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Occurrences and distribution of sulfonamide and tetracycline resistance genes in the Yangtze River Estuary and nearby coastal area
MPB-07142; No of Pages 7 Marine Pollution Bulletin xxx (2015) xxx–xxx
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Occurrences and distribution of sulfonamide and tetracycline resistance genes in the Yangtze River Estuary and nearby coastal area Lan Lin a, Ke Yuan b, Ximei Liang d, Xin Chen b,e,f, Zongshan Zhao c, Ying Yang b,e,f, Shichun Zou b,e,f, Tiangang Luan b,e,f, Baowei Chen b,e,f,⁎ a
Zhujiang Hospital of Southern Medical University, Guangzhou 510282, PR China MOE Key Laboratory of Aquatic Product Safety, School of Marine Sciences, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, PR China Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Qingdao 266100, PR China d College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang 330045, PR China e Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, School of Marine Sciences, Sun Yat-Sen University, Guangzhou 510275, PR China f South China Sea Resource Exploitation and Protection Collaborative Innovation Center, Sun Yat-Sen University, Guangzhou 510275, PR China b c
a r t i c l e
i n f o
Article history: Received 9 July 2015 Received in revised form 18 August 2015 Accepted 19 August 2015 Available online xxxx Keywords: Antibiotic resistance genes Estuary Integron Dissemination
a b s t r a c t The role of highly impacted estuaries needs to be examined with respect to the spread of antibiotic resistance genes in the environment. In the present study, sulfonamide resistance (sul), tetracycline resistance (tet) and class I integron (int1) genes were ubiquitous in the sediments of the Yangtze Estuary (YE) and nearby coastal area, and exhibited a declining trend from the inner estuary to the coast. Good relationships were only observed between int1 and sul1 genes, implying that int1 gene is essential to the proliferation of sul1 gene. A non-significant correlation between int1 and 16S rRNA genes indicated that the int1 gene came from pollution sources of ARGs instead of being intrinsic in environmental bacterial populations. Sulfonamides were rarely detected in the sediments of this region, so could not result in the production of sul genes in the local environment. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Antibiotic resistance genes (ARGs) have been identified as a newly emerging contaminant (Ashbolt et al., 2013; Baquero, 2012; Pruden et al., 2013). Although health risks of ARG pollution have not been accurately assessed (Ashbolt et al., 2013; Pruden et al., 2013), increasing body of evidence demonstrated that ARGs can be exchanged between environmental bacteria and human pathogens via horizontal gene transfer (HGT), or vice versa (Stecher et al., 2013; Willems et al., 2011). ARGs in environment bacteria are often identical to those carried by a diverse lineage of clinical pathogens (Forsberg et al., 2012). The evolution of pathogens with multiple antibiotic resistances poses a serious threat to the public health, such as a longer hospitalization period and treatment duration, and treatment failures of infectious disease (Chen and Huang, 2013; Pond et al., 2014). The aquatic environments are well recognized as one of reservoirs or sinks of ARGs, and their importance is established for the spread and dissemination of ARGs in the environment (Zhang et al., 2009b). ARGs originally occur in the natural environment as a mean of competing or ⁎ Corresponding author at: MOE Key Laboratory of Aquatic Product Safety, School of Marine Sciences, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, PR China. E-mail address: [email protected] (B. Chen).
combating for limited nutrients between microorganisms in terms of species or community (D'Costa et al., 2011; Toth et al., 2010). Nevertheless, ARGs often become be abundant in the human-impacted settings with elevated selective stress caused by various pollutants (Modi et al., 2013; Qiu et al., 2012), especially antibiotic use in clinic therapeutic and husbandry (Barraud et al., 2013; Zhu et al., 2013). Consequently, massive amounts of both antibiotic chemicals and antibiotic resistant bacteria from typical anthropogenic sources, e.g., sewage and wastewater treatment plants (Yang et al., 2012), and pharmaceutical manufacturing operations (Khan et al., 2013), are released into nearby aquatic environments. A representative trait of ARGs is that they can be horizontally transferred between microbes using mobile genetic elements (MGEs) as the carriers, e.g., conjugative plasmids and transposons. Integrons associated with these MGEs were initially found to be related to antibiotic resistance (Stokes and Hall, 1989). Integrase-catalyzed recombination leads to the insertion or excision of gene cassettes at certain sites (e.g., attI and attC), and ARGs resided in these gene cassettes are highly related to a large variety of antibiotics including aminoglycosides, β-lactams chloramphenicol, etc. (Partridge et al., 2009). Until now, integrons have been found in approximately 9% of sequenced bacterial genomes (Labbate et al., 2009), as well as carried by some MGEs (Hall and Collis, 1995; Partridge et al., 2009). Acting as an important gene-acquiring mode, integrons facilitate horizontal gene transfer (HGT) of ARGs between microbes under selective pressure (Di Conza and Gutkind, 2010;
http://dx.doi.org/10.1016/j.marpolbul.2015.08.036 0025-326X/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article as: Lin, L., et al., Occurrences and distribution of sulfonamide and tetracycline resistance genes in the Yangtze River Estuary and nearby coastal area, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.036
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L. Lin et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx
Gaze et al., 2011; Gootz, 2010), e.g., the sul1 gene is found in the conserved region of the class 1 integron (int1) sequence (Bennett, 2008; Nigro et al., 2013). Int1 genes are the most abundant gene capture and transmission system in clinical and environmental isolates (Partridge et al., 2009; Shah et al., 2012; Yan et al., 2010), and their abundance and structures were greatly influenced by anthropogenic contamination (Wright et al., 2008). The Yangtze delta is one of important industrial and economic centers in China, with a high population of more than 25 million. Intensive human activities and operation in this region lead to a huge input of various pollutants into the YE and nearby coastal area, including antibiotics (Shi et al., 2014b; Yan et al., 2013), polycyclic aromatic hydrocarbons (Yang et al., 2008), polychlorinated biphenyls (Zhang et al., 2011), trace metals (An et al., 2009), posing potential environmental risk to the local ecosystem. Although a few studies have been conducted on characterizing typical pollution sources of ARGs in the Yangtze Delta (e.g., drinking water treatment plants) (Guo et al., 2014; Jiang et al., 2013), limited information on the abundance of ARGs and resistance determinants (e.g., int1 gene) in ambient aquatic environment is available. Sulfonamides and tetracyclines are widely used in human therapeutics and husbandry, as well as in aquaculture (Tolls, 2001). The usages of sulfonamides and tetracyclines in China in 2013 were estimated to be 7890 and 6950 tons, respectively (Zhang et al., 2015). The objective of this study was to investigate the occurrence and spatial distribution of tetracycline and sulfonamide resistance genes and int1 gene in the sediments of the YE and adjacent coastal area, which would provide baseline data for source identification and pollution controls of ARGs in this region.
