Comprehensive Genome and Plasmidome Analysis of Antimicrobial Resistant Bacteria in Wastewater Treatment Plant Effluent of Tokyo.

To characterize environmental antimicrobial resistance (AMR) in urban areas, extended-spectrum ß-lactamase- (ESBL)/carbapenemase-producing bacteria (EPB/CPB, respectively) from urban wastewater treatment plant effluents in Tokyo were isolated on CHROMagar ESBL plate. Complete genome sequence analysis, including plasmids, indicated that 126 CTX-M-positive isolates (31%) were identified among the 404 obtained isolates. The CTX-M-9 group was predominant (n = 65, 52%), followed by the CTX-M-1 group (n = 44, 35%). Comparative genome analysis revealed that CTX-M-27-positive E. coli O16:H5-ST131-fimH41 exhibited a stable genome structure and clonal-global dissemination. Plasmidome network analysis revealed that 304 complete plasmid sequences among 85 isolates were grouped into 14 incompatibility (Inc) network communities (Co1 to Co14). Co10 consisted of primarily IncFIA/IncFIB plasmids harboring blaCTX-M in E. coli, whereas Co12 consisted primarily of IncFIA(HI1)/Inc FIB(K) plasmids harboring blaCTX-M, blaKPC, and blaGES in Klebsiella spp. Co11 was markedly located around Co10 and Co12. Co11 exhibited blaCTX-M, blaKPC, and blaNDM, and was mainly detected in E. coli and Klebsiella spp. from human and animal sources, suggesting a mutual role of Co11 in horizontal gene transfer between E. coli and Klebsiella spp. This comprehensive resistome analysis uncovers the mode of relational transfer among bacterial species, highlighting the potential source of AMR burden on public health in urban communities.

Metagenomic Analysis of Urban Wastewater Treatment Plant Effluents in Tokyo.

Purpose: Urban wastewater treatment plant (WWTP) effluents, even with proper treatment, may cause antimicrobial resistance (AMR) burden, with a high frequency of acquired antimicrobial resistance genes (ARGs). The dissemination of ARGs into the environment increases the risk of infectious diseases; however, there is little direct evidence regarding their epidemiological effects. This study aimed to assess effluents from urban WWTPs around the Tama River and Tokyo Bay using metagenomic analysis of (AMR) genes (ARGs) and heavy-metal resistance genes. Methods: Metagenomic DNA-seq analysis of water samples and resistome analysis were performed. Results: The most prevalent ARG was the sulfonamide resistance gene, sul1, followed by the quaternary ammonium compound resistance gene, qacE, suggesting that basic gene sets (sul1 and ∆qacE) in the class 1 integrons are the predominant ARGs. The aminoglycoside resistance genes, aadA and aph, and macrolide resistance genes, msr(E) and mph(E), were the predominant ARGs against each antimicrobial. bla OXA and bla GES were frequently detected, whereas the bla CTX-M cluster was faintly detected. Non-metric multidimensional scaling plot analysis and canonical correspondence analysis results suggested that marked differences in ARGs could be involved in the seasonal differences; qnrS2, aac(6')-Ib, and mef(C) increased markedly in summer, whereas msr(E) was more frequently detected in winter. Heavy-metal (Hg and Cu) resistance genes (HMRGs) were significantly detected in effluents from all WWTPs. Conclusion: We characterized a baseline level of the environmental ARG/HMRG profile in the overall community, suggesting that environmental AMR surveillance, particularly in urban WWTPs, is a valuable first step in monitoring the AMR dissemination of bacteria from predominantly healthy individuals carrying notable ARG/Bs.

Haplotype Explorer: an infection cluster visualization tool for spatiotemporal dissection of the COVID-19 pandemic.

The worldwide eruption of coronavirus disease 2019 (COVID-19) that began in Wuhan, China in late 2019 reached 10 million cases by late June 2020. In order to understand the epidemiological landscape of the COVID-19 pandemic, many studies have attempted to elucidate phylogenetic relationships between collected viral genome sequences using haplotype networks. However, currently available applications for network visualization are not suited to understand the COVID-19 epidemic spatiotemporally due to functional limitations that motivated us to develop Haplotype Explorer, an intuitive tool for visualizing and exploring haplotype networks. Haplotype Explorer enables to dissect epidemiological consequences via interactive node filters and provides the perspective on infectious disease dynamics depend on regions and time, such as introduction, outbreak, expansion, and containment. Here, we demonstrate the effectiveness of Haplotype Explorer by showing features and an example of visualization. The demo using severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes are available at https://github.com/TKSjp/HaplotypeExplorer/blob/master/Example/. There are several examples using SARS-CoV-2 genomes and Dengue virus serotype 1 E-genes sequence.

