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Antibiotic resistome and its association with bacterial communities during sewage sludge composting Jianqiang Su, Weiying Ouyang, Bei Wei, Fuyi Huang, Yi Zhao, Huijuan Xu, and Yong-Guan Zhu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b01012 • Publication Date (Web): 27 May 2015 Downloaded from http://pubs.acs.org on June 1, 2015
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TITLE
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Antibiotic resistome and its association with bacterial communities during sewage
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sludge composting
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Jian-Qiang Su1, Bei Wei1,2, Wei-Ying Ou-Yang1,2, Fu-Yi Huang1, Yi Zhao1, Hui-Juan, Xu1,2,
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Yong-Guan Zhu1*
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1. Key Lab of Urban Environment and Health, Institute of Urban Environment,
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Chinese Academy of Sciences, 1799 Jimei Road, Xiamen 361021, China.
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2. University of Chinese Academy of Sciences, Beijing 100049, China.
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*Correspondence: Yong-Guan Zhu, 1799 Jimei Road, Xiamen, 361021, China
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E-mail:
[email protected] 12
Phone: +86-592-6190997
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Fax: +86-6190977
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ABSTRACT
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Composting is widely used for recycling of urban sewage sludge to improve soil
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properties, which represents a potential pathway of spreading antibiotic resistant
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bacteria and genes to soils. However, the dynamics of antibiotic resistance genes
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(ARGs) and the underlying mechanisms during sewage sludge composting was not
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fully explored. Here, we used high-throughput quantitative PCR and 16S rRNA gene
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based illumina sequencing to investigate the dynamics of ARGs and bacterial
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communities during a lab-scale in-vessel composting of sewage sludge. A total of 156
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unique ARGs and mobile genetic elements (MGEs) were detected encoding
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resistance to almost all major classes of antibiotics. ARGs were detected with
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significantly increased abundance and diversity, distinct patterns, and were enriched
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during composting. Marked shift in bacterial community structures and compositions
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were observed during composting, with Actinobacteria being the dominant phylum
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at the late phase of composting. The large proportion of Actinobacteria may partially
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explain the increase of ARGs during composting. ARGs patterns were significantly
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correlated with bacterial community structures, suggesting that the dynamic of ARGs
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was strongly affected by bacterial phylogenetic compositions during composting.
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These results imply that direct application of sewage sludge compost on field may
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lead to the spread of abundant ARGs in soils.
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Key words: urban sewage sludge, composting, antibiotic resistance genes, bacterial
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community
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TOC art
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INTRODUCTION
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The increasing emergence and spread of antibiotic resistant bacteria (ARB) and
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resistance genes (ARGs) have caused intensive concern worldwide, threatening the
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achievements of modern medicines and posing risks to human health
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incomplete metabolism in human bodies and disposal of unused antibiotics have
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resulted in the release of large amounts of antibiotics into municipal wastewater 3.
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The presence of antibiotics and biological treatment processes in wastewater
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treatment plants (WWTPs) may potentially stimulate the development of antibiotic
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resistance 4. While, traditional WWTPs are not designed for the removal of
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antibiotics or ARGs, and ARB were frequently detected in WWTPs effluent, even after
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disinfection or mixed-media filtration
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hotspots for continually spreading antibiotics and antibiotic resistance into
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environments 4, 7.
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1, 2
. The
. WWTPs are considered as one of the
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Sewage sludge is one of the richest reservoirs of antibiotic resistance owing to the
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input of ARB and ARGs from human and veterinary waste water 8. It is characterized
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with high density and diversity of microbial flora, which may facilitate horizontal gene
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transfer (HGT) via mobile genetic elements (MGEs), such as plasmids
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integrons 11. By using culture-based method 12, quantitative PCR 13 and metagenomic
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investigation
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sludge, and that these antibiotic resistance factors were associated with clinical
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settings, raising a public health issue when disseminated in downstream
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environment of the sewage plant 15, 16.
