The Occurrence of Antibiotic Resistance Genes in an Urban River in Nepal

Urban rivers affected by anthropogenic activities can act as reservoirs of antibiotic resistance genes (ARGs). This study aimed to describe the occurrence of selected ARGs (blaTEM, ermF, mecA, and tetA) and a class 1 integron (intI1) in an urban river in Nepal. A total of 18 water samples were collected periodically from upstream, midstream, and downstream sites along the Bagmati River over a 1-year period. All ARGs except mecA and intI1 were consistently detected by a quantitative polymerase chain reaction in the midstream and downstream sites, with concentrations ranging from 3.1 to 7.8 log copies/mL. ARG abundance was significantly lower at the upstream site (p < 0.05), reflecting the impact of anthropogenic activities on increasing concentrations of ARGs at midstream and downstream sites. Our findings demonstrate the presence of clinically relevant ARGs in the urban river water of Nepal, suggesting a need for mitigating strategies to prevent the spread of antibiotic resistance in the environment.


Introduction
The majority of the antibiotics used to treat humans and animals are excreted and released into aquatic environments via sewage [1]. The release of antibiotics, even at lower concentrations, can promote the development and dissemination of antibiotic resistance genes (ARGs) [2]. ARGs can be horizontally transferred to different pathological bacterial strains by mobile genetic elements (MGEs), spreading antibiotic resistance [3]. Class 1 integrons are such bacterial MGEs with an established role in the transfer of ARGs [4].
Various studies have shown that urban rivers prone to anthropogenic activities and receiving effluents from wastewater treatment plants (WWTPs) act as reservoirs of ARGs and MGEs [5,6]. A cross-sectional study on the presence and removal of ARGs during wastewater treatment concluded that WWTPs reduce ARGs, but significant concentrations of ARGs are still discharged into wastewater-receiving water bodies [7]. ARGs, now considered as emerging contaminants [8], are more likely to pollute urban riverine systems in developing countries where wastewater is insufficiently treated or is directly discharged into rivers.
The Bagmati River in the Kathmandu Valley, Nepal, is an example of such an urban river polluted with untreated sewage [9]. It flows from north to south in the Kathmandu Valley, passing through the densely populated region. Previous studies have reported a degraded microbiological quality of water and showed that the Bagmati River is a reservoir of pathogenic bacteria, viruses, and protozoa [10][11][12]. However, studies regarding the detection of clinically relevant ARGs and MGEs have not been published.
Based on this knowledge, this study aimed to assess the presence of ARGs and a class 1 integron at different sites along the Bagmati River. Four clinically relevant ARGs (bla TEM , ermF, mecA, and tetA) conferring resistance to commonly used antibiotics in this region [13] were selected for this study.

Sample Collection and Extraction of Bacterial DNA
As reported previously [11,12], 100-mL river water samples were collected in sterile bottles once every 2 months between November 2015 and September 2016 from Sundarijal (upstream), Thapathali (midstream), and Chovar (downstream), as shown in Figure 1. Land cover usage details on the map were based on a previously conducted study [14]. Ten milliliters of each sample was filtered through a sterile disposable filter unit (pore size, 0.22 µm; diameter, 47 mm; Nalgene, Tokyo, Japan) and bacterial DNA was extracted from the membrane filter using CicaGeneus DNA extraction reagent (Kanto Chemical, Tokyo, Japan).
Water 2020, 12, x FOR PEER REVIEW 2 of 7 The Bagmati River in the Kathmandu Valley, Nepal, is an example of such an urban river polluted with untreated sewage [9]. It flows from north to south in the Kathmandu Valley, passing through the densely populated region. Previous studies have reported a degraded microbiological quality of water and showed that the Bagmati River is a reservoir of pathogenic bacteria, viruses, and protozoa [10][11][12]. However, studies regarding the detection of clinically relevant ARGs and MGEs have not been published.
Based on this knowledge, this study aimed to assess the presence of ARGs and a class 1 integron at different sites along the Bagmati River. Four clinically relevant ARGs (blaTEM, ermF, mecA, and tetA) conferring resistance to commonly used antibiotics in this region [13] were selected for this study.

Sample Collection and Extraction of Bacterial DNA
As reported previously [11,12], 100-mL river water samples were collected in sterile bottles once every 2 months between November 2015 and September 2016 from Sundarijal (upstream), Thapathali (midstream), and Chovar (downstream), as shown in Figure 1. Land cover usage details on the map were based on a previously conducted study [14]. Ten milliliters of each sample was filtered through a sterile disposable filter unit (pore size, 0.22 μm; diameter, 47 mm; Nalgene, Tokyo, Japan) and bacterial DNA was extracted from the membrane filter using CicaGeneus DNA extraction reagent (Kanto Chemical, Tokyo, Japan).

