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Nanopore-based long-read metagenomics uncover the resistome intrusion by antibiotic resistant bacteria from treated wastewater in receiving water body

Published in Water Research, 2022

Wastewater treatment plant (WWTP) effluent discharge could induce the resistome enrichment in the receiving water environments. However, because of the general lack of a robust antibiotic-resistant bacteria (ARB) identification method, the driving mechanism for resistome accumulation in receiving environment is unclear. Here, we took advantage of the enhanced ARBs recognition by nanopore long reads to distinguish the indigenous ARBs and the accumulation of WWTP-borne ARBs in the receiving water body of a domestic WWTP. A bioinformatic framework (named ARGpore2: https://github.com/sustc-xylab/ARGpore2) was constructed and evaluate to facilitate antibiotic resistance genes (ARGs) and ARBs identification in nanopore reads. ARGs identification by ARGpore2 showed comparable precision and recall to that of the commonly adopt BLASTP-based method, whereas the spectrum of ARBs doubled that of the assembled Illumina dataset. Totally, we identified 33 ARBs genera carrying 65 ARG subtypes in the receiving seawater, whose concentration was in general 10 times higher than clean seawater’s. Notably we report a primary resistome intrusion caused by the revival of residual microbes survived from disinfection treatment. These WWTP-borne ARBs, including several animal/human enteric pathogens, contributed up to 85% of the receiving water resistome. Plasmids and class 1 integrons were reckoned as major vehicles facilitating the persistence and dissemination of ARGs. Moreover, our work demonstrated the importance of extensive carrier identification in determining the driving force of multifactor coupled resistome booming in complicated environmental conditions, thereby paving the way for establishing priority for effective ARGs mitigation strategies.

Recommended citation: Ziqi WU et.al. (2022). "10.1016/j.watres.2022.119282." Water Research . 226(119282).
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Strategies and tools in illumina and nanopore‐integrated metagenomic analysis of microbiome data

Published in iMeta, 2022

Metagenomic strategy serves as the foundation for the ecological exploration of novel bioresources (e.g., industrial enzymes and bioactive molecules) and biohazards (e.g., pathogens and antibiotic resistance genes) in natural and engineered microbial systems across multiple disciplines. Recent advancements in sequencing technology have fostered rapid development in the field of microbiome research where an increasing number of studies have applied both illumina short reads (SRs) and nanopore long reads (LRs) sequencing in their metagenomic workflow. However, given the high complexity of an environmental microbiome data set and the bioinformatic challenges caused by the unique features of these sequencing technologies, integrating SRs and LRs is not as straightforward as one might assume. The fast renewal of existing tools and growing diversity of new algorithms make access to this field even more difficult. Therefore, here we systematically summarized the complete workflow from DNA extraction to data processing strategies for applying illumina and nanopore-integrated metagenomics in the investigation in environmental microbiomes. Overall, this review aims to provide a timely knowledge framework for researchers that are interested in or are struggling with the SRs and LRs integration in their metagenomic analysis. The discussions presented will facilitate improved ecological understanding of community functionalities and assembly of natural, engineered, and human microbiomes, benefiting researchers from multiple disciplines.

Recommended citation: Yu Xia, Ziqi Wu et.al. (2022). "doi.org/10.1002/imt2.72." iMeta. 1(2).
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Genome enrichment of rare and unknown species from complicated microbiomes by nanopore selective sequencing

Published in Genome Research, 2024

Rare species are vital members of a microbial community, but retrieving their genomes is difficult because of their low abundance. The ReadUntil (RU) approach allows nanopore devices to sequence specific DNA molecules selectively in real time, which provides an opportunity for enriching rare species. Despite the robustness of enriching rare species by reducing the sequencing depth of known host sequences, such as the human genome, there is still a gap in RU-based enriching of rare species in environmental samples whose community composition is unclear, and many rare species have poor or incomplete reference genomes in public databases. Therefore, here we present metaRUpore to overcome this challenge. When we applied metaRUpore to a thermophilic anaerobic digester (TAD) community and human gut microbial community, it reduced coverage of the high-abundance populations and modestly increased (∼2×) the genome coverage of the rare taxa, facilitating successful recovery of near-finished metagenome-assembled genomes (nf-MAGs) of rare species. The simplicity and robustness of the approach make it accessible for laboratories with moderate computational resources, and hold the potential to become the standard practice in future metagenomic sequencing of complicated microbiomes.

