Complex migration of antibiotic resistance in natural aquatic environments

Environmental Pollution xxx (2017) 1e9

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Complex migration of antibiotic resistance in natural aquatic environments* Hui Gao a, Linxiao Zhang a, b, Zihao Lu a, Chunming He a, c, Qianwei Li a, c, Guangshui Na a, * a

Key Laboratory for Ecological Environment in Coastal Areas (SOA), National Marine Environmental Monitoring Center, Dalian, China School of Biological Engineering, Dalian Polytechnic University, Dalian 116034, China c School of Marine Science, Shanghai Ocean University, Shanghai 201306, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 December 2016 Received in revised form 16 May 2017 Accepted 20 August 2017 Available online xxx

Antibiotic resistance is a worsening global concern, and the environmental behaviors and migration patterns of antibiotic resistance genes (ARGs) have attracted considerable interest. Understanding the long-range transport of ARG pollution is crucial. In this study, we characterized the dynamics of ARG changes after their release into aquatic environments and demonstrated the importance of traditional chemical contaminants in the transmission mechanisms of ARGs. We hypothesized that the main route of ARG proliferation switches from active transmission to passive transmission. This antibioticdominated switch is motivated and affected by non-corresponding contaminants. The effect of anthropogenic activities gradually weakens from inland aquatic environments to ocean environments; however, the effect of changes in environmental conditions is enhanced along this gradient. The insights discussed in this study will help to improve the understanding of the distribution and migration of ARG pollution in various aquatic environments, and provide a modern perspective to reveal the effect of corresponding contaminants and non-corresponding contaminants in the process of antibiotic resistance proliferation. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Antibiotic resistance Aquatic environments Pathogens Non-corresponding contaminants

1. Introduction A large number of studies have shown that the misuse and abuse of antibiotics has accelerated the emergence of drug resistance (Wollenberger et al., 2000; Hu et al., 2008; Rysz and Alvarez, 2004). The emergence and diffusion of antibiotic resistance within a wide range of infectious agents is a growing concern for global public health. In particular, the proliferation of antibiotic resistance genes (ARGs) and antibiotic resistance bacteria (ARB) will increase the migration, transformation and diffusion of antibiotic resistance among environmental bacterial groups, eventually leading to environmental bacteria becoming a natural reservoir for the various ARGs. Gene pollutants have the biological characteristics that “may be reproduced or transmitted” combined with the physicochemical characteristic of “environmental persistence,” resulting in ecological damage that is more direct than ordinary

* This paper has been recommended for acceptance by Dr. Harmon Sarah Michele. * Corresponding author. E-mail address: [email protected] (G. Na).

chemical pollutants, more easily spread, and more difficult to control and eliminate. In recent years, research into the persistence of antibiotic resistance in the environment has increased. ARGs and ARB have been isolated from the soil, sediment, urban sewage, aquaculture wastewater, rivers and even the marine and polar environments
ceres (Zhang et al., 2016; Yang et al., 2017; Joy et al., 2014; Calero-Ca et al., 2017; Na et al., 2014). These results were not only shown in developing countries with a larger use of antibiotics and a wider range of use but also in developed countries that have relatively stricter usages. The antibiotic usage rates in inpatient and surgical operations in China were 80%, and 95%, respectively, which were both far higher than in developed countries such as the Europe and the United States which exhibited rates of 22%e25%, as well as the international average of 30% according to the World Health Organization (Zhang, 2012). ARG pollution is released from its source pollution and can enter the natural aquatic environment both directly and indirectly. Important release sources include wastewaters from hospitals and agricultural livestock systems. For example, Szekeres et al. (2017) found that sul1 and sul2, which are related to sulfonamide, were

