Klebsiella pneumoniae is a major bacterial pathogen responsible for community- and hospital-acquired infections. Although strains associated with hospital-acquired disease have been well described, the genomic and phenotypic characteristics of community-acquired K. pneumoniae among community-acquired isolates from this setting remain insufficiently understood. This study aimed to characterize the molecular features, antimicrobial resistance patterns, and virulence-associated traits of community-acquired strains isolated from a tertiary hospital in Tianjin. A total of 27 community-acquired K. pneumoniae underwent whole-genome sequencing and comprehensive phenotypic assessment. Molecular typing revealed that the dominant lineages included ST11-KL64-O1/O2v1, ST268-KL20-O1/O2v1, and ST15-KL102-O1/O2v2. All isolates carried at least one virulence-associated gene as defined by established hvKp markers and produced high levels of siderophores (> 30 µg/mL). Most strains displayed multidrug-resistant phenotypes and simultaneously possessed virulence determinants, indicating the frequent detection of multidrug-resistant hypervirulent K. pneumoniae among the studied community-acquired isolates. Genomic analysis showed that the coexistence of resistance and virulence traits was largely associated with horizontal gene transfer mediated by mobile genetic elements such as plasmids, insertion sequences, and transposons. Replicons of the IncHI1B and IncFIB plasmid families were frequently linked to virulence-associated genes. Functional assays, including Galleria mellonella infection, biofilm formation, macrophage infection, and serum resistance testing, confirmed the pathogenic potential of these strains. Integrated genomic and phenotypic findings suggested that multiple virulence factors collectively enhance pathogenicity through immune evasion, increased biofilm production, and modulation of host responses. Community-acquired K. pneumoniae strains analyzed in this study exhibit a concerning combination of multidrug resistance and enhanced virulence. The convergence of these traits, largely associated with mobile genetic elements, represents an emerging public health concern and underscores the need for strengthened surveillance and targeted control measures.

BACKGROUND:Identifying the sources of pathogenic bacteria causing ventilator-associated pneumonia (VAP) in intensive care unit (ICU) patients is crucial for developing effective prevention and treatment strategies. However, the scarcity of reported cases with confirmed sources limits the ability to evaluate and manage VAP, which remains a major challenge for healthcare systems globally. METHODS:Pathogens were isolated from endotracheal aspirate (ETA) samples of VAP patients using conventional culture techniques. Whole-genome comparisons, based on average nucleotide identity (ANI), were performed to identify genetically identical strains by comparing pulmonary isolate genomes with gut metagenome-derived bacterial genomes. Mouse models of pneumonia and colitis were used to validate the translocation of pathogenic bacteria from the gut to the lungs. Metagenomic analysis was performed to characterize the gut microbiome and resistome. RESULTS:Pathogenic isolates were obtained from the ETA samples of seven VAP patients, with one isolate per sample. Among these, Escherichia coli (Ec1) and Burkholderia cenocepacia (Bc1) from two patients were genetically identical to strains in their respective gut microbiota, with ANI values above 99%, indicating gut-to-lung translocation. The Ec1 strain demonstrated increased resistance to cefazolin while remaining susceptible to gentamicin, amikacin, and kanamycin, compared to previously reported pneumonia-associated E. coli strains. The Bc1 strain showed elevated resistance to macrolides, chloramphenicols, and tetracyclines relative to pneumonia-associated B. cenocepacia strains. Metagenomic analysis revealed a highly individualized gut microbiota composition among VAP patients. Notably, the translocated bacteria were not dominant within their gut microbiota. Additionally, these patients showed a marked increase in the total abundance of antibiotic resistance genes (ARGs) in their gut microbiota. The translocation ability of the Ec1 strain was validated in a mouse pneumonia model, where it caused more severe lung damage. Furthermore, elevated levels of Escherichia-Shigella were detected in the lung tissues of colitis mice, suggesting that gut-to-lung bacterial translocation may occur in a severely inflamed host, potentially leading to pneumonia. CONCLUSIONS:This study demonstrates the gut-to-lung translocation of E. coli and B. cenocepacia, highlighting their role in the development and progression of VAP in ICU patients. These findings provide valuable insights for implementing targeted prevention and treatment strategies for VAP in ICU settings.