A Genome Epidemiological Study of SARS-CoV-2 Introduction into Japan.

After the first case of coronavirus disease 2019 (COVID-19) in Japan on 15 January 2020, multiple nationwide COVID-19 clusters were identified by the end of February. The Japanese government focused on mitigating the emerging COVID-19 clusters by conducting active nationwide epidemiological surveillance. However, an increasing number of cases continued to appear until early April 2020, many with unclear infection routes and no recent history of travel outside Japan. We aimed to evaluate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome sequences from the COVID-19 cases that appeared until early April 2020 and to characterize their genealogical networks in order to demonstrate possible routes of spread in Japan. Nasopharyngeal specimens were collected from patients, and reverse transcription-quantitative PCR tests for SARS-CoV-2 were performed. Positive RNA samples were subjected to whole-genome sequencing, and a haplotype network analysis was performed. Some of the primary clusters identified during January and February 2020 in Japan descended directly from the Wuhan-Hu-1-related isolates from China and other distinct clusters. Clusters were almost contained until mid-March; the haplotype network analysis demonstrated that the COVID-19 cases from late March through early April may have created an additional large cluster related to the outbreak in Europe, leading to additional spread within Japan. In conclusion, genome surveillance has suggested that there were at least two distinct SARS-CoV-2 introductions into Japan from China and other countries.IMPORTANCE This study aimed to evaluate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome sequences from COVID-19 cases and to characterize their genealogical networks to demonstrate possible routes of spread in Japan. We found that there were at least two distinct SARS-CoV-2 introductions into Japan, initially from China and subsequently from other countries, including Europe. Our findings can help understand how SARS-CoV-2 entered Japan and contribute to increased knowledge of SARS-CoV-2 in Asia and its association with implemented stay-at-home/shelter-in-place/self-restraint/lockdown measures. This study suggested that it is necessary to formulate a more efficient containment strategy using real-time genome surveillance to support epidemiological field investigations in order to highlight potential infection linkages and mitigate the next wave of COVID-19 in Japan.

High-throughput genotyping of a full voltage-gated sodium channel gene via genomic DNA using target capture sequencing and analytical pipeline MoNaS to discover novel insecticide resistance mutations.

In insects, the voltage-gated sodium channel (VGSC) is the primary target site of pyrethroid insecticides. Various amino acid substitutions in the VGSC protein, which are selected under insecticide pressure, are known to confer insecticide resistance. In the genome, the VGSC gene consists of more than 30 exons sparsely distributed across a large genomic region, which often exceeds 100 kbp. Due to this complex genomic structure, it is often challenging to genotype full coding nucleotide sequences (CDSs) of VGSC from individual genomic DNA (gDNA). In this study, we designed biotinylated oligonucleotide probes from CDSs of VGSC of Asian tiger mosquito, Aedes albopictus. The probe set effectively concentrated (>80,000-fold) all targeted regions of gene VGSC from pooled barcoded Illumina libraries each constructed from individual A. albopictus gDNAs. The probe set also captured all orthologous VGSC CDSs, except some tiny exons, from the gDNA of other Culicinae mosquitos, A. aegypti and Culex pipiens complex, with comparable efficiency as a result of the high nucleotide-level conservation of VGSC. To improve efficiency of the downstream bioinformatic process, we developed an automated pipeline-MoNaS (Mosquito Na+ channel mutation Search)-which calls amino acid substitutions in the VGSC from NGS reads and compares those to known resistance mutations. The proposed method and our bioinformatic tool should facilitate the discovery of novel amino acid variants conferring insecticide resistance on VGSC and population genetic studies on resistance alleles (with respect to the origin, selection, and migration etc.) in both clinically and agriculturally important insect pests.

Potential KPC-2 carbapenemase reservoir of environmental Aeromonas hydrophila and Aeromonas caviae isolates from the effluent of an urban wastewater treatment plant in Japan.