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present in the hospital wastewater of a Romanian study site. Further, studies have indicated that the aforementioned ARGs were present in the hospital wastewater of a Chinese study site, agricultural wastewater in Italy and swine wastewater in the United States (Li et al., 2016a; Luprano et al., 2016; Zhang et al., 2013). Antibiotic resistance contaminants can be directly released to the primary reception system, which is connected to the release source (e.g., via wastewater treatment plants [WWTPs]) (Szczepanowski et al., 2004; Schlüter et al., 2007). Furthermore, antibiotic resistance contaminants can leach to groundwater or be carried by runoff and erosion to the secondary reception system (groundwater and surface water; Tennstedt et al., 2005; Chen et al., 2007; Auerbach et al., 2007), and finally converge to the tertiary reception system (estuaries and nearby coastal and ocean systems) under the influence of hydrological dynamics. These release and reception systems ultimately lead to complex migration patterns of antibiotic resistance. Environmental reception systems at all levels run through the entire process of long-distance migration and diffusion of antibiotic resistance (from source to surface water to the ocean) (Fig. 4). The effect of anthropogenic activities becomes gradually weaker, whereas the effect of changes in environmental conditions (pollutants and the physical and chemical factors) is enhanced from the river to the ocean. Different environmental conditions affect the rate of contribution of each mode of transmission in the process of ARG migration, thereby affecting the characteristics of ARG pollution in the environmental medium. This review presents an overview of the studies on antibiotic resistance in different reception systems to reveal the large-scale diffusion of antibiotic resistance and subsequently study its globalization trend. Furthermore, we hypothesized that the main pathway of ARGs proliferation switches from active transmission, which is dominated by corresponding contaminants (antibiotics), to passive transmission, which is motivated and affected by non-corresponding contaminants (heavy metals, organic pollutants and physical and chemical factors). Overall, the insights discussed in this review will help improve the understanding of the distribution and fate of ARG pollution in various aquatic environments, and provide a modern perspective on the effect of corresponding contaminants and noncorresponding contaminants in the process of ARG proliferation. 2. Pollution levels of antibiotic resistance during their migration in various aquatic environments Since Pruden et al. (2006) put forward ARGs as a new type of environmental pollutant, they have attracted considerable attention in environmental research. Studies mainly in European countries, the US, and China have found multiple varieties of ARGs in wastewater, lakes, rivers, groundwater, and even coastal water (Joy et al., 2014; Yang et al., 2017; Calero-C
aceres et al., 2017; Na et al., 2014). ARGs are not only leached to groundwater or carried by runoff or erosion like typical chemical pollutants, but they can also be vertically transmitted to microbial offspring via proliferation dynamics, or passed among bacteria via horizontal transfer. These various complex transport processes have led to the complicated problem of assessing ARG pollution in the environment. 2.1. Pollution levels about the release source Applications of antibiotics in medical and aquaculture industries have accelerated the generation and discharge of ARB, eventually resulting in hospital and animal husbandry wastewaters becoming the main sources of ARG distribution into the environment. According to CHINET which surveys bacterial resistance in China (2015) statistics, among 17,309 clinically collected isolates of