2. Materials and methods 2.1. Chemicals and standards Analytical antibiotic standards were purchased from Sigma-Aldrich (St. Louis, MO, USA) for sulfadiazine (SDZ), sulfamethazine (SMZ), sulfamethoxazole (SMX), and sulfacetamide (SMMX) tetracycline (TC). 13C3-caffeine was purchased from Cambridge Isotope Labs (1 mg/mL in methanol, USA). Methanol (MeOH) and acetonitrile (ACN) were obtained from Merck (Darmstadt, Germany). Milli-Q water was prepared with a Milli-Q water purification system (Millipore, USA). Stock solutions of antibiotics (100 mg/L) were prepared in the methanol, and were stored in dark at −20 °C. Working solutions were freshly prepared daily for analysis.
2.2. Study areas and sample collection Sediment samples were collected from 13 strategic sites in the YE and nearby coastal area in July 2013, as shown in Fig. 1. The study area under investigation has been highly impacted by rapid urbanization and industrialization, as well as by intensive human activities. Four sampling sites (YZ1- YZ4) were located at the YE area. In the coastal area, four samples were collected from the north of the YE near the coast of Jiangsu province, two samples from the outer part of the YE, and three samples from the Hongzhou Bay in the south of the YE. Surface sediments (200 g) were collected from the top 10-cm layer using a grab sampler. The sediments were immediately mixed well and then frozen at −20 °C. All containers and tools were sterilized prior to use.
Fig. 1. Sampling sites in the YE and nearby coastal area.
Please cite this article as: Lin, L., et al., Occurrences and distribution of sulfonamide and tetracycline resistance genes in the Yangtze River Estuary and nearby coastal area, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.036
L. Lin et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx
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2.3. DNA extraction
2.7. Statistical analysis
DNA was extracted from the sediments using the PowerSoil® DNA Isolation Kit (MO BIO, CA) according to the manufacturer's protocol. In brief, approximately 0.25 g each of sediment samples was added into PowerSoil Bead tube, and then was votexed thoroughly. Following cell lysis and removals of organic and inorganic impurities, DNA-containing solutions were laden on spin filters. Spin filters binding with DNA were washed twice with ethanol, and thereafter were eluted with DNA-free water. The qualities and quantities of extracted DNA were measured using electrophoresis on 0.8% agarose gel and NanoDrop 2000 Spectrophotometer (Thermo Scientific, USA), respectively.
Linear fitting was conducted using SPSS® for Windows Release 16.0 (SPSS Inc. U.S.). Correlations above the level of p b 0.05 (95% confidence interval) were considered statistically significant.
2.4. Traditional polymerase chain reaction (PCR) The occurrences of ARGs in the water and sediments were first determined using traditional PCR. The sequences of the primers and the reaction conditions used in this study are presented in Table S1 in the Supporting Information (SI) section. Traditional PCR reactions were conducted in a 25-μL solution containing a 2 μL dNTP mixture (2.5 mM), 2.5 μL of 10× PCR buffer (Mg2+ Plus), 0.4 μL of each type of primer (20 μM), 0.2 μL of Taq polymerase (5 U/μL) (TaKaRa Biotechnology, China), and 1 μL of a DNA template. The thermal cycling program was carried out as follows: initially denaturing at 95 °C for 5 min, followed by 40 cycles of denaturing at 95 °C for 20 s, annealing at the corresponding temperature presented in Table S1 for 30–40 s, and finally elongating at 72 °C for 30 s. The final elongation was carried out at 72 °C for 10 min. The PCR products were run on the 3% agarose gel electrophoresis. 2.5. Real-time quantitative PCR The quantities of ARGs, int1, and 16S rRNA genes in the samples were measured using real-time quantitative PCR (q-PCR) (MJ Chromo4 Instrument, USA) using SYBR® Premix Ex Taq™ II (TaKaRa Biotechnology, China). The primers used for q-PCR were the same as those in Table S1 of the SI. The PCR reactions were performed in a 20-μL reaction mixture that comprised 10 μL of SYBR®Premix Ex Taq™ II (2×), 0.4 μL of each type of primer (20 μM), and 1 μL of a DNA template (5 ng). The thermocycling protocol consisted of an initial denaturation for 1 min at 95 °C, followed by 40 cycles of denaturation (95 °C, 10 s) and annealing at the temperatures in Tables S1. The specificity of the PCR products was checked by a melting curve analysis. The q-PCR standards were synthesized by cloning target genes into Escherichia coli DH5a using a PMD18-T vector Cloning Kit (TaKaRa Biotechnology, China). Vector concentrations were measured using NanoDrop, and the quantities of target genes per μL of solution were obtained basing on the length of plasmid and target gene sequence. Six-point calibration curves were generated by 10-fold serial dilutions of the plasmid carrying ARGs. All PCR reactions were run in parallel with serially diluted q-PCR standards and DNA-free water as the negative controls.
