This study identified the sources of fecal contamination in the groundwater of different land covers. A total of 300 groundwater samples were collected in the Kathmandu Valley, Nepal, in the dry (n = 152) and wet (n = 148) seasons of 2016. Fecal indicator bacteria were initially enumerated, and then fecal contamination sources were identified using human (BacHum), ruminant (BacR), and pig-associated (Pig2Bac) Bacteroidales quantitative polymerase chain reaction assays. Sixty-six percent (197/300) of the tested groundwater samples had Escherichia coli concentrations higher than the World Health Organization threshold for drinking (<1 most probable number/100 mL). The fecal contamination of the groundwater was of human (22%, 55/250), ruminant (11%, 28/250), and pig (3%, 8/250) origin. Deep tube wells were less likely to be positive for E. coli and fecal markers compared to shallow dug wells. The human fecal marker was more likely to be detected in sources from built-up as compared to agricultural areas (Adjusted odds ratio (AOR) = 3.60, p = 0.002). Likewise, the ruminant fecal marker was more likely to be detected in sources from agricultural as compared to built-up areas (AOR = 2.90, p = 0.018). These findings suggest the preparation of mitigation strategies for controlling fecal pollution based on land cover and well types.

Hospitals and wastewater treatment plants (WWTPs) are high-risk point sources of antibiotic-resistance genes (ARGs) and antibiotic-resistant bacteria. This study investigates the occurrence of clinically relevant ARGs (sul1, tet(B), bla_CTX-M, bla_NDM-1, qnrS) and a class one integron (intI1) gene in urban rivers, hospitals, and municipal wastewater in the Kathmandu Valley, Nepal. Twenty-five water samples were collected from three rivers, six hospitals, and a wastewater treatment plant to determine the concentrations of ARGs and intI1 using quantitative polymerase chain reactions. From the results, all tested ARGs were detected in the river water; also, concentrations of ARGs in WWTP and hospital effluents varied from 6.2 to 12.5 log₁₀ copies/L, highlighting the role of a WWTP and hospitals in the dissemination of ARGs. Except for bla_NDM-1, significant positive correlations were found between intI1 and other individual ARGs (r = 0.71–0.96, p < 0.05), indicating the probable implications of intI1 in the transfer of ARGs. Furthermore, this study supports the statement that the bla_NDM-1 gene is most likely to be spread in the environment through untreated hospital wastewater. Due to the interaction of surface water and groundwater, future research should focus on ARGs and factors associated with the increase/decrease in their concentration levels in drinking water sources of the Kathmandu Valley.

The aim of this study was to elucidate the inactivation effects on the antibiotic-resistance gene (vanA) of vancomycin-resistant enterococci (VRE) using chlorination, a disinfection method widely used in various water treatment facilities. Suspensions of VRE were prepared by adding VRE to phosphate-buffered saline, or the sterilized secondary effluent of a wastewater treatment plant. The inactivation experiments were carried out at several chlorine concentrations and stirring time. Enterococci concentration and presence of vanA were determined. The enterococci concentration decreased as chlorine concentrations and stirring times increased, with more than 7.0 log reduction occurring under the following conditions: 40 min stirring at 0.5 mg Cl₂/L, 20 min stirring at 1.0 mg Cl₂/L, and 3 min stirring at 3.0 mg Cl₂/L. In the inactivation experiment using VRE suspended in secondary effluent, the culturable enterococci required much higher chlorine concentration and longer treatment time for complete disinfection than the cases of suspension of VRE. However, vanA was detected in all chlorinated suspensions of VRE, even in samples where no enterococcal colonies were present on the medium agar plate. The chlorine disinfection was not able to destroy antibiotic-resistance genes, though it can inactivate and decrease bacterial counts of antibiotic-resistant bacteria (ARB). Therefore, it was suggested that remaining ARB and/or antibiotic-resistance gene in inactivated bacterial cells after chlorine disinfection tank could be discharged into water environments.