Sulfonamide and tetracycline resistance genes in nanjing lakes: effect of water quality and heavy metals

Due to high population density, anthropogenic activities and waste disposal have negatively affected artificial lakes in residential areas. These artificial lakes lack the capacity for auto-depuration to remove pollutants and contaminants; thus, they have the potential to act as reservoirs for antibiotic resistance genes (ARGs). In this study, we examined three urban artificial freshwater lakes in Nanjing to determine the abundance of sulfonamide and tetracycline resistance genes. Concerning water quality, the three lakes were found to be highly eutrophic, owing to their high levels of Total Nitrogen (TN), Phosphorous (TP), and Chlorophyll a (Chla). The average abundance of sulfonamide resistance genes detected in the three urban lakes was 42.446 log10 gene copies/100 ml, which was lower than the average abundance of tetracycline resistance genes (68.207 log10 gene copies/100 ml). Analysis by ANOVA revealed that all ARGs, except sul3, showed significant differences, probably due to varied anthropogenic influences in lakes. Pearson correlation and principal component analyses were performed to explore the correlation between ARGs, water quality markers, and heavy metals to understand the co-selection and drivers of ARGs propagation. tetM showed no correlation with any water quality markers, whereas Chla showed a positive correlation with all ARGs except tetM. tetM was the only gene observed to be unaffected by TN, TP, and Chla. The tet genes also showed strong associations with each other except tetM, especially tetA, tetQ, and tetG. The co-selection results between heavy metals and ARGs were insignificant (p > 0.05), with tetM being the most sensitive to the effects of heavy metals and As having the strongest effect on sul3 and tet genes. The results from this study provide basic but archival information on the effect of eutrophication and heavy metals such as Arsenic, showing the potential influence on the dissemination of certain sulfonamide and tetracycline ARGs in freshwater environments.