Escherichia coli from 20 Chinese hospitals, the prevalence of ampicillin resistance was as high as 85.5%. In addition, ciprofloxacin and sulfamethoxazole resistant strains accounted for an average of 57.8% and 58.1% of E. coli, respectively (Hu et al., 2015). With the increase in resistance levels, at least 700,000 people die due to antibiotic-resistant infections each year, and this figure is expected to exceed 10,000,000 in 2050 (Antimicrobial resistance global report on surveillance, 2014). In recent decades, researchers have detected 133 different ARGs in hospital, and animal husbandry wastewaters with activity towards 12 types of widely used antibiotics (Fig. 1, Table S1). Among all classes of ARGs detected in different hospital wastewaters, tetracycline resistance genes (tet genes) and sulfonamide resistance genes (sul genes) have the highest detection frequency. The average detection rate of both ARGs reached 100% (Fig. 1, Table S4). Furthermore, other ARGs also detected frequently include aminoglycoside resistance genes. The concentration and abundance of all different types of ARGs were 101e1.81
1011 copies/mL and 6.30
107e2.81
101 copies/16S rRNA, respectively, in hospital wastewaters (Fig. 2). Researchers have mainly focused on Enterococcus of gram-positive bacteria and P. aeruginosa of gram-negative bacteria among the ARB in hospital wastewaters (Varela et al., 2013; Hocquet et al., 2016). For ARB, the resistance to gentamicin and meropenem has received the most attention and the mean resistance rates were 24.1% and 11.8%, respectively (Table S5). Among all classes of ARGs detected in different animal husbandry wastewaters, sul genes and tet genes have the highest detection frequency with an average detection rate reaching 100% and 99.7%, respectively (Fig. 1, Table S4). The concentration and abundance of all kinds of ARGs were 100.94e2.43
1011 copies/mL and 108e6.30
101 copies/16S rRNA, respectively (Figs. 2 and 3). For ARB in the animal husbandry wastewaters, researchers have paid more attention to sulfamethoxazole resistance, for which the mean resistance rate is 46.4% (Table S5). This may be related to the extensive use of sulfamethoxazole in animal husbandry. Bacillus, Acinetobacter and Vibrio spp. were most common among the sulfamethoxazole resistant bacteria identified in animal husbandry (Gao et al., 2012a; Hoa et al., 2011). Overall, the concentration and abundance of all kinds of ARGs is mainly focused on 106e109 copies/mL and 103.1e101.6 copies/16S rRNA in the hospital and animal husbandry wastewaters, the median values were 107.1 copies/mL and 102.2 copies/16S rRNA, respectively. However, in some severely polluted areas, the concentration and abundance of ARGs reached 1010e1011 copies/mL and 102e101 copies/16S rRNA. Many researchers have indicated that the construction and abundance of antibiotic resistance is closely related to the usage of antibiotics in medical and aquaculture industries. For example, Peak et al. (2007) analyzed the correlation between ARGs and antibiotics in cattle wastewater of Kansas and found a strong correlation (r ¼ 0.67, p < 0.01). Chen et al. (2013) also found that the abundance of ARGs had a significant correlation with the concentration of antibiotics in the aquaculture areas of the Pearl River Estuary (winter, r ¼ 0.734, p < 0.0001; summer, r ¼ 0.740, p < 0.0001). Kerry et al. (1996) compared aquaculture ponds to a river without antibiotic pollution and found that the number of bacteria with tetracycline and chloramphenicol resistance was significantly higher in the aquaculture ponds. This may explain why the main type of antibiotic resistance is different from different release sources. The types of ARGs in the hospital wastewaters mainly correspond with the antibiotics used in human applications, such as tetracycline and aminoglycoside, but the types of ARGs in the animal husbandry wastewaters mainly correspond with the antibiotics used in animals, such as sulfonamides and tetracycline.

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Fig. 1. The detection frequency (%) of different ARGs.

Fig. 2. The concentration of ARGs (copies/mL).

2.2. Pollution levels in the primary reception system WWTPs are a receiving system for hospital, animal husbandry and urban wastewaters and their effluent is an important route through which ARGs enter aquatic environments. Researchers have detected 90 different ARGs in WWTP influents, including 12 types of antibiotics that are widely used (Fig. 1, Table S2). Among all classes of ARGs detected in different WWTP influents, sul genes and

tet genes have the highest detection frequency for which the average detection rate reached 100% in both cases (Fig. 1, Table S4). The concentration and abundance of all kinds of ARGs are 3.3
100e9.73
1010 copies/mL and 2.2
106e9.9
101 copies/ 16S rRNA, respectively, which mainly focused on 105e108 copies/ mL and 103.6-102 copies/16S rRNA, respectively (Figs. 2 and 3). The median values were 106 copies/mL and 103 copies/16S rRNA. However, in severely polluted areas, the concentration and

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Fig. 3. The abundance of ARGs (copies/16S rRNA).

Fig. 4. Conceptual representation of the key factors influencing the environmental fate and transport of ARGs. *Numbers correspond to enumerated items in the text, including: (1) Correspondence contaminants (antibiotics), (2) Non-correspondence contaminants (heavy metals, OPs), and (3) Physical and chemical factors (temperature, pH).

