Wastewater In Japan Research Articles

Detection of Legionella spp. in influent wastewater in Kobe City, Japan

ABSTRACT Legionella is an important waterborne pathogen that causes Legionnaires' disease (LD). Several outbreaks associated with wastewater treatment plants (WWTPs) have been reported in recent years; however, the prevalence of Legionella in WWTPs in Japan has rarely been investigated. In this study, we investigated the distribution of Legionella in influent wastewater collected from two WWTPs in Kobe, Japan between April 2023 and March 2024. The concentrations for Legionella in all seasons varied between 104 and 106 copies/100 mL in all seasons. Among the 10 Legionella species detected in the influent wastewater, Legionella pneumophila was the most commonly isolated. Genotyping revealed that pathogenic L. pneumophila strains were widely distributed in the influent wastewater in Japan with genetic diversity. LD is one of the most important infectious diseases during natural disasters. This study highlights the importance of influent wastewater as a potential source of LD in Japan, where natural disasters occur frequently.

Corallococcus caeni sp. nov., a novel myxobacterium isolated from activated sludge.

Two myxobacterial strains (KH5-1T and NO1) were isolated from the activated sludge tanks treating municipal sewage wastewater in Japan. These strains were recognised as myxobacteria based on their phenotypic characteristics of swarming colonies and fruiting bodies. Phylogenetic analyses using the 16S rRNA gene revealed that strains KH5-1T and NO1 were affiliated with the genus Corallococcus, with the closest neighbours being Corallococcus exercitusAB043AT (99.77% and 99.84%, respectively). Genome comparisons using orthologous average nucleotide identity (orthoANI) and digital DNA-DNA hybridisation similarity (dDDH) with strains KH5-1T and NO1 and their phylogenetically close relatives inCorallococcusspp. were below the thresholds. The major cellular fatty acids of strains KH5-1T and NO1 were iso-C15:0 (31.9%, 30.0%), summed feature 3(comprising C16:1ω7c and/or C16:1ω6c) (20.2%, 17.7%), and iso-C17:0 (12.1%, 14.8%), and the major respiratory quinone was found to be menaquinone (MK)-8. Based on the phenotypic, chemotaxonomic, and phylogenetic evidence, strains KH5-1T and NO1 represent a new species in the genusCorallococcus,for which the proposed nameisCorallococcus caenisp. nov. The type strain is KH5-1T (= NCIMB 15510T = JCM 36609T).

Research on the effects of ocean current on nuclear wastewater in Japan

According to reports, the Fukushima Daiichi Nuclear Power Station nuclear pollution water drainage project has been completed. Subsequently, Tokyo Electric Power will directly discharge nuclear wastewater into the ocean through a subsea tunnel connecting the land. These nuclear wastewater will be discharged to the waters near the Tropic of Cancer, which is greatly affected by the Japanese warm current, and nuclear wastewater will be taken around the world through ocean currents. German research institutions point out that within a month from the date of discharge of Fukushima nuclear wastewater, its radioactive material will spread to most of the Pacific Ocean, which will seriously affect global fish migration, ocean fisheries, human health and ecological security. Due to the impact of the global ocean current, China's East China Sea, the Yellow Sea and the Bohai Sea will also be affected by nuclear waste water pollution. Based on this, this text will start from the impact of the El Nino warm current on the flow of nuclear wastewater, and will soon explain the impact of Japan's dumping of nuclear wastewater on global security, and from a personal perspective on the current view of environmental protection and governance.

Research on the Transboundary Environmental Damage Caused by the Discharge of Nuclear Wastewater in Japan

Neighboring nations voiced severe protests when Japan unilaterally declared on August 23, 2023, that it intended to release radioactive effluent stockpiled since the Fukushima nuclear accident in 2011 into the ocean. Japan clearly breaches international law by acting in a way that is contrary to its duties for reasonable risk prevention, notification, and transboundary environmental impact assessment. In the international community, the question of transboundary injury accountability and compensation has long been of concern. International conventions addressing transboundary pollution are mostly framework agreements with soft law provisions, which means that their substantive impact on contracting parties' behavior is minimal. This article examines the national and personal obligations that the party in control of this nuclear wastewater discharge should have by combining concepts of international law, including the principle of attribution, state sovereignty, and international environmental protection. It examines the characteristics of international transboundary damage liability and compensation schemes from the standpoint of governments taking on international environmental responsibility.