Introduction
Antibiotics have been used extensively in medicine and agriculture to treat diseases and promote animal growth. However, with the release of antibiotics into the water ecosystem, antibiotic resistance genes (ARGs) have been developed by microorganisms in response to environmental changes (Yin et al 2013, Qiao et al 2018. These ARGs can spread in the environment through horizontal gene transfer, thereby creating antibiotic-resistant bacteria (ARB), posing new risks to global human health (Serwecińska 2020, Kraemer et al 2019. Natural and artificial lakes connected to major river bodies or industrialized cities receive antibiotics and ARGs through tributary connections and waste pipes (Liu et al 2018, Ohore et al 2020, Komijani et al 2021a, Guo et al 2022. The contamination is usually most severe in freshwater environments. Previous studies have tracked antibiotics and ARGs to municipal wastewater treatment plants (WWTP) or pond aquaculture effluents (Wang et al 2020a, Sabri et al 2020. Sabri et al (2020) analyzed the levels of antibiotics and ARGs upstream and downstream of the river where the WWTP discharge point is located and found. They found that the river acts as a dilution and transport agent for the incoming antibiotics and ARGs, with ARGs originating from the discharge point occurring in the water column and sediment downstream of the river up to 20km. Pham et al (2018) isolated and gained bacterial strains containing multiple resistance genes in the outlet channel of a prawn culture pond connected to the Vam Co River, from which 41 antibiotic resistance genes were detected. Research on the spread of ARGs in livestock farm wastewater (swine wastewater) revealed that soil,surface water, and groundwater in the vicinity of farms were heavily contaminated with ARGs from the wastewater and that the situation will exacerbate during the rainy season (He et al 2019, Huang et al 2019a). Thus the presence and transportation of ARGs in freshwater environments need to be investigated to understand their mechanism and co-occurrence with physical and chemical properties.
The transport and fate of ARGs in freshwater have received widespread attention; compared to the rapid transport (i.e., water movement and interconnection with other water bodies) of antibiotics and ARGs in rivers; lakes have different hydraulic characteristics with longer hydraulic residence time, thus having a higher potential for storage and accumulation of ARGs (Nnadozie and Odume 2019, Yang et al 2018). Although urban lakes as freshwater resources are usually used for landscaping and, in some cases, for irrigation purposes, some residents use them for recreation and also as drinking water sources. Thus, contact with urban lakes for recreational purposes (fishing, swimming, etc) is unavoidable (Adhikary et al 2021). A systematic review by (Shao et al 2018) reported that antibiotics such as ciprofloxacin, erythromycin, sulfamethoxazole, and trimethoprim disrupt the immune system of mollusks and impair the development of fish in their early life stages. Sulfonamide and tetracycline antibiotics were observed to be more highly toxic to algae growth than crustaceans and fish (Xu et al 2021, Duan et al 2022 . A systematic review by (Shao et al 2018) reported that antibiotics such as ciprofloxacin, erythromycin, sulfamethoxazole, and trimethoprim disrupt the immune system of mollusks and impair the development of fish in their early life stages. Sulfonamide and tetracycline antibiotics were observed to be more highly toxic to algae growth than crustaceans and fish (Xu et al 2021, Duan et al 2022. Therefore, investigating ARGs occurrence, abundance, and potential influencing factors in urban lakes is vital to maintaining ecological security and human health (Nappier et al 2020).
As the country with the highest production and consumption of antibiotics, high concentrations of antibiotics are often detected in the aquatic environment of lakes in China. The contamination of ARGs is most serious in the aquatic environment of economically developed and densely populated areas in the east, where the detection rate of ARGs is led by sulfonamide and tetracycline resistance genes (Liu et al 2018). The presence of antibiotics is not the only factor influencing the development of ARGs, where high concentrations of heavy metals in the environment can synergize with antibiotics to create cross-resistance and co-resistance in bacteria, leading to the co-selection of ARGs (Wang et al 2022b, Squadrone 2020b, Li et al 2017, Baker-Austin et al 2006. Xu et al (2017a) found a significant correlation between ARGs and heavy metals in a study on the effects of heavy metals on the fate and distribution of ARGs in the Xiangjiang River basin. Komijani et al (2021b) also found significant correlations between ARGs and heavy metals in a study of six lakes and wetlands in Iran, with vanadium showing a strong positive correlation with tetM. In addition to this, water quality markers, including temperature, pH, dissolved oxygen (DO), and nutrients (N/P), can significantly influence microbial abundance and community differences. These factors could cause the dilution, degradation, adsorption, and transport of ARGs in freshwater environments, thus also having a significant impact on the distribution and fate of ARGs (Eckert et al 2018). Several studies have recently focused on the effects of anthropogenic activities and environmental factors on the distribution of ARGs in freshwater lakes. Wang et al (2020c) studied the association between the distribution of ARGs and water quality factors in agriculturally disturbed Honghu Lake and obtained significant positive and negative correlations of ARGs with nutrients and DO levels, respectively. Yang et al (2017a) analyzed the effects of land use and environmental factors on sulfonamide and tetracycline resistance genes in lakes from the middle and lower reaches of the Yangtze River. However, more studies are needed to understand the interactions between ARGs, heavy metals and water quality markers. Exploration between these variables will provide more knowledge as to the occurrence, propogation and distribution of ARGs in freshwater lakes that are eutrophic and exposed to high levels of anthropogenic influence.
This study investigated three urban lakes in Nanjing: Baijia, Pipa, and Longchi Lake. We found no relevant studies on sul and tet genes distribution, water quality markers, and heavy metals among these lakes. In this study, we focused on the distribution and correlation of 9 ARGs belonging to sulfonamides (sul1, sul2, sul3) and tetracyclines (tetA, tetC, tetE, tetM, tetQ, tetG) resistance genes. We further analyzed correlations between the distribution of ARGs and the nine water quality markers (pH, Temperature, total dissolved solids (TDS), electrical conductivity (EC), dissolved oxygen (DO), total organic carbon (TOC), total nitrogen (TN), total phosphorous (TP), Chla) with four potentially toxic elements (Arsenic (As), Copper (Cu), Lead (Pb), Cadmium (Cd)). The objectives of this study are 1) to investigate the water quality in these three urban lakes, 2) to analyze the differences in the distribution of ARGs and whether they are related to eutrophication and water quality, and 3) to investigate the heavy metals and ARGs interactions on the propagation and co-selection of ARGs. The significance of this study is to reveal the potential factors affecting the spread and distribution of ARGs in urban freshwater lakes and the potential risk of ARG pollution in urban lakes to the health of local residents.

