Distribution characteristics of antibiotic resistant bacteria and genes in fresh and composted manures of livestock farms

Science of the Total Environment 695 (2019) 133781

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Distribution characteristics of antibiotic resistant bacteria and genes in fresh and composted manures of livestock farms Lanjun Wang, Jun Wang, Jinhua Wang ⁎, Lusheng Zhu, Lili Yang, Rui Yang National Engineering Laboratory for Efficient Utilization of Soil and Fertilizer Resources, Key Laboratory of Agricultural Environment in Universities of Shandong, College of Resources and Environment, Shandong Agricultural University, Taian 271018, People's Republic of China

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Chicken and swine manures have higher abundance of ARB and ARGs than cow manures. • The abundance of ARB and ARGs in fresh manures was significantly higher than that in composted manures. • Detected antibiotics and MGEs were significantly correlated with some of the target ARGs. • Acinetobacter lwoffii and Psychrobacter pulmonis are multiple resistant bacteria.

a r t i c l e

i n f o

Article history: Received 22 June 2019 Received in revised form 1 August 2019 Accepted 4 August 2019 Available online 06 August 2019 Editor: Jay Gan Keywords: Antibiotic resistant bacteria Antibiotic resistance genes Mobile genetic elements Fresh manure Composted manure

a b s t r a c t Livestock manure is a major reservoir of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). This study investigated the distribution characteristics of ARB, ARGs in fresh and composted manures of traditional breading industry in rural areas in China. Samples collected were naturally piled without professional composting, and will be applied to farmland. The real-time quantitative polymerase chain reaction (qPCR) results showed the presence of ten target ARGs and two mobile genetic elements (MGEs) in the tested manure samples. The relative abundance of tetracycline and sulfonamide resistance genes (TRGs and SRGs) was generally higher than that of macrolide resistance genes (MRGs), followed by quinolone resistance genes (QRGs). There were significant positive correlations between the abundance of sul1, sul2, tetW and MGEs (intl1, intl2). In addition, the distribution of target ARGs was associated with the residual concentrations of doxycycline (DOX), sulfamethazine (SM2), enrofloxacin (ENR) and tylosin (TYL). Overall, a total of 24 bacterial genera were identified. The resistance rates of ARB were 17.79%–83.70% for SM2, followed 0.40%–63.77% for TYL, 0.36%– 43.90% for DOX and 0.00%–13.36% for ENR, which showed a significant dose-effect. This study also demonstrated that the abundance of clinically relevant ARB and ARGs in chicken, swine and cow fresh manures significantly greater than that in composted manures, and chicken and swine manures had higher proportion of ARB and higher abundance of ARGs than that in cow manures. © 2019 Elsevier B.V. All rights reserved.

1. Introduction ⁎ Corresponding author at: College of Resources and Environment, Shandong Agricultural University, 61 Daizong Road, Taian 271018, People's Republic of China. E-mail addresses: [email protected] (J. Wang), [email protected] (J. Wang).

https://doi.org/10.1016/j.scitotenv.2019.133781 0048-9697/© 2019 Elsevier B.V. All rights reserved.

Antibiotics are widely used in the livestock and poultry industries to promote growth and control animal diseases (Sun et al., 2017; Zhang

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L. Wang et al. / Science of the Total Environment 695 (2019) 133781

et al., 2015). However, excessive use of antibiotics can speed the production of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in the gastrointestinal tract of animals (Looft et al., 2014; Holman and Chénier, 2014). When antibiotics are released into the environment through livestock manures, they exert certain selective pressure on microorganisms and promote the development of bacterial resistance in the environment (Barton, 2014). Generally, 25% 75% of antibiotics given to animals are excreted as passively in the form of prototypes or metabolites and eventually enter the natural environment (Luo et al., 2011). Previous studies have shown that High concentrations of tetracyclines (411.3–1453.4 μg/kg), fluoroquinolones, (3326.6–12,302.6 μg/kg), macrolides (1.4–4.8 μg/kg) and sulfonamides (170.6–1060.2 μg/kg) were found in amended soils and livestock manures (Hou et al., 2014; Ji et al., 2012). A study showed that ARGs detected in swine farm samples were closely associated with residues of sulfonamides, quinolones, and tetracyclines (Zhu et al., 2013). Mobile genetic elements (MGEs) are important vectors for the horizontal migration of ARGs, which may be correlated with the diversity and abundance of ARGs in various environments (Zhu et al., 2013). Due to the presence of antibiotics and the ability to transfer genes horizontally, ARGs are increasingly detected in livestock and poultry manures and may spread to other environments (Yang et al., 2014). Numerous studies have confirmed that tetracycline, sulfonamide, quinolone, and macrolide resistance genes (TRGs, SRGs, QRGs, MRGs) were frequently found in surface water, sewage treatment plants, farms and have received considerable attention (Zhang and Zhang, 2011; Qian et al., 2017). A study by Cheesanford et al. (2001) revealed that seven TRGs (tetM, tetB (P), tetO, tetQ, tetW, tetS, and tetT) were detected in two pig manure tanks. Li et al. (2012) detected QRGs (qnrD, qepA and oqxB) in seven pig farms and sewage-irrigated farm fields. Mu et al. (2015) found that the abundance of QRGs (oqxB) and SRGs (sul1 and sul2) in chicken manures reached 3.48 × 1010, 1.39 × 1010 and 7.53 × 109 copies/g, respectively. MRGs (ermB, ermC and ermF) concentrations in tylosin-treated pig manures were all N109 copies/g (Luby et al., 2016). Furthermore, the emergence of resistant bacteria and their spread in different environments has become a global public health problem. ARGs may also transmit to human pathogens through the food chain or other ways, thereby causing harm to human health (Sundin and Bender, 2010). Studies have reported that plasmidmediated myxomycin resistance gene MCR-1 is resistant to many antibiotics, which was found in E. coli isolates from farm. The emergence of MCR-1 indicates that ARGs destroys the last antibiotic defense line polymyxin (Liu et al., 2016). China is one of the largest livestock and poultry producers in the world, with N2 billion tons of livestock and poultry manures produced and discharged every year (Zhu et al., 2014). In traditional Chinese farming, N80% of animal manures on farms are usually applied to the fields as organic fertilizer, using simple compost (Xiong et al., 2010). To reveal the extent of ARGs in the traditional livestock in China, especially in small and medium-sized livestock farms in rural areas, the diversity and abundance of ARGs of fresh and composted manures in two states were determined in the present study. Ten ARGs (qepA, qnrB, qnrS, ermB, ermF, tetO, tetW, tetM, sul1and sul2) and 2 MGEs (intl1 and intl2) were detected by real-time quantitative polymerase chain reaction (qPCR) methods. We hypothesized that ARGs are contained in the collected manures and the abundance of ARGs is affected by the types and states of manures. 2. Materials and methods

