Environment International 133(2019)105203 Contents lists available at ScienceDirect e environment Environment International ELSEVIER journal homepage: www.elsevier.com/locate/envint Efficient reduction of antibiotic residues and associated resistance genes in tylosin antibiotic fermentation waste using hyperthermophilic composting Hanpeng Liao, Qian Zhao, Peng Cui, Zhi Chen", Zhen Yu, Stefan Geisen, Ville-Petri Friman Shungui Zhoua "Fujian Provincial Key Laboratory of Soil Environmental Health and Regulation, College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou, China " Guangdong Key Laboratory of Integrated Agro-environmental Pollution Control and Management, Guangdong Institute of Eco-environmental Science & Technology, Guangzhou 510650. China Department of Terrestrial Ecology, Netherlands Institute of Ecology, Wageningen, Netherlands Department of Biology, University of York, Wentworth Way, YO10 5DD York, UK ARTICLE INFO ABSTRACT Handling Editor: Zhen (Jason) He Insufficient removal of antibiotics and antibiotic resistance genes (ARGs) from waste products can increase the Keywords: risk of selection for antibiotic resistance in non-clinical environments. While composting is an efficient way to Antibiotic resistance genes reduce ARGs, most conventional methods are ineffective at processing highly contaminated antibiotic fermen Antibiotic residue tation waste. Here we explored the efficacy and underlying mechanisms of hyperthermophilic composting at Hyperthermophilic composting removing tylosin antibiotic fermentation residues (TFR) and associated ARGs and mobile genetic elements Horizontal gene transfer (MGEs; plasmids, integrons and transposon). Hyperthermophilic composting removed 95.0% of TFR, 75.8% of Plasmids ARGs and 98.5% of MGEs and this reduction mainly occurred after extended exposure to temperatures above 60"C for at least 6 days. Based on sequencing and culture-dependent experiments, reduction in ARGs and MGEs was strongly associated with a decrease in the number of bacterial taxa that were initially associated with ARGs and MGEs. Moreover, we found 94.1% reduction in plasmid genes abundances (ISCR1 and IncQ-oriV) that sig- nificantly correlated with reduced ARGs during the composting, which suggests that plasmids were the main carriers for ARGs. We verified this using direct culturing to show that ARGs were more often found in plasmids during the early phase of composting. Together these results suggest that hyperthermophilic composting is efficient at removing ARGs and associated resistance genes from antibiotic fermentation waste by decreasing the abundance of antibiotic resistance plasmids and associated host bacteria. 1.Introduction pose a major challenge to global public health through selection for multidrug resistant bacterial "superbugs" that are very difficult to treat Antibiotic fermentation residues are organic solid waste products (Povolo and Ackermann, 2019). Developing effective ways to treat created in the manufacturing process of antibiotics fermentation. They waste that contains antibiotics and ARGs is important for controlling contain fermentation media, antibiotics residues, heavy metals (zinc the development of antibiotic resistance in natural environments and copper etc.) sludge and multiple different antibiotic resistance (Bondarczuk et al., 2016). genes (ARGs)(Zhang et al., 2018a). Globally, millions of tons of anti Treatment of antibiotic fermentation residues using composting biotic fermentation residues are produced every year, and traditionally methods has recently received more research interest (Wang et al., these waste products are disposed in landfills (Chen et al., 2017b) 2016; Zhang et al., 2018a). Besides making waste safer, composting end However, this practice can cause a serious threat to the environment products contain high amounts of organic matter and mineral nutrients, through leaching of antibiotic pollutants into the natural environments which could allow it to be used as organic fertilizer. Despite attempts to including soils, groundwater and nearby waters (Chen et al, 2017a), assess the residual antibiotics and ARGs during the composting in la- where they could select for antibiotic resistant bacteria (Pepper et al. boratory conditions, traditional methods often fail to remove ARGs 2018). ARGs are thus considered emerging human-toxic pollutants that from the composting end products (Zhang et al., 2018a; Zhang et al., 'Corresponding author. E-mail address: sgzhou@soil.gd.cn (S. Zhou). https://doi.org/10.1016/j.envint.2019.105203 Received 14 April 2019; Received in revised form 17 September 2019; Accepted 19 September 2019 Available online 26 October 2019 0160-4120/2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http:/creativecommons.org/licenses/BY-NC-ND/4.0/)
H.Liao,et al. Environment International 133 (2019)105203 2018c).For example,it was recently shown that 40 days of composting composition)factors affecting ARG and MGE abundances during hy- only removed 39%of initial tylosin antibiotic residues in swine manure perthermophilic composting. (Zhang et al.,2018c).Moreover,Liu et al.(2018)recently reported that while the abundance of ARGs and mobile genetic elements (MGEs) 2.Materials and methods initially declined during composting of gentamicin fermentation re- sidues,their abundances rapidly recovered and even exceeded the in- 2.1.Full-scale experimental setup for hyperthermophilic composting itial concentrations during the later phases of composting.These fail- ures could be attributed to several factors.For example,MGEs,such as Hyperthermophilic composting experiments were conducted in a plasmids,could mobilize ARGs allowing them to move between suitable full-scale aerobic composting plant located in Henan district,Ningxia, bacterial hosts during the composting.Moreover,antibiotic fermenta- China as described by Liao et al.