abundance of ARGs reached 108e1010 copies/mL and 102e101 copies/16S rRNA, respectively. The concentration of ARB was between ND to 107.08 CFU/mL, which mainly focused on 104e105 CFU/ mL (Guo et al., 2013a, 2013b; Huang et al., 2015a; Munir et al., 2011; Makowska et al., 2016). Resistance to tetracycline and ciprofloxacin was frequently detected, with the mean resistance rate reaching 16.6% and 13.0%, respectively (Table S5). Although removal of ARGs occurs, most ARGs are still present in WWTP effluents at a certain concentration (Du et al., 2015; Li et al., 2017; Sharma et al., 2016). Researchers have detected 84 different ARGs in WWTP effluents covering 12 types of widely used antibiotics (Fig. 1, Table S2). The concentration and abundance of all kinds of ARGs are between 1.0
101 and 6.46
109 copies/mL and between 106 and 3.63
101 copies/16S rRNA, respectively, which

mainly focused on 103.7e107 copies/mL and 104e102.5 copies/16S rRNA, respectively (Figs. 2 and 3). The median values were 105 copies/mL and 103.1 copies/16S rRNA. Among all classes of ARGs detected in different WWTP effluents, sul genes and tet genes showed the highest detection frequency. The average detection rate reached 100% in both cases (Fig. 1, Table S4). Sul genes and tet genes have been reported to be more abundant than other ARGs in studies focusing on different types of WWTPs (Xu et al., 2015; Chen and Zhang, 2013a) and the same conclusion was obtained in this review. The concentration of ARB is between 5e2.0
105 CFU/mL, which mainly focused on 103e104 CFU/mL (Gao et al., 2012b; Munir et al., 2011; Huang et al., 2012; Makowska et al., 2016). The resistance to tetracycline and ciprofloxacin were detected frequently, the mean resistance rate reached 16.6% and 9.6%, respectively

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(Table S5). Among the resistant heterotrophs, Aeromonas spp., Escherichia spp, Staphylococcus aureus, Enterococcus faecalis and Salmonella were common (Everage et al., 2014; Zhang et al., 2015; Mahmoud et al., 2013). Researchers showed that the construction and abundance of antibiotic resistance are significantly correlated with antibiotic concentrations. For example, Xu et al. (2015) demonstrated that relative tet gene copies (tetB and tetW) were strongly correlated with the concentrations of tetracycline residues in water samples collected from a WWTP and its surrounding environment. In conclusion, there have been various ARGs and ARB detected in WWTP influents and effluents. The classes and concentration of ARGs in effluents are lower than in influents; however, no significant difference was shown for the abundance of ARGs. The ARG concentrations in effluents are one order of magnitude lower than that in influents. A comparison of the ARG pollution in the primary reception system with that in the release source indicated that the classes and concentrations of ARGs were reduced but that there was no significant difference in abundances. The concentration of ARGs in effluents is about two orders of magnitude lower than that in the release source. This may indicate that the treatment technology of WWTPs is at least partially successful in eliminating ARGs. However, the sul genes and tet genes both retain the highest detection frequency in the release source and in the primary reception system. 2.3. Pollution levels in the secondary reception system Although the treatment process of WWTPs can remove some of the ARGs and ARB, many ARGs and ARB are still released into the surface water. Researchers have shown that ARGs and ARB in septic storage of farms could enter the groundwater and surface water through infiltration and leakage (Chee-Sanford et al., 2009). In recent years, researchers have detected 59 different ARGs in the secondary reception system, covering 12 types of widely used antibiotics (Fig. 1, Table S3). Among all classes of ARGs detected in the secondary reception system, sul genes and tet genes had the highest detection frequency, reaching 98.1% and 90.1% on average, respectively (Fig. 1, Table S4). Overall, the concentration and abundance of all kinds of ARGs were 2.5
100e108.26 copies/mL, 9.65
107e100 copies/16S rRNA, respectively (Figs. 2 and 3). The concentration and abundance of all kinds of ARGs mainly focused on 102e105.2 copies/ mL, 103.7e101.7 copies/16S rRNA. The median values were 103.0 copies/mL and 102.8 copies/16S rRNA. The concentration of ARB was between 3 and 2.1
103 CFU/mL, which mainly focused on 102 CFU/mL (Harnisz et al., 2015; Ouyang et al., 2015; Ham et al., 2012; Yang et al., 2014a). Resistances to ampicillin, chloramphenicol, ciprofloxacin, gentamicin and tetracycline were frequently detected with a mean resistance rate of 51.2%, 20.8%, 21.1%, 30.2% and 33.7%, respectively (Table S5). Many researchers have shown that the construction and abundance of AR is also closely related to antibiotic concentrations. For example, Ling et al. (2013) detected the ARG pollution in Beijiang River of Guangzhou, China and found that the abundance of sul1 and sul2 were significantly correlated with the concentration of sulfonamide antibiotics. Recently, Jiang et al. (2013) detected the ARG pollution in the surface waters of the Huangpu River of Shanghai, China and also found that the sample sites with high sul genes had corresponding high levels of sulfonamide residues. Comparing the ARG pollution in the secondary reception system with the primary reception system indicates that the ARG concentration is two orders of magnitude lower in the secondary system, but there was no significant difference in abundance. Sul genes and tet genes have the highest detection frequency in both reception systems. This demonstrates that ARGs are easily passed