samples (fresh poultry manure, approx. 1 day: Ch-F; composted poultry manure for about 1 month: Ch-C) were collected from two representative chicken farms with production of 10,000 chickens or more per year (Shankou Town, Taian, China). Samples of swine manures (fresh swine manure for about 1 day: S-F; composted swine manure for about 7 days: S-C) were collected from two representative swine farms with production of 200 or more pigs per year (Beijipo Town, Taian, China). Three replicates of each manure sample were collected and all samples were stored on dry ice during transport and placed in −20 °C prior to analysis for antibiotics, ARGs, and ARB. 2.2. Antibiotics quantitation Four antibiotics, namely enrofloxacin (ENR), doxycycline (DOX), sulfadimidine (SM2) and tylosin (TYL) were extracted by solid phase extraction (SPE) and determined by high-performance liquid chromatography (HPLC) according to the method as described by Zhao et al. (2017). The test procedure was slightly modified. Firstly, antibiotics in the sample (2 g) were extracted with 15 ml extracting solution (prepared from Na2EDTA-McIlvaine buffer 7.5 ml, acetonitrile 3 ml, methanol 3 ml and acetone 1.5 ml). The extraction conditions were as follows: primarily, vortex-mixed for 5 min (200 rpm), then ultrasonically extracted for 20 min, centrifuged for 6 min (3000 rpm), and the supernatants was obtained. The above steps were repeated three times and the supernatants were combined and diluted to 450 ml. Secondly, the hydrophilic-lipophilic balance (HLB) (6 ml/150 mg, Waters, Ireland) solid phase extraction column was activated with 6 ml dichloromethane, 6 ml methanol and 0.5 g/l sodium ethylenediaminetetraacetate aqueous solution. The supernatants were then filtered through the HLB column at a rate of 3 ml/min. After that, the HLB column was washed successively with 6 ml ultrapure water, and vacuum-dried for 20 min. The antibiotics were eluted with 3 ml methanol and 5 ml methanol containing 1% formic acid. The eluted solution was collected in the calibration tube, blown to near-dry by nitrogen, and sealed in a brown sampling bottle at a constant volume of methanol to 1 ml. Finally, the samples were analyzed by HPLC (Agilent, 1260, USA), the mobile phase A is 0.05 mol/l phosphoric acid solution (pH, 3), B is acetonitrile, the volume ratio of A and B is 73:27, the column temperature is 25 °C, the injection volume is 10 μl, the flow rate is 1 ml/min, and the wavelength is 270 nm. The recoveries of four antibiotics were between 64.94 and 106.63%. 2.3. Count of cultivable bacteria and ARB in manures After gradient dilution, 100 μl of sample suspensions (cow manure 10−5, chicken manure 10−7 and swine manure 10−5) were applied to a sterilized Luria-Bertani (LB) plate, the final concentrations of four antibiotics ENR, TYL, DOX and SM2 on the LB plate were (0, 5, 10, 50 μg/kg), (0, 10, 50, 150 μg/kg), (0, 5, 10, 15 μg/kg) and (0, 50, 150, 250 μg/kg), respectively. The antibiotics concentrations on the plates were determined on the basis of the Clinical and Laboratory Standards Institute (CLSI) (2016). Three replications of each plate were performed, and E. coli (DH5α) was used instead of the suspensions as the negative control. The total number of cultiviable bacteria and ARB on the LB plate were counted after 72 h incubation at 37 °C. The bacterial resistance rates were calculated according to the formula: Bacterial resistance rate ¼ number of ARB=total cultivable bacteria  100%

2.1. Sampling sites and manure collection 2.4. Identification of ARB Cow manure samples (fresh cow manure approx. 1 day: C-F; composted cow manure, approx. 3 months: C-C) were collected from two representative cow farms with an animal intensity of 300 or more cows per year (Shengzhuang Town, Taian, China). Chicken manure

Based on a morphological observation, colonies with different color, morphology, and transparency were obtained from LB plate. After isolation and purification, isolates were inoculated into a sterilized liquid LB

L. Wang et al. / Science of the Total Environment 695 (2019) 133781

3

Fig. 1. Occurrence of ARGs in manures from the livestock farms. C-F, S-F and Ch-F represent fresh cow, swine and chicken manure, respectively; C-C, S-C and Ch-C represent composted cow, swine and chicken manure, respectively.

2.5. Quantitative PCR Using E.Z.N.Z.™ soil DNA kit (OMEGA, USA), DNA was extracted from 0.5 g fresh and composted manures. The DNA concentration test was then performed with Nanodrop3300 (Nanodrop, USA). ARGs detection was performed using ABI7500 (Applied Biosystems, USA). Based on the published literature, the sequences of all target ARGs primers and the PCR conditions are given in Table S1 (Supplementary Materials). The PCR reaction mixtures consisted of 1 μl template DNA, 0.6 μl forward and reverse primers (10 mM), 10 μl of SYBR Green I Master Mix and 9.5 μl of double distilled H2O. The thermal cycling conditions were 95 °C for 15 min; 45 cycles included 95 °C for 30 s, annealing at a selected temperature (Table S1) for 60 s, and 72 °C for 30 s, followed by a final melting curve stage, with a temperature ramp from 55 to 95 °C. To standardize ARGs in manures, the 16S rRNA gene was quantified as a housekeeping gene. RNA-free water were used as a negative control. All reactions were analyzed in three technical replicates. The standard curve was generated as described by (Zhao et al., 2017), using 10 fold serial dilutions of the plasmids carrying ARGs as standards. Copy numbers of the corresponding genes were calculated according to the curve. 2.6. Statistical analysis The relative abundance of ARGs was calculated by dividing the number of ARG copies by the number of copies of the 16S rRNA genes. The mean values and standard deviations of the experimental data were determined using Microsoft Excel 2010. A one-way analysis of variance (ANOVA) (p b 0.05) was performed with the SPSS software (version 22.0). The redundancy analysis (RDA) analysis was performed using Canoco software for Windows 4.5.