3. Results and discussion 3.1. Occurrences and quantities of ARGs and int1 in the YE and nearby coastal area Fig. 2 shows the occurrences and quantities of 16S rRNA genes, int1 gene and ARGs in the sediments of the YE and nearby coastal area. Sediments are considered as the major habitat of microbes in the aquatic environment (Gibbons et al., 2014; Lelieveld et al., 2003). Our results demonstrated that the mean level of 16S rRNA genes in the sediments of the YE and nearby coastal area was 1.34 × 109 copies per g sediment, which was approximately an order of magnitude lower than that in the Pearl River Estuary in China with identical primers being used (Chen et al., 2014). The int1, as one of mobile genetic carriers, was found in all samples, with a mean quantity of 3.7 × 106 copies per g sediment. Compared with other estuary and river in China (Chen et al., 2014; Luo et al., 2010a), the level of int1 gene in the YE and nearby coastal area was significantly lower as well. Sediments are also thought as an important reservoir and sink of ARGs in the aquatic environment (Chen et al., 2013; Luo et al., 2010a). Sulfonamide (sul) and tetracycline (tet) resistance genes were investigated for their occurrences in the sediments of the YE and adjacent coastal area. Fig. 2 showed that the total level of sul genes in the YE and adjacent coastal sediments was much higher than that of tet genes, as observed in typical pollution sources of ARGs in this region (Qu et al., 2012). The sul genes confer a resistance to sulfonamides by encoding alternative dihydropteroate synthase (DHPS) gene with lower affinity for this class of antibiotics (Skold, 2000). The sul genes were ubiquitous in the YE and nearby coastal area while the abundance of sul1 gene in the sediments was higher than that of sulII gene. Moreover, four subtypes of tet gene involved with different tetracycline resistance mechanisms were also analyzed, including tetA and tetE for efflux pump, tetM for ribosomal protection, and tetX for inactivating enzyme (Zhang et al., 2009a). Only tetA and tetM genes were detectable in the sediment samples from the YE and nearby coastal area, where tetA was more prevalent than tetM.
2.6. Analysis of antibiotics in the sediments Antibiotics were analyzed using an Agilent 1260 liquid chromatography (Agilent, Palo Alto, CA, USA) coupled with an Applied Biosystems 1200 Infinity Series tandem mass spectrometer equipped with an electrospray ionisation source operated in the positive mode. An Agilent Eclipse Plus C18 (3.0 × 150 mm; 5 μm) column was used for the separation of antibiotics. The mobile phase contained methanol (A) and 0.1% formic acid (B) and ran at a flow rate of 0.4 mL/min. A gradient elution program was described as follows: 0–5 min, 5% A; 5–10 min, 50% A; 10–18 min, 90% A; and 18–26 min, 5% A. A 20-μL aliquot of samples was injected. Details on the preparation method and MS/MS parameters are shown in the Supporting Information.
Fig. 2. Quantities of 16S rRNA, int1 and ARGs in the sediments of the YE and nearby coastal area. Error bar indicates one standard deviation (SD); n represents the number of sediment samples with the detection of genes. In total, thirteen sediment samples were collected from the YE and nearby coastal area in this study.
Please cite this article as: Lin, L., et al., Occurrences and distribution of sulfonamide and tetracycline resistance genes in the Yangtze River Estuary and nearby coastal area, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.036
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3.2. Spatial distribution of ARGs and int1 gene in the YE and nearby coastal area Fig. 3 shows the spatial distribution of total abundance of ARGs within the YE and nearby coastal area. Total abundance of ARGs was obtained from the total quantity of ARGs normalized to the quantity of 16S rRNA in the same sample. In comparison to other sites, the total abundance of ARGs was higher at the inner part of the YE and Hongzhou Bay close to Shanghai City. It implied that ARG pollution in the YE and nearby coast is probably due to extensive anthropogenic activities related to the use and production of antibiotics, e.g., municipal wastewater (Guo et al., 2013), hospitals (Dong et al., 2013), feedlots (Qu et al., 2012), etc. ARG pollution in the ambient aquatic environment is formed mainly via two pathways, viz. direct discharges from potential sources of pollution in intracellular or/and extracellular forms, or the selection under elevated stress caused by enormous inputs of various pollutants in the estuary (Chen et al., 2013). Spatial patterns of each of target genes (int1, sul and tet genes) in the sediments within the YE and nearby coastal area are shown in Fig. 4. The int1 gene obviously exhibited a similar spatial pattern with the sul and tet genes in the YE and adjacent coast, probably indicating that int1 gene plays an important role in the dissemination of ARGs. With respect to sul and tet genes, the abundance of sul genes was consistently higher at the locations near Shanghai City than other locations whereas tet genes were more enriched only at Site YZ3. Distinct spatial pattern of sul genes from tet genes was likely due to their difference in the discharges from type sources of pollution. In the drinking water treatment plants at this region, the levels of sul genes were significantly higher than those of tet genes (Guo et al., 2014). Previous study also demonstrated that sul genes were more recalcitrant to be eliminated than tet
genes along sewage and wastewater treatment processes (Gao et al., 2012). It is undoubted that sul genes could be presented at a higher level in the ambient aquatic environment influenced by the discharges of sewage and wastewater treatment plants. 3.3. Relationships of int1 gene and ARGs in the YE and nearby coastal area Integrons are able to horizontally transfer ARGs between microbes and/or incorporate ARGs into chromosomal DNA, which are considered as one of important dissemination mechanisms of ARGs (Di Conza and Gutkind, 2010; Gootz, 2010). They are structurally consisted of a gene that encodes a site-specific recombinase and a shuffled gene cassette that carries diverse resistance genes. The int1 gene is widely found in a large variety of human commensal microflora as well as modern environments (Di Conza and Gutkind, 2010; Ndi and Barton, 2011; Shah et al., 2012; Yan et al., 2010). Assuming that int1 gene occurs as the functional genes of maintaining regular metabolism in the bacterial genome, int1 gene abundance could have a good relationship with the whole population of bacteria in the environment. Fig. 5 explicitly demonstrated that int1 gene abundance was irrelevant with that of bacterial 16S rRNA genes in the sediments of the YE and nearby coastal area. Therefore, the occurrences and abundances of int1 genes could be closely associated with the functions expressed by their gene cassettes, which often fortify the adaptability of susceptible bacteria to the external stress such as antibiotics (Partridge et al., 2009). Consequently, good relationships between int1 gene and total concentration of antibiotics were frequently observed in the aquatic environments (Luo et al., 2010b). Fig. 6 shows a good correlation of int1 gene and sul1 gene at the level of p b 0.01 instead of sul2 gene. The sul1 gene is resided in one terminal
Fig. 3. Spatial distribution of total ARGs in the YE and nearby coastal area. ARG abundance was obtained by summing the abundance of each of ARGs including sulI, sulII, tetA, and tetM.
