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.

We evaluated the suitability of pulsed electric field (PEF) technology as a new disinfection option in the sewage treatment plants (STPs) that can inactivate antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). It was shown that PEF applied disinfection could inactivate not only vancomycin-resistant enterococci (VRE), but also vanA resistance gene. Cultivable VRE could be effectively inactivated by PEF applied disinfection, and were reduced to below the detection limit (log reduction value of VRE > 5 log). Although the vanA also showed a reduction of more than 4 log, it remained in the order of 10⁵ copies/mL, suggesting that ARGs are more difficult to be inactivated than ARB in PEF applied disinfection. Among parameters in each applying condition verified in this study, the initial voltage was found to be the most important for inactivation of ARB and ARGs. Furthermore, frequency was a parameter that affects the increase or decrease of the duration time, and it was suggested that the treatment time could be shortened by increasing the frequency. Our results strongly suggested that PEF applied disinfection may be a new disinfection technology option for STPs that contributes to the control of ARB and ARGs contamination in the aquatic environments.

Enterococci such as Enterococcus faecalis and E. faecium are considered as the most suitable indicators of fecal pollution in an aquatic environment, and new methods for Enterococcus determination have been developed, namely, membrane filtration (MF) using membrane-Enterococcus indoxyl-β-D-glucoside agar (mEI) and defined substrate technology (DST) using Enterolert®. This study used PCR analysis to identify E. faecalis and E. faecium among Enterococcus strains in river water isolated using both mEI plates and Enterolert® trays. There was a significantly high correlation between MF and DST in terms of enterococcal counts for river water samples. The combined percentages of E. faecalis and E. faecium with respect to the total number of all strains obtained using mEI plates and Enterolert® trays were approximately 30 % and >30 %, respectively. Other than E. faecalis and E. faecium, a large number of Enterococcus species were unspecified in the actual urban river samples. A comparison of the predominance of E. faecalis and E. faecium found that the abundance of a species depended on the sampling river and date. E. faecium is a non-predominant species in intestinal and fecal Enterococci, and it was one of the main Enterococcus species detected in surface water.

This study elucidated the distribution and fate of vancomycin (VCM)-resistant heterotrophic bacteria (HTB) and resistance genes, vanA and vanB, during each treatment unit process of a wastewater treatment plant (WWTP). Several bacterial counts as well as copy numbers of vanA and vanB genes were determined in each wastewater and sludge sample. In addition, HTB strains isolated from wastewater and sludge were analyzed for VCM susceptibility. Then, the fate and reduction ratios of each bacterial count, copy number of vanA and vanB genes, and the existence ratio of VCM-resistant HTB strains in the wastewater treatment unit process were evaluated. VCM-resistant HTB were detected in all wastewater and sludge samples, and their existence ratio decreased along the treatment process (92.9% in influent wastewater to 39.4% in chlorinated water). Notably, most of the HTB isolated from the influent wastewater were resistant to 8.0 µg/mL of VCM, strongly suggesting that a significant number of antibiotic-resistant bacteria are flowing into the WWTP from urban areas through the sewage system. The vanA and vanB genes were also detected in all wastewater and sludge, with high copy numbers (10²-10⁴ copies/mL) even in chlorinated water samples. Results revealed that residual VCM-resistant HTB, and resistance genes, which could not be completely removed, were ubiquitously released into the aquatic environment. Furthermore, a high existence ratio of VCM-resistant HTB and high copy numbers of resistance genes were also detected in the sludge, indicating that they are constantly circulating in the WWTP via the returned sludge.