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Effects of different composting methods on antibiotic-resistant bacteria, antibiotic resistance genes, and microbial diversity in dairy cattle manures

  • Minjia Tang
    Affiliations
    Key Laboratory of New Animal Drug Project, Gansu Province/Key Laboratory of Veterinary Pharmaceutical Development, Ministry of Agriculture and Rural Affairs/Lanzhou Institute of Husbandry and Pharmaceutical Sciences of the Chinese Academy of Agricultural Sciences, Lanzhou 730050, China
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  • Zhongyong Wu
    Affiliations
    Key Laboratory of New Animal Drug Project, Gansu Province/Key Laboratory of Veterinary Pharmaceutical Development, Ministry of Agriculture and Rural Affairs/Lanzhou Institute of Husbandry and Pharmaceutical Sciences of the Chinese Academy of Agricultural Sciences, Lanzhou 730050, China
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  • Wenzhu Li
    Affiliations
    Qilihe District People's Hospital of Lanzhou, Lanzhou 730050, China
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  • Muhammad Shoaib
    Affiliations
    Key Laboratory of New Animal Drug Project, Gansu Province/Key Laboratory of Veterinary Pharmaceutical Development, Ministry of Agriculture and Rural Affairs/Lanzhou Institute of Husbandry and Pharmaceutical Sciences of the Chinese Academy of Agricultural Sciences, Lanzhou 730050, China
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  • Amjad Islam Aqib
    Affiliations
    Department of Medicine, Cholistan University of Veterinary and Animal Sciences, Bahawalpur 63100, Pakistan
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  • Ruofeng Shang
    Affiliations
    Key Laboratory of New Animal Drug Project, Gansu Province/Key Laboratory of Veterinary Pharmaceutical Development, Ministry of Agriculture and Rural Affairs/Lanzhou Institute of Husbandry and Pharmaceutical Sciences of the Chinese Academy of Agricultural Sciences, Lanzhou 730050, China
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  • Zhen Yang
    Affiliations
    Key Laboratory of New Animal Drug Project, Gansu Province/Key Laboratory of Veterinary Pharmaceutical Development, Ministry of Agriculture and Rural Affairs/Lanzhou Institute of Husbandry and Pharmaceutical Sciences of the Chinese Academy of Agricultural Sciences, Lanzhou 730050, China
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  • Wanxia Pu
    Correspondence
    Corresponding author
    Affiliations
    Key Laboratory of New Animal Drug Project, Gansu Province/Key Laboratory of Veterinary Pharmaceutical Development, Ministry of Agriculture and Rural Affairs/Lanzhou Institute of Husbandry and Pharmaceutical Sciences of the Chinese Academy of Agricultural Sciences, Lanzhou 730050, China
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Open AccessPublished:November 01, 2022DOI:https://doi.org/10.3168/jds.2022-22193

      ABSTRACT

      Composting is a common practice used for treating animal manures before they are used as organic fertilizers for crop production. Whether composting can effectively reduce microbial pathogens and antibiotic resistance genes remain poorly understood. In this study, we compared 3 different dairy manure composting methods—anaerobic fermentation (AF), static compost (SC), and organic fertilizer production (OFP)—for their effects on antibiotic-resistant bacteria, antibiotic resistance genes, and microbial community diversity in the treated manures. The 3 composting methods produced variable and distinct effects on antibiotic-resistant bacteria, zoonotic bacteria, and resistance genes, some of which were decreased and others of which showed no significant changes during composting. Particularly, SC and OFP reduced chloramphenicol resistance gene fexA and opportunistic pathogen Vibrio fluvialis, whereas AF significantly reduced tetracycline resistance gene tetB and opportunistic pathogens Enterococcus faecium and Escherichia fergusonii. The compositions of microbial communities varied significantly during the composting processes, and there were significant differences between the 3 composting methods. In all 3 composts, the dominant phyla were Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. Interestingly, Firmicutes, Proteobacteria, and Bacteroidetes remained stable in the entire AF process, whereas they were dominated at the beginning, decreased at the early stage of composting, and rebounded at the later stage during SC and OFP. In general, SC and OFP produced a more profound effect than AF on microbial community diversities, pathogens, and dominant species. Additionally, Enterococcus aquimarinus was isolated from AF for the first time. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States function prediction analysis indicated that the genes related to membrane transport and amino acid metabolism were abundant in the 3 composts. The metabolism of amino acids, lipids, and carbohydrates increased as composting progressed. The biosynthesis of antibiotics was enhanced after fermentation in the 3 composting methods, and the increase in the SC was the most obvious. These results reveal dynamic changes in antibiotic-resistant bacteria, antibiotic resistance genes, microbial community composition, and function succession in different dairy manure composts and provide useful information for further optimization of composting practices.

      Key words

      INTRODUCTION

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      Fate of antibiotics in soil and their uptake by edible crops.
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      ). Due to the extensive use of antibiotics in livestock and poultry production systems, ARG were increasingly detected in livestock and poultry manures and may disseminate to other environmentally indigenous bacteria through horizontal gene transfer (
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      ). However, it is unknown whether manure treatments can effectively control the discharge of microbials and whether they can efficiently reduce ARB and ARG. To address these questions, we examined the ARB, ARG, and microbial diversity of 3 different dairy manure compost treatments in northwest China.

      MATERIALS AND METHODS

      Ethics

      This study did not involve endangered or protected species, animals, or clinical experiments.

      Sample Collection

      Three composting methods, anaerobic fermentation (AF), static compost (SC), and organic fertilizer production (OFP), were examined in this study because they are commonly used for treating cattle manure in China (
      • Meng Q.
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      Bacterial community succession in dairy manure composting with a static composting technique.
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      • Dong R.
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      • Guo J.
      Influence of anaerobic digestion on the labile phosphorus in pig, chicken, and dairy manure.
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      • Smith D.R.
      • Chaubey I.
      • Wu S.
      Long-term performance of three mesophilic anaerobic digesters to convert animal and agro-industrial wastes into organic fertilizer.
      ). The sampled dairy farms were located in Xinjiang Province and Qinghai Province of northwest China. All samples were collected in mid-March, and at that time the average temperature in both locations was around 10°C and the average daylight was 11 to 12 h. Compost samples were collected at 5 different sites corresponding to different stages of each composting method (discussed next). At each site, triplicate samples were obtained and each sample was collected by the 5-point sampling method (
      • Zhou L.
      • Zhang Y.
      • Xu Q.
      • Pang R.
      • Song B.
      Concentrations and health risk assessments of heavy metal contents in soil-corn system of Tongren, China.
      ). In total, 45 samples were collected from the 3 composting methods.
      At the Xinjiang site, composting was done by AF. Briefly, the raw manure (Z1) was separated into precipitated sand and suspension by stirring, suspending, and precipitating. The precipitated sand (Z2) was used as bedding for the dairy farm after more than 20 d of sun exposure, and the suspension was put into the fermenter for fermentation. After fermentation, products were separated into 3 parts. The air portion was used as bioenergy, the solid part (Z3) was used as bedding after sun exposure for more than 1 mo (Z4), and the liquid part (ZS) was used as fertilizer (Supplemental Figure S1A, https://doi.org/10.7910/DVN/WHS3EA ;
      • Tang M.
      • Wu Z.
      • Li W.
      • Shoaib M.
      • Aqib A.I.
      • Shang R.
      • Yang Z.
      • Pu W.
      Effects of different composting methods on antibiotic-resistant bacteria, antibiotic resistance genes, and microbial diversity in dairy cattle manures. Harvard Dataverse, V1.
      ). Samples were collected at the stages of Z1, Z2, Z3, Z4, and ZS for various analyses.
      The composting methods used at the Qinghai site were SC and OFP. For SC, raw manure from cowshed bedding (S9) was mechanically pressed and separated into liquid sewage (SS) and solid residue (S10). The liquid sewage was discharged into the lagoon and irrigated to the farm fields after over 1 mo of storage and natural fermentation. The solid residue was piled up and used as bedding for the dairy farm after more than 20 d of SC and sun exposure (S12) (Supplemental Figure S1B). During the SC, the pile was turned once every 7 d and the temperature in the manure pile was between 45°C and 50°C. The collected samples included raw manure (S9), liquid sewage in the lagoon (SS), solid residue (S10), and solid after 10 d (S11) and 20 d (S12) composting and sun exposure.
      For OFP, the raw manure, which was mainly from the playground and bedding (S13), was mixed with rice husk powder. The mixture (S14) was then put into a manure pit. For every 5 tons of mixed manure materials, 1 kg of fermentation agents was added, which contained approximately 1 billion viable bacteria per gram.
      The viable bacteria mainly included Myceliophthora thermophila, Streptomyces thermophilus, Bacillus stearothermophilus, Bacillus pallidus, and Bacillus subtilis. During the 1-mo fermentation period, the composts were turned and the temperature was measured every day. The fermentation temperature within the pit was approximately 60°C. After fermentation (S15), the compost material was crushed and screened, then it was combined with chemicals including nitrogen, phosphorus, and potassium to the total content of 4% (S16). The final product was processed into granular fertilizer after drying, dedusting, and cooling and was used as organic fertilizer (S17) (Supplemental Figure S1C). Samples of OFP were collected at S13, S14, S15, S16, and S17. For all 3 composting methods, a clear sampling scheme and sample information are shown in Supplemental Figure S1 and Supplemental Table S1 ( https://doi.org/10.7910/DVN/WHS3EA ;
      • Tang M.
      • Wu Z.
      • Li W.
      • Shoaib M.
      • Aqib A.I.
      • Shang R.
      • Yang Z.
      • Pu W.
      Effects of different composting methods on antibiotic-resistant bacteria, antibiotic resistance genes, and microbial diversity in dairy cattle manures. Harvard Dataverse, V1.
      ).