Helicobacter pylori infection is common in patients with alcohol-related liver disease (ALD), and bismuth quadruple therapy (BQT) is widely used for eradication. However, its impact on ALD remains unclear. This study aims to characterize BQT-induced gut microbiota alterations in asymptomatic H. pylori carriers and evaluate their effect on an ALD mouse model. Metagenomic sequencing was conducted to assess the gut microbiota composition of individuals before and after BQT. Fecal microbiota transplantation (FMT) from these donors was performed in an ALD mouse model. Gut microbiota in mice was analyzed by 16S rRNA sequencing. Liver and intestinal parameters were assessed using western blot, RT-qPCR, histopathology, ELISA, and flow cytometry. BQT treatment significantly altered the gut microbiota in H. pylori carriers, increasing the abundance of opportunistic pathogens, including Klebsiella pneumoniae, Escherichia coli, Klebsiella quasipneumoniae, and Klebsiella variicola, while decreasing beneficial bacteria such as Bifidobacterium, Eubacterium, Bacteroides, Faecalibacterium, and Blautia. In ALD mice receiving FMT from post-BQT donors, exacerbated gut dysbiosis was observed, marked by an enrichment of Enterobacteriaceae and Escherichia-Shigella. These microbiota changes were associated with impairment of intestinal barrier integrity, as evidenced by reduced levels of mucins, tight junction proteins, and antimicrobial peptides, along with a decrease in Treg cells and an increase in Th17 and Th1 cells. Additionally, this dysbiosis led to elevated serum lipopolysaccharide (LPS) levels, which activated the hepatic NLRP3 inflammasome pathway and subsequently increased IL-18 and IL-1β levels. Furthermore, liver function and oxidative stress markers, including ALT, AST, MDA, GSSG/GSH ratio, and SOD, were significantly elevated, indicating severe liver dysfunction and increased oxidative stress. Finally, probiotic supplementation effectively mitigated the negative effects of BQT-induced gut microbiota remodeling on ALD in mice. BQT markedly alters the gut microbiota in H. pylori carriers, promoting dysbiosis that exacerbates ALD in mice via LPS-mediated activation of hepatic inflammatory pathways. These findings highlight the need for careful consideration of BQT use in ALD patients. H. pylori infection and ALD frequently coexist in certain populations. BQT significantly alters the gut microbiota, increasing opportunistic pathogens and reducing beneficial bacteria in individuals. In ALD mouse models, FMT from post-BQT donors exacerbates gut dysbiosis, worsening ALD through the LPS-induced activation of the NLRP3 inflammasome pathway. This study highlights the potential for BQT to worsen ALD and underscore the need for careful consideration when administering this therapy to ALD patients.

Understanding the influence of environmental factors on the taxonomic and functional profiles of microbial communities is critical for assessing ecological health. In this study, we perform a large-scale field survey and microcosm experiment to investigate the effects of environmental heterogeneity on the microbial communities and functional profiles of oysters along the Chinese coastline. We found that salinity altered the spatial distribution of oyster-associated microorganisms and their functional profiles between the southern and northern regions. Specifically, the northern regions, with optimal salinity (18.3 part per thousand), exhibited a higher abundance of dominant functional microorganisms, more stable microbial networks, and enhanced carbon, nitrogen, and sulfur biogeochemical cycles than the southern regions. Moreover, metabolic mutualism among key taxa, such as Vibrio, Pseudomonas, and Shewanella, was identified as crucial for the coupled carbon, nitrogen, and sulfur cycles. These results suggest that salinity-driven microbial interactions and compositions play predominant roles in structuring the spatial heterogeneity of the functional profiles of oyster-associated microorganisms. Microcosm experiments further confirmed that moderate salinity, a crucial indicator of climate change, regulates and enriches the primary functional profiles of oyster-associated microorganisms. Overall, this study highlights how environmental conditions shape oyster-associated microbial and functional traits along the Chinese coastline, raising concerns about the impact of anthropogenic activities, such as climate change, on marine ecological functions.