Please cite this article as: Lin, L., et al., Occurrences and distribution of sulfonamide and tetracycline resistance genes in the Yangtze River Estuary and nearby coastal area, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.036
L. Lin et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx
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Fig. 4. Spatial distributions of int1 and ARGs in the sediments collected from YE and nearby coastal area.
conserved region of the int1 gene (Bennett, 2008; Nigro et al., 2013). It was not difficult to infer that the sul1 gene was enriched with transferring of the int1 genes between environmental microbes. Regardless of seasonal variations, sul1 abundance was always correlated well with int1 abundance in both the Haihe River and the Pearl River Estuary (PRE) (Chen et al., 2014; Luo et al., 2010b). On the contrary, the sul2 gene has not been found in the int1 gene sequence. Hence it is incurious that good correlation of sul2 and int1 genes was not observed in the YZ and adjacent coastal area. Differentiating relationships of sul genes with int1 gene probably imply that class I integron plays a significant role in the propagation of sul1 gene, whereas the dissemination of sul2 gene is mediated via other mechanisms such as mall plasmids of the IncQ family and pBP1 (Skold, 2000). The transposons (e.g., Tn402) that house the clinical class 1 integrons can often target resolution sites of larger genetic elements such as Tn21-like transposons and plasmids, which facilitate
the transposition of int1 genes within the genome and its transferring between bacteria (Partridge et al., 2009). Simultaneously, these translocations of int1 gene could also lead to the dissemination of sul1 gene in environmental bacteria. Most of sulfonamides were undetectable in the sediments of the YZ and nearby coastal area (Table S4) although analytical methods used in this study had comparable detection limits and recoveries of sulfonamides with previous studies (Liang et al., 2013; Shi et al., 2014a). SAs also exhibited the low detection frequency and average concentration in the sediments of other aquatic environments (Kim and Carlson, 2007). This phenomenon was likely attributed to the low Kd and Koc of sulfonamides, which indicates their low sorption affinities to soil and sediment particles (Thiele-Bruhn, 2003; Tolls, 2001). Hence it was questioned why a high level of sul genes conferring the resistance to sulfonamides was found in the YZ sediments. A sul genes-possessing
Fig. 5. Showing the relationship of int1 and 16S rRNA genes in the sediments of the YE and nearby coastal area.
Please cite this article as: Lin, L., et al., Occurrences and distribution of sulfonamide and tetracycline resistance genes in the Yangtze River Estuary and nearby coastal area, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.036
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L. Lin et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx
Fig. 6. Relationships between int1 and sul genes in the sediments of the YZ and nearby coastal area.
isolate was able to grow in the presence of 256 mg mL− 1 of sulfonamide, and even a susceptible strain had a minimum inhibitory concentration of 16 mg mL−1 (Gibreel and Skold, 1999). Obviously, sul genes could not be induced in situ by anthropogenic input of sulfonamides in the sediments. Alternatively, sul genes and their gene carriers (int1 gene), as well as antibiotics, were probably originated from typical sources of ARG pollution (Storteboom et al., 2010). ARGs including sul genes are more preferentially accumulated in the sediments than the overlaying water (Chen et al., 2014), yet sulfonamides were not (Thiele-Bruhn, 2003; Tolls, 2001), suggesting that different environmental behaviors led to distinct distributing patterns of antibiotics from their resistance genes in environmental compartments.
4. Conclusions The ARGs, including sul, tet and int1 genes, were widely distributed in the sediments of the YZ and nearby coastal regions, and exhibited a declining trend from the inner estuary to offshore area. Good correlation between int1 and sul1 genes implied that int1 gene played a significant role in the dissemination of sul1 gene rather than other ARGs. The absence of sulfonamides in the sediments elucidated that sul genes were mainly released from typical pollution sources of ARGs adjacent to the estuary, instead of being formed in situ. Effective regulation of pollution sources could ameliorate pollution status of ARGs in the aquatic environments.
Acknowledgments This work was supported by the Provincial Natural Science Foundation of Guangdong, China (2014A030313195), State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for EcoEnvironmental Sciences, Chinese Academy of Sciences (KF2014-01), and the National Natural Science Foundation of China (21177162, 41221004, 21407086, and 21477138).
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.marpolbul.2015.08.036.