      Detection of ARG in Manures by PCR

      The 3 samples from each sampling site were mixed, and the DNA of solid and liquid samples was extracted with the soil extraction kit (OMEGA Soil DNA Kit: D5625-01) and the liquid extraction kit (OMEGA Water DNA Kit: D5525-01) according to the manufacturer's instructions, respectively. In total, 32 ARG were analyzed by PCR including chloramphenicol/florfenicol resistance genes (floR and fexA), fluoroquinolones resistance genes [gyrA, gyrB, grlA, grlB, aac(6')-Ib-cr, and qnrA], β-lactam resistance genes (mecA, mecC, and femA), glycopeptide resistance genes (vanA, vanB, and vanC), sulfonamides resistance genes (sul1, sul2, and sul3), aminoglycoside resistance genes aac(6')/aph(2″), macrolides resistance genes (ermA, ermB, ermC, and ermF), tetracycline resistance genes (tetA, tetB, tetC, tetD, and tetM), carbapenem resistance genes (blaKPC and blaNDM-1), and multidrug resistance genes (norA, cfrB, and cfrC) (
      • Liu C.
      • Zhang Z.Y.
      • Dong K.
      • Yuan J.P.
      • Guo X.K.
      Antibiotic resistance of probiotic strains of lactic acid bacteria isolated from marketed foods and drugs.
      ,
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      • Fang F.G.
      • Pan L.
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      Resistance genetic testing and molecular typing of multidrug-resistant Staphylococcus aureus in poultry.
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      • Qu Z.N.
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      • Gai W.Y.
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      • Wang J.W.
      Isolation of Staphyloccus aureus from raw milk and its drug resistance analysis.
      ,
      • Wang L.H.
      • Lv D.H.
      • Zhang H.
      • Yang B.
      • Wei W.K.
      • Jiang H.X.
      Distribution of plasmid-mediated quinolone resistance genes in animal-derived Staphylococcus aureus..
      ;
      • He L.Y.
      Pollution characteristics and diffusion mechanism of antibiotic resistance genes in typical livestock and poultry breeding environment.
      ;
      • Yang S.S.
      • Wang J.
      • Fan K.W.
      • Yang X.Y.
      Antimicrobial resistance analysis and resistance genes detection in Staphylococcus isolated from swine in Fujian Province, China.
      ;
      • Zhang F.
      • Zhou X.J.
      • Song Q.F.
      • He S.K.
      • Xu J.Y.
      • Shi X.Q.
      • Zhou M.
      • Shi X.M.
      Drug resistance monitoring and virulence genes screening of Salmonella in Ningbo.
      ,
      • Zhang L.Y.
      • Lou H.H.
      • Hu Y.Q.
      • Shen L.Z.
      • Li Y.
      • Zhou H.J.
      Antibiotic resistance and molecular typing analysis of Klebsiella pneumoniae with carbapenem resistance.
      ;
      • Chen C.
      • Wan J.
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      • Chang J.
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      Analysis of enterotoxin genes, resistance genes and molecular typing of MRSA isolated from dairy cow.
      ;
      • Meng S.
      • Wang Y.L.
      • Liu C.G.
      • Yang J.
      • Yuan M.
      • Bai X.N.
      • Jin D.
      • Liang J.R.
      • Cui Z.G.
      • Li J.
      Genetic diversity, antimicrobial resistance, and virulence genes of Aeromonas isolates from clinical patients, tap water systems, and food.
      ;
      • Shen L.H.
      • Jin H.
      • Kong Q.X.
      • Wei L.Y.
      • Wang Y.H.
      • Chen B.B.
      • Wang H.M.
      • Cao Y.
      Investigation of common pathogenic bacteria on the hands of medical staff and detection of related drug resistance genes.
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      • Zhou Q.
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      • Zhang J.
      • Tang X.J.
      • Lu J.X.
      • Tang M.J.
      • Gao Y.S.
      Drug resistant and multilocus sequence typing of Campylobacter coli from pig source in Jiangsu province.
      ). Primer sequences and PCR reaction conditions are displayed in Supplemental Table S2 ( https://doi.org/10.7910/DVN/WHS3EA ;
      • Tang M.
      • Wu Z.
      • Li W.
      • Shoaib M.
      • Aqib A.I.
      • Shang R.
      • Yang Z.
      • Pu W.
      Effects of different composting methods on antibiotic-resistant bacteria, antibiotic resistance genes, and microbial diversity in dairy cattle manures. Harvard Dataverse, V1.
      ).

      Isolation and Identification of Cultivable ARB

      To a 500-mL conical flask containing 450 mL of 0.9% saline, a 50-g solid compost sample was added and mixed thoroughly. From the suspensions, 500 μL was plated onto an antibiotic-containing Luria-Bertani (LB) plate and triplicate plating was used for each sample suspension. The plates were aerobically incubated at 37°C for 18 to 24 h. The antibiotics used in the LB plates included kanamycin, oxacillin, sulfamethoxazole-trimethoprim (SXT), erythromycin (ERY), tetracycline (TET), vancomycin (VAN), florfenicol, and ciprofloxacin, and their concentrations were 64, 4, 8, 8, 16, 32, 8, and 4 μg/mL, respectively, based on the breakpoints defined by the
      • Clinical and Laboratory Standards Institute
      Performance Standards for Antimicrobial Susceptibility Testing M100.
      . According to visual observation, colonies with different colors, morphology, and opacity were isolated from the antibiotic-containing LB plates and subcultured onto LB plates until the colony morphology on the same plate was consistent. The isolated strains were identified by 16S rDNA sequencing using the universal bacterial primers 27F (5′-GAGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-CTACGGCTACCTTGTTACGA-3′) (
      • Popović T.
      • Ivanović Ž.
      • Živković S.
      • Trkulja N.
      • Ignjatov M.
      First report of Brenneria nigrifluens as the causal agent of shallow-bark canker on walnut trees (Juglans regia) in Serbia.
      ). Briefly, the total bacteria DNA of each isolate was extracted with the TIANamp Bacteria DNA Kit (Tiangen) following the manufacturer's instructions. Bacterial 16S rDNA was amplified by PCR and sequenced in Shanghai Personalbio Technology. The results of the sequence alignment were analyzed by the Basic Local Alignment Search Tool of the National Center for Biotechnology Information database.

      High-Throughput Sequencing Analysis

      The V3–V4 regions of bacterial 16S rDNA genes were amplified by primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) with the barcode (
      • Yang C.
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      Linkage between water soluble organic matter and bacterial community in sediment from a shallow, eutrophic lake, Lake Chaohu, China.
      ). The TruSeq DNA PCR-Free Sample Preparation Kit (Illumina), Qubit@ 2.0 Fluorometer (Thermo Scientific), Agilent Bioanalyzer 2100 system, and Illumina NovaSeq platform were used to build sequencing libraries, evaluate the library quality, and sequence the library. Sequence analysis was performed using Uparse software (Uparse v7.0.1001, http://drive5.com/uparse/ ), and MUSCLE software (version 3.8.31, http://www.drive5.com/muscle/ ) was used for multiple sequence alignment to determine the phylogenetic relationship of different operational taxonomic units and distinguish the dominant species. The α-diversity indices were calculated by the QIIME program (version 1.7.0) and visualized by R software (version 2.15.3). The β-diversity of weighted and unweighted UniFrac was calculated by QIIME software. Unweighted pair-group method with arithmetic means clustering was performed as a hierarchical clustering method to interpret the distance matrix using average linkage and was conducted by QIIME software. The linear discriminant analysis effect size (LEfSe, http://huttenhower.sph.harvard.edu/galaxy/ ) approach was used to determine the taxa that were enriched in a particular environment. The top 50 genera with the highest abundance were clustered, and their heatmaps were drawn by the vegan R package. Nonmetric multidimensional scaling was applied to analyze community variation with UniFrac distances. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt, http://huttenhower.sph.harvard.edu/galaxy/tool_runner?tool_id=PICRUSt_normalize ) was used for predicting the metabolic function of the bacterial community.