(2018).The hyperthermophilic com tion waste often also includes antibiotic residues and heavy metals that posting material (approximately 21 tons)consisted a mixture of TFR could reinforce selection for multidrug resistance plasmids that often waste (70%water content)and rice straw husk (15%water content, encode resistance genes for both antibiotics and heavy metals (Song provided by a local farm)in a ratio of 4:1 (w/w).TFR waste was ob- et al.,2017).Here we tested if hyperthermophilic composting that uses tained from a local biological pharmaceutical factory (Ningxia,China). relatively higher composting temperatures to traditional composting The main characteristics of the raw materials used for composting are methods (Liao et al.,2018)could be an effective way to treat tylosin shown in Table S1.Both raw waste materials were mixed thoroughly antibiotic fermentation waste. resulting in final moisture content of approximately 55%before loading Tylosin is one of the main macrolide antibiotics that is globally used into a fermentation compartment with the following dimensions:2.0 m in veterinary medicine and millions of tons of tylosin antibiotic fer- height,8.0 m length,and 4.0 m width.A forced ventilation system at mentation residue (TFR)waste is generated every year.TFR waste ty- the bottom of the compartment was used to ensure aerobic conditions. pically contains a high amount of antibiotic residues and heavy metals To mix the compost substrate well and to reduce pile-edge effects,a (Zhang et al.,2018a)that could act strong selection pressures for the mechanical turning of the hyperthermophilic composting material was transmission and prevalence of ARGs in waste-impacted bacterial performed every seven days using pile-specific forklifts.Fermentation communities (Pal et al.,2015).In addition to macrolide resistance temperature was daily monitored with automatic thermometers placed genes,antibiotic fermentation waste contains resistance genes to other at different depths of the hyperthermophilic composting piles and three antibiotics due to colocalization in multidrug resistance plasmids replicate piles were used for the composting experiment,which was run (Gonzalez-Plaza,2019).Our previous study focusing on composting of for 31 days. sewage sludge using hyperthermophilic composting (periodic tem- peratures reaching almost 90C)demonstrated efficient removal (89%) 2.2.Sample collection and physicochemical analysis of ARGs potentially due to a reduction in the horizontal transfer of ARGs in bacterial communities (Liao et al.,2018).However,it is un- To investigate the effect of time on the removal of ARGs during clear if hyperthermophilic composting is efficient at removing anti- composting,samples were collected at the beginning (DO)and after 4 biotic residues and ARGs in much more concentrated TRF waste.We (D4),7D7),13(D13),18(D18),25(D25)and31D31)days since the also lack a deeper and causal understanding of underlying mechanisms start of composting.This temporal sampling data was divided into early behind ARG removal by hyperthermophilic composting,or which spe- and late phases of composting based on temperature differences as re cific types of MGEs (plasmids,integrons or transposons)or host bac- ported earlier (An et al.,2012).Briefly,the early and late phases of terial taxa are important for the maintenance of ARGs during com- composting were split by samples before (D4 to D7)and after (D18 to posting.We hypothesized that,first,hyperthermophilic composting D31)day 13,which was considered as the 'middle point'based on the could be efficient at reducing ARGs by breaking down tylosin residues, maximum temperature reached during the composting which has been shown to occur faster at high temperatures (Yu et al. Samples were collected using a previously described protocol (Liao 2019).Second,high temperatures are likely to kill most of the non- et al.,2018).To obtain a uniform sampling distribution and re- thermophilic bacteria that carry ARGs leading to a reduction in ARG presentative samples at each time point,each pile was diagonally di- abundances.This process should also lead to a reduction in the abun- vided into 5 domains and each domain was sub-sampled (5000 g)from dance of MGEs,which could further decrease the horizontal transfer of upper,central and lower regions of the composting pile.After sampling, ARGs between remaining surviving bacterial taxa.As a result,we ex- each sample was mixed well and divided into two parts of which one pected to see changes in the composition and abundances of bacterial was shock-frozen in liquid nitrogen for biological analyses and the communities,ARGs and MGEs during the composting. other kept at 4'C for physicochemical analysis.The physicochemical To study these questions,we conducted a replicated full-scale hy- properties including pH,temperature (Temp),water content (WC), perthermophilic composting experiment of TFR waste,where we electrical conductivity (EC),total nitrogen content (TN),total carbon compared the dynamics of tylosin residues,heavy metals,ARGs and content (TC),total organic carbon content (TOC),ammonium (NH4), MGEs and changes in bacterial community composition during early and nitrate (NO3 concentrations were measured as described pre- (4-13 days)and late (18-31 days)phases of hyperthermophilic com- viously (Liao et al.,2018).DTPA-extractable heavy metals (nickel posting.We used temporal sampling followed by quantification of the (Ni2),copper (Cu2+),cobalt (Co2),zinc (Zn2),and plumbum abundance of tylosin residues and bio-active heavy metals,and quan- (Pb2+))were defined as bio-available heavy metals and analyzed as titative PCR to determine the abundances of 27 ARGs and three types of described previously (Guo et al.,2018).More detailed measurement MGEs (plasmids,integrons and transposon).Furthermore,we applied protocol for determining bio-availability of heavy metals is included in 16S rRNA gene amplicon sequencing to determine the composition of the supplementary file. the ARG and MGE-associated bacterial communities and used direct culture assays to validate the presence and location of ARGs in chro- 2.3.LC-MS/MS analysis of tylosin content mosomes and plasmids in the beginning and at the end of the com- posting experiment.The specific objectives of our study were (1)to Changes in tylosin residue quantities were determined using a investigate the feasibility of hyperthermophilic composting in removing previously described Liquid Chromatography-Mass Spectrometry (LC- tylosin residues and associated ARGs and MGEs at an industrial scale, MS/MS)method with some modifications (Zhang et al.,2018a).Briefly, (2)to understand the underlying mechanisms behind ARG removal and tylosin was extracted from 1.0 g composting samples using 5 mL 90% (3)to establish potential links between abiotic (composting properties, acetonitrile aqueous solution (v/v,pH 4.0)as follows.The tubes were tylosin residues,heavy metals)and biotic (bacterial community vortexed for 5 min and then sonicated in an ultrasonic bath for 30 min
