An increased abundance of antibiotic resistance genes (ARGs) in aquatic environments has been linked to environmental pollution. Mining polluted sites with high concentration of metals could favor the in situ coselection of ARGs, whereas wastewater discharges release fecal antibiotic resistant bacteria in the environment. To study the effect of human fecal contamination and mining pollution, water and sediment samples affected by mining activities and sewage discharges were collected from three lakes in Bolivia, the pristine Andean lake Pata Khota, the Milluni Chico lake directly impacted by acid mine drainage, and the Uru-Uru lake located close to Oruro city and highly polluted by mining activities and human wastewater discharges. Physicochemical parameters, including metal composition, were analyzed in water and sediment samples. ARGs were screened for and verified by quantitative polymerase chain reaction (PCR) together with the mobile element class 1 integron (intl1), as well as crAssphage, a marker of human fecal pollution. The gene intl1 was positively correlated with sul1, sul2, tetA, and blaOXA-2. CrAssphage was only detected in the Uru-Uru lake, and its tributaries and significantly higher abundance of ARGs were found in these sites. Multivariate analysis showed that crAssphage abundance, electrical conductivity, and pH were positively correlated with higher levels of intl1 and ARGs. Taken together, our results suggest that fecal pollution is the major driver of higher levels of ARGs and intl1 in environments contaminated by wastewater and mining activities.

26Water and sediment samples affected by mining activities were collected from 27 three lakes in Bolivia, the pristine Andean lake Pata Khota, the Milluni Chico lake 28 directly impacted by acid mine drainage, and the Uru-Uru lake located close to 29 Oruro city and highly polluted by mining activities and human wastewater 30 discharges. Physicochemical parameters, including metal compositions, were 31 analyzed in water and sediment samples. Antibiotic resistance genes (ARGs), 32 were screened for, and verified by quantitative PCR together with the mobile 33 element class 1 integron (intl1) as well as crAssphage, a marker of human fecal 34 pollution. The gene intl1 showed a positive correlation with sul1, sul2, tetA and 35 blaOXA-2. CrAssphage was only detected in Uru-Uru lake and its tributaries and 36 significantly higher abundance of ARGs were found in these sites. Multivariate 37 analysis showed that crAssphage abundance, electrical conductivity and pH were 38 positively correlated with higher levels of intl1 and ARGs. Taken together our 39 results suggest that fecal pollution is the major driver of higher ARGs and intl1 in 40 wastewater and mining contaminated environments. 41 3 48 can be incorporated and replicated in environmental microorganisms, thereby 49 increasing their concentration (3). 50 It has been reported that anthropogenic activities cause pollution of aquatic 51 environments with ARGs and MGEs (4). Wastewater discharges cause co-52 occurrence of MGEs and different ARGs in water and sediments (5). At a 53 continental scale, ARGs in sediments are strongly correlated with MGEs and 54antibiotic residues (6). Recently, it has been observed that microorganisms living in 55 aquatic microbial communities that came from wastewater were able to transfer 56 ARGs via horizontal gene transfer (HGT) after exposure to low levels of antibiotics 57 and biocides (7). Many of the ARGs that can be found in clinical settings have also 58 been found in the environment, suggesting the possibility of movement and 59 dissemination between these two scenarios (8). 60Mining activities cause contamination of downstream water with dissolved 61 metals (9) where heavy metals tend to accumulate in sediments (10). It has been 62 suggested that heavy metals can favor selection of ARGs via co-selection, i.e. the 63 simultaneous acquisition of both, ARG and metal resistance genes, where the 64 presence of metals constitutes the selective pressure (11). Several studies support 65 this relation. Urban soil samples of Belfast in Northern Ireland, exhibited a pattern 66 of co-occurrence between metals (Zn, Cu, Cd, Co, Ni, Hg, Cr and As) and many 67 ARGs. Moreover, the degree of metal toxicity was positively correlated with the 68 abundance of MGEs, and ARGs (12). Metals, as Cu and Zncan in some cases 69 exert stronger selection pressure over soil microbial communities for the selection 70 of resistant bacteria, even more than specific antibiotics (13). In the Dongying river 71 in China, the levels of Cu and Cr were positively c...

Watersheds contaminated with municipal, hospital, and agricultural residues are recognized as reservoirs for bacteria carrying antibiotic resistance genes (ARGs). The objective of this study was to determine the potential of environmental bacterial communities from the highly contaminated La Paz River basin in Bolivia to transfer ARGs to an Escherichia coli lab strain used as the recipient. Additionally, we tested ZnSO4 and CuSO4 at sub-inhibitory concentrations as stressors and analyzed transfer frequencies (TFs), diversity, richness, and acquired resistance profiles. The bacterial communities were collected from surface water in an urban site close to a hospital and near an agricultural area. High transfer potentials of a large set of resistance factors to E. coli were observed at both sites. Whole-genome sequencing revealed that putative plasmids belonging to the incompatibility group N (IncN, IncN2, and IncN3) were predominant among the transconjugants. All IncN variants were verified to be mobile by a second conjugation step. The plasmid backbones were similar to other IncN plasmids isolated worldwide and carried a wide range of ARGs extensively corroborated by phenotypic resistance patterns. Interestingly, all transconjugants also acquired the class 1 integron intl1, which is commonly known as a proxy for anthropogenic pollution. The addition of ZnSO4 and CuSO4 at sub-inhibitory concentrations did not affect the transfer rate. Metal resistance genes were absent from most transconjugants, suggesting a minor role, if any, of metals in the spread of multidrug-resistant plasmids at the investigated sites.

Enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of infant diarrhea in low- and middle-income countries (LMICs). Diarrheal pathogens are transmitted through environmental reservoirs; however, the bacterial clones that spread across the human-environment interphases remind unexplored. We aimed to determine the relationship and clonal dissemination of ETEC between children with diarrhea (> 5 years of age) and polluted water samples from local river in La Paz, Bolivia. Our study used whole genome sequencing and phenotypic fingerprinting system (PhenePlates) to analyze ETEC strains. We showed that ST218 and ST410 LT+STh CS23 ETEC were found with high frequency in both samples. The CS23 ETEC isolates were found within several STs, E. coli phylogroups A, B1, C, and D, and across ETEC lineages. Our comparative genomic analysis and PhenePlate screening of globally distributed clinical ETEC strains suggested that virulent CS23 plasmids acquisition occurs independently of the bacterial chromosomal background. Environmental strains were more often multidrug-resistant (MDR) than clinical isolates and harbored the class 1 integron-integrase gene intI1 next to the MDR cassettes. Retrospective analysis of antibiotic resistance in ETEC revealed a high frequency of MDR in clinical isolates. The LT+STh CS23 ETEC isolates showed an increased biofilm ability at environmental temperature, equal cytotoxicity, and significantly lower adherence to human epithelial cells compared to ETEC expressing other CFs. Together, our findings suggest that CS23 is more prevalent in ETEC than previously estimated, and the Choqueyapu River is a reservoir for LT+STh CS23 ETEC containing strains capable of causing diarrheal cases in children.