      RESULTS AND DISCUSSION

      Distribution of Cultivable ARB and Related ARG in the 3 Composts

      The PCR data showed that SC reduced the chloramphenicol resistance gene fexA and fluoroquinolone resistance gene gyrB (Figure 1A). The OFP method reduced the chloramphenicol resistance gene fexA, TET resistance gene tetB, macrolide resistance gene ermC, and multidrug resistance gene cfrB. The gyrB, aac(6')-Ib-cr, cfrC, and ermA genes existed in the OFP samples at the beginning, disappeared during processing, but rebounded at the later stage. These changes could be explained by differential death and survival of ARB and horizontal gene transfer of ARG between bacteria (
      • Miller J.H.
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      • Gu J.
      • Wang X.J.
      • Duan M.L.
      Mechanism and effect of temperature on variations in antibiotic resistance genes during anaerobic digestion of dairy manure.
      ) (Figure 1A). Anaerobic fermentation produced an obvious reduction in TET resistance gene tetB and fluoroquinolone resistance genes (gyrB and grlB). Interestingly, ermA, tetC, and cfrB first disappeared and then reappeared (Figure 1A). Previous studies showed that AF produced certain effects on ARB and ARG in livestock manures, but the specific effects were not consistent.
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      • Sun W.
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      Mechanism and effect of temperature on variations in antibiotic resistance genes during anaerobic digestion of dairy manure.
      found that thermophilic AF could effectively reduce the relative abundance of ARG in cow dung, but
      • Huang X.
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      • Topp E.
      • McAllister T.A.
      • Yu Z.
      Selected antimicrobial resistance during composting of manure from cattle administered sub-therapeutic antimicrobials.
      showed that most TET and ERY ARG were stable over a 126-d compost, whereas some ARG increased. In contrast,
      • Walczak J.J.
      • Xu S.
      Manure as a source of antibiotic-resistant Escherichia coli and Enterococci: A case study of a Wisconsin, USA family dairy farm.
      found a significant reduction of ARG during cattle manure composting. Other recent studies have noted significant reductions in the diversity and relative abundance of ARG and mobile genetic elements in actively managed and aerated cattle manure composts (
      • Gou M.
      • Hu H.W.
      • Zhang Y.J.
      • Wang J.T.
      • Hayden H.
      • Tang Y.Q.
      • He J.Z.
      Aerobic composting reduces antibiotic resistance genes in cattle manure and the resistome dissemination in agricultural soils.
      ).
      Figure thumbnail gr1
      Figure 1Abundances of antibiotic resistance genes (ARG) and antibiotic-resistant bacteria (ARB) in the 3 composts. (A) Detection of ARG by PCR. Red represents those genes that are detected, and gray represents those genes that are absent. (B) The number of bacterial species isolated with different antibiotics-containing plates. Color shading represents different numbers. (C)–(E) Relative abundances of ARB in the anaerobic fermentation (AF), static compost (SC), and organic fertilizer production (OFP), respectively. Color shading represents different abundances. Z1, Z2, Z3, Z4, and ZS are the sampled sites for AF; S9, S10, S11, S12, and SS are the sampled sites for SC; and S13, S14, S15, S16, and S17 are the sampled sites for OFP. Three samples were collected from each site and were pooled for ARG and ARB analyses.
      The plating results showed that all samples yielded colonies on the VAN-containing plates, and species isolated from them were gram-negative bacteria (Figure 1B). It is probably because VAN is not effective against gram-negative bacteria (
      • Wishart D.S.
      • Knox C.
      • Guo A.C.
      • Cheng D.
      • Shrivastava S.
      • Tzur D.
      • Gautam B.
      • Hassanali M.
      DrugBank: A knowledgebase for drugs, drug actions and drug targets.
      ). The isolation rates of ARB on kanamycin, ERY, SXT, and oxacillin plates were also relatively high. This is consistent with the result of ARG detection, as the detection rates of aac(6')/aph(2″) that mediate kanamycin resistance and ermABCF that mediate ERY resistance were high (Figure 1A). However, genes mecA, mecC, femA, blaKPC, and blaNDM-1 mediating β-lactam resistance and genes mediating SXT resistance were not detected by PCR. This could be explained by the possibility that the oxacillin and SXT resistance phenotypes were due to resistance mechanisms that were not targeted by the ARG-detecting PCR primers used in this study. The isolation rates of ARB on ciprofloxacin-, florfenicol-, and TET-containing plates were relatively low, suggesting that resistance to these antibiotics was low in the analyzed samples (Figure 1A, B).
      Single colonies identified from the 3 types of composts were classified into 64 different bacterial species, belonging to 27 genera. The majority of the isolated strains belonged to the genera Bacillus, Enterococcus, and Pseudomonas (Figure 1C, D, and E). As shown in Figure 1C, Alcaligenes faecalis was the most abundant, followed by Enterococcus aquimarinus and Alcaligenes aquatilis, during the AF process. Escherichia fergusonii was the most abundant, followed by A. faecalis, during the SC process (Figure 1D). A. faecalis was the most abundant, followed by E. fergusonii and A. aquatilis, during the OFP process (Figure 1E).
      A. faecalis existed in almost all samples analyzed in this study, which is in line with the findings reported by
      • Zhong X.Z.
      • Zeng Y.
      • Wang S.P.
      • Sun Z.Y.
      • Tang Y.Q.
      • Kida K.
      Insight into the microbiology of nitrogen cycle in the dairy manure composting process revealed by combining high-throughput sequencing and quantitative PCR.
      . Interestingly, the OFP composting significantly decreased its isolation rate (Figure 1C–E). This bacterium widely exists in natural environments (
      • Majewski P.
      • Majewska P.
      • Gutowska A.
      • Piszcz J.
      • Sacha P.
      • Wieczorek P.
      • Żebrowska A.
      • Radziwon P.
      • Tryniszewska E.
      Molecular characterisation of clinical pandrug-resistant Alcaligenes faecalis strain MUB14.
      ) and is considered an opportunistic pathogen highly resistant to antibiotics and a plant probiotic bacterial endophyte (
      • Tena D.
      • Fernández C.
      • Lago M.R.
      Alcaligenes faecalis: An unusual cause of skin and soft tissue infection.
      ;
      • Hasan M.J.
      • Nizhu L.N.
      • Rabbani R.
      Bloodstream infection with pandrug-resistant Alcaligenes faecalis treated with double-dose of tigecycline.
      ;
      • Puah S.M.
      • Puthucheary S.D.
      • Chua K.H.
      First report of extended-spectrum β-lactamases TEM-116 and OXA-10 in clinical isolates of Alcaligenes species from Kuala Lumpur, Malaysia.
      ;
      • Ngbede E.O.
      • Poudel A.
      • Kalalah A.
      • Yang Y.
      • Adekanmbi F.
      • Adikwu A.A.
      • Adamu A.M.
      • Mamfe L.M.
      • Daniel S.T.
      • Useh N.M.
      • Kwaga J.K.P.
      • Adah M.I.
      • Kelly P.
      • Butaye P.
      • Wang C.
      Identification of mobile colistin resistance genes (mcr-1.1, mcr-5 and mcr-8.1) in Enterobacteriaceae and Alcaligenes faecalis of human and animal origin, Nigeria.
      ). Recent studies have shown that A. faecalis can degrade a variety of toxic and harmful substances such as phenol (Jing et al., 2007), cyanide (
      • Pathak U.
      • Mogalapalli P.
      • Mandal D.D.
      • Mandal T.
      Biodegradation efficacy of coke oven wastewater inherent co-cultured novel sp. Alcaligenes faecalis JF339228 and Klebsiella oxytoca KF303807 on phenol and cyanide—kinetic and toxicity analysis.
      ), arsenite (
      • Boulanger M.J.
      • Murphy M.E.
      Directing the mode of nitrite binding to a copper-containing nitrite reductase from Alcaligenes faecalis S-6: Characterization of an active site isoleucine.
      ), and hydrogen sulfide. Numerous studies reported E. fergusonii as a newly identified human and animal pathogen that shows extensive resistance to antibiotics (
      • Lagacé-Wiens P.R.
      • Baudry P.J.
      • Pang P.
      • Hammond G.
      First description of an extended-spectrum-beta-lactamase-producing multidrug-resistant Escherichia fergusonii strain in a patient with cystitis.
      ;
      • Forgetta V.
      • Rempel H.
      • Malouin F.
      • Vaillancourt Jr., R.
      • Topp E.
      • Dewar K.
      • Diarra M.S.
      Pathogenic and multidrug-resistant Escherichia fergusonii from broiler chicken.
      ;
      • Glover B.
      • Wentzel J.
      • Jenkins A.
      • Van Vuuren M.
      The first report of Escherichia fergusonii isolated from non-human primates, in Africa.
      ;
      • Adesina T.
      • Nwinyi O.
      • De N.
      • Akinnola O.
      • Omonigbehin E.
      First detection of carbapenem-resistant Escherichia fergusonii strains harbouring beta-lactamase genes from clinical samples.
      ) and is commonly isolated from livestock manures (
      • Herráez P.
      • Rodríguez A.F.
      • Espinosa de los Monteros A.
      • Acosta A.B.
      • Jaber J.R.
      • Castellano J.
      • Castro A.
      Fibrino-necrotic typhlitis caused by Escherichia fergusonii in ostriches (Struthio camelus).
      ;
      • Hariharan H.
      • López A.
      • Conboy G.
      • Coles M.
      • Muirhead T.
      Isolation of Escherichia fergusonii from the feces and internal organs of a goat with diarrhea.
      ;
      • Oh J.Y.
      • Kang M.S.
      • An B.K.
      • Shin E.G.
      • Kim M.J.
      • Kwon J.H.
      • Kwon Y.K.
      Isolation and epidemiological characterization of heat-labile enterotoxin-producing Escherichia fergusonii from healthy chickens.
      ), beef, and cheese (
      • Fegan N.
      • Barlow R.S.
      • Gobius K.S.
      Escherichia coli O157 somatic antigen is present in an isolate of E. fergusonii..