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Please cite this article as: Lin, L., et al., Occurrences and distribution of sulfonamide and tetracycline resistance genes in the Yangtze River Estuary and nearby coastal area, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.036
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Please cite this article as: Lin, L., et al., Occurrences and distribution of sulfonamide and tetracycline resistance genes in the Yangtze River Estuary and nearby coastal area, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.036
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Occurrences and distribution of sulfonamide and tetracycline resistance genes in the Yangtze River Estuary and nearby coastal area Lan Lin a, Ke Yuan b, Ximei Liang d, Xin Chen b,e,f, Zongshan Zhao c, Ying Yang b,e,f, Shichun Zou b,e,f, Tiangang Luan b,e,f, Baowei Chen b,e,f,⁎ a
Zhujiang Hospital of Southern Medical University, Guangzhou 510282, PR China MOE Key Laboratory of Aquatic Product Safety, School of Marine Sciences, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, PR China Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Qingdao 266100, PR China d College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang 330045, PR China e Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, School of Marine Sciences, Sun Yat-Sen University, Guangzhou 510275, PR China f South China Sea Resource Exploitation and Protection Collaborative Innovation Center, Sun Yat-Sen University, Guangzhou 510275, PR China b c
a r t i c l e
i n f o
Article history: Received 9 July 2015 Received in revised form 18 August 2015 Accepted 19 August 2015 Available online xxxx Keywords: Antibiotic resistance genes Estuary Integron Dissemination
a b s t r a c t The role of highly impacted estuaries needs to be examined with respect to the spread of antibiotic resistance genes in the environment. In the present study, sulfonamide resistance (sul), tetracycline resistance (tet) and class I integron (int1) genes were ubiquitous in the sediments of the Yangtze Estuary (YE) and nearby coastal area, and exhibited a declining trend from the inner estuary to the coast. Good relationships were only observed between int1 and sul1 genes, implying that int1 gene is essential to the proliferation of sul1 gene. A non-significant correlation between int1 and 16S rRNA genes indicated that the int1 gene came from pollution sources of ARGs instead of being intrinsic in environmental bacterial populations. Sulfonamides were rarely detected in the sediments of this region, so could not result in the production of sul genes in the local environment. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Antibiotic resistance genes (ARGs) have been identified as a newly emerging contaminant (Ashbolt et al., 2013; Baquero, 2012; Pruden et al., 2013). Although health risks of ARG pollution have not been accurately assessed (Ashbolt et al., 2013; Pruden et al., 2013), increasing body of evidence demonstrated that ARGs can be exchanged between environmental bacteria and human pathogens via horizontal gene transfer (HGT), or vice versa (Stecher et al., 2013; Willems et al., 2011). ARGs in environment bacteria are often identical to those carried by a diverse lineage of clinical pathogens (Forsberg et al., 2012). The evolution of pathogens with multiple antibiotic resistances poses a serious threat to the public health, such as a longer hospitalization period and treatment duration, and treatment failures of infectious disease (Chen and Huang, 2013; Pond et al., 2014). The aquatic environments are well recognized as one of reservoirs or sinks of ARGs, and their importance is established for the spread and dissemination of ARGs in the environment (Zhang et al., 2009b). ARGs originally occur in the natural environment as a mean of competing or ⁎ Corresponding author at: MOE Key Laboratory of Aquatic Product Safety, School of Marine Sciences, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, PR China. E-mail address: [email protected] (B. Chen).
combating for limited nutrients between microorganisms in terms of species or community (D'Costa et al., 2011; Toth et al., 2010). Nevertheless, ARGs often become be abundant in the human-impacted settings with elevated selective stress caused by various pollutants (Modi et al., 2013; Qiu et al., 2012), especially antibiotic use in clinic therapeutic and husbandry (Barraud et al., 2013; Zhu et al., 2013). Consequently, massive amounts of both antibiotic chemicals and antibiotic resistant bacteria from typical anthropogenic sources, e.g., sewage and wastewater treatment plants (Yang et al., 2012), and pharmaceutical manufacturing operations (Khan et al., 2013), are released into nearby aquatic environments. A representative trait of ARGs is that they can be horizontally transferred between microbes using mobile genetic elements (MGEs) as the carriers, e.g., conjugative plasmids and transposons. Integrons associated with these MGEs were initially found to be related to antibiotic resistance (Stokes and Hall, 1989). Integrase-catalyzed recombination leads to the insertion or excision of gene cassettes at certain sites (e.g., attI and attC), and ARGs resided in these gene cassettes are highly related to a large variety of antibiotics including aminoglycosides, β-lactams chloramphenicol, etc. (Partridge et al., 2009). Until now, integrons have been found in approximately 9% of sequenced bacterial genomes (Labbate et al., 2009), as well as carried by some MGEs (Hall and Collis, 1995; Partridge et al., 2009). Acting as an important gene-acquiring mode, integrons facilitate horizontal gene transfer (HGT) of ARGs between microbes under selective pressure (Di Conza and Gutkind, 2010;
http://dx.doi.org/10.1016/j.marpolbul.2015.08.036 0025-326X/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article as: Lin, L., et al., Occurrences and distribution of sulfonamide and tetracycline resistance genes in the Yangtze River Estuary and nearby coastal area, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.036
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Gaze et al., 2011; Gootz, 2010), e.g., the sul1 gene is found in the conserved region of the class 1 integron (int1) sequence (Bennett, 2008; Nigro et al., 2013). Int1 genes are the most abundant gene capture and transmission system in clinical and environmental isolates (Partridge et al., 2009; Shah et al., 2012; Yan et al., 2010), and their abundance and structures were greatly influenced by anthropogenic contamination (Wright et al., 2008). The Yangtze delta is one of important industrial and economic centers in China, with a high population of more than 25 million. Intensive human activities and operation in this region lead to a huge input of various pollutants into the YE and nearby coastal area, including antibiotics (Shi et al., 2014b; Yan et al., 2013), polycyclic aromatic hydrocarbons (Yang et al., 2008), polychlorinated biphenyls (Zhang et al., 2011), trace metals (An et al., 2009), posing potential environmental risk to the local ecosystem. Although a few studies have been conducted on characterizing typical pollution sources of ARGs in the Yangtze Delta (e.g., drinking water treatment plants) (Guo et al., 2014; Jiang et al., 2013), limited information on the abundance of ARGs and resistance determinants (e.g., int1 gene) in ambient aquatic environment is available. Sulfonamides and tetracyclines are widely used in human therapeutics and husbandry, as well as in aquaculture (Tolls, 2001). The usages of sulfonamides and tetracyclines in China in 2013 were estimated to be 7890 and 6950 tons, respectively (Zhang et al., 2015). The objective of this study was to investigate the occurrence and spatial distribution of tetracycline and sulfonamide resistance genes and int1 gene in the sediments of the YE and adjacent coastal area, which would provide baseline data for source identification and pollution controls of ARGs in this region.