H.Liao,et al. Environment Intemational 133 C2019)105203 Subsequently,the mixture was centrifuged at 14,000g for 10 min and performed using a Ribosome Database Project Classifier provided by the the supernatant filtered through a 0.45 um filter.Samples were cleaned- Greengenes 13.8 16S rRNA gene database with 80%sequence similarity up by solid phase extraction (SPE)cartridges (HLB,6 cc/500 mg,Wa- threshold as described previously (McDonald et al.,2012).Differences ters,USA)and SPE eluents were concentrated until dry under a gentle in the diversity (beta-diversity)and composition between microbial nitrogen flow and then dissolved in 1.0 mL of methanol.The final communities (PCoA principal coordinate analysis)were analyzed using samples were filtered through a 0.22 um membrane filter (Millex, weighted Unifrac metric distances.Within microbial community di- Millipore Corp.,Billerica,MA),transferred to 1.5mL amber vials,and versity (alpha-diversity)was quantified using Chaol and Shannon di- stored at -20'C before the LC-MS/MS analysis.Quantity of tylosin was versity indexes.All sequences were deposited in the National Center for measured by liquid chromatography combined with ion trap mass Biotechnology Information Sequence Read Archive under the accession spectrometry system (XEVO-TQS,Waters,USA)as described previously number PRJNA551919. (Zhang et al.,2018a).The tylosin standard was obtained from Dr Eh- renstorfer GmbH (Germany).The average recovery rates of the blank 2.6.Isolation and identification of antibiotic resistant bacteria samples spiked with 10-100 ug/kg of tylosin were 93.6%and the de- tection limit of the tylosin was 1.5 ug/kg.All composting samples were Culture-based methods were used to identify potential ARG hosts at analyzed in triplicate to reduce measurement errors. different phases of composting.Isolation and identification culturable antibiotic resistant bacteria was performed as described by Zhang et al. 2.4.DNA extraction and quantitative PCR (qPCR)for determining (2018b).Samples from D4 to D7 and D25 to D31 were selected as re abundances of genes linked with antibiotic resistance and mobile genetic presentative samples of the early and late phases of composting.Briefly, elements 10 g of mixed sample was suspended into 90 mL phosphate-buffer by shaking at 200 rpm for 30 min.The sample was then serially diluted to Total genomic DNA was extracted using a Fast DNA spin kit (MP different concentrations and 100 uL of all dilutions spread on Luria- Biomedicals,Cleveland,OH,USA)from freeze-dried composting sam- Bertani (LB)agar plates containing four antibiotics:tetracycline,ery- ples according to the manufacturer's instructions.In addition to quan- thromycin,gentamicin and sulfadiazine at final concentrations of tifying tylosin macrolide antibiotic resistance gene abundances,three 16 mg/L,10 mg/L,16 mg/L,512 mg/L,respectively (Ren et al.,2018; other common ARGs found in organic waste (Liao et aL.,2018;Zheng Yang et al,2017).After 48 h incubation at 30'C,the number of colony- et al.,2019)were also measured including resistance genes to tetra- forming units (CFU)was determined for each type of agar plates.Based cycline,aminoglycoside and sulfonamide antibiotics.We chose several on colony color and morphology,a total of 29 culturable antibiotic variants of each type of ARGs including 10 tetracycline resistance genes resistant strains (including 10,9,8 and 2 isolates resistant to tetra- (tetA,tetB,tetC,tetG,tetL,tetM,tetQ,teto,tetw,and tetx),7 macrolide cycline,erythromycin,gentamicin,and sulfadiazine,respectively)were resistance genes (ermB,ermF,ermM,ermT,ermX,mefA,and ereA),7 isolated from the early phase composting samples.A total of 21 cul- aminoglycoside resistance genes (aacA4,aadA,aadB,aadE,aphAl,strA, turable antibiotic resistant strains (including 6,5,6 and 4 isolates re- and strB)and 3 sulfonamide resistance genes (sull,sul2,and sul3).From sistant to tetracycline,erythromycin,gentamicin,and sulfadiazine re- here on,all tetracycline,macrolide,aminoglycoside and sulfonamide sistant isolates,respectively)were isolated from late phase composting resistance genes are abbreviated as Tet,Mac,Amin,and Sul,respec- samples.To identify bacterial colonies,we extracted the genomic DNA tively.To investigate potential changes in the abundance of different from all isolates using Bacteria DNA Kit (Tiangen,Beijing,China)and types of MGEs,we chose the following commonly observed MGEs based amplified the 16S rRNA genes using the primers 27F(5-AGAGTTTGA on previously published studies (Ma et al.,2017):2 integrons (intll and TCCTGGCTCAG-3)and 1492R(5-GGCTACCTTGTTACGACTT-3). int2),2 conjugative plasmids (ISCRI and IncQ-oriv,abbreviated as IncQ)and one transposon (Tn916/1545,abbreviated as Tn916).All 2.7.Extraction of plasmid and genomic DNA from antibiotic resistant information about primers,annealing temperatures,reaction conditions bacteria and amplification used for all target genes were validated in a previous study (Liao et al.,2018)and are listed in the supplementary materials To detect if ARGs were located on chromosomes or plasmids in (Table S2 including information about positive and negative controls isolated antibiotic resistant bacteria,genomic and plasmid DNA were and standard curves).The absolute abundances of target genes are extracted using bacteria and plasmid DNA Kit (Tiangen,Beijing,China) presented as copy numbers per gram of sample.The relative abundance following manufacturer's protocol.Residual linear chromosomal DNA of different types of ARGs (Tet,Mac,Amin,and Sul)and MGEs (in- fragments were removed using plasmid-safe ATP-dependent DNase tegrons,plasmids,and transposon)are presented as the proportion of (Epicentre,Madison,WI,USA)treatment for 24 h to 48 h at 37C fol- all detected target genes. lowing a previous method (Kothari et al.,2019).The presence of chromosomal DNA was tested by PCR using 16S rRNA universal pri- 2.5.High-throughput sequencing and bioinformatics analysis exploring mers(BAC338F,5-ACTCCTACGGGAGGCAG-3,and BAC805R,5-GACT bacterial community diversity and composition ACCAGGGTATCTAATCC-3).If a 16S rRNA genes PCR product was visible on a 1%agarose gel,another overnight digestion reaction was The changes in bacterial community composition and diversity performed until the product could no longer be visualized.The DNase during hyperthermophilic composting were determined using 16S was inactivated at 70'C for 30 min.The chromosomal DNA-free rRNA gene amplicon sequencing on an Illumina HiSeq 2500 platform plasmid and