Antibiotic resistance gene (ARG) dynamics have been extensively studied in anthropogenically impacted environments. However, how ARGs evolve during natural succession in pristine ecosystems remains poorly understood. In this study, we utilized metagenomic sequencing to investigate ARG dynamics across a 12,000 year sedimentary chronosequence in an intertidal zone. ARG abundance and diversity showed a significant upward trend with sediment depth, peaking in the deepest, oldest layers, as corroborated by compositional shifts linked to sediment age (PERMANOVA, p < 0.001). This trend reflects the natural evolution of ARGs dominated by stochastic process, as supported by the low detection rates of three human activity-related indicators and the robust fit to the neutral community model (R2 = 0.576). Furthermore, ARG evolution was mainly attributed to alterations in microbial communities, reflected in the growing complexity of ARG-microbe interaction networks and significant procrustes correlations between microbial taxa and ARG profiles. Specially, Proteobacteriota and Actinobacteriota were identified as the key taxa driving ARG evolution, with their succession patterns closely mirroring ARG enrichment trends. Interestingly, the enrichment of MGEs in deeper sediments and their closer association with ARGs during natural succession underscore an enhanced HGT potential, highlighting the pivotal role of MGEs in driving ARG evolution. This study reveals the natural dynamics of ARGs during sedimentary processes, providing an important reference for understanding their independent natural evolution and strengthening our insight into the progression of environmental antibiotic resistance development.

To elucidate the characteristics and annual epidemic patterns of diarrheal viruses in municipal wastewater,this study conducted a comprehensive year-long investigation utilizing two sizeable municipal wastewater treatment plants in Tianjin.This study applied the electronegatively charged membranes and polyethylene glycol sodium chloride(PEG-NaCl)methods to concentrate viral particles efficiently.A real-time quantitative PCR method was employed to detect common diarrheal viruses within wastewater,specifically targeting Norovirus GI,GII,Rotavirus(RoV),Astrovirus(HAstV),Sapovirus(SaV),Enterovirus(EnV),Hepatitis A virus(HAV),and Adenovirus(HAdV).The goal of the study was to ascertain the annual prevalence of viral gastroenteritis within Tianjin's wastewater in China.The findings unveiled the seven targeted viruses across both A and B municipal wastewater treatment plants,with Norovirus GII and Sapovirus emerging as prevailing strains.Notably,no statistically significant disparity in virus detection between the two WWTPs was observed(P>0.05,Mann-Whitney U test),implying a uniform distribution within the studied regions.Furthermore,discernible seasonal fluctuations in the presence of diarrheal viruses within municipal wastewater treatment plants were identified,with Astrovirus exhibiting heightened detection rates in autumn,while Enterovirus prevalence peaked during the winter months.Conversely,Norovirus GII,Sapovirus,and Adenovirus displayed consistent levels across seasons(P>0.05).The notable frequency of diarrheal virus'detection within Tianjin's wastewater network suggests a significant presence of asymptomatic carriers within the local population.Consequently,further investigation is warranted to comprehensively evaluate the potential outbreak risk and prevalence dynamic across different temporal intervals.