Material and methods
2.1. Sampling sites, collection and analysis Water samples were collected from all the lakes mentioned in this study in Nanjing city, Jiangsu province, China. In our study, anthropogenic influence refers to recreational activities and garbage is thrown by residents and tourists. Figure 1 and table 1 provide the lake's geographical location, morphology, and eutrophication level. All lakes except Longchi are located in or around high population-density areas of the city, which leads to high levels of anthropogenic influence (figure 1). All three lakes are exposed to fly-tipping, occasional garbage disposal, residents swimming, fishing, and surface run-off. Longchi is independent, and Baijia is connected with the Qinhuai river. The water sample was collected as triplicates from all lakes in 2L sterilized High-Density Polyethylene (HDPE)bottles and stored on ice during transportation. The samples were transported to the university laboratory for further analysis. Further information about physical parameters, chemical parameters, heavy metal, and DNA analysis is mentioned in our previous paper (Rajasekar et al 2022)

qPCR assay for quantification
This study analyzed three sulfonamide resistance genes (sul1, sul2, and sul3) and six tetracycline resistance genes (tetA, tetC, tetE, tetM, tetQ, and tetG). Primers and PCR protocols for quantitative detection of these ARGs were obtained from previous sulfonamide and tetracycline resistance gene studies  Table S1). The assays were performed in 96 well plates in a 7500 Fast Real-Time PCR system (Applied Biosystems, USA). The R 2 values for all standard curves were 0.98 to 1, and the efficiency of our reactions ranged from 75% to 110%.

Statistical analysis
GraphPad Prism v9.0 was used for performing ANOVA and Pearson analysis. Canoco5 (Šmilauer and Lepš 2014) was used to conduct principal component analysis (PCA) between heavy metals, water quality markers, and ARGs.  Table 1. Description of lake morphology and water quality of the three urban lakes.