genomic DNA extracted from isolates were used to detect (Guangdong Magigene Biotechnology Co.Ltd,Guangzhou,China).The the ARGs and MGEs using specific primers using PCR (Table S2).Strains V4 region of the bacterial and archaeal 16S rDNA gene was amplified isolated from composting pile replicates at the same phases of com- using the primers 515F (5-GTGCCAGCMGCCGCGGTAA-3)and 806R posting were merged to analyze the data of ARGs and MGEs. (5'-GGACTACHVGGGTWTCTAAT-3)(Caporaso et al,2012).Raw Il- lumina sequence data was quality filtered using a pipeline coupling 2.8.Co-occurrence network analysis between different bacterial taxa,ARGs Trimmomatic (version 0.33)and QIIME (1.8.0)(Caporaso et al.,2010). and MGEs Primer sequences and low-quality reads that contained ambiguous nucleotides,mismatches in primer regions,or had a length shorter than Co-occurrence network analysis was used to explore pairwise cor- 100 bp were removed to obtain better sequence read data.Operational relations between bacterial taxa (based on genus level with abun- taxonomic units (OTUs)were defined at 97%sequence similarity level dance >0.1%)and different ARGs and MGEs during composting. using Uclust (Edgar,2010)and taxonomic assignment of OTUs was Pearson and Spearman correlations were extracted using PAST software
H.Liao,et al. Environment International 133 (2019)105203 v3.04 as described previously (Liu et al.,2019).Only relatively large Ni2+and Zn2+also with integron abundances (intll and intl2; correlation coefficients (p 0.8 and P0.05,Fig.S3a).However, early and late composting phase samples showed distinct clustering The initial composting TFR waste contained high levels of tylosin indicative of a difference in bacterial community composition (Adonis residues(85.0 mg/kg)and bio-available heavy metals (987.4 mg/kg). test,P=0.01,Fig.S3a).Similarly,total bacterial abundances (ob The hyperthermophilic composting method was effective at removing served OTUs),community richness (Chaol index)and community di- 95.0%of tylosin residues and 88.9%of bio-available heavy metals in versity (Shannon index)differed between early and late phases of 31 days (Fig.1a).The maximum composting temperature increased to composting (P 0.01,Fig.S3b-d),while no differences were found about 80'C after 13 days (Fig.1b)which coincided with the reduction between initial and early phase samples (P>0.05).Early and late of both tylosin residues and bio-available heavy metals (Fig.la-b).To phase composting samples clustered distinctly also at phylum and further understand how tylosin residues and heavy metals influenced genus levels (unsupervised hierarchical clustering based on the relative ARG and MGE abundances during composting,we explored their re- abundance of most prevalent taxa (>1%in any given sample)).Spe- lationships using correlation analysis.We found no significant corre- cifically,early phase communities had typically high relative abun- lation between the amount of tylosin residues and total ARGs abun- dances of Proteobacteria,Chloroflexi,OP11 and Thermi phyla and dances (P 0.05,Fig.1c).However,the amount of tylosin residues Psychrobacter,T78,and Methanosarcina,Ignatzschineria genera,whereas correlated positively with total MGE abundances and especially with late phase samples were enriched with Firmicute and Actinobacteria ISCRI and IncQ plasmid gene abundances (P 0.001,Fig.1d).Simi- phyla and Georgenia,Yaniella,Alcaligenes,Pseudomonas,Staphylococcus, larly,all heavy metals (Ni2+,Cu2+,Co2+,Zn2+,and Pb2+)sig- Bacillus genera (Fig.3).These bacterial community composition dif- nificantly correlated with MGE abundances (Spearman,P 0.05).In ferences were further confirmed using linear discriminant analysis ef- contrast,the majority of heavy metals (Ni2+,Cu2+,Co2+,Zn2+,and fect size analysis (Fig.S4).Together,these results suggest that changes Pb2)did not significantly correlate with total ARGs abundances in ARG and MGE abundances were linked with a reduction in bacterial (Spearman,P>0.05,Table 1).However,all detected heavy metals abundances and changes in the diversity and taxonomic composition of correlated significantly with plasmid abundances (ISCRI and IncQ)and composting communities
H.Liao,et al. Environment Internarional 133 (2019)105203 (a) (b) 100 100 --Tylosin residues Composting pile ·-Heavy metals 1000 ●-Amibent 80 E 80 800 E 60 600 (6x/6w) 40 40 400 6 20 20 200 0 0 (c) 0 510152025 30 (days) 0 510 15202530(days) Early phase Late phase d Early phase Late phase 6o 14.0 15 ●1SR1,R2=0.7100,P<0.0001 13.5 (601) ● ● 14 ▲ncQ,R2=0.6815.P<0.0001 13.0 13 12.5 seue6 plwseld ●●● 12 ●● ●● ● 12.0 44 11.5 10 AA 11.0 91 10.5 A Total MGEs,R2=0.7855,Pe0.0001 Total ARGs R2-0.2422,Pa0.C601 10.0 0 20 0 60 80 100 0 20 40 60 80 100 Amount of tylosin residues(mg/kg) Amount of tylosin residues(mg/kg) Fig.1.Changes in tylosin residues and bio-available heavy metals(a)and composting temperature during hyperthermophilic composting(b).Correlation analyses between tylosin content and the abundance of total ARGs and total MGEs (c)and plasmid genes(d)based on linear regression models.All absolute abundances of the target genes were normalized using logarithmic transformation (log10)and bars in panels (a)and (b)show1 standard error of mean. 3.4.Correlation between ARG,MGE and bacterial taxa abundances The co-occurrence patterns between ARGs,MGEs and bacterial taxa abundances were further compared using correlation-based co-occur- Based on procrustes analysis,changes in resistomes (based on all rence network analysis.The networks showed a clear shift between ARGs and MGEs)were significantly correlated with bacterial commu- early and late phases of composting mainly due to differences in bac- nity composition at genus level (P=0.0017,M2 0.5537,R 0.6681. terial diversity and community composition (Fig.4).The co-occurrence 999 permutations,Fig.S5a).Similarly,changes in MGEs (based on all network constructed at early phase of composting was larger and more types of MGEs)and bacterial community composition were sig- connected compared to the late phase network(Fig.4).Also,a larger nificantly correlated (P 0.0185,M=0.6940,R 0.5531,999 per- number of nodes and edges were included in early versus late com- mutations,Fig.S5b).As expected,all types of ARGs (tetracycline,sul- posting phase network and several network indices such as network fonamide,aminoglycoside and macrolide gene)and MGEs (plasmids, diameter,network density,network modularity,average path length, integrons and transposon)were