Environ Sci Pollut Res (2014) 21:7276–7284 DOI 10.1007/s11356-014-2613-5
RESEARCH ARTICLE
Occurrence and abundance of tetracycline, sulfonamide resistance genes, and class 1 integron in five wastewater treatment plants Jing Du & Hongqiang Ren & Jinju Geng & Yan Zhang & Ke Xu & Lili Ding
Received: 30 September 2013 / Accepted: 30 January 2014 / Published online: 26 February 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract To understand the transport and fate of antibiotic resistance genes in wastewater treatment plants, 12 resistance genes (ten tetracycline resistance genes, two sulfonamides genes) and class 1 integron gene (intI1) were studied in five wastewater treatment plants with different treatment processes and different sewage sources. Among these resistance genes, sulfonamides genes (sul1 and sul2) were of the most prevalent genes with detection frequency of 100 %. The effluent water contained fewer types of resistance genes than the influent in most selected plants. The abundance of five quantified resistance genes (tetG, tetW, tetX, sul1, and intI1) decreased in effluent of plants treating domestic or industrial wastewater with anaerobic/aerobic or membrane bioreactor (MBR) technologies, but tetG, tetX, sul1, and intI1 increased along the treatment units of plants treating vitamin C production wastewater by anaerobic/aerobic technology. In plant treating cephalosporins production wastewater by UASB/aerobic process, the quantities of tetG, tetX, and sul1 first decreased in anaerobic effluent water but then increased in aerobic effluent water.
Keywords Tetracycline resistance gene . Sulfonamide resistance gene . Class 1 integron . Sewage source . Treatment process
Responsible editor: Gerald Thouand J. Du : H. Ren (*) : J. Geng (*) : Y. Zhang : K. Xu : L. Ding State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, 210046 Jiangsu, People’s Republic of China e-mail:
[email protected] e-mail:
[email protected]
Introduction Over the past several decades, various antibiotics have been extensively used in the prophylaxis and therapy of human and animal infections, and also used as animal growth promoters, which have escalated the problem of emergence of antibiotic resistance genes (Chopra and Roberts 2001; Davies and Davies 2010). Resistance pathogens induced by antibiotic resistance genes (ARGs) seriously threaten both the human health and the ecological security (Pruden et al. 2006; Baquero et al. 2008). Various types of antibiotic resistance genes have been detected in aquaculture farms (Jun et al. 2004; Akinbowale et al. 2006), wastewater lagoons (Peak et al. 2007), farmland soils around swine feedlots (Huang et al. 2013), as well as in surface water environment in China (Zhang et al. 2009c), among which tetracycline resistance genes and sulfonamide resistance genes are of the most prevalent genes reported in the surveys (Luo et al. 2010; Zhang et al. 2009b). Therefore, it is particularly important to identify the sources, fate, and transport of these resistance genes in the environment. Wastewater treatment plant (WWTP) is thought as one of the main genetic reactors where antibiotic resistance evolves (Baquero et al. 2008). Auerbach et al. (2007) reported that ten tetracycline resistance genes were detected in the selected activated sludge treatment plants, and samples collected from each unit of the plant contained more than three kinds of these genes. Schmitt et al. (2006) quantified the release of tetracycline resistance genes (tetW and tetO) and sulfonamideresistant gene (Sul1) into the environment through effluent and biosolids of different wastewater treatment utilities. LaPara et al. (2011) demonstrated that tertiary-treated municipal wastewater was a significant point source of ARGs to the natural environment (Duluth-Superior Harbor) through quantifying tetracycline resistance genes (tetA, tetW, and tetX) and
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class 1 integrons (intI1) along St. Louis River to the DuluthSuperior Harbor. However, previous studies mostly focused on the fate of antibiotic resistance genes in single specific plants, the transport process and fate of these genes in wastewater treatment plants with different sewage resources (especially industrial wastewater) and different treatment technologies were seldom considered. In this study, occurrence of ten tetracycline resistance genes, two sulfonamides resistance genes and intI1 gene from five WWTPs were investigated. Furthermore, three tetracycline resistance genes (tetG, tetW, and tetX), one sulfonamides gene (sul1) and class 1 integron gene (intI1) were selected for quantitative detection, because these genes were frequently detected in wastewater treatment system, and the three tetracycline resistance genes encode proteins that confer tetracycline resistance via each of the three known mechanisms (LaPara et al. 2011). The purpose of this study is: (1) to detect the occurrence of selected resistance genes in each treatment unit of five wastewater treatment plants; and (2) to investigate the abundance of resistance genes in five treatment plants treating different types of sewage with different treatment technologies.