This study focuses on the characterization of human viral diversity within a major Wastewater Treatment Plant (WWTP) in the central area of Tianjin, China, with a particular emphasis on adenoviruses due to their robust survivability and potential health implications. By employing metagenomic sequencing, targeted sequencing, quantitative polymerase chain reaction (qPCR) analysis, and cell culture methodologies, we identified a broad spectrum of human viruses, encompassing 25 viral families and 45 viral genera, including enteric, respiratory, and contact viruses. Our findings demonstrate that adenoviruses were stably present in sewage from January to May and retained their infectious activity. The temporal analysis of viral loads across different months revealed no significant changes, suggesting a consistent risk of exposure. Additionally, our phylogenetic analysis of adenoviruses isolated from A549 cell cultures provides insights into their genotypic diversity and potential transmission dynamics. This comprehensive assessment underscores the critical need for improved viral surveillance in urban wastewater systems to mitigate public health risks and highlights the importance of advanced treatment technologies to address the challenges posed by adenoviruses and other pathogenic viruses in municipal wastewater.

The rising antibiotic resistance of Group B Streptococcus (GBS) threatens maternal and neonatal health globally. However, global temporal trends of GBS antibiotic resistance-spanning past, present, and future, remain poorly understood, which hinders the development of targeted control measures. We analyzed 1838 GBS genomes from the NCBI database have been analyzed to investigate trends in the abundance, diversity, and polymorphism of single-nucleotide polymorphisms (SNPs) in antibiotic resistance genes (ARGs) over the past 45 years. These findings indicate that GBS harbors ARGs conferring resistance to 9 antibiotic classes, predominantly macrolide, tetracycline, aminoglycoside, and lincosamide resistance genes. Over time, ARG abundance has increased 4-fold, from 1 to 4.67 copies per strain, accompanied by significant increases in ARG diversity and polymorphism. Through geospatial analysis, we identified Asia as a critical hotspot of antimicrobial resistance, exhibiting heightened resistance to aminoglycosides, tetracyclines, and lincosamides, alongside elevated genetic polymorphisms. A random forest prediction model projects a 187 % rise in global GBS resistance risk over two decades, with Asia facing an even sharper increase of 219 %. These findings underscore the urgent need for coordinated global interventions to mitigate resistance and protect vulnerable populations.

The rapid and multiplexed detection of waterborne viruses is crucial for infection prevention. However, current methods are limited by low-quality probes, instrument dependency, and time-consuming procedures. In this study, we developed a high-performance, nanobody pair-based, multichannel homogeneous platform for the simultaneous and rapid detection of three viruses─SARS-CoV-2, norovirus, and influenza A virus─in aquatic environments. To identify robust and sensitive multiepitope nanobody pairs for these viruses, we utilized pressure-assisted screening and docking techniques. For multichannel detection, we synthesized three distinct SiO2@TQD types with unique excitation wavelengths, each acting as an independent signal label. By integrating these multichannel antibody arrays and signal labels into a unified platform, we developed a homogeneous sensor capable of detecting all three viruses within 30 min. The system demonstrated detection limits of as low as 1.56 pg/mL for the SARS-CoV-2 antigen, 0.1 pg/mL for the norovirus antigen, and 0.39 pg/mL for the influenza A virus antigen, surpassing conventional antigen detection kits with a sensitivity enhancement of 160.26-6.25 × 104-fold. Notable advantages include exceptional specificity, accuracy, and stability. This work not only provides a transformative solution for monitoring waterborne pathogens but also establishes a versatile framework for developing multichannel detection platforms for other infectious agents.