The Impact of Japan's Discharge of Radioactive Water

Taking the global impact of Japan's nuclear sewage discharge as the starting point, this paper studies the harm caused by Japan's nuclear sewage flowing into the Pacific Ocean, the harm to the world's organisms, the impact on seaside workers, and investigates the harmful phenomenon of nuclear sewage to human beings. This paper is divided into four parts: the first part briefly introduces the harm of nuclear wastewater and how much nuclear wastewater needs to be discharged; the second part describes the source of nuclear wastewater in Japan; the third part summarizes the introduction literature of nuclear wastewater and the existing problems of nuclear wastewater in Japan; and the fourth part gives rise to the selfish thinking that Japan only concerns about its internal affairs. The research content of this paper mainly includes the nuclear sewage discharge incident in Japan, which caused immeasurable and serious consequences. Japan must also face consequences for its actions.

Biological-Activity-Based Prioritization of Antidepressants in Wastewater in England and Japan.

Antidepressants are one of the most commonly prescribed pharmaceuticals. Although they have been frequently detected in aquatic environments around the globe, little is known regarding their adverse effects on humans and aquatic organisms. Recently, an in vitro monoamine transporter inhibition assay was developed to detect transporter-inhibitory activities of antidepressants in wastewater in Japan. However, it was unclear which antidepressants were responsible for transporter-inhibitory activities in wastewater. Herein, the per capita consumption of 32 antidepressants, their excretion of unchanged parent compounds, per capita water consumption, removal rate during wastewater treatment processes, and potency values from the monoamine transporter inhibition assay were used to prioritize antidepressants of concern in effluent wastewater in England and Japan. In both countries, sertraline and O-desmethylvenlafaxine had the highest contribution to inhibitory activities against the human serotonin transporter (hSERT) and zebrafish SERT (zSERT), respectively. It was found that the antidepressants inhibited the zSERT more strongly than the hSERT. The inhibitory activities found against the zSERT in wastewater in England and Japan were higher than thresholds for abnormal behavior in fish. The antidepressants prioritized in this study provide insight into launching environmental monitoring and ecotoxicological studies of antidepressants.

Characterization of extended-spectrum β-lactamase-producing Escherichia coli isolated from municipal and hospital wastewater in Japan

The purpose of this study was to characterize the strains of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (ESBL-EC) isolated from municipal and hospital wastewater by detecting antibiotic resistance genes (ARGs) as well as antibiotic susceptibility. To identify the source of ESBL-EC, multi-locus sequence typing and typing plasmids that may carry ESBL-producing genes were conducted. Wastewater was sampled twice a month from February 2019 to February 2020 from a municipal wastewater treatment plant and hospital located in a city in northeastern Japan. Throughout the study period, 279 and 37 strains of ESBL-EC were isolated from municipal and hospital wastewater, respectively. All 316 isolates were resistant to ampicillin and cefotaxime and susceptible to imipenem and tigecycline. Almost all (98.1%) of the ESBL-EC isolates possessed blaCTX-M, and the blaCTX-M-9 group was detected most frequently (62.3%). Multi-locus sequence typing revealed a higher diversity of sequence types (STs) in the isolates from municipal wastewater than in those from hospital wastewater; although ST131, which recently caused nosocomial- and community-associated infections worldwide, was dominant in both types of wastewater. All ST131 isolates possessed the IncFII plasmid, which is often reported to carry blaCTX-M. These results demonstrated that healthy people carry clinically important antibiotic-resistant bacteria and ARGs, motivating routine monitoring of municipal wastewater to detect such antibiotic-resistant bacteria and ARGs from a variety of sources supported by the high diversity of STs in the present study.

Review of Antimicrobial Resistance in Wastewater in Japan: Current Challenges and Future Perspectives.

