yang et al. 2013). Statistical analysis was performed using Paleontological Statistics (PAST, version 2.17).

Results Digester performance Bench-scale anaerobic digesters were set up to study the response of ARGs to anaerobic digestion under thermophilic or mesophilic conditions fed with local AS at an SRT of 15 days. Digesters were operated in a semi-continuous way in that 20 % of the digester content was replaced every 3 days for

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both digesters. It took over 45 days for both digesters to stabilize with respect to the biogas production and total solid reduction rate (Figs. S1 and S2). Measurement showed that about 45.3±5.8 % of biogas was methane in the thermophilic digester and 55.6±7.8 % in the mesophilic digester when the performances of digesters were stable. Reduction of VSS in the thermophilic digester and mesophilic digester was 47.6± 4.1 % and 43.6±7.3 % in the stable phase.

Occurrence and abundance of ARGs in feed and digested sludge Abundance of ARGs in F-AS, T-ADS, and M-ADS ARG-like reads corresponding to 12 ARG types were identified in the F-AS with the sequencing depth of 2,389,000 reads applied consistently across the samples. The total abundance of ARG-like sequences was 24.1 ppm in FAS. Multidrug resistance genes (5.2 ppm, 21.6 %), betalactam resistance genes (3.7 ppm, 15.5 %), tetracycline resistance genes (3.7 ppm, 15.3 %), and bacitracin resistance genes (2.5 ppm, 10.4 %) (Fig. 1) were the major ARG types in F-AS. No obvious reduction of total ARG abundance was found by either thermophilic anaerobic digestion or mesophilic anaerobic digestion. The total ARG abundance in T-ADS and M-ADS was 22.9 and 24.4 ppm, respectively, which was well within the same order of magnitude as the F-AS. Furthermore, the numbers of identified ARG types in T-ADS and M-ADS were the same as that in F-AS. Among the 12 ARG types identified in each ADS dataset, 11 of them were shared by T-ADS and M-ADS, including ARGs corresponding to acriflavine, aminoglycoside, bacitracin, beta-lactam, chloramphenicol, multidrug, sulfonamide, tetracycline, MLS (macrolide-lincosamide-streptogramin), and vancomycin resistance as well as others (ARGs that could not be classified) (Fig. 1). Quinolone ARGs were only found in T-ADS, while polymyxin ARGs were only found in M-ADS under the current sequencing depth. However, the abundance of quinolone ARGs or polymyxin ARGs was low (