Quantitative Polymerase Chain Reaction (qPCR)
The extracted bacterial DNA was serially diluted in 10-fold dilutions (10 −1 and 10 −2 ) depending on the primer set and was subjected to qPCR using a StepOne Plus Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) and an ABI Power Sybr PCR mix (Applied Biosystems). Forward and reverse primers and thermal cycling conditions were followed according to previously published literature for total bacteria [15], intI1 [16], tetA [17], bla TEM [18], ermF [19], and mecA [20]. Positive controls were included in each assay using previously collected environmental or clinical isolates harboring the respective ARGs. Inhibition controls comprised the spiking of positive-control DNA into serial dilutions of template DNA. PCR-grade water was used to prepare negative controls. All assays were conducted in duplicates. Amplification products were subjected to melting-curve analysis and were quantified using a six-point 10-fold standard curve.

Discussion
To the best of our knowledge, this study is the first to report the presence of ARGs and MGE in an aquatic environment in Nepal. The abundance of microbial contaminants, such as blaTEM, tetA, and intI1, found in our study was comparatively higher than that reported in studies conducted in Japan [5] and China [21]. This could be explained by direct hospital and household discharges and drainage from nonfunctioning WWTPs into the Bagmati River. Interestingly, mecA was not detected in any of the river water samples tested. This result is in accordance with a previous study conducted in Germany that demonstrated detectable amounts of mecA only in clinical wastewater [22]. mecA is staphylococci-specific [23], and Staphylococcus belongs to phylum Firmicutes [24]. Sequencing results of the same samples demonstrate that Firmicutes accounts for only 6%-15% of the bacterial community [11]. The lower abundance of Staphylococcus in river water and the incapability of mecA to spread along wastewater, as reported previously [23], may have resulted in the failure to detect mecA in our samples. Lower concentrations of ARGs at the upstream site compared with mid and downstream sites may be attributed to the increase in the number of houses draining untreated sewage farther downstream. Fecal pollution resulting from sewage discharge has been found to be the major factor determining the abundance of ARGs [25]. These findings show that anthropogenic activities promote the dissemination of ARGs in the environment [26].

Correlation between Concentrations of Total Bacteria, E. coli, ARGs, and intI1
As shown in Table 2, significant positive correlations were observed between concentrations of intI1 and ARGs (r ≥ 0.94, p < 0.05) and between total bacteria and ARGs or intI1 (r ≥ 0.91, p < 0.05). Using data from Tandukar et al. (2018) [12], correlation analysis among concentrations of E. coli, total coliforms, and ARGs or intI1 also demonstrated a strong positive correlation (r ≥ 0.54, p < 0.05).

Discussion
To the best of our knowledge, this study is the first to report the presence of ARGs and MGE in an aquatic environment in Nepal. The abundance of microbial contaminants, such as bla TEM , tetA, and intI1, found in our study was comparatively higher than that reported in studies conducted in Japan [5] and China [21]. This could be explained by direct hospital and household discharges and drainage from nonfunctioning WWTPs into the Bagmati River. Interestingly, mecA was not detected in any of the river water samples tested. This result is in accordance with a previous study conducted in Germany that demonstrated detectable amounts of mecA only in clinical wastewater [22]. mecA is staphylococci-specific [23], and Staphylococcus belongs to phylum Firmicutes [24]. Sequencing results of the same samples demonstrate that Firmicutes accounts for only 6%-15% of the bacterial community [11]. The lower abundance of Staphylococcus in river water and the incapability of mecA to spread along wastewater, as reported previously [23], may have resulted in the failure to detect mecA in our samples. Lower concentrations of ARGs at the upstream site compared with mid and downstream sites may be attributed to the increase in the number of houses draining untreated sewage farther downstream. Fecal pollution resulting from sewage discharge has been found to be the major factor determining the abundance of ARGs [25]. These findings show that anthropogenic activities promote the dissemination of ARGs in the environment [26].
The significant positive correlation observed among ARGs, intI1, total bacteria, and E. coli was consistent with a study conducted in central Europe [27]. As discussed previously, this finding may indicate origination from a common source and release into the receiving river via a common carrier [27]. E. coli, with an ability to accumulate ARGs through horizontal gene transfer [28], could be the major bacterium harboring these ARGs and MGE. Likewise, strong positive correlations between intI1 and ARGs are also in agreement with a prior study that indicated that ARGs were possibly spread by the transfer of class I integrons [6]. This result further supports the use of intI1 as an indicator of antibiotic resistance in aquatic environments [29].
The people in the Kathmandu Valley use groundwater for domestic purposes [30] and river water for irrigation [31]. The interaction of groundwater and river water in Kathmandu [32] increases the probability of groundwater pollution with ARGs. Without any guidelines for the safe use of water polluted with ARGs, these sources of water are a probable source of antibiotic resistance in the Bagmati River basin. However, the detection of ARGs doesn't always mean their transfer occurs in the aquatic environment. Future studies should focus on assessing the suitability of the aquatic environment to transfer these ARGs to potential pathogenic bacteria.

Conclusions
In summary, three ARGs and intI1 were detected with concentrations ranging from 3.1 to 7.8 log copies/mL at three sites along the Bagmati River. The significant positive correlation between the abundance of ARGs and intI1 suggests the use of intI1 as an indicator of antibiotic resistance in the environment. In Nepal, studies regarding antibiotic resistance are limited to clinical human samples. The detection of clinically relevant ARGs and MGE in an urban river in Nepal indicates a need to monitor antibiotic resistance in the environment.