significantly correlated with their as- and average degree were greater for early compared to late phase sociated bacterial community (all P 0.05,999 permutations,Fig. network.Based on a previous study (Li et al.,2015),we hypothesized S6). that non-random co-occurrence patterns between ARGs and microbial Table 1 Correlation analysis between the amount of bio-available heavy metals,individual MGEs and total MGEs and ARGs during hyperthermophilic composting. MGEs type Ni2+ Cu2+ Co2+ Zn2+ P%3+ T-metals i Integron -0.68*t 0.33 -053 0.55* 0.38 0.55 int2 Integron 0.5744 -0.24 0.41 -0.66* -0.02 -0.66 Tn916 Transposon -0.6 0.63 -0.62 -0.49 0,81 0.49 ISCR1 Plasmid 0.774 -0.63 0.684 0.864 -0.48 0.86 IncQ Plasmid 0.78* -0.62* 0.71* 0.86* -0.52 0.85* T-MGEs 0.774 -0.64* 0.69*4 0.86*4 -0.5 -0.8644 T-ARGs -0.30 0.20 -0.27 0.49 0.05 0.39 Notes:Spearman's rank order correlation analysis was used based on absolute target gene abundances with following significances: Significant at P 0.05. *Significant at P 0.01. **Significant at P<0.001.T-metals,T-MGEs and T-ARGs denote for total concentration of bio-available heavy metals,total abundance of MGEs and total abundance of ARGs,respectively
H.Liao,et al. Environment Internarional 133 (2019)105203 (a) (b) 8E+12 +12 12 7E 12 6E+12 5E+12 +12 2 4E+12 3E+12 E+12 8 2 (c) Initial TFR Early phase Late phase (d) Initial TFR Early phase Late phase 100 8 100 Integrons 80 Plasmids Transposon 60 40 40 Tet 20 20 Sul Amir Mac 0 4 1 13 18 25 31(days) 0 47 1318 2531(days Early phase Late phase Early phase Late phase Fig.2.Temporal changes in the absolute abundances(a-b)and proportion (c-d)of target ARGs and MGEs during hyperthermophilic composting.Panels (a-b):Total absolute abundances of ARGs and MGEs in the initial TFR waste and during early and late phases of composting.Panels (c-d):Changes in the proportion of different types of ARGs (c)and MGEs (d)during composting.All data is based on target gene abundances and bars denote for1 standard error.One star ()significant at P 0.05,Two star (**)significant at P 0.01. taxa could be used to identify potential ARG hosts.By following this more often on plasmids than on chromosomes with early phase samples analysis,we identified 22 candidate bacterial genera as potential ARG (Fig.5a).However,the location of ARGs and MGEs was highly variable and MGE hosts at the early phase of composting (Fig.4a).Similarly,11 at the finer taxonomic level and in some cases a higher abundance of potential bacterial genera were non-randomly associated with ARGs target genes was observed on plasmids compared to chromosomes even and MGEs during the late phase of composting (Fig.4b).Crucially,the with late composting phase isolates(Fig.5b).These results are line with taxonomic composition of the potential host taxa differed between early our sequencing results demonstrating that culturable isolates carried and late phases of composting.The Psychrobacter,Morganella,and T78 fewer ARGs and MGEs at the end of the hyperthermophilic composting. were the dominant potential hosts at the early phase of composting and Furthermore,the high prevalence of antibiotic resistance genes during in the initial TFR waste accounting for 49.7%of the total 16S rRNA the early phase of composting was likely linked with a relatively high gene sequences.However,after 31 days of composting the abundance number of plasmids that might have carried multiple antibiotic re- of these taxa gradually decreased to 0.52%(Fig.S7)and Alcaligenes, sistance genes. Bacillus,and Staphylococcus bacteria became associated as potential ARG hosts during the late phase of composting.Moreover,two potential 3.6.Comparing the relative contributions of abiotic and biotic factors on ARG hosts,Lysobacter and Georgenia,were associated with ARGs only at ARG and MGE abundances during early and late phases of composting the late phase of composting.Together,these results suggest that as- sociations between ARGs,MGEs and their potential host bacterial taxa A total of 81.2%variance of ARG abundances could be explained by changed during the composting. composting properties (WC,TC,TN,EC,TOC,C/N,and NOs),the concentration of tylosin residues and bio-available heavy metals,bac- 3.5.Isolation of potential ARG host bacteria and identifying the location of terial community composition (based on OTUs)and MGE abundances ARGs in chromosomes and plasmids (CCA analysis,Fig.S9).To further study how ARGs were affected by abiotic and biotic factors at different phases of composting,we con- The number of cultivable antibiotic resistant strains conferring re. structed a Partial Least Squares Path Model (PLS-PM)describing direct sistance to tetracycline,erythromycin,gentamicin,and sulfadiazine and indirect relationships between biotic and abiotic factors.We found were significantly higher during the early versus late phase of com- that tylosin residue and heavy metal concentrations and other abiotic posting (9.1 x 10'vs 1.3 x 10'CFU/gram dry sample,P<0.05,Fig composting properties had no statistically significant effect on ARG S8).The antibiotic resistant strains isolated from the early phase of abundances during the early phase of composting (Fig.6a).However, composting belonged to 6 genera (Alcaligenes,Bacillus,Staphylococcus, tylosin residue concentrations had strong direct effects on the abun- Saccharopolyspora,Paenibacillus,and Vagococcus;Table S3).Only 2 dance of MGEs and bacterial community composition during the early genera (Alcaligenes and Staphylococcus)were found in the late phase of phase of hyperthermophilic composting.MGE abundances had a strong composting (Table S4).In line with our previous analyses,we detected direct positive effect on ARG abundances at the early phase of com- more ARGs and MGEs(61 vs 23 genes in total;average of 2.1 and 1.0 posting.However,this effect became much weaker during the late target genes per isolated strain)during early compared to the late phase phase of composting (Fig.6a).In contrast,bacterial community com of composting (Fig.5a).Interestingly,ARGs were on average located position affected ARG abundances during both the early and late phases