      ). This species existed in the raw manure samples (Z1, S9, S13) and disappeared in the later stages of AF and OFP (Figure 1C–E), suggesting that the composting treatments can effectively reduce opportunistic pathogen E. fergusonii. This finding is notable, as little is known about the effect of composting on E. fergusonii. E. aquimarinus existed in almost all samples of AF in the present study (Figure 1C–E). However, there are few studies on E. aquimarinus, which was first isolated from seawater (
      • Švec P.
      • Vancanneyt M.
      • Devriese L.A.
      • Naser S.M.
      • Snauwaert C.
      • Lefebvre K.
      • Hoste B.
      • Swings J.
      Enterococcus aquimarinus sp. nov., isolated from sea water.
      ). To our knowledge, this is the first isolation of this bacterium from AF. Many other opportunistic pathogens were also detected in this study, including Acinetobacter indicus, Enterococcus gallinarum, Streptococcus uberis, Aerococcus viridans, Citrobacter murliniae, Enterococcus faecium, Enterococcus faecalis, Staphylococcus lentus, Vagococcus fluvialis, Vagococcus lutrae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter lwoffii, Raoultella terrigena, and Microbacterium paraoxydans. Among these opportunistic pathogens, Enterococcus faecium decreased in the late stage of AF, and V. fluvialis decreased in the late stage of SC and OFP. There was no significant difference in other opportunistic pathogens among the 3 composting treatments (Figure 1C–E).
      Most of the isolated species were nonpathogenic, and some even contributed to plant growth by providing protection against the hypersaline environment or enhancing the usability of nutrients. Thermophilic bacterium A. aquatilis is rare but was with a high isolation rate in the 3 composting methods in this study (Figure 1C–E). A. aquatilis has a wide range of beneficial effects (
      • Haouas A.
      • El Modafar C.
      • Douira A.
      • Ibnsouda-Koraichi S.
      • Filali-Maltouf A.
      • Moukhli A.
      • Amir S.
      Alcaligenes aquatilis GTE53: Phosphate solubilising and bioremediation bacterium isolated from new biotope “phosphate sludge enriched-compost”.
      ). It can use aromatic hydrocarbons as sources of carbon and nitrogen (
      • Durán R.E.
      • Barra-Sanhueza B.
      • Salvà-Serra F.
      • Méndez V.
      • Jaén-Luchoro D.
      • Moore E.R.B.
      • Seeger M.
      Complete genome sequence of the marine hydrocarbon degrader Alcaligenes aquatilis QD168, isolated from crude oil-polluted sediment of Quintero Bay, Central Chile.
      ) and dissolve inorganic phosphates, which contributes to the growth of maize plants (
      • Pande A.
      • Pandey P.
      • Kaushik S.
      Co-inoculation of Burkholderia cepacia and Alcaligenes aquatilis enhances plant growth of maize (Zea mays) under green house and field condition.
      ). Other beneficial bacteria were also detected in this study, such as Staphylococcus equorum, Achromobacter piechaudii, Enterobacter ludwigii, and Enterobacter tabaci, which can enhance the tolerance of plants to salt (
      • Mayak S.
      • Tirosh T.
      • Glick B.R.
      Plant growth-promoting bacteria confer resistance in tomato plants to salt stress.
      ;
      • Lee J.H.
      • Heo S.
      • Jeong D.W.
      Genomic insights into Staphylococcus equorum KS1039 as a potential starter culture for the fermentation of high-salt foods.
      ;
      • Khan M.A.
      • Asaf S.
      • Khan A.L.
      • Adhikari A.
      • Jan R.
      • Ali S.
      • Imran M.
      • Kim K.M.
      • Lee I.J.
      Plant growth-promoting endophytic bacteria augment growth and salinity tolerance in rice plants.
      ). These species were only detected in SC and OFP (Figure 1D, E), suggesting that these 2 treatments were conducive to the growth or survival of high-salt-tolerant species. Some bacteria that can remove heavy metals or provide plants with the ability to resist heavy metals were analyzed in the present study, including Pseudomonas tuomuerensis (
      • Zhang W.
      • Zhang M.
      • An S.
      • Xiong B.
      • Li H.
      • Cui C.
      • Lin K.
      Ecotoxicological effects of decabromodiphenyl ether and cadmium contamination on soil microbes and enzymes.
      ), Providencia vermicola (
      • Tan L.
      • Wu H.
      • Cui H.
      • Xu H.
      • Xu M.
      • Xiao Y.
      • Qiu G.
      • Liu X.
      • Dong H.
      • Xie J.
      Selective adsorption of palladium and platinum from secondary wastewater using Escherichia coli BL21 and Providencia vermicola..
      ), Citrobacter murliniae (
      • Boechat C.L.
      • Giovanella P.
      • Amorim M.B.
      • de Sá E.L.
      • de Oliveira Camargo F.A.
      Metal-resistant rhizobacteria isolates improve Mucuna deeringiana phytoextraction capacity in multi-metal contaminated soils from a gold mining area.
      ), Pseudomonas indoloxydans (
      • Shahid M.J.
      • Ali S.
      • Shabir G.
      • Siddique M.
      • Rizwan M.
      • Seleiman M.F.
      • Afzal M.
      Comparing the performance of four macrophytes in bacterial assisted floating treatment wetlands for the removal of trace metals (Fe, Mn, Ni, Pb, and Cr) from polluted river water.
      ), Enterobacter ludwigii (
      • Wang Q.
      • Li Q.
      • Lin Y.
      • Hou Y.
      • Deng Z.
      • Liu W.
      • Wang H.
      • Xia Z.
      Biochemical and genetic basis of cadmium biosorption by Enterobacter ludwigii LY6, isolated from industrial contaminated soil.
      ), Bacillus wiedmannii (
      • Chen Y.
      • Chen Y.
      • Wu J.
      • Zhang J.
      The effect of biotic and abiotic environmental factors on Pd(II) adsorption and reduction by Bacillus wiedmannii MSM.
      ), and Proteus hauseri (
      • Khalilian M.
      • Zolfaghari M.R.
      • Soleimani M.
      High potential application in bioremediation of selenate by Proteus hauseri strain QW4.
      ). These species were most commonly distributed in SC, followed by OFP and AF (Figure 1C–E). Some bacteria detected in this study possess biodegradation functions. For example, Pusillimonas noertemannii can degrade substituted salicylates (
      • Stolz A.
      • Bürger S.
      • Kuhm A.
      • Kämpfer P.
      • Busse H.J.
      Pusillimonas noertemannii gen. nov., sp. nov., a new member of the family Alcaligenaceae that degrades substituted salicylates.
      ), Enterococcus mundtii can convert hexose and pentose sugars (
      • Collins M.D.
      • Farrow J.A.E.
      • Jones D.
      Enterococcus mundtii sp. nov.
      ), and Enterobacter ludwigii can biodegrade and detoxify chlorimuron-ethyl (
      • Pan X.
      • Wang S.
      • Shi N.
      • Fang H.
      • Yu Y.
      Biodegradation and detoxification of chlorimuron-ethyl by Enterobacter ludwigii sp. CE-1.
      ) (Figure 1C–E).
      Based on the results from this study, there were considerable variations and changes in ARG associated with the 3 composting methods. According to the bacterial isolation result, AF and OFP significantly reduced opportunistic pathogen E. fergusonii. Additionally, AF significantly reduced opportunistic pathogen Enterococcus faecium, and SC and OFP significantly reduced opportunistic pathogen V. fluvialis. However, changes in ARB and ARG across the 3 composting methods were not uniform, and it is difficult to state which method is superior to the others. Variables other than compost management also affect compost resistomes and their fate. These include the concentration of residual antibiotics, the co-occurrence of ARG and mobile genetic elements, and growth-modulating factors such as nutrient and heavy metal contents, which may influence microbial population shifts and gene regulation and transfer (
      • Qian X.
      • Sun W.
      • Gu J.
      • Wang X.J.
      • Sun J.J.
      • Yin Y.N.
      • Duan M.L.
      Variable effects of oxytetracycline on antibiotic resistance gene abundance and the bacterial community during aerobic composting of cow manure.
      ;
      • Qian X.
      • Gu J.
      • Sun W.
      • Wang X.J.
      • Su J.Q.
      • Stedfeld R.
      Diversity, abundance, and persistence of antibiotic resistance genes in various types of animal manure following industrial composting.
      ). Additionally, the inability to correlate ARB and ARG could be related to the timing of sampling (
      • Singer R.S.
      • Patterson S.K.
      • Wallace R.L.
      Effects of therapeutic ceftiofur administration to dairy cattle on Escherichia coli dynamics in the intestinal tract.
      ;
      • Chambers L.
      • Yang Y.
      • Littier H.
      • Ray P.
      • Zhang T.
      • Pruden A.
      • Strickland M.
      • Knowlton K.
      Metagenomic analysis of antibiotic resistance genes in dairy cow feces following therapeutic administration of third generation cephalosporin.
      ), substrain level differences (
      • McConnel C.S.
      • Stenkamp-Strahm C.M.
      • Rao S.
      • Linke L.M.
      • Magnuson R.J.
      • Hyatt D.R.
      Antimicrobial resistance profiles in Escherichia coli O157 isolates from northern Colorado dairies.
      ), and horizontal gene transfer complexities (
      • Dolejska M.
      • Jurcickova Z.
      • Literak I.
      • Pokludova L.
      • Bures J.
      • Hera A.
      • Kohoutova L.
      • Smola J.
      • Cizek A.
      IncN plasmids carrying blaCTX-M-1 in Escherichia coli isolates on a dairy farm.
      ;
      • Gonggrijp M.A.
      • Santman-Berends I.
      • Heuvelink A.E.
      • Buter G.J.
      • van Schaik G.
      • Hage J.J.
      • Lam T.
      Prevalence and risk factors for extended-spectrum β-lactamase- and AmpC-producing Escherichia coli in dairy farms.
      ). However, because the detection of ARG in this experiment was carried out by a limited number of PCR detections, it was not sufficient to detect all changes in ARG during the composting processes. Another limitation of the present study was that ARB were only obtained by aerobic culture, not other culture conditions. Therefore, it is likely that the number of different ARB identified in the composts was underestimated and incomplete.