2. Materials and methods 2.1. Chemicals and standards Analytical antibiotic standards were purchased from Sigma-Aldrich (St. Louis, MO, USA) for sulfadiazine (SDZ), sulfamethazine (SMZ), sulfamethoxazole (SMX), and sulfacetamide (SMMX) tetracycline (TC). 13C3-caffeine was purchased from Cambridge Isotope Labs (1 mg/mL in methanol, USA). Methanol (MeOH) and acetonitrile (ACN) were obtained from Merck (Darmstadt, Germany). Milli-Q water was prepared with a Milli-Q water purification system (Millipore, USA). Stock solutions of antibiotics (100 mg/L) were prepared in the methanol, and were stored in dark at −20 °C. Working solutions were freshly prepared daily for analysis.
2.2. Study areas and sample collection Sediment samples were collected from 13 strategic sites in the YE and nearby coastal area in July 2013, as shown in Fig. 1. The study area under investigation has been highly impacted by rapid urbanization and industrialization, as well as by intensive human activities. Four sampling sites (YZ1- YZ4) were located at the YE area. In the coastal area, four samples were collected from the north of the YE near the coast of Jiangsu province, two samples from the outer part of the YE, and three samples from the Hongzhou Bay in the south of the YE. Surface sediments (200 g) were collected from the top 10-cm layer using a grab sampler. The sediments were immediately mixed well and then frozen at −20 °C. All containers and tools were sterilized prior to use.
Fig. 1. Sampling sites in the YE and nearby coastal area.
Please cite this article as: Lin, L., et al., Occurrences and distribution of sulfonamide and tetracycline resistance genes in the Yangtze River Estuary and nearby coastal area, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.036
L. Lin et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx
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2.3. DNA extraction
2.7. Statistical analysis
DNA was extracted from the sediments using the PowerSoil® DNA Isolation Kit (MO BIO, CA) according to the manufacturer's protocol. In brief, approximately 0.25 g each of sediment samples was added into PowerSoil Bead tube, and then was votexed thoroughly. Following cell lysis and removals of organic and inorganic impurities, DNA-containing solutions were laden on spin filters. Spin filters binding with DNA were washed twice with ethanol, and thereafter were eluted with DNA-free water. The qualities and quantities of extracted DNA were measured using electrophoresis on 0.8% agarose gel and NanoDrop 2000 Spectrophotometer (Thermo Scientific, USA), respectively.
Linear fitting was conducted using SPSS® for Windows Release 16.0 (SPSS Inc. U.S.). Correlations above the level of p b 0.05 (95% confidence interval) were considered statistically significant.
2.4. Traditional polymerase chain reaction (PCR) The occurrences of ARGs in the water and sediments were first determined using traditional PCR. The sequences of the primers and the reaction conditions used in this study are presented in Table S1 in the Supporting Information (SI) section. Traditional PCR reactions were conducted in a 25-μL solution containing a 2 μL dNTP mixture (2.5 mM), 2.5 μL of 10× PCR buffer (Mg2+ Plus), 0.4 μL of each type of primer (20 μM), 0.2 μL of Taq polymerase (5 U/μL) (TaKaRa Biotechnology, China), and 1 μL of a DNA template. The thermal cycling program was carried out as follows: initially denaturing at 95 °C for 5 min, followed by 40 cycles of denaturing at 95 °C for 20 s, annealing at the corresponding temperature presented in Table S1 for 30–40 s, and finally elongating at 72 °C for 30 s. The final elongation was carried out at 72 °C for 10 min. The PCR products were run on the 3% agarose gel electrophoresis. 2.5. Real-time quantitative PCR The quantities of ARGs, int1, and 16S rRNA genes in the samples were measured using real-time quantitative PCR (q-PCR) (MJ Chromo4 Instrument, USA) using SYBR® Premix Ex Taq™ II (TaKaRa Biotechnology, China). The primers used for q-PCR were the same as those in Table S1 of the SI. The PCR reactions were performed in a 20-μL reaction mixture that comprised 10 μL of SYBR®Premix Ex Taq™ II (2×), 0.4 μL of each type of primer (20 μM), and 1 μL of a DNA template (5 ng). The thermocycling protocol consisted of an initial denaturation for 1 min at 95 °C, followed by 40 cycles of denaturation (95 °C, 10 s) and annealing at the temperatures in Tables S1. The specificity of the PCR products was checked by a melting curve analysis. The q-PCR standards were synthesized by cloning target genes into Escherichia coli DH5a using a PMD18-T vector Cloning Kit (TaKaRa Biotechnology, China). Vector concentrations were measured using NanoDrop, and the quantities of target genes per μL of solution were obtained basing on the length of plasmid and target gene sequence. Six-point calibration curves were generated by 10-fold serial dilutions of the plasmid carrying ARGs. All PCR reactions were run in parallel with serially diluted q-PCR standards and DNA-free water as the negative controls.