Lakes
Longitude 3. Results and discussion

Abundance and distribution of ARGs in three urban lakes
The abundance with the standard error of the nine sulfonamide and tetracycline resistance genes in Baijia, Pipa, and Longchi lakes are represented in figure 2 and table 2, respectively. The ∑ARG recorded the highest value in Pipa lake (37.752 log 10 gene copies/100 ml), followed by Baijia (36.573 log 10 gene copies/100 ml) and Longchi (36.328 log 10 gene copies/100 ml). In addition, we observed that tetracycline resistance genes tetA (3.864 ± 0.0162 log10 gene copies/100 ml), tetC (3.888 ± 0.0056 log10 gene copies/100 ml), tetE (3.984 ± 0.0090 log10 gene copies/100 ml), tetQ (3.887 ± 0.0085 log10 gene copies/100 ml) and tetG (5.296 ± 0.0024 log10 gene copies/100 ml) in Pipa lake were significantly (p<0.05) more abundant than those from Baijia and Longchi. However, the abundance of tetM in Baijia lake (3.342 ± 0.0458 log10 gene copies/100 ml) was significantly (p<0.05) higher than that in Pipa (2.642 ± 0.0522 log10 gene copies/100 ml) and Longchi (2.662 ± 0.0715 log10 gene copies/100 ml). From our reconnaissance study, Pipa lake is a tourist attraction on the hills above the city center and is surrounded by lush vegetation; a combination of fly-tipping, fallen leaves, and surface rainwater movement could have contributed to the high eutrophication levels (Jiao et al 2018). Among the physicochemical parameters of the three lakes, Pipa lake had significantly higher (p < 0.05) levels of TN (1.25 mg l −1 ) and Chla (57.7 mg l −1 ) than Baijia (TN with 0.87 mg l −1 ; Chla with 21.2 mg l −1 ) and Longchi (TN with 0.92 mg l −1 ; Chla with 49.7 mg l −1 ). In addition, Baijia Lake is located in a residential area and is more severely affected by human activities as a receptor of domestic sewage and road-cleaning wastewater.
The results of the abundance of ARGs in the three lakes showed that the dominant resistance genes in the three lakes were sul2 (5.522 log 10 gene copies/100 ml), sul1 (5.271 log 10 gene copies/100 ml), and tetG (5.247 log 10 gene copies/100 ml), while the two ARGs with the lowest average abundance were tetM (2.882 log 10 gene copies/100 ml) and sul3 (3.357 log 10 gene copies/100 ml) in turn, which is similar to the research on Honghu lake and east Dongting lake by Yang et al (2016). Commonly, freshwater lakes tend to be dominated by sul1 and sul2, and the abundance of sul1 tends to be higher than that of sul2, but the abundances are not significantly different (Yang et al 2018). However, comparing the average ∑sulfonamide resistance genes with the average ∑tetracycline resistance genes among the three lakes revealed that the tetracycline genes (68.207 log 10 gene copies/100 ml) were, in fact, the dominant ARGs, significantly higher than the sulphonamide genes (42.446 log 10 gene copies/100 ml). In contrast to previous studies in the middle and lower reaches of the Yangtze River, Bosten lake, and Poyang lake, sulfonamide resistance genes were detected as the dominant ARGs in surface water or sediment from these freshwater lakes, and the reason was primarily attributed to the abundance of sulfonamide class antibiotics in these lakes (Zhou et al 2014, Yang et al 2017a, Liang et al 2020. Most studies have indicated multiple reasons behind the abundance of sulfonamide and tetracycline resistance genes in freshwater Figure 2. The abundance of sul1, sul2, sul3, tetA, tetC, tetE, tetM, tetQ and tetG in the three lakes. environments; these include antibiotics and mechanisms leading to the resistant gene development induced by unavoidable bacterial chronic exposure to biocides, heavy metals, detergents, and disinfectants (Baker-Austin et al 2006). The relatively higher abundance found in our study is of concern since most agricultural areas and recreational activities use reclaimed freshwater or freshwater, thus potentially introducing these ARGs into a particular environment. Therefore, people can be exposed to ARGs through swimming, aquatic sports, occupational exposure, and the consumption of food crops irrigated with reclaimed water (Ben et al 2017, Amarasiri et al 2020). Table 3 shows that all ARGs except sul3 showed significant differences in abundance in the three urban lakes, ranked from strong to weak according to significance as tetG, tetQ, tetA, tetC, sul1, tetM, tetE, and sul2. The difference in abundance of tetG was most significant in these three urban lakes (F = 631.7), followed by tetQ (F = 492.9), which is likely due to the varying water quality levels, eutrophication level, and heavy metal influence. We believe the concentration and presence of antibiotics in different urban lakes could potentially explain the variations in ARGs among these lakes. Previous studies on sulfonamide and tetracycline genes have also observed them to be dominant in urban lakes located in dense urban areas since they are exposed to heavy metal pollution, nutrient pollution, and antibiotic waste (Tong et al 2020, Su et al 2020, Komijani et al 2021a. Guo et al (2022) also found that ARG concentrations were significantly higher downstream than upstream, influenced by urban sewage discharge from studying the spatial and temporal distribution of ARGs and antibiotics in the East Dongting Lake watershed.
3.2. Interactions of physicochemical parameters, heavy metals, and ARGs on the ARGs distribution 3.2.1. Relationship between ARGs and physicochemical parameters Water quality, i.e., the physicochemical parameters of the water body, has received much attention as it can influence the community structure of waterborne ARGs-carrying microorganisms and the spread and transport of ARGs (Zainab et al 2020). The results of the correlation analysis between the ARGs and the physicochemical parameters of the water column for the selected urban lakes are shown in table 4 and figure 3.
The PCA combined with Pearson correlation coefficients showed that the physicochemical parameters showed varying degrees of association between the physicochemical parameters with the tetracycline and sulfonamide genes. The results showed that for sulfonamide genes, both sul2 and sul3 showed positive correlations with physicochemical parameters except TP and TOC. At the same time, sul2 had an overall stronger response to physicochemical parameters and was positively correlated with TN, DO, Chla, Temperature, TDS, and pH, with TN (r = 0.974) and DO (r = 0.931) showing the strongest correlations. Unlike sul2 and sul3, sul1 showed an overall weaker correlation with physicochemical parameters and was strongly  positively correlated with TOC (r = 0.957); however, sul1 also showed a positive correlation with Temperature, DO, and Chla, implying that high Temperature, Chla, and DO levels contribute to the propagation of sulfonamide genes. Similar to sul2 and sul3, for tetracycline genes except for tetM, pH, Temperature, TDS, EC, DO, TN and Chla also showed and presented even stronger positive correlations, with TN showing significantly positively linear correlations with tetA, tetC, tetQ, and tetG, TN was significantly strongly positively correlated with tetC (r = 0.998, p < 0.05). In contrast, tetM was only positively correlated with EC (r = 0.402) and TP (r = 0.970) and was strongly negatively correlated with Temperature and DO, which could be because the tetM-carrying bacteria could be more adapted to an anaerobic environment (Tong et al 2020, Zhang et al 2020a. In addition, it is worth mentioning that temperature, DO, and Chla showed positive correlations for both sulfonamide and tetracycline genes except for tetM. Chla levels usually reflect the extent of water pollution from human activities, implying that human activity has contributed to the spread of ARGs (Zhang et al 2017). Zhao et al (2022), in a study on the correlation between ARGs with antibiotic distribution and water quality in the urban river Itong, found that elevated levels of Chla in the river caused by human activities lead to increased concentrations of antibiotics, resulting in the production of antibiotic-induced ARGs. Huang et al (2019b) also found a positive correlation between ARGs and Chla in their study of the distribution of ARGs in Qingcaosha Reservoir, Shanghai.