      Sequencing Analysis

      High-throughput sequencing of bacterial 16S rDNA (V3-V4 region) was performed to determine the differences and changes in microbial community diversity (MCD) of the 3 different manure composts. A total of 1,069,318 high-quality sequences were obtained and were classified as 5,790 operational taxonomic units, which were classified into 985 phyla, 983 classes, 958 orders, 800 families, 383 genera, and 40 species on average. The average high-quality sequences generated per sample were 71,288 for the bacterial community. The sequence information of each sample was shown in Supplemental Table S3 ( https://doi.org/10.7910/DVN/WHS3EA ;
      • Tang M.
      • Wu Z.
      • Li W.
      • Shoaib M.
      • Aqib A.I.
      • Shang R.
      • Yang Z.
      • Pu W.
      Effects of different composting methods on antibiotic-resistant bacteria, antibiotic resistance genes, and microbial diversity in dairy cattle manures. Harvard Dataverse, V1.
      ).

      MCD

      The rarefaction curve based on the Chao1 index and observed species shows that all the curves tend to be flat in the current sequencing amount, and the sequencing depth is sufficient to capture the MCD in the samples (Figure 2A, B). The α diversity index for each manure compost stage was compared. Chao1 and Shannon diversity indices are important to measure distribution richness and evenness of species in compost ecology (
      • Huang K.
      • Li F.
      • Wei Y.
      • Chen X.
      • Fu X.
      Changes of bacterial and fungal community compositions during vermicomposting of vegetable wastes by Eisenia foetida.
      ;
      • Du J.
      • Zhang Y.
      • Qu M.
      • Yin Y.
      • Fan K.
      • Hu B.
      • Zhang H.
      • Wei M.
      • Ma C.
      Effects of biochar on the microbial activity and community structure during sewage sludge composting.
      ).
      Figure thumbnail gr2
      Figure 2Rarefaction curves and α diversity index in the 3 composts. (A) Chao1 richness. (B) Observed species. (C) Chao1 index. (D) Shannon index. Z1, Z2, Z3, Z4, and ZS are the sampled sites for anaerobic fermentation; S9, S10, S11, S12, and SS are the sampled sites for static compost; and S13, S14, S15, S16, and S17 are the sampled sites for organic fertilizer production. Three samples were collected from each site, and each data point is shown as the mean ± SD (n = 3).
      As Figure 2C–D shows, the Chao1 and Shannon indices decreased first and then increased continuously during the 3 compost treatments. The lowest points of AF, SC, and OFP were Z2, S11, and S15, respectively. This indicated that the microbial community richness and evenness of the solid after solid-liquid separation (SLS), the solid after 10 d of compost, and the solid after fermentation were the lowest during the 3 composting processes. The α-diversity results showed that there was no significant difference in richness and evenness of microbial communities at different stages of AF (P > 0.05), indicating that microbial dynamics and abundance are no different throughout the AF process, which is consistent with the findings by
      • Resende J.A.
      • Godon J.J.
      • Bonnafous A.
      • Arcuri P.B.
      • Silva V.L.
      • Otenio M.H.
      • Diniz C.G.
      Seasonal variation on microbial community and methane production during anaerobic digestion of cattle manure in Brazil.
      . On the contrary, the richness and evenness of microbial communities after fermentation during SC and OFP were significantly reduced (P < 0.05). Both processes were in the early stages of composting, when the temperature was relatively high. The variation trend was consistent with the notion that the quantity and diversity of community would decrease significantly with the increase of temperature and would increase with the depletion of organic matter (
      • Tian W.
      • Sun Q.
      • Xu D.
      • Zhang Z.
      • Chen D.
      • Li C.
      • Shen Q.
      • Shen B.
      Succession of bacterial communities during composting process as detected by 16S rRNA clone libraries analysis.
      ). We also noticed that the richness and diversity of the microflora increased after a long period of storage (Figure 2C, D). This might be due to the presence of degraded organic substances that promoted the rapid evolution of microbes and the decrease of temperature in the later compost stage (Gannes et al., 2013;
      • Meng Q.
      • Xu X.
      • Zhang W.
      • Men M.
      • Xu B.
      • Deng L.
      • Bello A.
      • Jiang X.
      • Sheng S.
      • Wu X.
      Bacterial community succession in dairy manure composting with a static composting technique.
      ;
      • Sun Y.
      • Men M.
      • Xu B.
      • Meng Q.
      • Bello A.
      • Xu X.
      • Huang X.
      Assessing key microbial communities determining nitrogen transformation in composting of cow manure using Illumina high-throughput sequencing.
      ).
      Nonmetric multidimensional scaling was conducted based on weighted UniFrac to elucidate the differences in microbial composition among different manure composts. Samples after AF (Z2, Z3, and Z4) were still close to the raw manure sample (Z1), indicating that AF had little effect on the MCD (Figure 3), which is similar to observations made by
      • Wei S.
      • Guo Y.
      Comparative study of reactor performance and microbial community in psychrophilic and mesophilic biogas digesters under solid state condition.
      . On the other hand, our data showed that the distance matrix of the samples after SC (S11 and S12) was far from the samples before fermentation (S9 and S10), and the distance matrix of the samples in OFP showed the same trend (Figure 3). These results demonstrated that the SC and OFP composting had a greater influence on MCD. Given the ecological and physiological diversity in microbial communities (
      • Delgado-Baquerizo M.
      • Eldridge D.J.
      • Ochoa V.
      • Gozalo B.
      • Singh B.K.
      • Maestre F.T.
      Soil microbial communities drive the resistance of ecosystem multifunctionality to global change in drylands across the globe.
      ;
      • Mooshammer M.
      • Hofhansl F.
      • Frank A.H.
      • Wanek W.
      • Hämmerle I.
      • Leitner S.
      • Schnecker J.
      • Wild B.
      • Watzka M.
      • Keiblinger K.M.
      • Zechmeister-Boltenstern S.
      • Richter A.
      Decoupling of microbial carbon, nitrogen, and phosphorus cycling in response to extreme temperature events.
      ), various species have different ecological and physiological characteristics and respond to compost in different ways. Therefore, distinct microbial taxa predominated different compost piles, and each member adapted to specific environmental conditions that varied at different stages (
      • Zhao X.
      • Wei Y.
      • Fan Y.
      • Zhang F.
      • Tan W.
      • He X.
      • Xi B.
      Roles of bacterial community in the transformation of dissolved organic matter for the stability and safety of material during sludge composting.
      ;
      • Qiao C.
      • Ryan Penton C.
      • Liu C.
      • Shen Z.
      • Ou Y.
      • Liu Z.
      • Xu X.
      • Li R.
      • Shen Q.
      Key extracellular enzymes triggered high-efficiency composting associated with bacterial community succession.
      ;
      • Wu N.
      • Xie S.
      • Zeng M.
      • Xu X.
      • Li Y.
      • Liu X.
      • Wang X.
      Impacts of pile temperature on antibiotic resistance, metal resistance and microbial community during swine manure composting.
      ). In addition, the feed may affect microbial diversity in manure. Background information showed that although the total nutritional values of feed formula in the 2 locations were roughly the same, the proportions of various feed ingredients were different. The proportions of cottonseed meal and silage alfalfa were higher in the feed used for Xinjiang dairy farms, whereas the proportions of soybean meal and whole-crop corn silage were higher in the feed used for Qinghai dairy farms, which may also account for some of the differences in microbial compositions after fermentation.
      Figure thumbnail gr3
      Figure 3Nonmetric multidimensional scaling (NMDS) ordination of microbial communities in the 3 composts (n = 3). Each composting method is represented by a different shape, and the different colors represent the different sampled sites in each composting process. The square represents anaerobic fermentation, the circle represents static compost, and the triangle represents organic fertilizer production. Z1, Z2, Z3, Z4, and ZS are the sampled sites for anaerobic fermentation; S9, S10, S11, S12, and SS are the sampled sites for static compost; and S13, S14, S15, S16, and S17 are the sampled sites for organic fertilizer production.
      To determine the functional communities in samples, LEfSe was applied to identify the distinct groups across the different compost treatments, which are shown in a cladogram (Figure 4). The bacterial taxa varied during the 3 compost processes. There were 15 significantly abundant bacterial taxa, including Armatimonadetes, Actinobacteria, Acidobacteria, TM7, and Alphaproteobacteria, in the SC process. Phylum TM7 had only 16S rDNA sequence characteristics without known pure culture representatives but is widely distributed in the environment (
      • Hugenholtz P.
      • Goebel B.M.
      • Pace N.R.
      Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity.
      ,
      • Hugenholtz P.
      • Tyson G.W.
      • Webb R.I.
      • Wagner A.M.
      • Blackall L.L.
      Investigation of candidate division TM7, a recently recognized major lineage of the domain Bacteria with no known pure-culture representatives.
      ). Studies have shown that TM7 is associated with human inflammatory mucosal diseases (
      • Marcy Y.
      • Ouverney C.
      • Bik E.M.
      • Lösekann T.
      • Ivanova N.
      • Martin H.G.
      • Szeto E.
      • Platt D.
      • Hugenholtz P.
      • Relman D.A.
      • Quake S.R.
      Dissecting biological “dark matter” with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth.
      ;
      • Kuehbacher T.
      • Rehman A.
      • Lepage P.
      • Hellmig S.
      • Fölsch U.R.
      • Schreiber S.
      • Ott S.J.
      Intestinal TM7 bacterial phylogenies in active inflammatory bowel disease.
      ) and oral disease (
      • Brinig M.M.
      • Lepp P.W.
      • Ouverney C.C.
      • Armitage G.C.
      • Relman D.A.
      Prevalence of bacteria of division TM7 in human subgingival plaque and their association with disease.
      ). The presence of TM7 in the SC suggests that it poses a potential health risk when used as farm fertilizer. There were 3 bacterial taxa including CFB_26 (order) that were significantly abundant in the OFP process. Meanwhile, only one taxon, Deltaproteobacteria, was enriched in AF. We also carried out LEfSe analysis (linear discriminant analysis score greater than 2) among each stage of AF, SC, and OFP. No biomarkers were identified between stages of the different compost processes (Figure 4).
      Figure thumbnail gr4
      Figure 4Linear discriminant analysis effect size analysis of the 3 composting methods (linear discriminant analysis score greater than 4.0). Cladogram of the linear discriminant analysis scores computed for differentially abundant predicted functions among the 3 composting methods. Linear discriminant analysis scores of 2.0 or higher are considered significant in Kruskal–Wallis. AF = anaerobic fermentation; SC = static compost; OFP = organic fertilizer production.