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to the aquatic environment. 2.4. Pollution levels in the tertiary reception systems As previously described, the concentration of ARGs progressively decreases through the reception systems. However, the ARGs detected frequently in the upper degree can still be detected frequently in the next degree. This demonstrates that ARG pollution from the release source is passed to the primary reception system (WWTPs), then could migrate to the secondary reception system (groundwater and surface water), and finally converge to the tertiary reception system (estuaries and nearby coastal and ocean systems) under the influence of hydrological dynamics. Researchers have detected 30 different ARGs in the tertiary reception system, covering eight types of widely used antibiotics (Fig. 1, Table S3). Among all classes of ARGs detected in the tertiary reception system, sul genes and tet genes also have the highest detection frequency and the average detection rate reached 100% and 87.9%, respectively (Fig. 1, Table S4). The concentration and abundance of all different ARGs was between 4.43
102 and 1.1
108 copies/mL and 105.4e101 copies/16S rRNA, respectively, which mainly focused on 103e105 copies/mL and 104e102.1 copies/16S rRNA, respectively (Figs. 2 and 3). The median values were 104 copies/mL and 103 copies/16S rRNA. There was no significant difference in concentrations and abundances between the secondary and tertiary reception systems. The limited amount of research regarding tertiary reception systems may explain this finding. The concentration of ARB was between 1 and 9
102 CFU/ mL (Meena et al., 2015; Leonard et al., 2015; Dang et al., 2007). The resistance to tetracycline was detected frequently and the mean resistance rate reached 39.3% (Table S5). However, unlike the release source, primary and secondary reception system, the correlation between antibiotic resistance and antibiotics was lessened in the tertiary system. Researchers have shown that the construction and abundance of antibiotic resistance may be closely related to the antibiotic concentration; however, other studies have indicated no significant correlation. For example, Na et al. (2014) revealed that the relative abundance of sul1 and sul2 was positively correlated with the total antibiotic concentration in the Northern Yellow Sea, China (sul1, r2 ¼ 0.707, p ¼ 0.001; sul2, r2 ¼ 0.907, p ¼ 0.001). Chen et al. (2015b) also found that the relative abundance of individual sul gene types (sul1 and sul2) was significantly correlated with the total concentration of sulfonamides in the Pearl River and Pearl River Estuary (sul1, r ¼ 0.69, p < 0.01; sul2, r ¼ 0.76, p < 0.01). However, Lu et al. (2015) researched sul genes and sulfonamide in Daliaohe and Liaohe river estuaries and found that sul genes were not related to the sulfonamide antibiotics. Lin et al. (2015) researched the occurrence and distribution of sul genes in the Yangtze River Estuary and the nearby coastal area, and found that sulfonamides were rarely detected in the sediments of this region. Thus, it is unlikely that the production of sul genes occurred in the local environment. 3. The influencing factors on ARGs transmission in various aquatic environments In recent years, researchers have focused on the process of proliferation for ARGs and the mechanisms of antibiotic resistance. Results from these studies have indicated that bacteria rapidly obtaining antibiotic resistance cannot be explained by gene mutation alone. In the open natural environment, vertical transmission and HGT play an important role in the diffusion process of ARGs. Because ARGs have both the biological characteristics of replication and the physical and chemical characteristic of environmental persistence, the process may be described as a multi-dimensional

