Water is one of the most precious resources on earth. For humans, it is one of the basic needs used for food and other ancillary needs. It is therefore agreed that its availability in quantity and quality is essential for life on earth. Among these resources, streams represent one of the most important resources after the oceans. It is widely used for many human activities, such as fishing, agriculture, transportation, and many others. However, it is subject to numerous contaminations. This contamination can come from various sources related to human activities, such as industrial, agricultural, domestic discharges and hospital effluents (Adelowo et al., 2018; Kayembe et al., 2018; Zhou and Wang, 2019; Laffite et al., 2020; Yeh et al., 2020; Duan et al., 2021). The latter is one of the most important sources of contamination. Indeed, the contamination of streams can be of various natures that are chemical, microbiological, and sometimes radioactive (Pietryczuk et al., 2018; Carles et al., 2019; Mandaric et al., 2019; Yeh et al., 2020). Given that hospitals are environments par excellence where we witness the presence of large numbers of microbes of various kinds (bacteria, parasites, fungi, and viruses), these hospital effluents therefore contribute strongly to the microbiological contamination of streams (Verburg et al., 2019; Suzuki et al., 2020). In addition to microbes, the most important of which are bacteria, hospital effluents also contain antibiotic residues and resistance genes. Among the priority antibiotic-resistant pathogens, carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa, carbapenem-resistant and ESBL-producing Enterobacteriaceae are at the top of the WHO list and represent the greatest threat to human health (Sheu et al., 2019). In addition, Colistin regained global interest as a consequence of the rising prevalence of multidrug-resistant Gram-negative Enterobacteriaceae. In parallel, colistin resistant bacteria emerged in response to the unregulated and increased use of this antibiotic, which is a last resort drug due to failure of carbapenems, has possibly contributed to the development and spread of resistance to colistin among Enterobacteriaceae (Gogry and Siddiqui, 2019; Gogry et al., 2021, 2022). Indeed, many studies have shown the presence of these priority resistant pathogens in stream and hospital effluents (Adelowo et al., 2018; Bartley et al., 2019; Posada-Perlaza et al., 2019; Niest?pski et al., 2020; Suzuki et al., 2020). Even if other activities, such as livestock farming and domestic water, can bring these elements into watercourses, hospital effluents are suspected to be one of the main sources (Adelowo et al., 2018).

Bacteria are known to be the most prevalent in the hospital environment and in these effluents (Giannakis et al., 2017; Mittelman and Jones, 2018). These bacteria are most often multidrug-resistant to antibiotics due to the presence of antibiotics in the environment and the strong transmission of resistance genes (Giannakis et al., 2017; Mittelman and Jones, 2018; Laffite et al., 2020). Additionally, the presence of heavy metals in the medical environment exerts pressure favoring the selection of opportunistic pathogens resistant to antibiotics. Heavy metal resistance (HMR) associated with antibiotic resistance (AR) in hospital effluents makes them potentially dangerous (Chen et al., 2019; Dahanayake et al., 2019; Turner et al., 2020). Moreover, several studies have shown the presence of antibiotic and heavy metal resistance genes (ARGs and HMRGs) in wastewater, sewage sludge, river water, and Black sea (Sabatino et al., 2020; Hubeny et al., 2021; Martin et al., 2021). Other studies have established the correlation between these two types of resistance (Di Cesare et al., 2016; Yuan et al., 2018; Ohore et al., 2019). This is why it is advisable to have a water treatment and purification system in every hospital or city. Even if industrialized countries have these types of systems, this is not the case in developing countries. In developing countries, the metabolites of products used in hospitals or their byproducts, accompanied by a bacterial load (ARGs and HMRGs), are potentially found in hospital effluents treated in situ or collected by urban sewage systems, which are themselves connected to a water treatment plant and discharge the treated effluent into the natural environment (Laffite et al., 2016). In Benin, National Hospital and University Center of Cotonou is the only one hospital with a purification and treatment system for hospital effluents but this system remains moderately efficient (Todedji et al., 2020). Some studies have looked at the microbiological quality of hospital effluents and streams and the isolated bacterial strains have been characterized (Deguenon et al., 2022; Gbotche et al., 2023).

However, none of these studies have evaluated the presence of ARGs and HMRGs in the genome of pathogenic in these different matrices. The present study was undertaken to address this lack of data through. This nationwide study was therefore conducted to determine the current status of ARGs and HMRGs in hospital effluents and waterways in Benin.

Genomic DNA was extracted from the identified multiresistant drug-resistant bacterial strains using a Quick-DNA TM miniprep kit (Zymo Research Corp, United Stat) according to the manufacturer's instructions. In all, 12 ARGs (cmlA, catII, bla_TEM-1, sulI, sulII, tetA, tetQ, tetX, tetG, tetW, ermG, ermB) and six HMRGs (zntA, pbrT, czcA, czcC, czcD, copA) were researched. cmlA, catII, bla_TEM-1, sulI, and sulII were researched in gram-negative bacilli, and cmlA, catII, tetA, tetQ, tetX, tetG, tetW, ermG and ermB were researched in gram-positive cocci. Real-time PCR was run using a LineGene9600 Plus Fluorescent Quantitative Detection System (Hangzhou Bioer Technology, China) with the following program: 95°C for 60 s, 40 cycles consisting of (i) 95°C for 15 s, (ii) annealing temperature for 15 s, and a melting stage consisting of (i) 95°C for 15 s, (ii) melting temperature for 60 s and (iii) 95°C for 15 s. Cycle thresholds (CT) were reported, and positive samples were isolated with CT below 30. Primer sequences and annealing temperatures are displayed in (Table 1). Positive controls for antibiotic resistance genes were clinical isolates that carry those genes and the detection was done by standard PCR. For heavy metal genes, to none positive controls were used, but the experiments were ruled twice to confirm the true positive. All negative controls were RNA/DNA free water.