      Taxonomic Analysis of Microbial Composition Profiles

      Microbial Community Composition at the Phylum Level

      Seventeen known phyla were identified in all samples: Firmicutes, Proteobacteria, Chloroflexi, Actinobacteria, Bacteroidetes, Gemmatimonadetes, Cyanobacteria, Synergistetes, Acidobacteria, [Thermi], Verrucomicrobia, Spirochaetes, Tenericutes, Planctomycetes, Nitrospirae, Chlorobi, and Fibrobacteres (Supplemental Figure S2, https://doi.org/10.7910/DVN/WHS3EA ;
      • Tang M.
      • Wu Z.
      • Li W.
      • Shoaib M.
      • Aqib A.I.
      • Shang R.
      • Yang Z.
      • Pu W.
      Effects of different composting methods on antibiotic-resistant bacteria, antibiotic resistance genes, and microbial diversity in dairy cattle manures. Harvard Dataverse, V1.
      ). The major phyla among the microbial communities in the 3 composting methods were Firmicutes, Proteobacteria, Actinobacteria, Chloroflexi, and Bacteroidetes. Numerous studies have shown that they are related to the degradation of lignocellulose (
      • de Gannes V.
      • Eudoxie G.
      • Hickey W.J.
      Prokaryotic successions and diversity in composts as revealed by 454-pyrosequencing.
      ;
      • Zhang L.
      • Ma H.
      • Zhang H.
      • Xun L.
      • Chen G.
      • Wang L.
      Thermomyces lanuginosus is the dominant fungus in maize straw composts.
      ;
      • Awasthi M.K.
      • Zhang Z.
      • Wang Q.
      • Shen F.
      • Li R.
      • Li D.S.
      • Ren X.
      • Wang M.
      • Chen H.
      • Zhao J.
      New insight with the effects of biochar amendment on bacterial diversity as indicators of biomarkers support the thermophilic phase during sewage sludge composting.
      ;
      • Meng Q.
      • Xu X.
      • Zhang W.
      • Men M.
      • Xu B.
      • Deng L.
      • Bello A.
      • Jiang X.
      • Sheng S.
      • Wu X.
      Bacterial community succession in dairy manure composting with a static composting technique.
      ). Firmicutes were the dominant microflora in anaerobically fermented solids, which are most common in biogas reactors and are responsible for organic degradation and fermentation (
      • McGarvey J.A.
      • Miller W.G.
      • Zhang R.
      • Ma Y.
      • Mitloehner F.
      Bacterial population dynamics in dairy waste during aerobic and anaerobic treatment and subsequent storage.
      ;
      • Liu F.H.
      • Wang S.B.
      • Zhang J.S.
      • Zhang J.
      • Yan X.
      • Zhou H.K.
      • Zhao G.P.
      • Zhou Z.H.
      The structure of the bacterial and archaeal community in a biogas digester as revealed by denaturing gradient gel electrophoresis and 16S rDNA sequencing analysis.
      ;
      • Kampmann K.
      • Ratering S.
      • Kramer I.
      • Schmidt M.
      • Zerr W.
      • Schnell S.
      Unexpected stability of Bacteroidetes and Firmicutes communities in laboratory biogas reactors fed with different defined substrates.
      ;
      • Bengelsdorf F.R.
      • Gerischer U.
      • Langer S.
      • Zak M.
      • Kazda M.
      Stability of a biogas-producing bacterial, archaeal and fungal community degrading food residues.
      ). Actinobacteria play a dominant role in the late compost stage, which can release inorganic nutrients related to humus formation and may inhibit pathogens by secreting antibiotics, as well as play an important role in the degradation of cellulose, hemicellulose, lignin, and chitin (
      • Steger K.
      • Jarvis A.
      • Vasara T.
      • Romantschuk M.
      • Sundh I.
      Effects of differing temperature management on development of Actinobacteria populations during composting.
      ;
      • Franke-Whittle I.H.
      • Knapp B.A.
      • Fuchs J.
      • Kaufmann R.
      • Insam H.
      Application of COMPOCHIP microarray to investigate the bacterial communities of different composts.
      ;
      • Neher D.A.
      • Weicht T.R.
      • Bates S.T.
      • Leff J.W.
      • Fierer N.
      Changes in bacterial and fungal communities across compost recipes, preparation methods, and composting times.
      ). Proteobacteria are environmental organisms that have no relationship with animal diseases (
      • Coenye T.
      • Vanlaere E.
      • Samyn E.
      • Falsen E.
      • Larsson P.
      • Vandamme P.
      Advenella incenata gen. nov., sp. nov., a novel member of the Alcaligenaceae, isolated from various clinical samples.
      ). Firmicutes and Actinobacteria were usually higher in compost piles, suggesting that compost fertilization might transmit Firmicutes and Actinobacteria into soils (
      • Sivasakthi S.
      • Usharani G.
      • Saranraj P.
      Biocontrol potentiality of plant growth promoting bacteria (PGPR)—Pseudomonas fluorescens and Bacillus subtilis: A review.
      ;
      • Chowdhury S.P.
      • Hartmann A.
      • Gao X.
      • Borriss R.
      Biocontrol mechanism by root-associated Bacillus amyloliquefaciens FZB42—A review.
      ;
      • Rybakova D.
      • Cernava T.
      • Köberl M.
      • Liebminger S.
      • Etemadi M.
      • Berg G.
      Endophytes-assisted biocontrol: Novel insights in ecology and the mode of action of Paenibacillus.
      ;
      • Shafi T.
      • Tian H.
      • Ji M.
      Bacillus species as versatile weapons for plant pathogens: a review.
      ;
      • Chaurasia A.
      • Meena B.R.
      • Tripathi A.N.
      • Pandey K.K.
      • Rai A.B.
      • Singh B.
      Actinomycetes: An unexplored microorganisms for plant growth promotion and biocontrol in vegetable crops.
      ).
      There were differences in dominant phyla at different stages among the 3 composting methods. As shown in Supplemental Figure S2, Firmicutes and Proteobacteria were always dominant in the AF process. Both of them were dominant in samples before fermentation and showed a downward trend at the early stage but rebounded at the later stage during SC and OFP. Chloroflexi were dominant in samples after fermentation during SC and OFP. Actinobacteria showed a downward trend in the late stage of SC but remained stable in OFP. Bacteroidetes were stable during the AF process and decreased during the SC and OFP processes but rebounded at the later stage of SC. A possible explanation is that the manure temperature was high at the early stage of compost, which could inhibit the growth of Proteobacteria, Firmicutes, and Bacteroidetes (
      • Chroni C.
      • Kyriacou A.
      • Manios T.
      • Lasaridi K.E.
      Investigation of the microbial community structure and activity as indicators of compost stability and composting process evolution.
      ). However, Chloroflexi was ubiquitous in the composting process (
      • Tian W.
      • Sun Q.
      • Xu D.
      • Zhang Z.
      • Chen D.
      • Li C.
      • Shen Q.
      • Shen B.
      Succession of bacterial communities during composting process as detected by 16S rRNA clone libraries analysis.