Antimicrobial resistance (AMR) circulates through humans, animals, and the environments, requiring a One Health approach. Recently, urban sewage has increasingly been suggested as a hotspot for AMR even in high-income countries (HICs), where the water sanitation and hygiene infrastructure are well-developed. To understand the current status of AMR in wastewater in a HIC, we reviewed the epidemiological studies on AMR in the sewage environment in Japan from the published literature. Our review showed that a wide variety of clinically important antibiotic-resistant bacteria (ARB), antibiotic resistance genes (ARGs), and antimicrobial residues are present in human wastewater in Japan. Their concentrations are lower than in low- and middle-income countries (LMICs) and are further reduced by sewage treatment plants (STPs) before discharge. Nevertheless, the remaining ARB and ARGs could be an important source of AMR contamination in river water. Furthermore, hospital effluence may be an important reservoir of clinically important ARB. The high concentration of antimicrobial agents commonly prescribed in Japan may contribute to the selection and dissemination of AMR within wastewater. Our review shows the importance of both monitoring for AMR and antimicrobials in human wastewater and efforts to reduce their contamination load in wastewater.

Intertypic reassortment of mammalian orthoreovirus identified in wastewater in Japan

Mammalian orthoreovirus (MRV), a non-enveloped virus with a ten-segmented double-stranded RNA genome, infects virtually all mammals, including humans. Human infection with MRV seems to be common in early childhood, but is rarely symptomatic. Despite the ubiquitous presence of MRV in mammals as well as in environmental waters, the molecular characterisation of the MRV genome remains to be fully elucidated. In this study, two novel strains, MRV-2 THK0325 and MRV-1 THK0617, were unintentionally isolated from wastewater in Japan via an environmental surveillance of enteric viruses. Homology and phylogenetic analysis demonstrated that all the segments of THK0325 were closely related to the MRV-2 Osaka strains, which were recently proposed to have existed for at least two decades in Japan. Most of the segments in THK0617 also showed a close relationship with the MRV-2 Osaka strains, but the M2, S1, and S3 segments belong to another MRV cluster. According to the S1 sequence, the determinant of serotype THK0617 was classified as MRV-1, and both the M2 and S3 segments were closely related to MRV-1 and -3 from the tree shrew in China. These results suggest that the MRV-2 Osaka-like strain spread widely throughout Japan, accompanied by intertypic reassortment occurring in East Asia.

Development of UASB–DHS System for Anaerobically-Treated Tofu Processing Wastewater Treatment under Ambient Temperature

Tofu is widely processed in East and Southeast Asian countries. During the production, highly polluted wastewater is discharged. This wastewater is commonly treated using a high-rate anaerobic wastewater treatment process; however, several organic compounds and nitrogen remain in the anaerobic effluent. The aim of this study was to develop a combined upflow anaerobic sludge blanket (UASB) and downflow hanging sponge (DHS) biosystem that that serves as a post-treatment for an expanded granular sludge blanket reactor used for treating tofu-processing wastewater in Japan for 699 days. The UASB reactor played a role in treating of COD, with 58 ± 16% and 74 ± 20% of total COD and soluble COD removed anaerobically. Besides, methane was recovered from removed soluble COD were 63 ± 28% and 87 ± 64% at winter and summer. Meanwhile, the DHS reactor showed its potential in treatment of BOD and TSS. The final effluents were recorded as 67 ± 38 mg·L-1, 50 ± 26 mg·L-1, and 22 ± 16 mg·L-1 of total COD, BOD and total suspended solids, respectively. This indicates that the proposed UASB-DHS system has proven its suitability as post-treatment system for anaerobically treated tofu-processing wastewater

New impacts and countermeasures of nuclear waste water discharge from Japan on China’s aquatic products trade

On April 13, 2021, the Japanese government officially decided to release Fukushima Daiichi Nuclear Power Plant into the sea, which is harmful to the marine environment. Japan is adjacent to the East China Sea. The East China Sea is China’s main marine ecological aquaculture area. The discharge of nuclear waste water in Japan will inevitably have an impact on the status of China’s aquatic product industry and aquatic product trade. Based on the current situation of China’s aquatic products industry and the competitiveness of China’s aquatic products in foreign trade, this paper predicts the impact of Japan’s nuclear waste water discharge on China’s aquatic products trade. Therefore, China should actively use technological innovation, regulatory upgrading, optimization of fisheries technology and other policies in advance to deal with the impact of this nuclear waste water discharge.

Synthesis of metaettringite from blast furnace slag and evaluation of its boron adsorption ability.

