Antimicrobial resistome and mobilome in the urban river affected by combined sewer overflows and wastewater treatment effluent.

The dissemination of antimicrobial resistance in the environment is an emerging global health problem. Wastewater treatment effluent and combined sewer overflows (CSOs) are major sources of antimicrobial resistance in urban rivers. This study aimed to clarify the effect of municipal wastewater treatment effluent and CSO on antimicrobial resistance genes (ARGs), mobile gene elements, and the microbial community in an urban river. The ARG abundance per 16S-based microbial population in the target river was 0.37-0.54 and 0.030-0.097 during the CSO event and dry weather, respectively. During the CSO event, the antimicrobial resistome in the river shifted toward a higher abundance of ARGs to clinically important drug classes, including macrolide, fluoroquinolone, and ß-lactam, whereas ARGs to sulfonamide and multidrug by efflux pump were relatively abundant in dry weather. The abundance of intI1 and tnpA genes were highly associated with the total ARG abundance, suggesting their potential application as an indicator for estimating resistome contamination. Increase of prophage during the CSO event suggested that impact of CSO has a greater potential for horizontal gene transfer (HGT) via transduction. Consequently, CSO not only increases the abundance of ARGs to clinically important antimicrobials but also possibly enhances potential of HGT in urban rivers.

Development of two microbial source tracking markers for detection of wastewater-associated Escherichia coli isolates.

Escherichia coli has been used as an indicator of fecal pollution in environmental waters. However, its presence in environmental waters does not provide information on the source of water pollution. Identifying the source of water pollution is paramount to be able to effectively reduce contamination. The present study aimed to identify E. coli microbial source tracking (MST) markers that can be used to identify domestic wastewater contamination in environmental waters. We first analyzed wastewater E. coli genomes sequenced by us (n = 50) and RefSeq animal E. coli genomes of fecal origin (n = 82), and identified 144 candidate wastewater-associated marker genes. The sensitivity and specificity of the candidate marker genes were then assessed by screening the genes in 335 RefSeq wastewater E. coli genomes and 3318 RefSeq animal E. coli genomes. We finally identified two MST markers, namely W_nqrC and W_clsA_2, which could be used for detection of wastewater-associated E. coli isolates. These two markers showed higher performance than the previously developed human wastewater-associated E. coli markers H8 and H12. When used in combination, W_nqrC and W_clsA_2 showed specificity of 98.9 % and sensitivity of 25.7 %. PCR assays to detect W_nqrC and W_clsA_2 were also developed and validated. The developed PCR assays are potentially useful for detecting E. coli isolates of wastewater origin in environmental waters, though users should keep in mind that the sensitivity of these markers is not high. Further studies are needed to assess the applicability of the developed markers to a culture-independent approach.

Occurrence of E. coli and antibiotic-resistant E. coli in the southern watershed of Lake Biwa, including in wastewater treatment plant effluent and inflow rivers.

The emergence of antibiotic-resistant bacteria (ARB) and their antibiotic resistance genes (ARGs) poses a serious challenge to human, animal, and environmental health worldwide. ARB can spread into the environment via various sources and routes. In this study, we investigated the occurrence of antibiotic-resistant E. coli in the southern watershed of Lake Biwa. Two-year monitoring of antibiotic-resistant E. coli was carried out in the southern part of Lake Biwa and inflow rivers and at three WWTPs around the southern part of the lake. Concentrations of E. coli in waters that are resistant to ampicillin (AMP), cefotaxime (CTX), ceftazidime (CAZ), levofloxacin (LVFX), tetracycline (TC), and amikacin (AMK) were measured using the culture method. Of these antibiotic-resistant E. coli, AMP-resistant E. coli were found at the highest prevalence, followed by LVFX, CTX, CAZ, TC, and AMK-resistant in both the influent and effluent of WWTPs. These resistance patterns in wastewater are the same as those in clinical samples in Japan. The numbers of antibiotic-resistant E. coli decreased by around a factor of 1000 during the wastewater treatment processes, but the rates clearly increased, suggesting that selection for antibiotic resistance might occur during the wastewater treatment process. AMP-resistant and TC-resistant E. coli were also detected in Lake Biwa and inflow rivers, which suggests that antibiotic resistance might come from not only WWTPs but also livestock farms and small-scale wastewater treatment facilities located in the river catchment.

Emergence of rare carbapenemases (FRI, GES-5, IMI, SFC and SFH-1) in Enterobacterales isolated from surface waters in Japan.

OBJECTIVES: Carbapenemase-producing Enterobacterales (CPE) pose serious threats to public health. Compared with clinical CPE, the genetic characteristics of environmental CPE are not well understood. This study aimed to characterize the genetic determinants of carbapenem resistance in CPE isolated from environmental waters in Japan. METHODS: Eighty-five water samples were collected from rivers and a lake in Japan. CPE were identified using selective media, and genome sequencing was performed for the obtained isolates (n = 21). RESULTS: Various rare/novel carbapenemases were identified: GES-5 in Raoultella planticola (n = 1), FRI-8 and FRI-11 in Enterobacter spp. (n = 8), IMI-22 and IMI-23 in Serratia ureilytica (n = 3), and SFC-1, SFC-2 and SFH-1 in Serratia fonticola (n = 9). Genomes of 11 isolates could be closed, allowing the elucidation of the genetic contexts of the carbapenemase genes. The blaGES-5 gene was located within a class 1 integron, In2071 (cassette array, blaGES-5-aacA3-aadA16), on a 33 kb IncP6 plasmid. The blaFRI-8 genes were carried on IncFII(Yp) plasmids ranging in size from 191 kb to 244 kb, and the blaFRI-11 genes were carried on 70 kb and 74 kb IncFII(pECLA)/IncR plasmids. The blaIMI-22 and blaIMI-23 genes were co-located on a 107 kb plasmid. The blaSFC and blaSFH-1 genes were found on putative genomic islands inserted at tRNA-Phe genes in chromosomes. CONCLUSIONS: This study revealed the presence of rare/novel carbapenemases among CPE in aquatic environments, suggesting that the environment may act as a potential reservoir of these minor carbapenemases.

Characterization of nitrosamines and nitrosamine precursors as non-point source pollutants during heavy rainfall events in an urban water environment.

N-nitrosodimethylamine (NDMA) and N-nitrosomorpholine (NMOR) and their specific precursors (N,N-dimethylformamide [DMF] for NDMA and morpholine [MOR] for NMOR) were widely identified in runoff of urban area around the southern Lake Biwa basin, Japan. It was thought that this runoff might constitute a non-point source of the four compounds in rivers and sewage treatment plants (STPs) during heavy rainfall events. We investigated the spatiotemporal patterns of NDMA, NMOR, DMF and MOR in runoff and rivers in rainy days. NDMA and NMOR were detected in concentrations of up to 295 ng/L, while DMF and MOR were detected in concentrations of up to 33.7 µg/L. Continuous sequential sampling over periods of 24 or 48 h at the largest STP in the study area revealed that the four compounds in the primary effluent (PE) each had higher mass fluxes during heavy rainfall events than on dry days. This phenomenon might be contributed to non-point sources (e.g., runoff) from infiltration/inflow related to rainwater into sanitary sewers. Moreover, the four compounds were confirmed to have higher mass fluxes in the final effluent of the STP during periods of PE bypass (1.3-1.7 times for NDMA, NMOR, and MOR; over 200 times for DMF; on average) than that on dry days because of increasing inflow during heavy rain than during periods without PE bypass in dry weather.