3. Results and discussion 3.1. Distribution characteristics of ARGs and MGEs in manures Ten ARGs (tetW, tetO, tetM, sul1, sul2, qepA, qnrB, qnrS, ermB, and ermF) were all detected in chicken, swine and cow manures (Fig. 1). Many studies have also confirmed that animal manures are important sources of ARGs (Heuer et al., 2011; Jechalke et al., 2013). Of the four types of ARGs, the most abundant genes were SRGs and TRGs, which were the dominant resistance genes in chicken, cow and swine manures, and these ARGs were easily detected in animal manures (Aminov et al., 2001; Sergeant et al., 2014). Moreover, the relative abundance of SRGs was higher than that of TRGs, consistent with previous findings (McKinney et al., 2010). In addition, the relative abundance of sul1 was slightly higher than that of sul2, and the relative abundance of sul1 in the Ch-F sample was as high as 3.13E+01 (Fig. 1). The difference in the abundance of detection of sul1 and sul2 genes may be due to their different diffusion mechanisms. The SRGs encode the sul1 protein as a dihydrofolate synthetase, while the sul2 gene is often located on the small plasmid of the IncQ family. As a result, sul1 has a broader spectrum of host bacteria and a more abundant transfer diffusion space (Sköld, 2000; Sköld, 2001). TRGs were also have high abundance in most samples (Fig. 1). Besides the fact that the relative abundance of tetM in chicken manures was greater than that of tetO, the order of

Relative abundances of MGEs in manure samples 1.00E-03

1.00E-02

1.00E-01

1.00E+00

1.00E+01

C-C C-F

intl1 intl2

Sample

medium and placed in shaking incubator at 150 rpm and 37 °C. The DNA used for PCR amplification of the ARB was extracted with BacteriaGen DNA Kit (CWBIO, Beijing, China), the primers were 27F (AGAGTTTGA TCCTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT). The PCR reaction system contains 1 μl of template DNA, 0.5 μl of primer (10 Mm), 12.5 μl of 2 × PCR Taq Master Mix and 10.5 μl of double distilled H2O. The thermal cycling conditions of the PCR were performed at 94 °C for 5 min; 30 cycles included: 94 °C for 1 min, 56 °C for 1 min, 72 °C for 2 min; and the extension was performed at 72 °C for 10 min. Then the PCR product was tested with agarose gel electrophoresis (1%) and was sequenced. The results of the sequences alignment were analyzed by the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI).

S-C S-F

Ch-C Ch-F

Fig. 2. Occurrence of MGEs in manures from the livestock farms. C-F, S-F and Ch-F represent fresh cow, swine and chicken manure, respectively; C-C, S-C and Ch-C represent composted cow, swine and chicken manure, respectively.

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L. Wang et al. / Science of the Total Environment 695 (2019) 133781

abundance was tetW N tetO N tetM, and the relative abundance of tetW in S-F was as high as 3.86E+00 (Fig. 1). The results were similar to previous studies in which high concentrations of TRGs were detected in environmental media such as chicken manure, swine farm sewage and soil near swine farm (Cheng et al., 2013; Wu et al., 2010). TRGs can be diffused and transmitted to not only gram-negative but also grampositive bacteria. ARGs that alter antibiotic target sites, in particularly, such as tetM, tetW, and tetQ are believed to originate from grampositive bacteria and can be transferred between species and genera (Chopra and Roberts, 2001). Furthermore, our results confirm those of previous study detected that tetW and sul1 genes at high frequencies and high levels in livestock contaminated media and identified those genes as indicators of the traceability of ARGs in the environment (McKinney et al., 2010; Cheng et al., 2013; Pruden et al., 2012; Storteboom et al., 2010). The relative abundance of MRGs was really lower than that of TRGs and SRGs. In most samples, the relative abundance of ermF was higher

than that of ermB (Fig. 1). The relative abundance of ermF in S-F samples was 9.96E–01. The ermF and ermB genes were MLSB genes that are common in isolated strains. A study reported that the rates of ermB and ermF carriers were 100% and 69% in multiple resistant enterococci isolated from the indoor air of large pig farms (Sapkota et al., 2006). Chen et al. (2007) also revealed that ermB and ermF were abundant in cow, swine, composted swine manure, swine farm wastewater, and biological filters. The QRGs have the lowest abundance in our study (Fig. 1). The qnrS gene was the dominant QRG gene in S-C (1.38E–02) and S-F (7.97E–02) samples (Fig. 1). Although the frequency of detection of QRGs in our samples was lower than other ARGs, it has been detected in many other environments (Luo et al., 2011; Hou et al., 2014). Fig. 1 shows that swine and chicken manures had higher abundance of ARGs than cow manures. In several, SRGs had the highest abundance in chicken manures, TRGs had the highest abundance in swine manures, and MRGs and QRGs levels in swine and chicken manures were all higher than those in cow manures. Exceptions were that the abundance

A 3.50E+01

Relative abundances of intl1

3.00E+01 2.50E+01

tetW

R² = 0.8757

sul1

R² = 0.7496

sul2

R² = 0.8682

2.00E+01 1.50E+01 1.00E+01 5.00E+00 0.00E+00 0.00E+00

5.00E-01

-5.00E+00

B

1.50E+00

2.00E+00

2.50E+00

Relative abundances of ARGs

3.50E+01 3.00E+01

Relative abundances of intl2

1.00E+00

2.50E+01

tetW

R² = 0.9186

sul1

R² = 0.8445

sul2

R² = 0.9607

2.00E+01 1.50E+01 1.00E+01 5.00E+00 0.00E+00 0.00E+00 5.00E-01 1.00E+00 1.50E+00 2.00E+00 2.50E+00 -5.00E+00

Relative abundances of ARGs

Fig. 3. Correlation of intl (intl1 (A) and intl2 (B)) abundances and tetW, sul1 and sul2 abundances in manures.

L. Wang et al. / Science of the Total Environment 695 (2019) 133781

70 Content of antibiotics in manures (mg/kg)

of qnrB was higher than that in Ch-C; the abundance of qepA in cow manures was greater than that in S-C, S-F and Ch-C; the abundance of tetO in C-F was higher than that in Ch-C and Ch-F; the relative abundance of sul1 in C-F was higher than that in S-C; and the relative abundance of ermB in Ch-C was the lowest (8.39E–05). Studies also indicated that the risk of transmission of ARGs in chicken and swine manures was higher than in cow manures (Cheesanford et al., 2001; Wang et al., 2011). Compared composted and fresh manures, the latter has higher proportion of ARGs, which was particularly pronounced in chicken and swine manures (Fig. 1). Some studies have shown that the concentrations of tetB and tetM in cow manures gradually decreased during the composting process (Storteboom et al., 2007). Aerobic composting has been shown to eliminate the SRGs sul1, sul2, dfrA1, and dfrA7 genes, the QRGs gyrA genes and TRGs tetW, tetC, tetZ, tetG, tetQ and tetY genes from swine manures (Selvam et al., 2012). However, the difference in ARGs levels between fresh and composted manures was not significant in cow manures. It is possible that the background value of ARGs in the intestinal microorganisms of the cows was low. Other studies have reported that background values of ARGs in microorganisms in swine and chicken manures were higher than that in cow manures (Zhu et al., 2013; Looft et al., 2012).