Blast furnace slag (BFS) is generated as a by-product in the ironmaking process, and hence, the development of a recycling system for BFS waste is important. In this study, the calcium component of BFS obtained from a steel company in Japan was successfully used as raw material of the synthesis of metaettringite. Metaettringite has been recently considered for as an adsorbent for boron, which is toxic to humans and animals. The BFS used was amorphous, and mainly consisted of CaO, SiO2, Al2O3, and MgO; in particular, it contained 42.82 mass % CaO. The X-ray diffraction pattern and Fourier transform infrared spectrum of the synthesized sample indicated the formation of metaettringite. Field-emission scanning electron microscopy observations revealed its needle-like morphology. The synthesized metaettringite adsorbed 1.189mg-B/g-adsorbent in 240min, which was approximately 25 times greater than that achieved using the parent BFS. The metaettringite can reduce the boron concentration to below the uniform effluent standard in industrial wastewater in Japan (16mg/L).

Detection of SARS-CoV-2 in wastewater in Japan during a COVID-19 outbreak

The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater samples has been documented in several countries. Wastewater-based epidemiology (WBE) is potentially effective for early warning of a COVID-19 outbreak. In this study, presence of SARS-CoV-2 RNA in wastewater samples was investigated and was compared with the number of the confirmed COVID-19 cases in the study area during COVID-19 outbreak in Japan. In total, 45 influent wastewater samples were collected from five wastewater treatment plants in Ishikawa and Toyama prefectures in Japan. During the study period, the numbers of confirmed COVID-19 cases in these prefectures increased from 0.3 and 0 to >20 per 100,000 people. SARS-CoV-2 ribonucleic acid (RNA) in the samples was detected using several PCR-based assays. Of the 45 samples, 21 were positive for SARS-CoV-2 according to at least one of the three quantitative RT-PCR assays. The detection frequency increased when the number of total confirmed SARS-CoV-2 cases in 100,000 people exceeded 10 in each prefecture; however, SARS-CoV-2 could also be detected at a low frequency even when the number was below 1.0. SARS-CoV-2 in wastewater could be detected in the early stage of the epidemic, even if the number of confirmed cases potentially underestimates the actual numbers of cases. This suggests that WBE approach can potentially act as an early warning of COVID-19 outbreaks in Japan.

Microalgae Oil Production Using Wastewater in Japan—Introducing Operational Cost Function for Sustainable Management of WWTP

A comparative evaluation of economic efficiency was performed for native polyculture microalgae oil production in an oxidation ditch (OD) process wastewater treatment plant (WWTP). A cost function was developed for the process. The operational cost per 1 m3 of wastewater (w.w.) was 1.34 $/m3-w.w. in the existing scenario, 1.29 $/m3-w.w. in algal scenario A (no cost for CO2 and waste heat) and 1.36 $/m3-w.w. in algal scenario B (no cost for CO2). The conditions were set as follows: hydraulic retention time (HRT): 4 days, microalgal productivity: 0.148 g/L and daily treatment volume: 81.6 m3-w.w./d. The cost differences were related to the increase in polymer flocculants for algae separation (+0.23 $/m3-w.w), carbon credits from CO2 absorption (−0.01 $/m3-w.w), the sales of biocrude (−0.04 $/m3-w.w) and sludge disposal (−0.18 $/m3-w.w). Hence, the introduction of the algae scenario was the same cost-effective as the existing scenario. Microalgae oil production in an OD process WWTP can serve as a new energy system and reduce the environmental load in a society with a declining population.

First environmental surveillance for the presence of SARS-CoV-2 RNA in wastewater and river water in Japan

Wastewater-based epidemiology is a powerful tool to understand the actual incidence of coronavirus disease 2019 (COVID-19) in a community because severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, can be shed in the feces of infected individuals regardless of their symptoms. The present study aimed to assess the presence of SARS-CoV-2 RNA in wastewater and river water in Yamanashi Prefecture, Japan, using four quantitative and two nested PCR assays. Influent and secondary-treated (before chlorination) wastewater samples and river water samples were collected five times from a wastewater treatment plant and three times from a river, respectively, between March 17 and May 7, 2020. The wastewater and river water samples (200–5000 mL) were processed by using two different methods: the electronegative membrane-vortex (EMV) method and the membrane adsorption-direct RNA extraction method. Based on the observed concentrations of indigenous pepper mild mottle virus RNA, the EMV method was found superior to the membrane adsorption-direct RNA extraction method. SARS-CoV-2 RNA was successfully detected in one of five secondary-treated wastewater samples with a concentration of 2.4 × 103 copies/L by N_Sarbeco qPCR assay following the EMV method, with sequence confirmation of the qPCR product, whereas all the influent samples were tested negative for SARS-CoV-2 RNA. This result could be attributed to higher limit of detection for influent (4.0 × 103–8.2 × 104 copies/L) with a lower filtration volume (200 mL) compared to that for secondary-treated wastewater (1.4 × 102–2.5 × 103 copies/L) with a higher filtration volume of 5000 mL. None of the river water samples tested positive for SARS-CoV-2 RNA. Comparison with the reported COVID-19 cases in Yamanashi Prefecture showed that SARS-CoV-2 RNA was detected in the secondary-treated wastewater sample when the cases peaked in the community. This is the first study reporting the detection of SARS-CoV-2 RNA in wastewater in Japan.