Human bocavirus (HBoV) is an emerging health concern worldwide, associated with range of clinical manifestations, including gastroenteritis and respiratory infections. Therefore, it is crucial to comprehend and minimize their prevalence in different systems. In this study, we conducted regular sampling throughout the year in two different sizes and work processes of wastewater treatment plants (WWTPs) in Tianjin, China. Our objective was to investigate the occurrence, prevalence, and endurance of HBoV in wastewater, while also evaluating the efficacy of amplicon target sequencing in directly detecting HBoV in wastewater. At two WWTPs, HBoV2 (45.51 %-45.67 %) and HBoV3 (38.30 %-40.25 %) were the most common genotypes identified, and the mean concentration range of HBoV was 2.54-7.40 log10 equivalent copies/l as determined by multiplex real-time quantitative PCR assay. A positive rate of HBoV was found in 96.6 % (29/30) samples of A-WWTP, and 96.6 % (26/27) samples of B-WWTP. The phylogenetic analysis indicated that the nucleotide similarity between the HBoV DNA sequences to the reference HBoV sequences published globally ranged from 90.14 %-100 %. A significant variation in the read abundance of HBoV2 and HBoV3 in two wastewater treatment plants (p < 0.05) was detected, specifically in the Winter and Summer seasons. The findings revealed a strong correlation between the genotypes detected in wastewater and the clinical data across various regions in China. In addition, it is worth mentioning that HBoV4 was exclusively detected in wastewater and not found in the clinical samples from patients. This study highlights the high prevalence of human bocavirus in municipal wastewater. This finding illustrates that amplicon target sequencing can amplify a wide variety of viruses, enabling the identification of newly discovered viruses.

Antibody pairs-based immunoassay platforms served as essential and effective tools in the field of pathogen detection. However, the cumbersome preparation and limited detection sensitivity of antibody pairs challenge in establishment of a highly sensitive detection platform. In this study, using COVID-19 testing as a case, we utilized readily accessible nanobodies as detection antibodies and further proposed an accurate design concept with a more scientific and efficient screening strategy to obtain ultrasensitive antibody pairs. We employed nanobodies capable of binding different antigenic epitopes of the nucleocapsid (NP) or receptor-binding domain (RBD) antigens sandwich as substitutes for monoclonal antibodies (mAbs) sandwich in fast detection formats and utilized time-resolved fluorescence (TRF) microspheres as the signal probe. Consequently, we developed a multi-epitope nanobody sandwich-based fluorescence lateral flow immunoassay (FLFA) strip. Our results suggest that the NP antigen had a detection limit of 12.01 pg/mL, while the RBD antigen had a limit of 6.51 pg/mL using our FLFA strip. Based on double mAb sandwiches, the values presented herein demonstrated 4 to 32-fold enhancements in sensitivity, and 32 to 256-fold enhancements compared to commercially available antigen lateral flow assay kits. Furthermore, we demonstrated the excellent characteristics of the proposed test strip, including its specificity, stability, accuracy, and repeatability, which underscores its the prospective utility. Indeed, these findings indicate that our established screening strategy along with the multi-epitope nanobody sandwich mode provides an optimized strategy in the field of pathogen detection.

S-amlodipine, a commonly prescribed antihypertensive agent, is widely used in clinical settings to treat hypertension. However, the potential adverse effects of long-term S-amlodipine treatment on the liver remain uncertain, given the cautionary recommendations from clinicians regarding its administration in individuals with impaired liver function. To address this, we conducted a study using an eight-week-old male rat model and administered a daily dose of 0.6 similar to 5 mg/kg of S-amlodipine for 7 weeks. Our findings demonstrated that 1.2 similar to 5 mg/kg of S-amlodipine treatment induced liver inflammation and associated dysfunction in rats, further in vitro experiments revealed that the observed liver inflammation and dysfunction were not attributable to direct effects of S-amlodipine on the liver. Metagenome sequencing analysis revealed that S-amlodipine treatment led to alterations in the gut microbiome of rats, with the bloom of E. coli (4.5 similar to 6.6-fold increase) and a decrease in A. muciniphila (1,613.4 similar to 2,000-fold decrease) and B. uniformis (20.6 similar to 202.7-fold decrease), subsequently causing an increase in the gut bacterial lipopolysaccharide (LPS) content (1.4 similar to 1.5-fold increase in feces). S-amlodipine treatment also induced damage to the intestinal barrier and increased intestinal permeability, as confirmed by elevated levels of fecal albumin; furthermore, the flux of gut bacterial LPS into the bloodstream through the portal vein resulted in an increase in serum LPS content (3.3 similar to 4-fold increase). LPS induces liver inflammation and subsequent dysfunction in rats by activating the TLR4 pathway. This study is the first to show that S-amlodipine induces liver inflammation and dysfunction by perturbing the rat gut microbiome. These results indicate the adverse effects of S-amlodipine on the liver and provide a rich understanding of the safety of long-term S-amlodipine administration.

