As a first step for assessing the risk to human health posed by vancomycin-resistant enterococci (VRE) in the aquatic environment, we screened sewage and urban river water samples from Miyazaki, Japan for VRE. Because vancomycin-resistant organisms are not as prevalent in sewage and river water as vancomycin-susceptible organisms, the samples were screened by minimum inhibitory concentration test using the vancomycin-supplemented membrane-Enterococcus indoxyl-β-d-glucoside (mEI) agar. The isolates, presumed to be enterococci, were identified using 16S rRNA sequencing analysis. The percentages of VRE isolates screened using 4 μg mL(-1) vancomycin-supplemented mEI agar from sewage and urban river water samples were 12% and 24%, respectively. The vancomycin-resistant genes vanC1 and vanC2/3 were detected in the isolates from both samples by PCR analysis. All enterococci isolates containing vanC1, which is a specific gene for vanC-type of VRE, were identified as Enterococcus casseliflavus/gallinarum. Further, 92% enterococci isolates containing vanC2/3 were identified as E. casseliflavus/gallinarum, the remaining isolates containing vanC2/3 were E. faecium (4%) and E. faecalis (4%). Thereafter, the distribution of E. faecium and E. faecalis, which are the major types of enterococci in humans containing vanC2/3, was observed in the water samples collected.

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Rotavirus is one of the major causes of infectious gastroenteritis among infants and children, and live attenuated vaccines for rotavirus A (RVA), namely Rotarix and RotaTeq, have become recently available in Japan. Rotavirus is known to be excreted from patients and accumulated in oysters similar to norovirus; however, the vaccine strains in aquatic environments or oysters have not yet been analyzed. In this study, we focused on wild-type RVA, which is highly important in considering the risk of infectious diseases. We quantified total RVA, Rotarix, and RotaTeq strains in oyster and sewage samples collected between September 2014 and July 2016 to assess the contamination levels of wild-type RVA by subtracting the quantitative value of rotavirus vaccine strains from that of total RVA. The positive rates of wild-type RVA, Rotarix, and RotaTeq in oysters were 54, 14, and 31%, respectively. These rates were comparable with those of wild-type RVA (57%) and RotaTeq (35%) in sewage; however, Rotarix was not detected in any sewage samples. The comparison of viral concentrations in oysters and sewage suggested more efficient accumulation of the vaccine strains in oysters than the wild-type RVA. The concentration of wild-type RVA in oysters was significantly correlated with that in sewage with a lag-time of -6 to 0 weeks which is required for viral transportation from wastewater treatment plants to oysters. On the other hand, no significant correlation was observed between wild-type RVA concentration in sewage and the number of rotavirus-associated gastroenteritis cases, implying the existence of asymptomatic RVA-infected individuals. Importance We quantified rotavirus A (RVA), Rotarix, and RotaTeq strains in oyster and sewage samples during two gastroenteritis seasons, and revealed the exact contamination of wild-type RVA by subtracting the quantitative value of rotavirus vaccine strains from that of RVA. The concentration of wild-type RVA was significantly correlated between oysters and sewage, although no significant correlation was seen between wild-type RVA concentration in sewage and the number of rotaviruses detected in patients with gastroenteritis. This finding suggested the existence of asymptomatic patients and that monitoring of rotavirus vaccine strain could be useful to understand the trend of wild-type RVA and rotavirus outbreak in detail. We believe that our study makes a significant contribution to the literature because it is the first report of detection of rotavirus vaccine strains in oysters.

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Disease outbreaks attributed to monsoon flood-induced pathogen exposure are frequently reported, especially in developing cities with poor sanitation. Contamination levels have been monitored in past studies, yet the sources, routes, and extents of contamination are not always clear. We evaluated pollution from municipal wastewater (MWW) discharge and investigated fecal contamination by Escherichia coli (E. coli) in three agricultural fields on the outskirts of Hue City, Vietnam. After E. coli concentration was determined in irrigation water (IRW), MWW, soil, vegetables (VEG), and manure, its dispersion from MWW was tracked using multilocus sequence typing (MLST) and phylogenetic analyses during the wet and dry seasons. IRW was severely contaminated; 94% of the samples were positive with E. coli exceeding the stipulated standards, while VEG contamination was very low in both seasons. The confirmed total number of isolates was comparable between the seasons; however, results from MLST and phylogenetic clustering revealed more links between the sites and samples to MWW during the wet season. The wet season had four mixed clusters of E. coli isolates from multiple locations and samples linked to MWW, while only one mixed cluster also linking MWW to IRW was observed during the dry season. The most prevalent sequence type (ST) complex 10 and two others (40 and 155) have been associated with disease outbreaks, while other STs have links to major pathotypes. Irrigation canals are significant routes for E. coli dispersion through direct links to the urban drainage-infested river. This study clarified the genotype of E. coli in Hue city, and the numerous links between the samples and sites revealed MWW discharge as the source of E. coli contamination that was enhanced by flooding.