Multiple β-Lactam Resistance Gene-Carrying Plasmid Harbored by Klebsiella quasipneumoniae Isolated from Urban Sewage in Japan.

The continuous emergence of carbapenemase-producing Enterobacteriaceae (CPE) presents a great public health challenge. Mitigation of CPE spread in the environment is crucial, particularly from a One Health perspective. Here we describe the isolation of CPE strain SNI47 from influent water of a sewage treatment plant in Japan. SNI47 was identified as Klebsiella quasipneumoniae subsp. quasipneumoniae by phylogenetic analysis and was resistant to β-lactams, including carbapenems. Of four plasmids detected from SNI47, the 185,311-bp IncA/C2 plasmid (pTMSNI47-1), which carried 10 drug resistance genes, including genes for four β-lactamases (blaCTX-M-2, blaDHA-1, blaKHM-1, and blaOXA-10), was transferred to Escherichia coli J53 via conjugation. The MICs of all tested β-lactams for the transconjugant were higher than for the recipient. We constructed recombinant plasmids, into which each β-lactamase gene was inserted, and used them to transform E. coli DH5α cells, demonstrating that KHM-1 enhanced carbapenem resistance. In addition, these β-lactamases were responsible for a wide-spectrum β-lactam resistance acquisition with mutual compensation. KHM-1, recognized as a rare type of metallo-β-lactamase, was detected in a transferable plasmid, from a sewage treatment plant, involved in horizontal gene transfer. The detection of such plasmids raises a health risk alarm for CPE dissemination.IMPORTANCE In our investigation of urban wastewater in Japan, carbapenem-resistant Klebsiella quasipneumoniae subsp. quasipneumoniae was isolated that carried the pTMSNI47-1 plasmid, which carries four β-lactamase genes and has transferability among Enterobacteriaceae pTMSNI47-1 was found to encode a rarely reported carbapenemase, KHM-1. Cooperative effects of β-lactamases encoded by pTMSNI47-1 appeared to have broad-spectrum resistance to β-lactams. The detection of the KHM-1 gene in urban wastewater suggests that such a rare antimicrobial resistance (AMR) gene can be pooled in the environment, potentially emerging as an AMR determinant in a pathogen. When the number of β-lactamase resistance genes is increased in one plasmid, the transfer of this plasmid can confer broad-spectrum resistance to β-lactams, even if the individual gene confers narrow-spectrum resistance. The present study adds important information about the potential risk of sewage treatment plants as reservoirs and environmental suppliers of AMR genes, contributing to the public health from a One Health perspective.

Complete Genome Sequence of a Novel Myoviridae Phage, SfΦ01, Infecting Shigella spp.

The Shigella bacterium is one of the most significant causes of waterborne and foodborne bacterial dysentery. A lytic bacteriophage infecting Shigella flexneri was isolated from wastewater in Japan. We report here the complete genome sequence of this bacteriophage, revealing that it belongs to the Myoviridae family and possesses linear genomic DNA.