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from sample collection location to the laboratory was typically less than 4 h, always less than 8 h. Five physicochemical parameters (NH4+–N, COD, TP, TN, and TOC) were measured for all the wastewater samples (Table 1). The analysis processes were conducted according to the national standard method of China (Wei 2002). Sample processing and DNA extraction After collection, water samples were passed through 0.45 μm mixed cellulose ester filters (Xinya, Shanghai, China) to concentrate microbial biomass (Pruden et al. 2006; Munir et al. 2011; Ma et al. 2011). Then the filters were immersed in 50 % ethanol for DNA protection, and stored at −20 °C for extracting DNA. DNA was extracted using FastDNA™ Spin Kit for soil (MP Biomedicals, Santa Ana, CA). Single DNA extraction was performed for each water sample. Extraction yield and quality of the extracted DNA were verified by spectrophotometry and agarose gel electrophoresis. Each DNA template was diluted to 20 ng/μL to perform the qualitative PCR, and diluted to 2 ng/μL to satisfy the quantitative PCR (qPCR). Qualitative PCR
Materials and methods Wastewater treatment plants and sampling sites Four plants in Jiangsu province, China, and one in Shanghai, China, were studied in this paper, and they were referred as plant A to plant E. Among them, plant A (AO, DW) adopted anaerobic/aerobic (A/O) process treating domestic wastewater; plant B (MBR, MW) adopted anaerobic/anoxic/aerobic (A2/O) - MBR process treating mixed wastewater of 56 % industrial wastewater (mainly electronics industry, machinery industry and pharmaceuticals industry wastewater) and 44 % domestic wastewater; plant C (AO, VW) adopted A/O process treating vitamin C production wastewater; plant D (UO, CW) adopted two levels up-flow anaerobic sludge bed (UASB)/ aerobic process treating cephalosporins production wastewater; and plant E (AO, PW) adopted A/O process treating petroleum chemical wastewater. The average daily flow rate of plant A to plant E were 580,000, 30,000, 8,000–9,000, 9,000–10,000, and 24,000 m3, respectively. Water samples were collected using sterile containers from influent water (after primary settling tank), anaerobic effluent, aerobic effluent and secondary effluent of five WWTPs in November 6th, 2012, to November 15th, 2012. The each water sample was collected in roughly equal volumes (about 2 L) per hour for 5 h, and well-mixed samples were used for subsequent ARG determination. All samples were kept in a bunker filled with ice packs and transported to the laboratory for immediate analysis. The duration of the transportation
Ten tetracycline resistance genes (five efflux pump genes: tetA, teB, tetC, tetE, tetG; four ribosomal protection genes: tetM, tetO, tetQ, tetW; and one enzymatic modification gene: tetX), two sulfonamides genes (sul1 and sul2), and class I integron gene (intI1) in the samples were investigated using PCR with the primers listed in Table 2. All PCR assays were conducted in 25 μL reactions containing 2.5 μL 10× PCR Buffer (TaKaRa, Japan), 2 μL MgCl2 (25 mM), 2 μL dNTP Mixture (each 2.5 mM), 0.2 μL of each forward and reverse primer (20 μM), and 0.125 μL Ex Taq DNA polymerase (5 U/ μL), 2 μL DNA template (∼20 ng/μL). The thermal cycles were performed on an Applied Biosystems Veriti Thermal Cycler (Life Technologies, USA) using the