Recommended citation: Yuhong Sun, Ziqi WU et.al. (2024). "doi/10.1101/gr.277266.122." Genome Research.
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Plasmid-driven clonal expansion of multidrugresistant monophasic Salmonella Typhimurium in a Global Food Trade Hub

Published in Emerging Microbes & Infections, 2025

Shenzhen, a major port city with a heavily imported food supply, offers a critical setting to examine the spread and adaptation of multidrug-resistant Salmonella 1,4,[5],12:i: – (S. 1,4,[5],12:i:-). This study integrates 17 years of genomic, epidemiological, and food safety data. We explored the serovar’s population structure, antibiotic resistance gene (ARG) patterns, and transmission dynamics locally and globally. Our analyses revealed substantial rise in S. 1,4,[5],12:i: – prevalence among non-typhoidal Salmonella isolates over the past 17 years, from 2.27% in 2007 to 24.79% in 2023. S. 1,4,[5],12:i: – was predominated by ST34 (97.9%), with high genotypic resistance to aminoglycosides (100%), tetracyclines (96.6%), β-lactams (89.3%), and sulphonamides (88.5%). Phylogenetic analysis separated S. 1,4,[5],12:i: into four clades. Clade 4, first detected in Shenzhen in 2013, emerged as the predominant lineage by 2023 (56.9%). This clade exhibited minimal genetic diversity (≤ 38 core SNPs), with adaptive traits linked to the acquisition of resistance-associated plasmids. Notably, plasmid-driven ARGs, including carbapenem resistance genes, have emerged as a growing concern. Transmission analysis identified two key transmission dynamics: transient outbreaks primarily involving food handlers and persistent lineages sustained through local and international spread, often facilitated by the food supply chain. These findings underscore the role of occupational carriers and imported food products in the dissemination of ARGs, emphasizing the need for enhanced surveillance and improved health and hygiene practices for food handlers. This study provides a comprehensive molecular epidemiological framework for addressing multidrug-resistant Salmonella in globalized urban food hubs, offering a foundation for future surveillance and control efforts.

Recommended citation: Dingjie Huang, Ziqi WU et.al. (2025). "doi.org/10.1080/22221751.2025.2542251." Emerging Microbes & Infections.
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Beyond borders: plasmids drive a shared antibiotic resistome in European urban water systems

Published in Microbiome, 2026

This study applies a multi-omics approach, including metagenomic and direct plasmidome sequencing, high-throughput qPCR array, and whole genome sequencing of plasmid isolates, to comprehensively profile the microbial plasmidome and resistome on 78 samples across UWSs in Denmark, Spain, and the UK. We successfully uncovered an extensive plasmid and ARG diversity that could not be fully captured by a single method, especially identified 78,574 plasmids, including 20,925 plasmids previously unreported. We also observed that plasmids carried a disproportionate share of clinically relevant ARGs, particularly beta-lactamase resistance genes; most importantly, they were preferentially located on transmissible plasmids. Furtherly, plasmids harbor ARG can enhance their persistence in wastewater ecosystems, especially harboring multiple types of ARGs. Moreover, Bacteroides emerged as a unique persistent ARG reservoir not only for harboring and disseminating diverse resistance genes especially in residential-relevant areas, but also emerged as a major driver of antimicrobial resistance dynamics across different wastewater treatment processes.

Recommended citation: Wanli He, Ziqi Wu et.al. (2024). "doi.org/10.1186/s40168-025-02253-0." Microbiome . 14:39.
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London Calling 2018