TYL

5

DOX

SMZ

ENR

60 50 40 30 20 10 0 C-F

C-C

Ch-F Ch-C Sample

S-F

S-C

Fig. 4. Profiles of antibiotic concentrations in manures from the livestock farms. C-F, S-F and Ch-F represent fresh cow, swine and chicken manure, respectively; C-C, S-C and ChC represent composted cow, swine and chicken manure, respectively.

3.2. Correlations between target MGEs and ARGs Two MGEs (intl1 and intl2) were both detected to varying degrees in chicken, swine and cow manures (Fig. 2). The relative abundance of intl1 ranged from 1.34E–03 to 2.46E+00, and that of intl2 ranged from 2.05E–03 to 2.30E+00. The correlation between ARGs and MGEs are shown in Fig. 3. Sul1 was significantly positive correlated with intl1 and intl2 (R2 = 0.7496 and 0.8445, respectively, P b 0.05), sul2 was significantly positive correlated with intl1 and intl2 (R2 = 0.8682 and 0.9607, respectively, P b 0.05) and tetW was significantly positive correlated with intl1 and intl2 (R2 = 0.8757 and 0.9186, respectively, P b 0.05). The correlation between these ARGs and integrase genes in livestock manures suggests that intl1 and intl2 could play an important role in the transmission of ARGs. Horizontal gene transfer is an important mechanism for the spread of ARGs in the environment (Thomas and Nielsen, 2005). In general, intl1 is located on transposon of Tn21 and intl2 on transposon of Tn7 and its derivatives (Hall and Stokes, 1993; Hall, 1997) so that they can spread among bacteria with transposons. Many studies have shown that intl1 and intl2 genes are widespread in manure, field and greenhouse soils and that the widespread presence of integrase genes may promote the accumulation and persistence of ARGs through horizontal gene transfer (Li et al., 2017; Zhang et al., 2017). Zhang et al. (2009) showed that intl1 coexisted with tetracycline ARGs in sludge samples from five wastewater treatment plants. And Zhao et al. (2019) reported that the total relative abundance of intl genes was positively correlated with tetW, tetO, sul1 and sul2 in soils amended with manure, perhaps due to ARGs transmission and metastasis.

concentrations of ciprofloxacin, fleroxacin and norfloxacin in chicken manures were between 45.60 and 225.00 mg/kg (Zhao et al., 2010). Similar finding was reported by Pan et al. (2011), tetracycline and sulfonamide antibiotics were detected in 126 samples of pig manures from 21 large-scale pig farms, with the highest residual concentrations being 764.40 mg/kg and 28.70 mg/kg, respectively. However, a study by Arikan et al. (2006) found that the concentration of oxytetracycline was 0.55 mg/kg at the start of composting and 0.84 mg/kg at the end of composting. This result can be explained by the fact that some antibiotics present in composted manures have been converted into substances via microorganism metabolism and are easily extracted and detected. RDA shows the relationship between ARGs and antibiotic residues in samples (Fig. 5). Significant correlations between DOX and tetM, sul1, sul2, qepA, qnrB, ermB, and ermF were found. Furthermore, ENR and TYL had a significant correlation with tetW, tetO, qnrS. In contrast, there was no significant correlation between SM2 and ARGs. Previous

3.3. Effects of antibiotic residuals on ARGs distribution Antibiotic residue concentrations in manures were also determined. The ranges of concentrations of ENR, SMZ, DOX and TYL in the manures were 1.01–11.79, 3.03–26.31, 0.59–33.00 and 1.46–19.39 mg/kg, respectively (Fig. 4). Among them, the concentrations of SMZ and TYL in composted chicken manures were lower than those in fresh chicken manures, but SMZ and TYL in C-C were higher than that of C-F. SM2 and TYL concentrations in the S-C were higher than in S-F. Overall, SM2 and DOX concentrations were higher than those of ENR and TYL. Livestock manure is one of the main sources of environmental contamination with antibiotics. Previous studies have shown that concentrations of ciprofloxacin, ENR, tetracycline and chlortetracycline in swine and cow manures were between 21.00 and 59.60 mg/kg and

Fig. 5. RDA compares the abundance of antibiotics and ARGs. 1, 3 and 5 represent fresh cow (C-F), chicken (Ch-F) and swine (S-F) manure, respectively; 2, 4 and 6 represent composted cow (C-C), chicken (Ch-C) and swine (S-C) manure, respectively.

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Table 1 Abundance (CFU/g) of ARB exposed to different concentrations of ENR, TYL, DOX and SM2 (μg/ml).

TYL

DOX

SM2

C-C

C-F

Ch-C

Ch-F

S-C

S-F

(1.80 ± 0.02) × 108 0 0 0 (1.63 ± 0.02) × 107 (7.00 ± 0.01) × 106 (6.67 ± 0.01) × 105 (4.67 ± 0.01) × 106 (2.33 ± 0.01) × 106 (6.67 ± 0.01) × 105 (1.40 ± 0.02) × 108 (1.15 ± 0.04) × 108 (8.83 ± 0.06) × 107

(3.59 ± 0.08) × 108 0 0 0 (3.70 ± 0.02) × 107 (1.43 ± 0.02) × 107 (2.67 ± 0.01) × 106 (2.07 ± 0.02) × 107 (1.07 ± 0.02) × 107 (4.67 ± 0.01) × 106 (2.08 ± 0.06) × 108 (1.56 ± 0.08) × 108 (1.24 ± 0.08) × 108

(1.55 ± 0.06) × 1010 (8.67 ± 0.02) × 108 (2.00 ± 0.01) × 108 0 (1.30 ± 0.03) × 108 (8.33 ± 0.01) × 108 (3.67 ± 0.02) × 108 (5.67 ± 0.01) × 108 (3.67 ± 0.01) × 108 (1.33 ± 0.01) × 108 (1.25 ± 0.05) × 1010 (1.12 ± 0.03) × 1010 (1.01 ± 0.03) × 1010