Simultaneous removal and recovery of boron from waste water by multi-step bipolar membrane electrodialysis

We have developed bipolar membrane electrodialysis (BPED) for simultaneous removal and recovery of boron from waste water. BPED is performed with a two-cell electrodialysis unit composed of an anion exchange membrane (AEM) sandwiched between two bipolar membranes (BPM). A semi-batch type operation was performed by circulating solutions through the cells, namely, the removal and concentration cells. The volume ratio of the reserve tanks for the feed stream to the cells was set to 4:1 (removal: concentratation). Tetrahydroxyborate ions were transported from the removal cell to the concentration cell through the AEM. In the ideal case, the boron concentration in the concentrate should be concentrated to 5 times the initial concentration, when the solutions of the same initial concentration were supplied to both cells of concentration and removal. The concentrated solution was then used for second step to further concentrate the solution. This concentrated solution was then used for third step, and so forth. The operation was continued step-by-step until the boron concentration was sufficiently high to easily recover boric acid. The experimental results with a stack of five cells for the sodium borate solutions (initial concentration: 100 mg/L) showed that the boron concentration in the concentrate was about 4, while that in the diluate was less than 10 mg/L, which is below the regulatory value for waste water in Japan. After four steps, the boron concentration in the concentrate reached close to the solubility of boric acid (ca 10,000 mg/L), at which boron can be recovered for recycling.

Characteristics of Carbapenemase-Producing Enterobacteriaceae in Wastewater Revealed by Genomic Analysis.

Wastewater is considered a major source of antibiotic-resistant bacteria released into the environment. Here, we characterized carbapenemase-producing Enterobacteriaceae (CPE) in wastewater by whole-genome analysis. Wastewater samples (n = 40) were collected from municipal wastewater treatment plants and hospital wastewater in Japan and Taiwan. Samples were screened for CPE using selective media, and the obtained isolates were sequenced using an Illumina MiSeq. The isolates (n = 45) included the following microorganisms: Klebsiella quasipneumoniae (n = 12), Escherichia coli (n = 10), Enterobacter cloacae complex (n = 10), Klebsiella pneumoniae (n = 8), Klebsiella variicola (n = 2), Raoultella ornithinolytica (n = 1), Citrobacter freundii (n = 1), and Citrobacter amalonaticus (n = 1). Among the 45 isolates, 38 harbored at least one carbapenemase-encoding gene. Of these, the blaGES (blaGES-5, blaGES-6, and blaGES-24) genes were found in 29 isolates. The genes were situated in novel class 1 integrons, but the integron structures were different between the Japanese (In1439 with blaGES-24 and In1440 with blaGES-5) and Taiwanese (In1441 with blaGES-5 and In1442 with blaGES-6) isolates. Other carbapenemase-encoding genes (blaVIM-1, blaNDM-5, blaIMP-8, blaIMP-19, and blaKPC-2) were found in one to three isolates. Notably, class 1 integrons previously reported among clinical isolates obtained in the same regions as the present study, namely, In477 with blaIMP-19 and In73 with blaIMP-8, were found among the Japanese and Taiwanese isolates, respectively. The results indicate that CPE with various carbapenemase-encoding genes in different genetic contexts were present in biologically treated wastewater, highlighting the need to monitor for antibiotic resistance in wastewater.

Temporal dynamics of norovirus determined through monitoring of municipal wastewater by pyrosequencing and virological surveillance of gastroenteritis cases

Norovirus is a leading etiological agent of viral gastroenteritis. Because of relatively mild disease symptoms and frequent asymptomatic infections, information on the ecology of this virus is limited. Our objective was to examine the genetic diversity of norovirus circulating in the human population by means of genotyping the virus in municipal wastewater. We investigated norovirus genogroups I and II (GI and GII) in municipal wastewater in Japan by pyrosequencing and quantitative PCR (qPCR) from November 2012 to March 2013. Virological surveillance for gastroenteritis cases was concurrently conducted in the same area. A total of fourteen distinct genotypes in total (GI.1, 3, 4, 6, 7, GII.2, 4, 5, 6, 7, 12, 13, 14, and 17), with up to eight genotypes detected per sample, were observed in wastewater using pyrosequencing; only four genotypes (GI.6, GII.4, 5, and 14) were obtained from clinical samples. Seventy-eight percent of norovirus-positive stool samples contained GII.4, but this genotype was not dominant in wastewater. The norovirus GII.4 Sydney 2012 variant, which appeared and spread during our study period, was detected in both the wastewater and clinical samples. These results suggest that an environmental approach using pyrosequencing yields a more detailed distribution of norovirus genotypes/variants. Thus, wastewater monitoring by pyrosequencing is expected to provide an effective analysis of the distribution of norovirus genotypes causing symptomatic and asymptomatic infections in human populations.