Aeromonas hydrophila and Aeromonas caviae adapt to saline water environments and are the most predominant Aeromonas species isolated from estuaries. Here, we isolated antimicrobial-resistant (AMR) Aeromonas strains (A. hydrophila GSH8-2 and A. caviae GSH8M-1) carrying the carabapenemase blaKPC-2 gene from a wastewater treatment plant (WWTP) effluent in Tokyo Bay (Japan) and determined their complete genome sequences. GSH8-2 and GSH8M-1 were classified as newly assigned sequence types ST558 and ST13, suggesting no supportive evidence of clonal dissemination. The strains appear to have acquired blaKPC-2 -positive IncP-6-relative plasmids (pGSH8-2 and pGSH8M-1-2) that share a common backbone with plasmids in Aeromonas sp. ASNIH3 isolated from hospital wastewater in the United States, A. hydrophila WCHAH045096 isolated from sewage in China, other clinical isolates (Klebsiella, Enterobacter and Escherichia coli), and wastewater isolates (Citrobacter, Pseudomonas and other Aeromonas spp.). In addition to blaKPC-2 , pGSH8M-1-2 carries an IS26-mediated composite transposon including a macrolide resistance gene, mph(A). Although Aeromonas species are opportunistic pathogens, they could serve as potential environmental reservoir bacteria for carbapenemase and AMR genes. AMR monitoring from WWTP effluents will contribute to the detection of ongoing AMR dissemination in the environment and might provide an early warning of potential dissemination in clinical settings and communities.

Complete Genome Sequence of a blaKPC-2-Positive Klebsiella pneumoniae Strain Isolated from the Effluent of an Urban Sewage Treatment Plant in Japan.

Antimicrobial resistance genes (ARGs) and the bacteria that harbor them are widely distributed in the environment, especially in surface water, sewage treatment plant effluent, soil, and animal waste. In this study, we isolated a KPC-2-producing Klebsiella pneumoniae strain (GSU10-3) from a sampling site in Tokyo Bay, Japan, near a wastewater treatment plant (WWTP) and determined its complete genome sequence. Strain GSU10-3 is resistant to most ß-lactam antibiotics and other antimicrobial agents (quinolones and aminoglycosides). This strain is classified as sequence type 11 (ST11), and a core genome phylogenetic analysis indicated that strain GSU10-3 is closely related to KPC-2-positive Chinese clinical isolates from 2011 to 2017 and is clearly distinct from strains isolated from the European Union (EU), United States, and other Asian countries. Strain GSU10-3 harbors four plasmids, including a blaKPC-2-positive plasmid, pGSU10-3-3 (66.2 kb), which is smaller than other blaKPC-2-positive plasmids and notably carries dual replicons (IncFII [pHN7A8] and IncN). Such downsizing and the presence of dual replicons may promote its maintenance and stable replication, contributing to its broad host range with low fitness costs. A second plasmid, pGSU10-3-1 (159.0 kb), an IncA/C2 replicon, carries a class 1 integron (containing intI1, dfrA12, aadA2, qacEΔ1, and sul1) with a high degree of similarity to a broad-host-range plasmid present in the family Enterobacteriaceae The plasmid pGSU10-3-2 (134.8 kb), an IncFII(K) replicon, carries the IS26-mediated ARGs [aac(6')Ib-cr,blaOXA-1, catB4 (truncated), and aac(3)-IId], tet(A), and a copper/arsenate resistance locus. GSU10-3 is the first nonclinical KPC-2-producing environmental Enterobacteriaceae isolate from Japan for which the whole genome has been sequenced.IMPORTANCE We isolated and determined the complete genome sequence of a KPC-2-producing K. pneumoniae strain from a sampling site in Tokyo Bay, Japan, near a wastewater treatment plant (WWTP). In Japan, the KPC type has been very rarely detected, while IMP is the most predominant type of carbapenemase in clinical carbapenemase-producing Enterobacteriaceae (CPE) isolates. Although laboratory testing thus far suggested that Japan may be virtually free of KPC-producing Enterobacteriaceae, we have detected it from effluent from a WWTP. Antimicrobial resistance (AMR) monitoring of WWTP effluent may contribute to the early detection of future AMR bacterial dissemination in clinical settings and communities; indeed, it will help illuminate the whole picture in which environmental contamination through WWTP effluent plays a part.