Relationship between ARGs
The results of the correlation analysis between ARGs are shown in table 5. The analysis of correlations between ARGs can reflect the linkage and mechanism of action between ARGs, which few previous studies have done before.
The results show that the sulfonamide genes sul1 with sul2 and sul2 with sul3 are positively correlated with each other, with sul2 having a stronger positive correlation with sul3 than sul1 with sul2. Apart from the weak negative correlation between tetM and other tetracycline genes, tet ARGs also showed positive correlations between each other, with tetA, tetQ, and tetG showing significantly strong positive correlations (r = 1.000), tetC also showed significant correlations with them (r = 0.999, p < 0.05). The weak negative correlation between sul1 and tet ARGs implies no direct link between sul1 and tetracycline genes, which may be caused by different transport mechanisms or external pressures, such as the use of antibiotics being different in various human activities. Previous studies have shown that tetracycline genes are distributed across different microbial communities, with tetA and tetC usually found in Gram-negative bacteria, while tetM and tetQ tend to be found in Gram-positive bacteria (Cheng et al 2019, Zhang et al 2017. This may explain why tetA and tetC are significantly positively correlated. The reason why tetQ also shows a significant positive correlation with them because they are both present in the same genetic components, such as transposons, plasmids, and integrins (Zhou et al 2017).
Similarly, this can also explain the positive correlation between sulfonamide genes. The insignificant correlations of tetM with other tet genes and sul1 with sul3 imply that they may go through horizontal transfer in different ways or may be influenced by other environmental stresses. Ohore et al (2019) and Dang et al (2017) also found significant positive correlations between sul1 and sul2 in the Taihu and Haihe studies, respectively, and both sul1 and sul2 were found to be present on conjugative plasmids in Haihe.