H.Liao,et al. Environment International 133 (2019)105203 (a) (b ☐Late phase ☐Early phase nitial TFR Initial TFR Early phase ☐Late phase abundance (% 0.029.057.9 Yaniella Bacillus Georgenia Brachybacterium Staphylococcus Pseudomonas (c) Alcaligenes ☐Earlyphase ☐Late phase confidence intervals T78 172e.4 Methanosarcina 110-3 Morganella The间t 258e.3 Streptomyces Proteobacteria 209e.3 4510-3 Ignatzschineria Firmicutes日 499e-3 Jeotgalicoccus Euryarchao 836a.3 01 Lysobacter Oceanobacillus 53 40 60 Virgibacillus (d) Mean proportion】 n proportions Providencia ☐Early phase Proteiniclasticum Georgenia 26e-4 Sporosarcina T70日 234-3 Gracilibacillus JJeotgaNeoccus 73 Corynebacterium 744 Sphingobacterium 50.2 Caldicoprobacter Tepidimicrobium Sporanaerobacter SHD-231 B-42 0025 0025 Psychrobacter 0.031 0.050 星程程8889888明 330-30-25106402030405061 n陶 ortions Fig.3.Changes in bacterial community composition during early and late phases of hyperthermophilic composting at different taxonomic levels.Panels (a-b):the relative bacterial abundances at the genera (a)and phyla (b)level between initial TFR and early and late phase composting samples.Panels (c-d):the difference analysis of relative bacterial abundances at the phyla(c)and genera(d)level between early and late phase composting samples using STAMP software.Corrected P. values were calculated using the Benjamini-Hochberg false discovery rate (P 0.01). of composting (Fig.6a-b).Association between bacterial community become antibiotic-resistant in order to survive in antibiotic and heavy composition and MGEs were positively correlated only during the early metal-rich environment(Baker-Austin et al.,2006).Despite the initially phase of composting,which suggests that this relationship was lost high ARG concentrations,hyperthermophilic composting was effective during the late phase of composting.Together these results suggest that at reducing the amount of ARGs (75%),MGEs (98%),tylosin residues tylosin residues and heavy metal concentrations were not directly (95%)and bioactive heavy metals(89%)in 31 days.This was likely due linked with ARG abundances,but instead,had highly significant effects degradation of antibiotic residues at high temperatures (Yu et al.,2019) on MGE abundances,which were strongly linked with ARGs and and change in the bioavailability of heavy metals into unavailable form changes in bacterial community composition (Fig.6b). during the composting (Chen et al.,2019;Zhou et al.,2018). No clear difference in ARG and MGE abundances was found be- 4.Discussion tween initial and early phase composting samples when the composting temperature did not differ much from ambient temperature(mean of 4.1.Hyperthermophilic composting efficiently reduces the amount of tylosin 23C).However,changes became pronounced during the late phase of residues and bio-active heavy metals and associated antibiotic resistance composting when the temperatures reached -60'C (Fig.1b).This genes suggests that removal of ARGs happened during the late phase of composting due to prolonged exposure to high temperatures.Although In this study,we explored the efficiency of hyperthermophilic most ARGs decreased during the composting,few ARGs such as tetL and composting at treating antibiotic fermentation waste in a full-scale in- sull increased towards the end.This is in line with previous studies dustrial composting experiment.High abundances of diverse ARGs(27 (Zhang et al.,2016),suggesting a potential enrichment of these ARGs in ARGs and 5 MGEs with 8.1 x 1012 gene copies per gram)were found in thermophilic bacteria that can survive high composting temperatures. the initial TFR waste.These ARG abundances are 1-2 orders of mag- However,this hypothesis needs to be tested experimentally in the fu- nitude higher than previously reported in other kinds of waste such as ture. food waste (Liao et al.,2019),sewage sludge (Liao et al.,2018)and Presence of heavy metals in composting waste can induce selection animal manure (Munir and Xagoraraki,2011). for metal resistance genes.Because metal resistance genes are often This is likely attributed to a strong selection pressure for bacteria to located in multidrug-resistance plasmids,high heavy metal
H.Liao,et al. Environment International 133 (2019)105203 (a)Early phase (b)Late phase Gedrgenia s01 tetA mefA Lysobacter adA ISR1 Alcalig strA tetw Bacillus IncQ Jeotg.alicoccus tetx strB 842 tetC a边A1 Pseudomonas Gra liba tetQ Staphylococcus Parameters Early phase Late phase Module I(13) ● odule1(12☒ ○ModuleV() Number of total edges 116 35 ● Number of total nodes 46 Module ll (13) 28 Module ll(6) ○ Module VI(☒ Network diameter 7 3 ● Module (12) Network density 0.112 0.093 ● Module Ill (3) Network modularity 0.693 0.489 Module IV(3) Module IV(B) Average path length 2.266 1.742 Average degree 5.043 2.5 Fig.4.Network analysis exploring candidate bacterial hosts (genus level)associated with ARGs and MGEs based on gene co-occurrence analysis during early (a)and late phases(b)of hyperthermophilic composting (only strong and highly significant correlations based on both Spearman and Pearson's correlation coefficient were included to the analysis p >0.8,P <0.01).The different colors represent different modules and the node sizes and edge widths are proportional to the correlation coefficient values. concentrations can indirectly co-select for ARGs in soils and in animal (89%),which was strongly correlated with the reduction in the abun- guts (Ding et al.,2019;Zhao et al.,2018).In line with this,we observed dance of plasmids.Together these results suggest that hyperthermo- that Zn2+concentration(927 mg/kg)was significantly linked with philic composting was efficient at removing ARGs by reducing the total ARGs abundances during the composting-an association that has concentrations of both antibiotics and heavy metals and the strength of previously been reported in dairy farms (Zhou et al.,2016).Notably,we selection for ARGs and MGEs. found that most heavy metals were more strongly associated with MGE abundances,which suggests that they mainly affected the mobilization of ARGs via MGEs (Hu et al.,2017).Importantly,also the concentra- tions of bio-available heavy metals decreased during composting (a) (b) Target genes on plasmids Target genes on plasmids Target genes on chromosomes Target genes on chromosomes Alcangenes faecals GX28 Staphylococcus lontus NEAU-TD63 alis LHW 1748 Late 00443020 phase Staphylococcus lentus FJAT-hcl-24 Paenibacillus cineris Early c2c BacMlus coveus CCM 201 phase tus JCM 2426 2. .10 Number of ARGs and MGEs Early phase Late phase Fig.5.The total number of ARGs and MGEs detected on chromosomes or plasmids of all culturable isolates from early and late phase composting samples (a).Panel (b)shows the number of resistance genes on plasmids and chromosomes at the taxa level