(1.51 ± 0.02) × 1010 (1.10 ± 0.01) × 109 (7.33 ± 0.01) × 108 (2.00 ± 0.01) × 108 (2.00 ± 0.01) × 109 (1.57 ± 0.01) × 109 (1.37 ± 0.01) × 109 (2.30 ± 0.02) × 109 (1.77 ± 0.01) × 109 (9.33 ± 0.01) × 108 (1.27 ± 0.04) × 1010 (1.13 ± 0.03) × 1010 (9.10 ± 0.03) × 109

(2.02 ± 0.16) × 108 (8.33 ± 0.02) × 106 (2.67 ± 0.01) × 106 (6.67 ± 0.01) × 105 (7.37 ± 0.03) × 107 (6.27 ± 0.05) × 107 (3.83 ± 0.02) × 107 (4.73 ± 0.03) × 107 (3.03 ± 0.02) × 107 (2.43 ± 0.01) × 107 (1.19 ± 0.03) × 108 (5.50 ± 0.11) × 107 (3.23 ± 0.09) × 107

(2.00 ± 0.08) × 108 (2.67 ± 0.04) × 107 (1.20 ± 0.03) × 107 (1.33 ± 0.01) × 106 (1.27 ± 0.08) × 108 (9.23 ± 0.10) × 107 (4.80 ± 0.06) × 107 (8.77 ± 0.06) × 107 (5.87 ± 0.02) × 107 (3.90 ± 0.02) × 107 (1.64 ± 0.03) × 108 (1.48 ± 0.04) × 108 (1.08 ± 0.04) × 108

studies have shown that the abundance of ARGs may be positively correlated with antibiotic residues in agricultural soils and livestock manures (Zhu et al., 2013; Wu et al., 2013). In fact, previous studies have shown that residual antibiotics in the soil can exert selective pressure on soil bacterial communities (Peng et al., 2015; Peng et al., 2014). The addition of antibiotics such as tetracycline, sulfamethazine, penicillin, and bacitracin to livestock industry not only altered the intestinal microbial community of farm animals but also significantly increased the abundance and diversity of ARGs (Zhu et al., 2013; Looft et al., 2012). Therefore, antibiotic residues in livestock manures may provide insights into the distribution of ARGs in animal manures. 3.4. Analysis of antibiotic resistant bacteria (ARB) in manures ENR, DOX, TYL, and SM2, which are commonly used in livestock farms, were selected as antibiotics for ARB screening. The concentrations of cultivable bacteria and ARB in chicken, swine and cow manures are shown in Table 1. The total concentrations of cultivable bacteria in CC, C-F, CH-C, CH-F, S-C, and S-F were 1.80 × 108, 3.59 × 108, 1.55 × 1010, 1.51 × 1010, 2.20 × 108, 2.00 × 108 CFU/g, respectively. Chicken manures contained the highest concentrations of cultivable bacteria.

Drug resistance (%)

A 100

c bbc c

80 60

b

a

a

cc c

a

cc c b a

40

C-C C-F Ch-C Ch-F S-C S-F

d d

20

Furthermore, the abundance of ARB in chicken manures was highest, followed by swine manures, and the abundance of ARB in cow manures was lowest. In addition, ENR-resistant bacteria were not found in cow manures. ARB in manures might be due to the fact that some antibiotic-sensitive strains had been eliminated by antibiotic residues (Yang et al., 2014). On the other hand, microorganisms in animal intestines may develop resistance by mutation during long-term feeding of antibiotics under the selective pressure of antibiotics (Heuer et al., 2011). In this study, chicken, swine, and cow manures from different farms all had ARB that could be associated with long-term feeding with antibiotics. The reason for the relatively high abundance of ARB in chicken manures may be related to the ability of an animal to metabolize drugs or to the dosage and mode of feeding of antibiotics (Zhao et al., 2010). In addition, the digestive system of chicken is not perfect compared to mammals, resulting in higher indigestible nutrient residues in chicken manures, which provide good survival conditions for the growth and reproduction of ARB (Lanyasunya et al., 2006). The bacterial resistant rates are shown in Fig. 6. The resistance rates of bacteria to ENR ranged from 0 to 13.36%, which was considerably lower than that of other antibiotics (SM2 (17.79%–83.70%), TYL (0.40%–63.77%) and DOX (0.36%–43.90%)). The results may be due to

B 80 Drug resistance (%)

ENR

0 5 10 50 10 50 150 5 10 15 50 150 250

d 60

20

C-C

f

C-F

aa

40

Ch-C

e

30

Ch-F

e

20

e

d b a c

c a bb

d

d c a bab

5 10 15 Concentration of DOX (µg/kg)

S-C S-F

D 15 Drug resistance (%)

C 50 Drug resistance (%)

c

b a aba

a

b aa

a

d

b

10 50 150 Concentration of TYL (µg/kg)

50 150 250 Concentration of SM2 (µg/kg)

0

c

0

0

10

d c

40

C-C C-F Ch-C Ch-F S-C S-F

C-C

c

C-F Ch-C

10 ab 5

Ch-F

a b

b a a

S-C

b

S-F

a a b

0 5 10 50 Concentration of ENR (µg/kg)

Fig. 6. The resistance rate of bacteria exposed to the different concentration antibiotics (SM2 (A), TYL (B), DOX (C), ENR (D)) in manures. C-F, S-F and Ch-F represent fresh cow, swine and chicken manure, respectively; C-C, S-C and Ch-C represent composted cow, swine and chicken manure, respectively.

L. Wang et al. / Science of the Total Environment 695 (2019) 133781

7

Compositions of AMB at the genus level (%)

100% Wohlfahrtiimonas

Sporosarcina

Staphylococcus

Pseudomonas

80%

Psychrobacter

Planococcus

70%

Ochrobactrum

Micrococcus

60%

Lysinibacillus

Lactobacillus

50%

Klebsiella

Kurthia

Jeotgalicoccus

Ignatzschineria

Escherichia

Enterococcus

Empedobacter

Corynebacterium

20%

Brevibacterium

Bacillus

10%

Alcaligenes

Acinetobacter

Aerococcus

Arthrobacter

90%

40% 30%

0% SMZ

TYL DOX Antibiotics

ENR

Fig. 7. Distribution characteristics of antibiotic resistant bacteria (ARB) at the genus level.