Relationship between ARGs and heavy metals
Antibiotic residue in the environment is often taken as an important factor in analyzing the cause of ARG distribution, but it is not the only factor that determines the fate of ARGs (Manaia et al 2018, Zheng et al 2021). The link between heavy metals and antibiotic resistance and the role of co-selection has recently received extensive attention (Zhang et al 2022, Zhang et al 2018. In our previous study on the distribution of ARGs in three urban lakes in Nanjing, we found that four heavy metals (As, Cu, Pb, Cd) had a strong influence on the selection of ARGs and that Cu, Pb, and Cd were all positively correlated with strB, with the strongest correlation being with Cu (Rajasekar et al 2022). Han et al (2022) also found a significant positive correlation between Cu content and ARGs abundance in a recent study of factors influencing ARGs in soils from the Dexing copper mining area. In addition, Lu et al (2022) reached consistent conclusions through redundancy analysis in a study of the spread of ARGs in the agriculturally influenced Jialing River. In this study, we also focused on the effect of four heavy metals, As, Cu, Pb, and Cd, on the selection of ARGs. Unlike the ARGs we previously focused on, here we focused on the resistance genes of two antibiotic related genes, sulfonamides (sul) and tetracyclines (tet). The results of Pearson correlation analysis and principal component analysis of ARGs with heavy metals are shown in table 6 and figure 4, respectively.
Pearson correlation analysis showed that four heavy metals affected different antibiotic-resistance genes. For tet ARGs except for tetM (tetA, tetC, tetE, tetQ, and tetG), all heavy metals except As showed a negative correlation, with Pb showing an overall strongest negative correlation and was strongly negatively correlated with tetA (r = −0.854). Similarly, all four heavy metals also negatively correlated with sul1 and sul2. However, sul3, As, Cu, and Cd all showed positive correlations, with As showing the strongest correlation (r = 0.733). Thus, sul3 was more susceptible to heavy metal stress compared to other sul ARGs. In addition, As, Cu, Pb and Cd were all positively correlated with tetM, with Cu showing the overall strongest correlation coefficient, indicating that tetM was most susceptible to heavy metal stress among all sulfonamide and tetracycline genes. Unlike what we found in our previous study, Cu had a weaker effect on the spread of ARGs than the other heavy metals. Environmental stress was commonly observed to be the primary mechanism for ARG formation (Jiang et al 2022, Xu et al 2017b). Zhao et al (2020), in their study of the effects of As and Cd on paddy soil ARGs distribution, found a stronger effect of As than Cd on ARGs and attributed this to the stronger toxicity of As. He et al (2017a) used a similar principle to explain the stronger co-selection effect of Zn than Cu on ARGs when studying ARG formation under heavy metal and antibiotic stresses. However, this does not seem to explain why As and Cd, especially As had a stronger effect on tet ARGs than Cu in this study. If the , more negligible contribution of Cu to the spread of ARGs is attributed to Cu's relatively weak oxidative stress on microorganisms, then this cannot explain the more significant contribution of Cu to the spread of ARGs, especially strB, that we previously found. Previous studies have indicated that ARG enrichment could be attributed to the concentration of heavy metals since they can induce efflux pumps (Mao et al 2015, Amarasiri et al 2020). This theory could explain the As influence on certain sul and tet genes in these lakes. We found a study that identified that As can enrich the abundance of multidrug-resistant bacteria by 75% through long term exposure (Seiler and Berendonk 2012). Therefore, further molecular-level studies are still needed on the mechanism of co-selection of sul and tet ARGs with heavy metals. PCA analysis between heavy metals and ARGs also pointed out that most heavy metals were positively correlated with tetM and sul3 resistance genes, in addition to As being positively correlated with all tet genes, implying that tet genes could be more susceptible to As in these three freshwater lakes. In general, sul1 is positively correlated with sul2 and tends to be positively correlated with heavy metals in freshwater lakes. In addition to our findings in previous research, Zhou et al (2017), in a study on the occurrence and distribution of sul and tet genes in agricultural soils, also found a higher association between heavy metals and sul genes compared to tet genes using principal component analysis and attributed this to the greater sensitivity of sul genes to heavy metal contamination. Heavy metals are known to either induce efflux pumps or accelerate Horizontal Gene Transfer by conjugation (Wu et al 2015, He et al 2017b. Wu et al (2015) similarly found a significant positive correlation between sul1 and sul2 with heavy metals in a study of surface sediments from Chaohu Lake. However, in this study, not only sul1 and sul2 genes were found to be negatively correlated with most heavy metals, indicating that most heavy metals did not contribute significantly to the spread of sul1 and sul2 genes, but also no significant positive correlation was shown between sul1 and sul2, the exact reason of which still needs to be further investigated. The lack of experimental studies on certain heavy metals and their potential for ARG enrichment needs further investigation. From our study, the data indicate that apart from As, none of the other heavy metals positively affect ARG enrichment or propagation. We believe heavy metals such as Zinc and Cobalt could have helped enrich these ARGs.