H.Liao,et al. Environment Internarional 133 (2019)105203 a Early phase Late phase Composting -0.93" Composting -0.53 properties R2=0.88 R2=086 0.13 11 0.98 0 0.16 0.66 0.09 0.93 -0.29 0.85 0 -0.50 Heavy metals MGEs 1.31 community Heavy metals -0.79 MGEs 0.67 R2=0.97 R2=0.76 R2=09 R2=0.86 R2=0.81 R2-0.97 0.24 0.43 0.08 0.92 Goodness-Of-Fit ARGs Goodness-Of-Fit 0.7196 ARGs R2=0.99 0.6992 R2=0.98 (b) Direct effect Direct effect Indirect effec Total effect a Fig.6.Partial least squares path models(PLS-PM)showing direct and indirect effects of different factors on absolute ARG abundances during early and late phases of hyperthermophilic composting.Panel (a-b):PLS-PM describing the relationships between composting properties,tylosin residues,heavy metals,bacterial community composition(based on OTUs)and MGEs on ARGs abundances during early and late phases of hyperthermophilic composting.Arrow widths describe the magnitude of path coefficients and red and blue colors indicate positive and negative effects,respectively.Path coefficients and coefficients of determination (R)were calculated after999 bootstrap replicates and significance levels are indicated by*(P<0.05),**(P<0.01)and***(P<0.001)levels.Panel (b):The standardized direct, indirect and total(direct indirect)effects derived from the partial least squares path models. 4.2.The potential mechanisms underlying the removal of ARGs and MGEs high levels during the early phase of composting while they almost during hyperthermophilic composting completely vanished during the late phase of composting.Plasmids,in particular,are known to incur large fitness costs for bacteria (San We found three interrelated mechanisms for ARG removal in our Millan and MacLean,2017)because they often carry multiple ARGs and experiment:reduction in the strength of selection for ARGs and MGEs, heavy metal resistance genes (Gullberg et al.,2014)leading to a high reduced horizontal transfer of ARGs via MGEs and loss of suitable hosts metabolic burden (Andersson and Hughes,2010).Reduction in the for ARGs.First,the reduction in tylosin and heavy metal concentrations frequency of plasmids likely reduced the horizontal gene transfer of (bioavailable phase)was positively linked with a reduction in total ARGs between different host bacteria.For example,horizontal gene MGE abundances (specifically,ISCRI and IncQ plasmid genes).While transfer mediated by a conjugative plasmid RP4 that carries multiple the relationship between tylosin and heavy metal concentrations and antibiotic resistance genes has been shown to maintain antibiotic re ARG abundances was non-significant,the total MGE and ARG abun- sistance in the presence of heavy metals and non-antibiotic pollutions dances were significantly positively correlated (Fig.S2).This suggests (Klumper et al.,2017;Lu et al.,2018).In support for this,we found that that hyperthermophilic composting weakened the strength of selection larger number of resistance genes were located on plasmids during the for ARGs and MGEs by reducing the concentrations of tylosin and heavy early versus phase of composting when the selective pressure by anti- metals in the compost substrate.The mechanistic explanation for this biotics and heavy metals was stronger (Fig.5).These results suggest might be that ARGs and MGEs often impose severe costs for host bac- that the elimination of ISCR1/IncQ plasmid genes played a key role in teria in terms of reduced growth and competitive ability (Bjorkman and reducing ARG abundances during hyperthermophilic composting. Andersson,2000).As a result,a reduction in antibiotic and metal In addition to affecting the strength of selection and the relative concentrations likely decreased the relative benefit of ARGs and fa- abundance of MEGs,hyperthermophilic composting could have af- vored bacteria that did not carry costly ARGs and MGEs (Baker-Austin fected the ARG abundances by changing the bacterial communities.We et al.,2006).The relative contribution of various types of MGEs (in- observed a clear decrease in bacterial abundances and community di- tegrons,plasmids and transposon)varied during composting,sug- versity during the experiment,which is consistent with previous com- gesting that different types of MGEs could have been responsible for the posting studies (Zhang et al,2018c).The high composting temperature dissemination and prevalence of ARGs at different phases of com- was likely detrimental for many bacteria during the composting,which posting.Specifically,ISCR1 and IncQ plasmid genes were maintained at could have also led to a loss of associated resistance genes.The
H.Liao,et al. Environment International 133 (2019)105203 reduction in bacterial densities could have also indirectly reduced ARG Acknowledgements abundances by lowering the horizontal transfer of ARGs via less fre. quent bacterial encounter rates (Liao et al.,2018).In line with this,we This work was supported by the National Key Technologies R&D observed clear correlations between bacterial abundances and com- Program of China (2017YFD0800203),Key Research Development munity composition and the prevalence of ARGs and MGEs based on Plan of Fujian Province (2017NZ0001-1).Natural Science Foundation our sequencing data.Furthermore,less abundant and less diverse of China and Fujian Province (31972521 and 2018J01709)and Fujian community of antibiotic resistant bacteria could be isolated and cul- Agriculture and Forestry University Program for Distinguished Young tured from the late compared to early phase composting samples (Table Scholar (No.XJQ2017001).Ville-Petri Friman is supported by the S3-S4). Wellcome Trust [ref:105624]through the Centre for Chronic Diseases We also found that some taxa were non-randomly linked with and Disorders (C2D2)and Royal Society Research Grants changes in ARG and MGE abundances.For example,the Chloroflexi and (RSG\R1\180213 and CHL\R1\180031)at the University of York. Proteobacteria taxa,which are often linked with ARG carriage (Wu Stefan Geisen is supported by a NWO-VENI grant from the Netherlands et al.,2017),had high relative abundances during the early but low Organization for Scientific Research (016.Veni.181.078).We would like abundances during the late phase of composting.To study this in more to thank Beijing Geogreen Innotech Co.,Ltd for offering the experi- detail,we performed a network analysis exploring associations between mental condition and Huan Liu for help in sampling ARGs and different bacterial taxa.We found that early and late phase composting networks