3. Results and discussion 3.1. Occurrences and quantities of ARGs and int1 in the YE and nearby coastal area Fig. 2 shows the occurrences and quantities of 16S rRNA genes, int1 gene and ARGs in the sediments of the YE and nearby coastal area. Sediments are considered as the major habitat of microbes in the aquatic environment (Gibbons et al., 2014; Lelieveld et al., 2003). Our results demonstrated that the mean level of 16S rRNA genes in the sediments of the YE and nearby coastal area was 1.34 × 109 copies per g sediment, which was approximately an order of magnitude lower than that in the Pearl River Estuary in China with identical primers being used (Chen et al., 2014). The int1, as one of mobile genetic carriers, was found in all samples, with a mean quantity of 3.7 × 106 copies per g sediment. Compared with other estuary and river in China (Chen et al., 2014; Luo et al., 2010a), the level of int1 gene in the YE and nearby coastal area was significantly lower as well. Sediments are also thought as an important reservoir and sink of ARGs in the aquatic environment (Chen et al., 2013; Luo et al., 2010a). Sulfonamide (sul) and tetracycline (tet) resistance genes were investigated for their occurrences in the sediments of the YE and adjacent coastal area. Fig. 2 showed that the total level of sul genes in the YE and adjacent coastal sediments was much higher than that of tet genes, as observed in typical pollution sources of ARGs in this region (Qu et al., 2012). The sul genes confer a resistance to sulfonamides by encoding alternative dihydropteroate synthase (DHPS) gene with lower affinity for this class of antibiotics (Skold, 2000). The sul genes were ubiquitous in the YE and nearby coastal area while the abundance of sul1 gene in the sediments was higher than that of sulII gene. Moreover, four subtypes of tet gene involved with different tetracycline resistance mechanisms were also analyzed, including tetA and tetE for efflux pump, tetM for ribosomal protection, and tetX for inactivating enzyme (Zhang et al., 2009a). Only tetA and tetM genes were detectable in the sediment samples from the YE and nearby coastal area, where tetA was more prevalent than tetM.
2.6. Analysis of antibiotics in the sediments Antibiotics were analyzed using an Agilent 1260 liquid chromatography (Agilent, Palo Alto, CA, USA) coupled with an Applied Biosystems 1200 Infinity Series tandem mass spectrometer equipped with an electrospray ionisation source operated in the positive mode. An Agilent Eclipse Plus C18 (3.0 × 150 mm; 5 μm) column was used for the separation of antibiotics. The mobile phase contained methanol (A) and 0.1% formic acid (B) and ran at a flow rate of 0.4 mL/min. A gradient elution program was described as follows: 0–5 min, 5% A; 5–10 min, 50% A; 10–18 min, 90% A; and 18–26 min, 5% A. A 20-μL aliquot of samples was injected. Details on the preparation method and MS/MS parameters are shown in the Supporting Information.
Fig. 2. Quantities of 16S rRNA, int1 and ARGs in the sediments of the YE and nearby coastal area. Error bar indicates one standard deviation (SD); n represents the number of sediment samples with the detection of genes. In total, thirteen sediment samples were collected from the YE and nearby coastal area in this study.
Please cite this article as: Lin, L., et al., Occurrences and distribution of sulfonamide and tetracycline resistance genes in the Yangtze River Estuary and nearby coastal area, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.036
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3.2. Spatial distribution of ARGs and int1 gene in the YE and nearby coastal area Fig. 3 shows the spatial distribution of total abundance of ARGs within the YE and nearby coastal area. Total abundance of ARGs was obtained from the total quantity of ARGs normalized to the quantity of 16S rRNA in the same sample. In comparison to other sites, the total abundance of ARGs was higher at the inner part of the YE and Hongzhou Bay close to Shanghai City. It implied that ARG pollution in the YE and nearby coast is probably due to extensive anthropogenic activities related to the use and production of antibiotics, e.g., municipal wastewater (Guo et al., 2013), hospitals (Dong et al., 2013), feedlots (Qu et al., 2012), etc. ARG pollution in the ambient aquatic environment is formed mainly via two pathways, viz. direct discharges from potential sources of pollution in intracellular or/and extracellular forms, or the selection under elevated stress caused by enormous inputs of various pollutants in the estuary (Chen et al., 2013). Spatial patterns of each of target genes (int1, sul and tet genes) in the sediments within the YE and nearby coastal area are shown in Fig. 4. The int1 gene obviously exhibited a similar spatial pattern with the sul and tet genes in the YE and adjacent coast, probably indicating that int1 gene plays an important role in the dissemination of ARGs. With respect to sul and tet genes, the abundance of sul genes was consistently higher at the locations near Shanghai City than other locations whereas tet genes were more enriched only at Site YZ3. Distinct spatial pattern of sul genes from tet genes was likely due to their difference in the discharges from type sources of pollution. In the drinking water treatment plants at this region, the levels of sul genes were significantly higher than those of tet genes (Guo et al., 2014). Previous study also demonstrated that sul genes were more recalcitrant to be eliminated than tet
genes along sewage and wastewater treatment processes (Gao et al., 2012). It is undoubted that sul genes could be presented at a higher level in the ambient aquatic environment influenced by the discharges of sewage and wastewater treatment plants. 3.3. Relationships of int1 gene and ARGs in the YE and nearby coastal area Integrons are able to horizontally transfer ARGs between microbes and/or incorporate ARGs into chromosomal DNA, which are considered as one of important dissemination mechanisms of ARGs (Di Conza and Gutkind, 2010; Gootz, 2010). They are structurally consisted of a gene that encodes a site-specific recombinase and a shuffled gene cassette that carries diverse resistance genes. The int1 gene is widely found in a large variety of human commensal microflora as well as modern environments (Di Conza and Gutkind, 2010; Ndi and Barton, 2011; Shah et al., 2012; Yan et al., 2010). Assuming that int1 gene occurs as the functional genes of maintaining regular metabolism in the bacterial genome, int1 gene abundance could have a good relationship with the whole population of bacteria in the environment. Fig. 5 explicitly demonstrated that int1 gene abundance was irrelevant with that of bacterial 16S rRNA genes in the sediments of the YE and nearby coastal area. Therefore, the occurrences and abundances of int1 genes could be closely associated with the functions expressed by their gene cassettes, which often fortify the adaptability of susceptible bacteria to the external stress such as antibiotics (Partridge et al., 2009). Consequently, good relationships between int1 gene and total concentration of antibiotics were frequently observed in the aquatic environments (Luo et al., 2010b). Fig. 6 shows a good correlation of int1 gene and sul1 gene at the level of p b 0.01 instead of sul2 gene. The sul1 gene is resided in one terminal
Fig. 3. Spatial distribution of total ARGs in the YE and nearby coastal area. ARG abundance was obtained by summing the abundance of each of ARGs including sulI, sulII, tetA, and tetM.