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and multi-mechanism complex communication process. This process is affected by complicated environmental factors. Therefore, the identification and detection of critical points in aquatic environments, which can provide and enhance the transfer and dissemination of ARGs, are highly important (Marti et al., 2014). Comprehensive understanding of the influencing factors of ARG transmission in various water environments will help to clarify the trend of change in antibiotic resistance, which can contribute to improvements in human health and ecological safety. 3.1. Corresponding contaminants The generation and propagation of ARGs are directly connected to the widespread use of antibiotics. In theory, environmental behaviors such as the migration, transformation and fate of antibiotics should be consistent with the ARGs induced by antibiotics. As we have described before, the construction and abundance of antibiotic resistance is closely related to the usage of antibiotics in the release source. A consistent phenomenon has also been observed in primary and secondary reception systems. However, unlike the release source and the primary and secondary reception system, the correlation between antibiotic resistance and antibiotics was lessened in the tertiary reception system. This may be caused by the high variability in the concentration of antibiotics in different natural aquatic environments. In the release sources, the antibiotic concentrations are high (several to several hundred mg/L) (Hsu et al., 2014; Zhang et al., 2013; Wang et al., 2016). Moreover, in the polluting pharmaceutical industries or wastewaters from hospitals and livestock farms, the antibiotic concentrations are even higher (at the level of g/L) (Khan et al., 2013; Ujam et al., 2013). In primary and secondary reception systems, the antibiotic concentrations are lower (ng/Lemg/L) (Xu et al., 2015, 2016; Gao et al., 2012b). Finally, the levels of antibiotics are much lower in tertiary reception systems (ng/L) (Lu et al., 2015; Guan et al., 2017). In conclusion, the relationship between antibiotic resistance and antibiotics becomes weaker when the concentration of antibiotics is lower. Other factors may share or substitute the role of antibiotics and contribute to the propagation of ARGs. 3.2. Non-corresponding contaminants Non-corresponding contaminant pressures can select bacteria with an elevated mutation rate along the lines of antibiotic pressure, thereby indirectly selecting genes with an increased probability of resistance to antibiotics (Taddei et al., 1997). Consequently, the fate of ARGs is susceptible to ambient pressures from noncorresponding contaminants. Heavy metals are one of the important non-corresponding contaminants. Unlike antibiotics, metals are not subject to degradation and can subsequently represent a long-term selection pressure (Stepanauskas et al., 2005). Heavy metals used as feed supplements are elevated in the manures, thus indicating the potential for co-selection of resistance traits (Zhu et al., 2013). Research has indicated that heavy metals influence the fate of ARGs through co-selection (Chapman, 2003), including the mechanisms of co-resistance (the genes specifying resistant phenotypes are located together on the same genetic element) and cross-resistance (same genetic determinant responsible for resistance to antibiotics and other contaminants). Significant correlations exist between the ARGs and metals (Cu, Zn and As), suggesting that the contamination of ARGs is related to the chemical pollution of the sediment (Su et al., 2014), and the presence of heavy metals provides another coselection pressure for ARG (Zhu et al., 2013; Bondarczuk et al., 2016). Lu et al. (2015) also indicated that several metals (Cr, Co, Ni, Cu, Zn, and Pb) are significantly and positively correlated with