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9, 10
and
, diverse and abundant ARB and ARGs had been detected in sewage
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The high antibiotic resistance and large amount of sewage sludge produced by
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WWTPs pose great economic and environmental challenges for safe treatment and
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recycling. It is estimated that more than 600 wastewater treatment plants have been
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established by the year 2007 in China, producing around 5,000 million kg of sewage
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sludge every year 17. Composting is aerobic digestion of sewage sludge to create an
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end product that can be applied as soil conditioner and fertilizer to improve soil
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physical prosperities, especially texture, water holding capacity and soil fertility 18-20.
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About 120,000 tonnes of class B-equivalent biosolids are applied to soil annually in
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Ontario, Canada
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continue to grow because of constraints and environmental concerns of land-filling
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and incineration
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metals, bacterial pathogens and organic pollutants other than pharmaceutical
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residues and ARGS. Land application of biosoilds and compost could lead to the input
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of antibiotics into the soil
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tetracyclines
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soils and further to vegetables
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resistance genes and integrons
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integrons were much slower than previous reports 25. These findings suggested that
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mitigating the spread of ARGs should pay more attention to the shift of ARGs during
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sewage sludge treatment.
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21
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. The interest of land application of treated sewage sludge
. However, recycling of sewage sludge mainly focused on heavy
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, including fluoroquinolones, macrolides, and
. The emergence of ARGs was also detected in biosolids-amended 21
, exhibiting enrichment of tetracycline, macrolides
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, and that the decay rates for ARGs and class 1
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Degradation of antibiotics and decline of ARGs during manure composting have been
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reported previously
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digestion of sewage sludge on specific types of ARGs, such as tetracyclines,
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sulfonamides, marcrolides resistance genes 29. Although sewage sludge is recognized
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as a rich reservoir of ARGs
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compost
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sludge is not well characterized, and the mechanisms underlying the shift of ARGs
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has not been fully explored. By combining high throughput quantitative PCR (295
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primer sets targeting almost all major classes of ARGs and MGEs) and illumina
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sequencing of bacterial 16S rRNA gene, this study aimed: 1) to characterize the shift
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26-28
. However, only a few studies focus on the effect of various
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and diverse ARGs are frequently detected in sludge
, the temporal change of ARGs during aerobic composting of sewage
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of the abundance and patterns of ARGs during a lab-scale composting of urban
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sewage sludge; and 2) to investigate the dynamics of bacterial communities to
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address the factors affecting the ARGs variation.
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EXPERIMENTAL SECTION
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Composting experiment set-up
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A lab-scale of urban sewage sludge composting was carried out with dewatered
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sewage sludge collected from the Tong’an Sewage Treatment Plant, Xiamen, China.
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Raw materials for composting, sawdust and rice straw (Table S1) were crushed in a
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mill (2 mm mesh size), autoclaved and were thoroughly mixed with sewage sludge
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using a blender at the ratio of 1:2:2 (v/v). The composting experiment was conducted
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for 50 days in quadruplicate, each containing ca. 35 kg of mixture in a PVC box (65 cm
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in length, 50 cm in width and 40 cm in height). Moisture was maintained between 50%
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and 65% by adding ddH2O irregularly during the composting 31, 32.
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Sample collection and DNA extraction
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Samples were collected on days 0, 1, 2, 8, 20 and 50, including the following stages: (i)
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municipal sewage sludge before composting was collected as control sample, day 0;
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(ii) mesophilic phase (calefactive phase) at 45 0C, day 1; (iii) thermophilic phase
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above 55 0C, day 2 (60 0C), this phase maintained over 55 0C for 2 days; (iv) cooling
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phase at 45 0C, day 8; (v) maturation phase at 35 0C and 30 0C, day 20 and 50,
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respectively. Samples were collected by mixing subsamples from the upper, central
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and lower portion of the compost uniformly to achieve high representativeness 31. All
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the samples were stored at -80 0C for further analysis.