Please cite this article as: Lin, L., et al., Occurrences and distribution of sulfonamide and tetracycline resistance genes in the Yangtze River Estuary and nearby coastal area, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.036
L. Lin et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx
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Fig. 4. Spatial distributions of int1 and ARGs in the sediments collected from YE and nearby coastal area.
conserved region of the int1 gene (Bennett, 2008; Nigro et al., 2013). It was not difficult to infer that the sul1 gene was enriched with transferring of the int1 genes between environmental microbes. Regardless of seasonal variations, sul1 abundance was always correlated well with int1 abundance in both the Haihe River and the Pearl River Estuary (PRE) (Chen et al., 2014; Luo et al., 2010b). On the contrary, the sul2 gene has not been found in the int1 gene sequence. Hence it is incurious that good correlation of sul2 and int1 genes was not observed in the YZ and adjacent coastal area. Differentiating relationships of sul genes with int1 gene probably imply that class I integron plays a significant role in the propagation of sul1 gene, whereas the dissemination of sul2 gene is mediated via other mechanisms such as mall plasmids of the IncQ family and pBP1 (Skold, 2000). The transposons (e.g., Tn402) that house the clinical class 1 integrons can often target resolution sites of larger genetic elements such as Tn21-like transposons and plasmids, which facilitate
the transposition of int1 genes within the genome and its transferring between bacteria (Partridge et al., 2009). Simultaneously, these translocations of int1 gene could also lead to the dissemination of sul1 gene in environmental bacteria. Most of sulfonamides were undetectable in the sediments of the YZ and nearby coastal area (Table S4) although analytical methods used in this study had comparable detection limits and recoveries of sulfonamides with previous studies (Liang et al., 2013; Shi et al., 2014a). SAs also exhibited the low detection frequency and average concentration in the sediments of other aquatic environments (Kim and Carlson, 2007). This phenomenon was likely attributed to the low Kd and Koc of sulfonamides, which indicates their low sorption affinities to soil and sediment particles (Thiele-Bruhn, 2003; Tolls, 2001). Hence it was questioned why a high level of sul genes conferring the resistance to sulfonamides was found in the YZ sediments. A sul genes-possessing
Fig. 5. Showing the relationship of int1 and 16S rRNA genes in the sediments of the YE and nearby coastal area.
Please cite this article as: Lin, L., et al., Occurrences and distribution of sulfonamide and tetracycline resistance genes in the Yangtze River Estuary and nearby coastal area, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.036
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L. Lin et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx
Fig. 6. Relationships between int1 and sul genes in the sediments of the YZ and nearby coastal area.
isolate was able to grow in the presence of 256 mg mL− 1 of sulfonamide, and even a susceptible strain had a minimum inhibitory concentration of 16 mg mL−1 (Gibreel and Skold, 1999). Obviously, sul genes could not be induced in situ by anthropogenic input of sulfonamides in the sediments. Alternatively, sul genes and their gene carriers (int1 gene), as well as antibiotics, were probably originated from typical sources of ARG pollution (Storteboom et al., 2010). ARGs including sul genes are more preferentially accumulated in the sediments than the overlaying water (Chen et al., 2014), yet sulfonamides were not (Thiele-Bruhn, 2003; Tolls, 2001), suggesting that different environmental behaviors led to distinct distributing patterns of antibiotics from their resistance genes in environmental compartments.
4. Conclusions The ARGs, including sul, tet and int1 genes, were widely distributed in the sediments of the YZ and nearby coastal regions, and exhibited a declining trend from the inner estuary to offshore area. Good correlation between int1 and sul1 genes implied that int1 gene played a significant role in the dissemination of sul1 gene rather than other ARGs. The absence of sulfonamides in the sediments elucidated that sul genes were mainly released from typical pollution sources of ARGs adjacent to the estuary, instead of being formed in situ. Effective regulation of pollution sources could ameliorate pollution status of ARGs in the aquatic environments.
Acknowledgments This work was supported by the Provincial Natural Science Foundation of Guangdong, China (2014A030313195), State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for EcoEnvironmental Sciences, Chinese Academy of Sciences (KF2014-01), and the National Natural Science Foundation of China (21177162, 41221004, 21407086, and 21477138).
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.marpolbul.2015.08.036.
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Please cite this article as: Lin, L., et al., Occurrences and distribution of sulfonamide and tetracycline resistance genes in the Yangtze River Estuary and nearby coastal area, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.036