sul genes. Consistent correlations exist between the ARG levels and Cu in Cuba (Graham et al., 2011). Overall, heavy metals might be one of the dominant factors in off-setting the effect of decreasing vertical transmission in estuary and marine environments and they can also have an important role in the maintenance and proliferation of ARGs when antibiotic-selective pressure is weak (Lu et al., 2015). Besides heavy metals, other environmental factors can also affect the maintenance and proliferation of ARGs. For example, Dealtry et al. (2014) detected a clear enhancement in the relative abundance of bacteria carrying IncP-1b plasmids, accompanied by the increasing concentrations of various pesticides, thereby suggesting the role of these organic pollutants (OPs) in the stimulation of the mobile genetic element expression. Furthermore, Sun et al. (2015) also investigated a stronger positive relationship between the ARG abundance and bioaccessible OP content than the total OP content. Mckinney et al. (2010) reported that tetO, tetW, and total tet genes (tetO, tetW) were positively correlated (P  0.01) with chemical oxygen demand (tetO, r ¼ 0.5328; tetW, r ¼ 0.5489; total tet genes, r ¼ 0.5463), total N (tetO, r ¼ 0.6679; tetW, 0.6977; total tet genes, r ¼ 0.6929), ammonia (tetO, r ¼ 0.6647; tetW, r ¼ 0.7040; total tet genes, r ¼ 0.6797), nitrate (tetO, r ¼ 0.4160; tetW, r ¼ 0.4329; total tet genes, r ¼ 0.4313), and phosphate (tetO, r ¼ 0.3905; tetW, r ¼ 0.4240; total tet genes, r ¼ 0.4116), which are all indicators of poor water quality. Similar results were reported by Chen and Zhang (2013b), who confirmed that water contamination levels can be analyzed based on tet genes. Yang et al. (2014a) suggested that an acidic (pH ¼ 4) water environment not only affects the community composition of ARB but also the pH and average pore size of the medium affect the relative abundances of ARGs in the effluent. The abundance of sul genes is also significantly reduced with increased salinity (Lu et al., 2015). Several studies have shown that ARGs are present in the areas with low antibioticselective pressure or even no antibiotic-selective pressure (Tamminen et al., 2011; Bhullar et al., 2012; Di Cesare et al., 2012; Ling et al., 2013). A series of studies have shown that noncorresponding contaminants can affect the maintenance and proliferation of ARGs. A reasonable hypothesis that they may share or substitute the role of antibiotics when the antibiotic concentration is low. In conclusion, a demarcation point may exist such that the spread of ARGs is mainly determined by the corresponding antibiotic when antibiotic concentration is sufficiently high (such as at the release source). In contrast, non-corresponding contaminants (e.g., heavy metals, organic pollutants and physical and chemical factors) may share or substitute the role of antibiotics and contribute to the propagation of ARGs when the antibiotic concentration is low, as in the tertiary reception systems. During the transfer process, the main pathway of ARGs proliferation may switch from active transmission, which is dominated by antibiotics, to passive transmission, which is motivated and affected by noncorresponding contaminants. 4. Conclusion The emergence and proliferation of antibiotic resistance within a wide range of infectious agents is a growing concern for global public health. The ARG pollution levels in the release source are significantly higher than in the reception systems. The diversity classes and concentration of ARGs progressively reduce from the source to the tertiary reception system. Among all the ARGs so far identified, sul genes and tet genes are the most commonly observed. The spread of ARGs is mainly determined by the corresponding contaminants when the antibiotic concentration is sufficiently high. In contrast, non-corresponding contaminants may

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share or substitute the role of antibiotic concentration and contribute to the propagation of ARGs when the antibiotic concentration is lower. Therefore, the main pathway of ARGs proliferation may switch from active transmission to passive transmission. Researchers should focus on the role of metal exposure as a mechanism for increasing the incidence and propagation of ARG pollution within a framework of ecological relevance. It is necessary to provide a cohesive and rigorous understanding of the processes of ARG pollution to comprehensively assess the role of corresponding contaminants and non-corresponding contaminants as selective forces in maintaining and propagating the pool of ARGs in the environment. Acknowledgements This research was supported by the National Natural Science Foundation of China (No.41406088, No.21377032), Chinese Polar Environment Comprehensive Investigation and Assessment Programs (2016-02-01, 2016-04-01, 2016-04-03), Marine public welfare scientific research projects (201105013), Project of China Scholarship Council (CSC201504180002), Liaoning BaiQianWan Talents Program (201723) and Foundation of polar science key laboratory, SOA, China (KP201208). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.envpol.2017.08.078. References Auerbach, E.A., Seyfried, E.E., McMahon, K.D., 2007. Tetracycline resistance genes in activated sludge wastewater treatment plants. Water Res. 41, 1143e1151. Bhullar, K., Waglechner, N., Pawlowski, A., Koteva, K., Banks, E.D., Johnston, M.D., Barton, H.A., Wright, G.D., 2012. Antibiotic resistance is prevalent in an isolated cave microbiome. PLoS One 7, 282, 282. Bondarczuk, K., Markowicz, A., Piotrowska-Seget, Z., 2016. The urgent need for risk assessment on the antibiotic resistance spread via sewage sludge land application. Environ. Int. 87, 49e55.
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Please cite this article in press as: Gao, H., et al., Complex migration of antibiotic resistance in natural aquatic environments, Environmental Pollution (2017), http://dx.doi.org/10.1016/j.envpol.2017.08.078