The problem of liquid effluent management remains a concern in developing countries like Benin. The objective of this study was to assess the presence of antibiotic and metal resistance gens in Benin hospital effluents and streams. The detection of resistance genes in the extracted DNA of the different bacterial strains isolated showed the presence as well as of ARGs (tetA, bla_TEM-1, ermB, sulI, sulII) and HMRGs (zntA, czcA, copA). These genes are found both in hospital effluents (tetA, bla_TEM-1, ermB, sulI, sulII, zntA, czcA, copA) and in waterways (cmlA, bla_TEM-1, sulI, sulII, zntA, czcA, copA). In hospital effluents, the resistance genes were found in strains of Staphylococcus aureus, coagulase-negative staphylococci, Acinetobacter spp., Escherichia coli, Klebsiella spp., Pseudomonas spp. and Yersinia enterolitica. Bacterial strains of Acinetobacter spp., Yersinia spp., Klebsiella spp., Staphylococcus aureus, and Pseudomonas spp. isolated from wastewater at a sewage treatment plant in western Massachusetts, USA, contained resistance genes to β-lactams, sulfonamides, tetracyclines, zinc, and copper (Martin et al., 2021). Sewage treatment plants receive wastewater from the entire community, including hospital effluents. Therefore, we can say that the results obtained in the present study are in line with those of (Martin et al., 2021). Several studies have shown the presence of antibiotic resistance genes in hospital effluents (Hara et al., 2018; Paul et al., 2018; Yousfi et al., 2019; Fadare and Okoh, 2021), but very few have focused on the presence of HMRGs in effluents. This study provides new scientific data on the presence of heavy metals in hospital effluents. This can be explained by the different human activities practiced and the important flow of humans in hospitals (kitchen, medical care, discharge of heavy metal residues through urine, and feces). These results also support the fact that there is a correlation between the presence of antibiotic and heavy metal resistance genes (Di Cesare et al., 2016). Furthermore, it noted a low presence of bla_TEM-1 gene, while it is known that penicillin and cephalosporins are widely used in the country, as indicated by the studies of Dougnon et al. (2020). It would therefore be interesting to update the scientific data on the consumption of antibiotic molecules in Benin. However, it should be noted that the bla_TEM-1 genes represent only one of the many genes coding for cephalosporin resistance. It should also be noted that other origins may contribute to the presence of these genes in rivers, including migratory birds in which the same genes have been noted (Yuan et al., 2018). In Poland, and more precisely in the Warmia and Mazury regions, the resistance genes bla_TEM-1, sulI, and sulII were detected in river, wastewater and sewage sludge samples (Hubeny et al., 2021). These antibiotic resistance genes were correlated with heavy metals found in variousconcentrations in the same samples (Hubeny et al., 2021). These results are consistent with what have been obtained in this with those in this study, where the presence of bla_TEM1, sulI, sulII, and heavy metal resistance genes zntA, czcA, and copA) has been detected. Similar results were obtained by Sabatino et al. (2020) in samples from the Black Sea, where an abundance of tetA, sulII and czcA genes were detected. Hubeny et al. (2021) has reported that wastewater and sewage sludge are discharged into the river. This supports our argument that antibiotic and heavy metal resistance genes are transferred from hospital effluents and community wastewater to Benin's streams. Al Salah et al. (2021) have shown in their studies that the co-occurrence of heavy metals, antibiotic resistant bacteria (ARB), and ARGs in hospital effluent spreading in riverine receiving systems and the assessment of the associated risks are topics of scientific interest and are still little studied in developing countries under tropical conditions.

All these results show the involvement of hospital effluents in the contamination of rivers. It is therefore important that other studies showing the flow of this dissemination should be carried out to identify the treatment and purification sites of hospital effluents before their discharge into streams. This will contribute to the conservation of water resources and help prevent the spread of antimicrobials through a One Health approach.

Antibiotic and heavy metal resistance genes are environmental pollutants that contribute significantly to the emergence of multidrug resistance. In the present study, the presence of multidrug-resistant bacteria in hospital effluents was linked to the main streams of Benin. Similar antibiotic resistance genes were found in hospital effluents and streams. These results indicate that hospital effluents are a potential source of dissemination of these hazardous contaminants into water sources. However, it is urgent that these results be used as a basis for monitoring both hospital effluents and streams and for setting up treatment and purification systems for these waters. DNA sequencing to characterize resistance genes and phylogenetic analysis will help to understand and track the flow of antibiotics and metal resistance genes between hospital effluents and streams. However, in the present study, we were not able to carry out these techniques due to the unavailability of the necessary equipment in Benin.

The authors have not declared any conflicts of interests.

The authors are very grateful to The World Academy of Sciences (TWAS) and the Islamic Development Bank (ISDB), which funded this study. They are finally grateful to the Minister of the Living Environment and Sustainable Development in Benin, his Excellency M. Jose ? TONATO, and all his staff.

The authors are very grateful to The World Academy of Sciences (TWAS) and the Islamic Development Bank (ISDB). These two institutions have made this research possible through research funding allocated to the research team under the IsDB-TWAS Grants for Research Collaboration in Sustainability Sciences (506808). They reviewed the research protocol and validated the design of the study and collection, analysis, and interpretation of data.