Implications for the freshwater lake, ecological and human health
The presence of ARGs in freshwater lakes is considered a hazardous issue for ecological and human health (Squadrone 2020a, Komijani et al 2021a, Ana et al 2021, Wang et al 2022a. These lakes should be of the highest priority since they are vulnerable to diverse anthropogenic activities such as fishing, swimming, etc These activities could cause long-term damage, requiring remediation measures that are cost-ineffective. Our present results show that the sulfonamide and tetracycline ARGs are relatively high, even in independent lakes that are relatively smaller in size. However, recent studies on ARG detection in freshwater lakes and rivers have revealed the presence of higher amounts of ARGs (Wang et al 2021, Zheng et al 2021, Zhao et al 2022, Rajasekar et al 2022 which poses a severe threat to ecological and human health. Notably, most citizens perform multiple recreational activities in these lakes; fishing and swimming are the most common and consistent activities. Thus, we propose a rigorous approach for ARG abatement through policies that prevent/minimize such activities in these lakes. Multiple studies, including ours, have pointed out that most Chinese freshwater lakes are mesotrophic to highly eutrophic (Wang et al 2019, Wang et al 2020b, Lin et al 2021, which could cause the dissemination of certain ARGs (Zhang et al 2020b. The government should conduct comprehensive and systematically in-depth surveys regarding antibiotics usage, ARG formation, and tailwater discharge. Although the results of this study are relatively basic, they provide a scientific basis in displaying the focus that's highly required for the management of freshwater lakes, which could prove to be relatively cleaner water sources for the future.

Conclusion
As a contaminant, the spread of ARGs in water bodies is influenced by physicochemical factors in the water column and strongly linked to heavy metals. The study results show significant differences in the spatial distribution of ARGs affected by anthropogenic activities in the three urban lakes in Nanjing. The correlation analysis yielded that the water quality index had different effects on the spread of sulphonamide and tetracycline resistance genes, with Chla contributing to the spread of almost all ARGs. In addition, apart from tetM, there is some linkage in the spread patterns of the tetracycline resistance genes. Finally, this study also found that the spread of tet ARGs and heavy metals were closely related, with As showcasing a potential impact on the spread of ARGs and tetM being the most sensitive to heavy metals, the exact mechanism of which remains to be further investigated. We propose future studies to compare Antibiotic and ARG pollution in freshwater lakes, sediments, and groundwater to explore contamination issues caused by freshwater lake movement. Heavy metal-induced resistance gene formation caused by specific bacteria also needs further investigation. The information and data gathered during this study will provide vital information to the government and stakeholders in policy-making in this sector.

Data availability statement
All data that support the findings of this study are included within the article (and any supplementary files).

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