      ), and its relative proportion increased when the abundance of other bacteria decreased. Firmicutes and Proteobacteria increased at various levels at the later stage of SC and OFP, when the temperature of the compost decreased (Supplemental Figure S2). Another note is that the content of Gemmatimonadetes increased by approximately 20 times in the later stage of OFP. The sharp increase of Gemmatimonadetes was possibly due to the decrease of moisture in the compost, which is consistent with the report by
      • DeBruyn J.M.
      • Nixon L.T.
      • Fawaz M.N.
      • Johnson A.M.
      • Radosevich M.
      Global biogeography and quantitative seasonal dynamics of Gemmatimonadetes in soil.
      .

      Microbial Community Composition at the Genus Level

      The abundance of genera in each sample is shown in Figure 5. The relatively abundant genera of the samples before fermentation (Z1, S9, S13, and S14) included Facklamia, Unclassified_Aerococcaceae, Unclassified_Lachnospiraceae, Erysipelothrix, Unclassified_Ruminococcaceae, Unclassified_Erysipelotrichaceae, Unclassified_Porphyromonadaceae, Psychrobacter, and Unclassified_[Tissierellaceae]. In Z2 and S10, the relatively abundant genera included Unclassified_Pseudomonadaceae, Unclassified_Moraxellaceae, Corynebacterium, Acinetobacter, and Arthrobacter. The abundance of Unclassified_Peptostreptococcaceae, Clostridium, Unclassified_Clostridiaceae, Sedimentibacter, and Clostridiales was relatively high in ZS and SS. Notably, the clustering relationship of S9 and S10 is close, and so is that of S11 and S12. The same clustering relationship was also found in OFP. Those results indicate that SC and OFP yielded a major impact on dominant species. We also observed that the abundant genera of OFP changed significantly after fermentation, which may be due to the addition of viable bacteria in the compost, indicating that the addition of live bacteria can produce more obvious effects on fermentation and abundant genera. Other studies also showed that the addition of thermophilic bacteria and Bacillus were beneficial to the decomposition of litter and improved the fermentation process (
      • Shen Q.
      • Sun H.
      • Yao X.
      • Wu Y.
      • Wang X.
      • Chen Y.
      • Tang J.
      A comparative study of pig manure with different waste straws in an ectopic fermentation system with thermophilic bacteria during the aerobic process: Performance and microbial community dynamics.
      ;
      • Huang B.
      • Jia H.
      • Han X.
      • Gou J.
      • Huang C.
      • Wang J.
      • Wei J.
      • Wang J.
      • Zhang C.
      Effects of biocontrol Bacillus and fermentation bacteria additions on the microbial community, functions and antibiotic resistance genes of prickly ash seed oil meal-biochar compost.
      ).
      Figure thumbnail gr5
      Figure 5Heat map of bacterial community compositions at the genus level (n = 3 per sampled site). (A) Anaerobic fermentation (AF). (B) Static compost (SC). (C) Organic fertilizer production (OFP). Green represents low abundance, and red represents high abundance. The lines on the left of each panel represent the clustering relationship at the genus level, and the lines on top of each panel represent the clustering relationship of samples collected at different composting stages with unweighted pair-group method with arithmetic means clustering. Only the most abundant taxa (top 50 genera) are displayed. Z1, Z2, Z3, Z4, and ZS are the sampled sites for AF; S9, S10, S11, S12, and SS are the sampled sites for SC; and S13, S14, S15, S16, and S17 are the sampled sites for OFP.
      Facklamia is a bacteremia-related pathogen (
      • Gahl M.
      • Stöckli T.
      • Fahrner R.
      Facklamia hominis bacteremia after transurethral resection of the prostate: A case report.
      ) and can also lead to joint infection (
      • Corona P.S.
      • Haddad S.
      • Andrés J.
      • González-López J.J.
      • Amat C.
      • Flores X.
      Case report: First report of a prosthetic joint infection caused by Facklamia hominis..
      ). Rhusiopathiae in Erysipelothrixis is potentially zoonotic and can be transmitted to humans, causing erysipelas (
      • Opriessnig T.
      • Forde T.
      • Shimoji Y.
      Erysipelothrix spp.: Past, present, and future directions in vaccine research.
      ). Porphyromonas in Porphyromonadaceae is a periodontitis-related pathogen (
      • Zhang X.
      • Ma C.
      • Zhang W.
      • Li W.
      • Yu J.
      • Xue D.
      • Wu X.
      • Deng G.
      Shifts in microbial community, pathogenicity-related genes and antibiotic resistance genes during dairy manure piled up.
      ). Psychrobacter immobilus is an opportunistic pathogen that has been found in eyes, brain tissue, urethra, cerebrospinal fluid, and blood (
      • Shao K.
      • Yao X.
      • Wu Z.
      • Jiang X.
      • Hu Y.
      • Tang X.
      • Xu Q.
      • Gao G.
      The bacterial community composition and its environmental drivers in the rivers around eutrophic Chaohu Lake, China.
      ). All these species existed in the raw manure, indicating there are many zoonotic or opportunistic pathogens in untreated manure. If untreated manure is used as an organic fertilizer on farmland, the persistence of these bacteria in soil may enhance the likelihood of these bacteria entering the food chain through contaminated crops and becoming active ARG donors (
      • Leclercq S.O.
      • Wang C.
      • Sui Z.
      • Wu H.
      • Zhu B.
      • Deng Y.
      • Feng J.
      A multiplayer game: Species of Clostridium, Acinetobacter, and Pseudomonas are responsible for the persistence of antibiotic resistance genes in manure-treated soils.
      ). Pseudomonas in Pseudomonadaceae is a pathogen associated with septicemia and lung infections, Acinetobacter causes nosocomial infection, and Corynebacterium is a potential human pathogen (
      • Soltan Mohammadi N.
      • Mafakheri S.
      • Abdali N.
      • Bárcena-Uribarri I.
      • Tauch A.
      • Benz R.
      Identification and characterization of the channel-forming protein in the cell wall of Corynebacterium amycolatum..
      ). These species declined at the late stage of compost, suggesting that the 3 composting practices examined in this study were able to reduce these pathogens, which is in line with the findings by
      • Li Y.B.
      • Jin P.F.
      • Liu T.T.
      • Lv J.H.
      • Jiang J.S.
      A novel method for sewage sludge composting using bamboo charcoal as a separating material.
      . However, pathogens such as Unclassified_Porphyromonadaceae, Unclassified_Erysipelotrichaceae, and Unclassified_[Tissierellaceae] remained in the liquid after AF. If the liquid is used to fertilize farmland, pathogens may flow into the environment, which might pose direct or indirect threats to human and animal health. In general, the 3 composting methods yielded a certain level of reduction in pathogens, and the effect of AF was weaker than that of SC and OFP (Figure 5).