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DNA was extracted from 0.5 g fresh samples using FastDNA Spin Kit for soil (MP
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Biomedical, France) according to the manufacturer’s instructions. The total DNA
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were eluted with 100 μL of provided DES solution and stored at -20 0C until use. The
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concentration and quality of extracted DNA were checked by spectrophotometric
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analysis using NanoDrop ND-1000 (Nanodrop, USA).
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High-throughput quantitative PCR (HT-qPCR) and data analysis
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To evaluate the abundance of ARGs in samples, high-throughput quantitative PCR
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(HT-qPCR) of ARGs were performed using the SmartChip Real-time PCR (Warfergen
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Inc. USA) as described previously with a slight modification 7. A total of 296 primer
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sets (Table S2) were used including 293 validated and used primer sets targeting
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284 ARGs conferring resistance to major classes of antibiotics, 8 transposase and 1
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16S rRNA gene
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(intI) 35 and 1 for clinical class 1 integron-integrase gene (cintI) 36. HT-qPCR data was
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preprocessed as described previously, for each primer set, amplifications efficiency
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beyond the range (90% - 110%) were discarded, and amplification was confirmed
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with more than two positive replicates
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figure out ARGs’ fold change (FC value) of compost samples compared to the control
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absolute copy numbers by normalizing to 16S rRNA gene copy numbers which was
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quantified separately from the Wafergen platform 37, 39. The average number of 16S
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rRNA-encoding genes per bacterium is currently estimated at 4.1 based on the
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Ribosomal RNA Operon Copy Number Database (rrnDB version 4.3.3) 40. Bacterial cell
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numbers was then estimated by dividing 16S rRNA gene copy numbers by this value
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and normalized copy number of ARGs per bacterial cell was calculated 41.
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, 1 for blaNDM-1 34, 1 for universal class I integron-integrase gene
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. Comparative CT method was used to
. Relative copy number of ARGs and MGEs was calculated and transformed to
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16S rRNA gene amplication, sequencing and data processing
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To investigate the bacterial community structures and compositions, the V3 region of
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bacterial 16S rRNA gene were amplified, purified, quantified, pooled and sequenced
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on an Illumina Hiseq2000 platform at BGI, Shenzhen, China
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. Raw pair-end reads
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were assembled after filtering adaptor, low-quality reads, ambiguous N and barcode
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to generate clean joined reads capturing the complete V3 region of the 16S rRNA
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gene by BGI. The generated high quality sequences were processed and analyzed
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using Quantitative Insights Into Microbial Ecology (QIIME)
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operational taxonomic unit (OTU) picking was performed following the online
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instruction of QIIME. OTUs were defined at the 97% similarity level using UCLUST
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clustering 44. Representative sequence of each OTU was selected by default method,
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which was assigned to taxonomy using RDP classifier with a confidence threshold of
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0.80 (80%)
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Sequences were aligned using PyNAST aligner
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using the Fasttree algorithm 47 for downstream analysis. The levels of alpha diversity
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were estimated using the metrics observed species (OTU), chao1 and PD Whole tree,
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and rarefaction curves were generated to compare the level of bacterial OTU
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diversity. The difference between microbial communities was compared using the
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weighted and unweighted Unifrac metric and Bray-Curtis distances followed by
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principal coordinate analysis (PCoA) and Adonis test. All sequences have been
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deposited in the National Center for Biotechnology Information Sequence Read
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Archive under the accession number SRP052220.
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. The open-reference
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. Global singletons were removed prior to downstream analysis. 46
and a phylogenetic tree was built
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Statistical analysis
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Averages, standard deviations, and fold change values of ARGs were determined
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using Excel 2013 (Microsoft Office 2013, Microsoft, USA). ARGs were considered
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statistically enriched or decreased if the range created by two standard deviations of
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the mean fold change was entirely >1 or