a higher SM2 residue than other antibiotics in these sampling sites. Zhao et al. (2019) reported that the addition of sulfonamides (60–100 g/1000 kg feed) is much higher than that of tetracyclines (7.5 g/1000 kg feed) and the price of sulfonamides is lower than that of tetracyclines. There was a correlation between antibiotic exposure and ARB production. With the increase of antibiotic concentration in feed, the resistance rate of bacteria has decreased. Previous studies have shown that there was a significant and positive correlation between antibiotics doses in livestock feed and the occurrence of ARB in surface waters contaminated with animal manures (McKinney et al., 2010). Under high exposure to tetracycline or oxytetracycline, the proportion of ARB in the wheat rhizosphere soil was significantly higher than in low concentration treatments (Yang et al., 2009; Yang et al., 2010). Therefore, the occurrence of high proportion of ARB in livestock was significantly associated with high dose use of antibiotics over a longer period of time. In addition, the resistant rates of bacteria in swine and chicken manures were much higher than in cow manures. As in a previous report, the proportion of ARG-carrying bacteria in swine manures was significantly higher than in cow and sheep manures due to a large number of antibiotics used in pig farming (McKinney et al., 2010). Our study also indicated that the abundance of resistant bacteria in fresh manures was higher than in composted manures. A previous study showed that composting can decompose complex organic matter in livestock manure into humus by microbial fermentation, the high temperature of 50–65 °C during the decomposition process can kill pathogenic microorganisms and eliminate some antibiotic residues (Hakk et al., 2005). The distribution of bacteria among the genera in the manures is shown in Fig. 7. A total of 24 bacterial genera were identified. As shown in Fig. 7 and Table S2 (Supplementary materials), there were a total of 12 bacterial genera with 19 bacterial close species of SM2resistant bacteria, 12 bacterial genera with 14 close bacterial species of DOX-resistant bacteria, 10 bacterial genera with 15 close bacterial species of TYL-resistant bacteria and 10 bacterial genera with 11 close bacterial species of ENR-resistant bacteria. Bacillus (21.05%), Kurthia (14.28%), Psychrobacter (14.28%), Enterococcus (20%) and Sporosarcina (19%) were the most abundant genera that were resistant to SM2, DOX, TYL, and ENR, respectively. Moreover, the species Acinetobacter lwoffii and Psychrobacter pulmonis were simultaneously resistant to SM2, TYL, and DOX. As many as 33 multiple ARB have been detected in chicken manures, the genera Alcaligenes, Myroides, Ignatzschineria, Escherichia, Proteus, Providencia, Enterococcus and Acinetobacter being the most common cultivable multiple ARB (Yang et al., 2017). Studies

have elucidated the mechanism for the transfer and diffusion of some ARB and their ARGs from cultured animals to humans, such as Enterococci, Escherichia coli, Campylobacter and Salmonella, pose a potential threat to public health (Barton, 2014; Poulsen et al., 2012). There are also many opportunistic pathogens in manure samples. For example, Acinetobacter lwoffii is a non-fermentative gram-negative bacteria that is widespread in nature, it is also one of the important pathogens of nosocomial infection (Turton et al., 2010; Li and Wang, 2010). As a result, the existence and spread of ARB in the environment has become a delicate global problem that could pose a threat to human health. The presence of multiple ARB entering the natural environment through livestock manures should be sufficiently taken into account. 4. Conclusions Target ARGs and MGEs were all detected in different types of livestock manures. Compared with MRGs and QRGs, SRGs and TRGs had higher abundance. Significant correlations were found between sul1, sul2, tetw with intl1, and intl2, indicating that these ARGs in manures have the possibility of diffusion in the environment. These residual antibiotics, which may exert selective pressure on bacterial communities, were significantly correlated with most of the target ARGs. About ARB, a total of 24 bacterial genera were identified, Acinetobacter lwoffii and Psychrobacter pulmonis were identified as apparently resistant to all three antibiotics. In addition, with increasing concentration of antibiotics, the abundance of ARB decreased. Overall, chicken and swine manures had higher proportion of ARB and higher abundance of ARGs than that in cow manures, and the abundance of ARB and ARGs in fresh manures was considerably higher than that in composted manures. In order to reduce the transmission of ARB and ARGs from livestock and poultry manure to the agricultural environment, effective measures to reduce ARB and ARGs should be taken in composting process. Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2019.133781. Acknowledgments This work was supported by the National Science and Technology Major Project of the Ministry of Science and Technology of China [Nos. 2017YFD0800703 and 2016YFD0201203]; Natural Science Foundation of Shandong Province, China [Nos. JQ201711 and ZR2016JL029], and the Special Funds of Taishan Scholar of Shandong Province, China.