      Prediction of Functional Profiles of the Bacterial Community

      PICRUSt is designed to estimate the gene families contributed to a metagenome by bacteria or archaea identified and to predict functional profiles of bacterial communities based on 16S rDNA sequences (
      • Langille M.G.
      • Zaneveld J.
      • Caporaso J.G.
      • McDonald D.
      • Knights D.
      • Reyes J.A.
      • Clemente J.C.
      • Burkepile D.E.
      • Vega Thurber R.L.
      • Knight R.
      • Beiko R.G.
      • Huttenhower C.
      Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences.
      ). We performed PICRUSt to analyze the differences in bacterial functions among 3 manure composts. A total of 328 Kyoto Encyclopedia of Genes and Genomes pathways were obtained in the 3 different composts by comparing with the Kyoto database. Among these pathways, gene sequences in the 3 treatments were primarily involved in metabolism, followed by genetic information processing and environmental information processing. In addition, the analysis of the second functional layer of the predicted genes showed that it consisted of 41 subfunctions, and the gene abundance of membrane transport, replication and repair, and carbohydrate, energy, and amino acid metabolism was high among the 3 composting methods (Supplemental Figure S3, https://doi.org/10.7910/DVN/WHS3EA ;
      • Tang M.
      • Wu Z.
      • Li W.
      • Shoaib M.
      • Aqib A.I.
      • Shang R.
      • Yang Z.
      • Pu W.
      Effects of different composting methods on antibiotic-resistant bacteria, antibiotic resistance genes, and microbial diversity in dairy cattle manures. Harvard Dataverse, V1.
      ), which is consistent with results reported by others (
      • Bello A.
      • Han Y.
      • Zhu H.
      • Deng L.
      • Yang W.
      • Meng Q.
      • Sun Y.
      • Egbeagu U.U.
      • Sheng S.
      • Wu X.
      • Jiang X.
      • Xu X.
      Microbial community composition, co-occurrence network pattern and nitrogen transformation genera response to biochar addition in cattle manure-maize straw composting.
      ;
      • Liang J.
      • Tang S.
      • Gong J.
      • Zeng G.
      • Tang W.
      • Song B.
      • Zhang P.
      • Yang Z.
      • Luo Y.
      Responses of enzymatic activity and microbial communities to biochar/compost amendment in sulfamethoxazole polluted wetland soil.
      ). It is worth noting that the abundance of amino acid and carbohydrate functional genes was predominant among the 3 compost treatments, and the metabolism of amino acids, lipids, and carbohydrates increased as the composting process advanced. It is reported that amino acid metabolism promotes the growth and activity of bacteria by providing carbon and energy sources (
      • López-González J.A.
      • Suárez-Estrella F.
      • Vargas-García M.C.
      • López M.J.
      • Jurado M.M.
      • Moreno J.
      Dynamics of bacterial microbiota during lignocellulosic waste composting: Studies upon its structure, functionality and biodiversity.
      ;
      • Liang J.
      • Tang S.
      • Gong J.
      • Zeng G.
      • Tang W.
      • Song B.
      • Zhang P.
      • Yang Z.
      • Luo Y.
      Responses of enzymatic activity and microbial communities to biochar/compost amendment in sulfamethoxazole polluted wetland soil.
      ). Membrane transport and carbohydrate metabolism increased, whereas amino acid and energy metabolism remained stable after fermentation during SC and OFP (Supplemental Figure S3). This was probably owing to the degradation of energy-rich wastes contained in the compost, including crude proteins and sugars (
      • Zhong X.Z.
      • Li X.X.
      • Zeng Y.
      • Wang S.P.
      • Sun Z.Y.
      • Tang Y.Q.
      Dynamic change of bacterial community during dairy manure composting process revealed by high-throughput sequencing and advanced bioinformatics tools.
      ). The metabolism of carbohydrates may produce various compounds through cellulose and hemicellulose degradation (
      • Toledo M.
      • Gutierrez M.C.
      • Siles J.A.
      • Garcia-Olmo J.
      • Martin M.A.
      Chemometric analysis and NIR spectroscopy to evaluate odorous impact during the composting of different raw materials.
      ). Additionally, amino acids serve as a carbon and energy source for bacterial metabolism, which is continuously produced at different stages of the composting process.
      As shown in Supplemental Figure S4 ( https://doi.org/10.7910/DVN/WHS3EA ;
      • Tang M.
      • Wu Z.
      • Li W.
      • Shoaib M.
      • Aqib A.I.
      • Shang R.
      • Yang Z.
      • Pu W.
      Effects of different composting methods on antibiotic-resistant bacteria, antibiotic resistance genes, and microbial diversity in dairy cattle manures. Harvard Dataverse, V1.
      ), the activity of the biosynthesis gene of streptomycin was the highest in all antibiotic biosynthesis genes. The activity of VAN, butirosin and neomycin, penicillin and penicillin, ansamycins, and TET biosynthesis genes was moderate, whereas the activity of clavulanic acid and β-lactam resistance related biosynthesis genes was low but stable. It is worth noting that the biosynthesis genes of ansamycins, VAN, butirosin, penicillin and cephalosporin, streptomycin, TET, and neomycin antibiotics were enriched at the later stage of the 3 composting processes. The activity of antibiotic biosynthesis genes in the late stage of SC was the highest, followed by OFP and AF (Supplemental Figure S4). The proportion of Firmicutes and Actinobacteria, which are traditionally thought to be antibiotics producers, showed a downturn in the late stage of SC and OFP, whereas the activity of antibiotic biosynthesis genes was increased at the same time. Whether there is a correlation between these 2 changes needs further investigation.

      CONCLUSIONS

      The 3 composting methods examined in this study produced variable and distinct effects on ARB, zoonotic bacteria, and ARG as well as microbial communities. Some ARB and ARG decreased, whereas others showed no significant difference during the composting processes. All 3 composting methods reduced the richness and evenness of microbial microflora. Among the 3 composting methods, the effects of SC and OFP on MCD, including zoonotic and opportunistic pathogens, and dominant species were more profound than those of AF. The addition of viable bacteria can produce more obvious effects on fermentation and abundant genera. As shown by PICRUSt function prediction analysis, the genes encoding membrane transport, carbohydrate metabolism, and amino acid metabolism were abundant among the 3 composting methods. The biosynthesis of antibiotics was enhanced after fermentation in the 3 composting methods, and the increase in SC was the most obvious. These results provide useful information for further optimizing composting practices.

      ACKNOWLEDGMENTS

      This work was supported by the National Key Research and Development Program (2016YFD0501305) and the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (25-LZIHPS-03). Wanxia Pu: conceptualization, writing—review and editing, funding acquisition. Minjia Tang: data curation, writing—original draft and formal analysis. Zhongyong Wu: conceptualization, investigation, formal analysis. Wenzhu Li: investigation. Muhammad Shoaib: investigation. Amjad Islam Aqib: writing—review and editing. Ruofeng Shang: writing—review and editing. Zhen Yang: writing—review and editing. The authors have not stated any conflicts of interest.

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