Published: May 20, 2018

Prokaryotic Pathogen Seminar Series

Published: March 01, 2020

The International Conference on Bacteria and Global Health

Published: February 17, 2024

• Poster title: “Conjugative plasmids-associated to shape the antibiotic resistance dissemination prevalence in European wastewater sewers”
• Abstract: Antibiotic resistance (AR) surveillance is a key objective in the World Health Organization’s global action plan. Urban wastewater sewer systems serve as crucial reservoirs for environmental AR. The widespread prevalence of AR is partly attributed to the horizontal transfer of antibiotic resistance genes (ARGs), typically mediated by conjugative plasmids. However, large-scale investigation on conjugative plasmid-mediated ARGs in urban wastewater sewer systems remain scarce. In this study, we conducted an international surveillance of conjugative plasmids, obtained from wastewater sewers in hospital and residential settings in three medium-sized cities: Denmark, Spain, and the United Kingdom. A total of 797 conjugative plasmids were collected and their antibiotic susceptibilities were assessed using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Based on results of AST, about 99% (790/797) of the 797 conjugative plasmids were found to be resistant to multiple of antibiotics. The conjugative plasmids’ resistance phenotypes in different countries are influenced to some extent by the antibiotic consumption of those countries. The analysis revealed distinct phenotypes of antibiotic resistance between plasmids from residential and hospital sewers at local and global scale. Among them, 96 specific plasmids exhibiting resistance to multiple antibiotic classes were selected for whole-genome sequencing using nanopore technologies. The sequencing analysis identified five types of mobilization systems (MOBs), seventeen types of incompatibility groups (Inc), and seventy-four types of ARGs among these plasmids. These broad-host-range conjugative plasmids were also as a critical role in HGT of β-lactamases genes, multidrug resistance genes and macrolides inside the bacteria communities in wastewater. This surveillance represents the most comprehensive profiling to date of the antibiotic resistance phenotype associated with conjugative plasmids in clinical and residential sewer systems across multiple countries. The findings emphasize the critical importance of enhancing surveillance of sewage-based antibiotic resistance and contribute to a deeper understanding of the complex dynamics of conjugative plasmid-mediated antibiotic resistance, paving the way for informed strategies to combat this global challenge.

American Chemical Society(ACS) Spring 2025

Published: March 21, 2025

• Title: Conjugative plasmids-associated to shape the antibiotic resistance dissemination prevalence in European wastewater sewers
• Abstract: Antibiotic resistance (AR) surveillance is a key objective in the World Health Organization’s global action plan. Urban wastewater sewer systems serve as crucial reservoirs for environmental AR. The widespread prevalence of AR is partly attributed to the horizontal transfer of AR genes, typically mediated by conjugative plasmids. However, a comprehensive investigation on phenotypic antibiotic resistance of conjugative plasmid in urban wastewater sewers remains scarce. In this study, we applied culture-enriched and -selected phenotypic whole genomics to investigate the AR phenotypes and genotypes of conjugative plasmids in three medium-sized cities of Denmark, Spanish, and the United Kingdom. We collected 797 conjugative plasmids and assessed their antibiotic susceptibilities based on the European Committee on Antimicrobial Susceptibility Testing (AST) guidelines. Almost all conjugative plasmids were found to be resistant to multiple antibiotics. The AR phenotypes of conjugative plasmids were distinct from residential and hospital sewers in the cities of Spanish and the United Kingdom, but the phenotypic result was the opposite in the city of Denmark. Conjugative plasmids from hospital sewers possessed a stronger ability of trans-conjugation than those from residential sewers. There were 96 conjugative plasmids exhibiting resistance to more than three types of antibiotics selected for whole-genome sequencing using nanopore technologies. There were five types of mobilization systems (MOBs), seventeen types of incompatibility groups (Inc), and seventy-four types of AR genes identified among these plasmids. AR genes carried by these plasmids were conferred to their corresponding resistance phenotypes. The β-lactamases and aminoglycoside resistance genes were mainly encoded on these plasmids. About half of these plasmids carried multidrug-resistant genes. Our surveillance represents a profile to date of the AR phenotype associated with conjugative plasmids in clinical and residential sewers. It emphasizes the importance of enhancing surveillance of sewage-based AR and contributes to a deeper understanding of conjugative plasmid-mediated AR.

Nanopore Workshop

Technical training, Southern University of Science and Technology, College of Env, 2018

Nanshan CDC Workshop

Workshop, Nanshan CDC, Shenzhen, 2026

Research on the transmission and spread of drug-resistant pathogenic bacteria and genes in urban wastewater monitoring

• Based on metagenomic sequencing “short fragments”, the resistance genes and microbial community composition in the sewage of Shenzhen City were revealed
• Based on metagenomic sequencing “assembly fragments”, the composition of drug resistance genes and their hosts in the sewage of Shenzhen City was revealed
• Based on metagenomic sequencing at the “MAGs” level, the composition of drug resistance genes and their hosts in the sewage of Shenzhen was revealed