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Declaration of competing interest None declared. Submission declaration The work has not been published previously. References Aminov, R.I., Garrigues-Jeanjean, N., Mackie, R.I., 2001. Molecular ecology of tetracycline resistance: development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins. Appl. Environ. Microb. 67, 22–32. Arikan, O.A., Sikora, L.J., Mulbry, W., Khan, S.U., Rice, C., Foster, G.D., 2006. The fate and effect of oxytetracycline during the anaerobic digestion of manure from therapeutically treated calves. Process Biochem. 41, 1637–1643. Barton, M.D., 2014. Impact of antibiotic use in the swine industry. Curr. Opin. Microbiol. 19, 9–15. Cheesanford, J.C., Aminov, R.I., Krapac, I.J., Garriguesjeanjean, N., Mackie, R.I., 2001. Occurrence and diversity of tetracycline resistance genes in lagoons and groundwater underlying two swine production facilities. Appl. Environ. Microbiol. 67, 1494–1502. Chen, J., Yu, Z.T., Michel, F.C., Wittum, T., Morrison, M., 2007. Development and application of real-time PCR assays for quantification of erm genes conferring resistance to macrolides-lincosamides-streptograminb in livestock manure and manure management systems. Appl. Environ. Microb. 73, 4407–4416. Cheng, W., Chen, H., Su, C., Yan, S., 2013. Abundance and persistence of antibiotic resistance genes in livestock farms: a comprehensive investigation in eastern China. Environ. Int. 61, 1–7. Chopra, I., Roberts, M., 2001. Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol. Mol. Biol. R. 65, 232–260. Hakk, H., Millner, P., Larsen, G., 2005. Decrease in water-soluble 17β‑estradiol and testosterone in composted poultry manure with time. J. Environ. Qual. 34, 943–950. Hall, R.M., 1997. Mobile gene cassettes and integrons: moving antibiotic resistance genes in gram-negative bacteria. CIBA Found. Symp. 207, 192–202. Hall, R.M., Stokes, H.W., 1993. Integrons: novel DNA elements which capture genes by site-specific recombination. Genetica 90, 115–132. Heuer, H., Schmitt, H., Smalla, K., 2011. Antibiotic resistance gene spread due to manure application on agricultural fields. Curr. Opin. Microbiol. 14, 236–243. Holman, D.B., Chénier, M.R., 2014. Temporal changes and the effect of subtherapeutic concentrations of antibiotics in the gut microbiota of swine. FEMS Microbiol. Ecol. 90, 599–608. Hou, J., Wan, W.N., Mao, D.Q., Wang, C., Mu, Q.H., Qin, S.Y., Luo, Y., 2014. Occurrence and distribution of sulfonamides, tetracyclines, quinolones, macrolides, and nitrofurans in livestock manure and amended soils of northern China. Environ. Sci. Pollut. Res. 22, 4545–4554. Jechalke, S., Kopmann, C., Rosendahl, I., Groeneweg, J., Weichelt, V., Krogerrecklenfort, E., Brandes, N., Nordwig, M., Ding, G.C., Siemens, J., Heuer, H., Smalla, K., 2013. Increased abundance and transferability of resistance genes after field application of manure from sulfadiazine-treated pigs. Appl. Environ. Microb. 79, 1704–1711. Ji, X.L., Shen, Q.H., Liu, F., Ma, J., Xu, G., Wang, Y.L., Wu, M.H., 2012. Antibiotic resistance gene abundances associated with antibiotics and heavy metals in animal manures and agricultural soils adjacent to feedlots in Shanghai; China. J. Hazard. Mater. 235236, 178–185. Lanyasunya, T.P., Rong, W.H., Abdulrazak, S.A., 2006. Factors limiting use of poultry manure as protein supplement for dairy cattle on smallholder farms in Kenya. J. Poult. Sci. 5, 75–80. Li, X., Wang, H.H., 2010. Tetracycline resistance associated with commensal bacteria from representative ready-to-consume deli and restaurant foods. J. Food Protect. 73, 1841–1848. Li, J., Wang, T., Shao, B., Shen, J.Z., Wang, S.C., Wu, Y.N., 2012. Plasmid-mediated quinolone resistance genes and antibiotic residues in wastewater and soil adjacent to swine feedlots: potential transfer to agricultural lands. Environ. Health. Persp. 120, 1144–1149. Li, J.J., Xin, Z.H., Zhang, Y.Z., Chen, J.W., Yan, J.X., Li, H.J., Hu, H.W., 2017. Long-term manure application increased the levels of antibiotics and antibiotic resistance genes in a greenhouse soil. Appl. Soil Ecol. 121, 193–200. Liu, Y.Y., Wang, Y., Walsh, T.R., Yi, L.X., Zhang, R., Spencer, J., et al., 2016. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect. Dis. 16, 161–168. Looft, T., Johnson, T.A., Allen, H.K., Bayles, D.O., Alt, D.P., Stedtfeld, R.D., Chai, B.l., Cole, J.R., Hashsham, S.A., Tiedje, J.M., Stanton, T.B., 2012. In-feed antibiotic effects on the swine intestinal microbiome. P. Natl. Acad. Sci. Usa. 109, 1691–1696. Looft, T., Allen, H.K., Cantarel, B.L., Levine, U.Y., Bayles, D.O., Alt, D.P., Henrissat, B., Stanton, T.B., 2014. Bacteria, phages and pigs: the effects of in-feed antibiotics on the microbiome at different gut locations. ISME J. 8, 1566–1576. Luby, E.M., Moorman, T.B., Soupir, M.L., 2016. Fate and transport of tylosin-resistant bacteria and macrolide resistance genes in artificially drained agricultural fields receiving swine manure. Sci. Total Environ. 550, 1126–1133. Luo, Y., Xu, L., Rysz, M., Wang, Y.Q., Zhang, H., Alvarez, P.J., 2011. Occurrence and transport of tetracycline, sulfonamide, quinolone, and macrolide antibiotics in the Haihe River basin, China. Environ. Sci. Technol. 45, 1827–1833.