were very different,which was most likely driven Declaration of Competing Interest by a reduction and loss of several bacterial taxa (Li et al.,2015).Im- portantly,ARGs were associated with different bacterial taxa during The authors declare no conflict of interest. early and late phases of composting.For instance,the Psychrobacter, Morganella,and T78 were the most potential ARG-associated hosts in Appendix A.Supplementary material the initial TFR waste and during the early phase of composting.In contrast,Alcaligenes,Bacillus,and Staphylococcus were the most likely Supplementary data to this article can be found online at https:// potential ARG-associated hosts during the late phase of composting. doi.org/10.1016/j.envint.2019105203. Specifically,we identified Georgenia as a candidate bacterial host genus for the sull resistance gene.This taxon could only be isolated at the late References phase of composting when it had 38 times higher abundance compared to the initial TFR waste.Together these results suggest that associations An,C.J.,Huang,G.H.,Yao,Y.,Sun,W.,An,K.,2012.Performance of in-vessel com- between ARGs and their potential host bacterial taxa changed during posting of food waste in the presence of coal ash and uric acid.J.Hazard.Mater. 203204.3845. composting and that certain antibiotic resistant bacterial taxa might be Andersson,D.I.,Hughes,D.,2010.Antibiotic resistance and its cost:is it possible to re. difficult to eradicate even with hyperthermophilic composting. verse resistance?Nat.Rev.Microbiol.8,260. Finally,we explored complex relationships between abiotic and Baker-Austin,C.,Wright,M.S.,Stepanauskas,R.,McArthur,J.V.,2006.Co-selection of antibiotic and metal resistance.Trends Microbiol 14 (4).176-182. biotic drivers on ARG removal using partial least squares path model- Bondarczuk,K.,Markowicz,A.,Piotrowska-Seget,Z.,2016.The urgent need for risk ling.We found that variation in ARGs abundances was mainly ex assessment on the antibiotic resistance spread via sewage sludge land application. plained by MGE abundances during the early phase of composting.In Environ.Int.87.49-55. contrast,bacterial community composition had a significant and strong Bjorkman,J..Andersson.D.I.,2000.The cost of antibiotie resistance from a bacterial perspective.Drug Resist Update 3,237-245. influence on MGE and ARG abundances throughout composting.The Caporaso,J.G.,Kuczynski,J.,Stombaugh,J.,Bitting er,K.,Bushman.F.D..Costello.E.K. tylosin residue and bio-available heavy metal concentrations indirectly Fierer,N..Pena,A.G.,Goodrich,J.K.,Gordon,J.I.,2010.QIIME allows analysis of affected ARGs through direct effects on MGEs.Interestingly,the direct high-throughput community sequencing data.Nat.Methods 7,335-336. Caporaso,J.G.,Lauber,C.L,Walters,W.A.,Berg-Lyons,D.,Huntley,J.,Fierer,N.,Owens effect of bacterial community composition on ARG abundances became S.M.,Betley,J.,Fraser,L.Bauer,M.,Gormley,N.,Gilbert,J.A,Smith,G.,Knight,R.. stronger during the late phase of composting.This finding is supported 2012.Ultra-high-throughput microbial community analysis on the Illumina HiSeq by the above results showing that most ARGs observed at the end of the and MiSeq platforms.ISME J.6,1621. Chen,Q.L.Li.H.,Zhou,X.Y.,Zhao,Y..Su,J.Q.,Zhang.X.,Huang.F.Y..2017a.An experiment were located on bacterial chromosomes instead of plasmids. underappreciated hotspot of antibiotic resistance:The groundwater near the muni- In the future,it will be important to study if hyperthermophilic com- cipal solid waste landfill.Sci.Total Environ.609,966-973. posting favors certain type of plasmids and if it can directly select for de Chen,W.,Geng,Y.,Hong,J.,Kua,H.W,Xu,C,Yu,N.,2017b.Life cycle assessment of novo antibiotic resistance via rapid bacterial evolution. antibiotic mycelial residues management in China.Renew.Sust.Energy Rev.79, 830838. Chen,Y.,Chen,Y.,Li,Y.,Wu,Y.,Zeng,Z,Xu,R.,Wang,S.,Li,H,Zhang,J.,2019. Changes of heavy metal fractions during co-composting of agricultural waste and 4.3.Conclusions river sediment with inoculation of Phanerochaete chrysosporium.J.Hazard.Mater 378.120757 Cui,P.,Fan,F.,Yin,C.,Song,A.,Huang,P.,Tang,Y.,Zhu,P.,Peng,C,Li,T.,Wakelin. In conclusion,our study shows that hyperthermophilic composting S.A.,2016.Long-term organic and inorganic fertilization alters temperature sensi- is efficient at removing tylosin antibiotic residues,heavy metals and tivity of potential N 2 O emissions and associated microbes.Soil Biol.Biochem.93. 131-141 associated ARGs and MGEs from tylosin antibiotic fermentation waste. Ding.J.,Zhu,D.,Hong,B.,Wang,H.T.,Li,G..Ma,Y.B..Tang,Y.T.,Chen,Q.L.2019. Mechanistically,this was driven by a reduction in the abundance of Long-term application of organic fertilization causes the accumulation of antibiotic plasmid genes (ISCRI and IncQ-oriv)during the late phase of com- resistome in earthworm gut microbiota.Environ.Int.124,145-152. Edgar,R.C..2010.Search and clustering orders of magnitude faster than BLAST. posting.These plasmid genes were highly correlated with the abun- Bioinformatics 26,2460-2461. dance of ARGs and thus likely acted as carriers of the resistance genes. Gullberg.E,Albrecht,L.M.,Karlsson,C.,Sandegren,L.Andersson,D.L.2014.Selection Co-occurrence network analysis and culture-dependent experiment ofa multidrug resistance plasmid by sublethal levels of antibiotics and heavy metals. mB105.e01918-01914. further revealed that the potential hosts for ARGs were effectively re Gonzalez-Plaza,J.J.,2019.Antibiotic-manufacturing sites are hot-spots for the release duced during the composting,which likely reduced opportunities for and spread of bacteria carrying antibiotic resistance genes and mobile genetic ele. horizontal gene transfer of ARGs.Together these results suggest that ments in receiving aquatic environments.Environ.Int.130,104735. hyperthermophilic composting can be a successful strategy for treating Guo,T.,Lou,C.,Zhai,W.,Tang,X.,Hashmi,M.Z,Murtaza,R.,Li,Y.,Liu,X.,Xu,J.,2018. Increased occurrence of heavy metals,antibiotics and resistance genes in surface soil highly concentrated antibiotic fermentation waste at industrial scale by after long-term application of manure.Sci.Total Environ.635,995-1003. having negative effects on bacterial hosts associated with multidrug- Hu,H.,Wang,J.-T.,Li,J.,Shi,X.,Ma,Y.,Chen,D.,He,J.-Z.,2017.Long-term nickel contamination increases the occurrence of antibiotic resistance genes in agricultural resistance plasmids. soils.Environ.Sci.Technol.51,790-800