following conditions: initial denaturation at 94 °C 5 min, followed by 35 cycles of 94 °C for 45 s, annealing (varied-Table 2) for 45 s, 72 °C for 90 s, with a final extension of 72 °C for 10 min. PCR products was analyzed by electrophoresis on 1 % (w/v) agarose gel with ethidium bromide in 1× TAE buffer at 120 V for 15 min. To ensure reproducibility, duplicate PCR reactions were performed for each sample. Ultrapure water was used as the negative control in every run. Quantitative PCR TetG, tetW, tetX, intI1, and sul1 were selected for quantitative detection using the SYBR Green method (LaPara et al. 2011). qPCR reactions were conducted in 96-well plates with a final volume of 25 μL, containing 12.5 μL 2×power SYBR®
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Table 1 Chemical parameters of the water samples collected from five wastewater treatment plants
CODa
NH4+–N
TP
TN
TOC
Influent Secondary effluent Influent MBR effluent Influent
83.67 13.33 93.33 33.33 2,200.00
13.9 0.12 19.73 0.97 11.09
20.34 14.46 28.92 15.23 29.18
2.92 1.86 3.84 0.37 6.31
57.62 18.00 10.69 10.00 112.64
Anaerobic effluent Aerobic effluent Influent Anaerobic effluent Aerobic effluent Influent Secondary effluent
373.33 266.67 3,880.00 3,533.00 213.33 506.67 360.00
1.04 0.05 23.74 0.40 0.07 9.45 0.09
11.73 13.73 40.34 1.89 6.34 28.97 11.81
7.22 4.04 0.64 0.02 0.19 0.50 0.54
91.85 61.94 67.13 65.61 44.54 78.48 14.76
Sample sites Plant A Plant B
Plant C
Plant D Plant E a
Units of all the parameters are in milligram per liter
Green PCR Master Mix (Life Technologies, USA), 0.2 μL each primer (20 μM) and 2 μL template DNA (2 ng/μL). The thermal cycles were carried out on an Applied Biosystems 7500 (Life Technologies, USA) using the following procedure: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C or 65 °C for 1 min. Following
each run, melting curve was generated and analyzed to verify that nonspecific amplification did not occur. Each reaction was run in triplicate for each permutation of sample and primer set, as well as the negative controls in which ultrapure water was added instead of DNA template.
Table 2 Primers and annealing temperature Target
Sequences (5′–3′)
Annealing temperature (°C)
Amplicon size (bp)
Reference
Ng et al. 2001
TetA
F-W R-V
GCTACATCCTGCTTGCCTTC CATAGATCGCCGTGAAGAGG
55
210
TetB
F-W R-V F-W R-V F-W R-V F-W R-V F-W R-V F-W R-V F-W R-V F-W R-V F-W R-V F-W R-V F-W R-V F-W R-V
TTGGTTAGGGGCAAGTTTTG GTAATGGGCCAATAACACCG CTTGAGAGCCTTCAACCCAG ATGGTCGTCATCTACCTGCC AAACCACATCCTCCATACGC AAATAGGCCACAACCGTCAG GCAGAGCAGGTCGCTGG CCYGCAAGAGAAGCCAGAAG GTGGACAAAGGTACAACGAG CGGTAAAGTTCGTCACACAC AACTTAGGCATTCTGGCTCAC TCCCACTGTTCCATATCGTCA TTATACTTCCTCCGGCATCG ATCGGTTCGAGAATGTCCAC GAGAGCCTGCTATATGCCAGC GGGCGTATCCACAATGTTAAC AGCCTTACCAATGGGTGTAAA TTC TTA CCT TGG ACA TCC CG CGCACCGGAAACATCGCTGCAC TGAAGTTCCGCCGCAAGGCTCG TCCGGTGGAGGCCGGTATCTGG CGGGAATGCCATCTGCCTTGAG CCT CCC GCA CGA TGA TC TCC ACG CAT CGT CAG GC
55
659
55
418
55
278
65
134
Zhang et al. 2009b
55
406
Ng et al. 2001
50
515
55
904
60
168
Aminov et al. 2001
60
278
Schmitt et al. 2006
PCR:55, Q-PCR:65
163
Pei et al. 2006
55
191
60
280
TetC TetE TetG TetM TetO TetQ TetW TetX Sul1 Sul2 IntI1
Schmitt et al. 2006