McKinney, C.W., Loftin, K.A., Meyer, M.T., Davis, J.G., Pruden, A., 2010. Tet and sul antibiotic resistance genes in livestock lagoons of various operation type, configuration, and antibiotic occurrence. Environ. Sci. Technol. 44, 6102–6109. Mu, Q.H., Li, J., Sun, Y.X., Mao, D.Q., Wang, Q., Luo, Y., 2015. Occurrence of sulfonamide-, tetracycline-, plasmid-mediated quinolone- and macrolide-resistance genes in livestock feedlots in northern China. Environ. Sci. Pollut. Res. 22, 6932–6940. Pan, X., Qiang, Z.M., Ben, W.W., Chen, M.X., 2011. Residual veterinary antibiotics in swine manure from concentrated animal feeding operations in Shandong province, China. Chemosphere 84, 695–700. Peng, F.J., Zhou, L.J., Ying, G.G., Liu, Y.S., Zhao, J.L., 2014. Antibacterial activity of the soilbound antimicrobials oxytetracycline and ofloxacin. Environ. Toxicol. Chem. 33, 776–783. Peng, F.J., Ying, G.G., Liu, Y.S., Su, H.C., He, L.Y., 2015. Joint antibacterial activity of soiladsorbed antibiotics trimethoprim and sulfamethazine. Sci. Total Environ. 506-507, 58–65. Poulsen, L.L., Bisgaard, M., Son, N.T., Trung, N.V., An, H.M., Dalsgaard, A., 2012. Enterococcus faecalis clones in poultry and in humans with urinary tract infections, Vietnam. Emerg. Infect. Dis. 18, 1096–1100. Pruden, A., Arabi, M., Storteboom, H.N., 2012. Correlation between upstream human activities and riverine antibiotic resistance genes. Environ. Sci. Technol. 46, 11541–11549. Qian, X., Gu, J., Sun, W., 2017. Diversity, abundance, and persistence of antibiotic resistance genes in various types of animal manure following industrial composting. J. Hazard. Mater. 344, 716–722. Sapkota, A.R., Ojo, K.K., Roberts, M.C., Schwab, K.J., 2006. Antibiotic resistance genes in multidrug-resistant enterococcus spp. and streptococcus spp. recovered from the indoor air of a large-scale swine-feeding operation. Lett. Appl. Microbiol. 43, 534–540. Selvam, A., Xu, D., Zhao, Z., Wong, J.W.C., 2012. Fate of tetracycline, sulfonamide and fluoroquinolone resistance genes and the changes in bacterial diversity during composting of swine manure. Bioresour. Technol. 126, 383–390. Sergeant, M.J., Constantinidou, C., Cogan, T.A., Bedford, M.R., Penn, C.W., Pallen, M.J., 2014. Extensive microbial and functional diversity within the chicken cecal microbiome. PLoS One 9, e91941. Sköld, O., 2000. Sulfonamide resistance: mechanisms and trends. Drug Resist Update 3, 155–160. Sköld, O., 2001. Resistance to trimethoprim and sulfonamides. Vet. Res. 32, 261–273. Storteboom, H.N., Kim, S.C., Doesken, K.C., Carlson, K.H., Davis, J.G., Pruden, A., 2007. Response of antibiotics and resistance genes to high-intensity and low-intensity manure management. J. Environ. Qual. 36, 1695–1703. Storteboom, H., Arabi, M., Davis, J.G., Crimi, B., Pruden, A., 2010. Identification of antibiotic-resistance-gene molecular signatures suitable as tracers of pristine river, urban, and agricultural sources. Environ. Sci. Technol. 44, 1947–1953. Sun, W., Qian, X., Gu, J., Wang, X.J., Zhang, L., Guo, A.Y., 2017. Mechanisms and effects of arsanilic acid on antibiotic resistance genes and microbial communities during pig manure digestion. Bioresour. Technol. 234, 217–223. Sundin, G.W., Bender, C.L., 2010. Dissemination of the stra-strb streptomycin-resistance genes among commensal and pathogenic bacteria from humans, animals, and plants. Mol. Ecol. 5, 133–143. Thomas, C.M., Nielsen, K.M., 2005. Mechanisms of, and barriers to, horizontal gene transfer between bacteria. Nat. Rev. Microbiol. 3, 711–721. Turton, J.F., Shah, J., Ozongwu, C., Pike, R., 2010. Incidence of acinetobacter species other than A. baumannii among clinical isolates of acinetobacter: evidence for emerging species. J. Clin. Microbiol. 48, 1445–1449. Wang, Y., Zhang, W.J., Wang, J., Wu, C.M., Shen, Z.J., Fu, X., Yan, Y., Zhang, Q.J., Schwarz, S., Shen, J.Z., 2011. Distribution of the multidrug resistance gene cfr in staphylococcus species isolates from swine farms in China. Antimicrob. Agents Ch. 56, 1485–1490. Wu, N., Qiao, M., Zhang, B., Cheng, W.D., Zhu, Y.G., 2010. Abundance and diversity of tetracycline resistance genes in soils adjacent to representative swine feedlots in China. Environ. Sci. Technol. 44, 6933–6939. Wu, L.H., Pan, X., Like, C., Huang, Y.J., Teng, Y., Luo, Y.M., Christie, P., 2013. Occurrence and distribution of heavy metals and tetracyclines in agricultural soils after typical land use change in east China. Environ. Sci. Pollut. Res. 20, 8342–8354. Xiong, X., Li, Y.X., Li, W., Lin, C.Y., Han, W., Yang, M., 2010. Copper content in animal manures and potential risk of soil copper pollution with animal manure use in agriculture. Resour. Conserv. Recy. 54, 985–990. Yang, Q.X., Zhang, J., Zhu, K.F., Zhang, H., 2009. Influence of oxytetracycline on the structure and activity of microbial community in wheat rhizosphere soil. J. Environ. Sci. 21, 954–959. Yang, Q.X., Zhang, J., Zhang, W.Y., Wang, Z., Xie, Y.S., Zhang, H., 2010. Influence of tetracycline exposure on the growth of wheat seedlings and the rhizosphere microbial community structure in hydroponic culture. J. Environ. Sci. Heal. A, Part B. 45, 190–197. Yang, Q.X., Ren, S., Niu, T.Q., Guo, Y.H., Qi, S.Y., Han, X.K., 2014. Distribution of antibioticresistant bacteria in chicken manure and manure-fertilized vegetables. Environ. Sci. Pollut. Res. 21, 1231–1241. Yang, Q.X., Tian, T.T., Niu, T.Q., Wang, P., 2017. Molecular characterization of antibiotic resistance in cultivable multidrug-resistant bacteria from livestock manure. Environ. Pollut. 229, 188–198. Zhang, X.X., Zhang, T., 2011. Occurrence, abundance, and diversity of tetracycline resistance genes in 15 sewage treatment plants across China and other global locations. Environ. Sci. Technol. 45, 2598–2604. Zhang, T., Zhang, M., Zhang, X., Fang, H.H., 2009. Tetracycline resistance genes and tetracycline resistant lactose-fermenting enterobacteriaceae in activated sludge of sewage treatment plants. Environ. Sci. Technol. 43, 3455–3460. Zhang, Q.Q., Ying, G.G., Pan, C.G., Liu, Y.S., Zhao, J.L., 2015. Comprehensive evaluation of antibiotics emission and fate in the river basins of China: source analysis, multimedia modeling, and linkage to bacterial resistance. Environ. Sci. Technol. 49, 6772–6782.

L. Wang et al. / Science of the Total Environment 695 (2019) 133781 Zhang, Y.J., Hu, H.W., Gou, M., Wang, J.T., Chen, D., He, J.Z., 2017. Temporal succession of soil antibiotic resistance genes following application of swine, cattle and poultry manures spiked with or without antibiotics. Environ. Pollut. 231, 1621–1632. Zhao, L., Dong, Y.H., Wang, H., 2010. Residues of veterinary antibiotics in manures from feedlot livestock in eight provinces of China. Sci. Total Environ. 408, 1069–1075. Zhao, X., Wang, J.H., Zhu, L.S., Ge, W.L., Wang, J., 2017. Environmental analysis of typical antibiotic-resistant bacteria and ARGs in farmland soil chronically fertilized with chicken manure. Sci. Total Environ. 593-594, 10–17.

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Zhao, X., Wang, J.H., Zhu, L.S., Wang, J., 2019. Field-based evidence for enrichment of antibiotic resistance genes and mobile genetic elements in manure-amended vegetable soils. Sci. Total Environ. 654, 906–913. Zhu, Y.G., Johnson, T.A., Su, J.Q., Qiao, M., Guo, G.X., Stedtfeld, R.D., 2013. Diverse and abundant antibiotic resistance genes in Chinese swine farms. P. Natl. Acad. Sci. Usa. 110, 3435–3440. Zhu, J.C., Zhang, Z.Q., Fan, Z.M., Li, H.R., 2014. Biogas potential, cropland load and total amount control of animal manure in China. J. Agrometeorol. 33, 435–445.