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PCR product of each gene was purified using the Axyprep PCR Cleanup Kit (Axygen Scientific, USA), and cloned using pMD® 18-T Vector (TaKaRa, Japan). Plasmids carrying the corresponding genes were extracted and purified using MiniBest Plasmid Purification Kit Ver.3.0 (TaKaRa, Japan). Six- or five-point calibration curves were generated using tenfold serial dilution of the plasmids carrying the target genes. The PCR efficiency was 93–103 % and the R2 was 0.997–0.999. Statistical analysis In this study, removal of resistance genes in water samples by different treatment processes of five treatment plants was estimated by the following formula: ! c ij Removal of specific gene ð jÞ ¼ lg j ce
Here, j indicates specific gene, which includes tetG, tetW, tetX, sul1, and intI1. cji indicates the concentration of specific gene (j) in influent water of the treatment unit, copies/mL cje indicates the concentration of specific gene (j) in effluent water of the treatment unit, copies/mL. Independent t tests were conducted to test whether the log10 removal regarding to five quantified resistance genes were different between plants, and p0.05). Surprisingly, the quantities of resistance genes increased along the process of treatment in plant C (AO, VC) (Fig. 1b). For instance, the quantity of tetG increased from 102.37 copies/mL in influent to 102.97 copies/mL in anaerobic effluent, and finally increased to 104.38 copies/mL in aerobic effluent. TetX, intI1, and sul1 exhibited similar trends with tetG. Overall, the concentration of above four resistance genes increased 0.6–2.3 log10 after sludge treatment (Table 5). And it is generally accepted that pharmaceutical wastewater is an important source of antibiotic resistance genes into the environment (Lim et al. 2013; Saussereau et al. 2013). In plant D (UO, CW), trends of tetG, tetX, intI1, and sul1 were consistent, descending firstly in anaerobic effluent, then rising up in aerobic effluent (Fig. 1b). But the concentration of tetW decreased along the path of treatment. Similar result was found in previous study that tetG increased along the plant, while tetL, tetM, tetQ, and tetW decreased (Liu et al. 2012). The increase of resistance genes may be attributed to selection pressure provided by the cephalosporins and/or the more
Table 5 Log10 removal of antibiotic resistance genes and class 1 gene by different treatment units of five plants Log10 removal
Treatment process
TetG
TetW
TetX
IntI1
Sul1
Plant D
Anaerobic/aerobic Anaerobic/anoxic/aerobic—MBR Anaerobic Aerobic UASB
1.93 2.20 −0.55a −1.47 1.18
2.48 2.90 −0.48 0.16 1.15
1.41 1.71 −0.15 −2.18 1.13
1.93 2.15 −0.10 −0.82 1.01
1.88 2.07 −0.23 −0.37 0.83
Plant E
Aerobic Anaerobic/aerobic
−1.64 0.97
1.23 1.56
−1.60 1.67
−0.71 0.24
−0.06 0.63
Plant A Plant B Plant C
“log10” was calculated by the formula provided in “Statistical analysis” section a
“−” represent that resistance genes increased after treatment process
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extensive elimination of susceptible bacteria during wastewater treatment (Figueira et al. 2011). In plant E (AO, PW), the effluent contained lower concentrations of five determined genes than influent (Fig. 1c). The removal log10 of tetG, tetW, tetX, intI1, and sul1 were 0.97 log10, 1.56 log10, 1.67 log10, 0.24 log10, and 0.63 log10, respectively (Table 5). Compared with plant A (AO, DW) which also adopted AO treatment process and had all the determined genes reduced, plant E achieved less gene removal (p