Environmental viruses in wastewater and sludge are widely recognized for their roles in waterborne diseases. However, previous studies mainly focused on RNA viruses, and little is known about the diversity of DNA viral communities and their driving factors in municipal wastewater treatment environments. Herein, we conducted a pilot study to explore DNA virus profiles in municipal wastewater and recycled sludge by metagenomics method, and track their temporal changes in northern China. Results showed that 467 viral species were co-shared among all the samples. We identified six families of human viruses with a prevalence of 0.1%, which were rare but relatively stable in wastewater and sludge for six months. Adenoviridae, Parvoviridae, and Herpersviridae were the most dominant human viral families in municipal wastewater and recycled sludge. A time series of samples revealed that the dynamic changes of human DNA viruses were stable based on qPCR results, particularly for high-risk fecal-oral transmission viruses of adenovirus, bocavirus, polyomavirus, human gamma herpesvirus, human papillomavirus, and hepatitis B virus. Concentrations of Adenovirus (5.39-7.48 log10 copies/L) and bocavirus (4.36-7.48 log10 copies/L) were observed to be the highest in these samples compared to other viruses. Our findings demonstrated the DNA viruses' high prevalence and persistence in municipal wastewater treatment environments, highlighting the value of enhancing public health responses based on wastewater-based epidemiology.

The inappropriate use of antibiotics is widely recognized as the primary driver of bacterial antibiotic resistance. However, less attention has been given to the potential induction of multidrug-resistant bacteria through exposure to disinfectants. In this study, Klebsiella pneumonia, an opportunistic pathogen commonly associated with hospital and community-acquired infection, was experimentally exposed to NaClO at both minimum inhibitory concentration (MIC) and sub-MIC levels over a period of 60 days. The result demonstrated that NaClO exposure led to enhanced resistance of K. pneumonia to both NaClO itself and five antibiotics (erythromycin, polymyxin B, gentamicin, tetracycline, and ciprofloxacin). Concurrently, the evolved resistant strains exhibited fitness costs, as evidenced by decreased growth rates. Whole population sequencing revealed that both concentrations of NaClO exposure caused genetic mutations in the genome of K. pneumonia. Some of these mutations were known to be associated with antibiotic resistance, while others had not previously been identified as such. In addition, 11 identified mutations were located in the virulence factors, demonstrating that NaClO exposure may also impact the pathogenicity of K. pneumoniae. Overall, this study highlights the potential for the widespread use of NaClO-containing disinfectants during the COVID-19 pandemic to contribute to the emergence of antibiotic-resistant bacteria.Environmental ImplicationConsidering the potential hazardous effects of disinfectant residues on environment, organisms and biodiversity, the sharp rise in use of disinfectants during COVID-19 pandemic has been considered highly likely to cause worldwide secondary disasters in ecosystems and human health. This study demonstrated that NaClO exposure enhanced the resistance of K. pneumonia to both NaClO and five antibiotics (erythromycin, polymyxin B, gentamicin, tetracycline, and ciprofloxacin), highlighting the widespread use of NaClO-containing disinfectants during the COVID-19 pandemic may increase the emergence of antibiotic-resistant bacteria in the environment.

Immunoassays have become essential tools for detecting infectious viruses. However, traditional monoclonal antibody-dependent immunoassays are costly, fragile, and unstable, especially in complex media. To overcome these challenges, we have developed cost-effective, robust, and high-affinity nanobodies as alternatives to monoclonal antibodies for rapid detection applications. We engineered dual-epitope nanobody (NB) pairs and incorporated them into a sandwich immunosensor design to detect transmitted rotaviruses in rectal swabs and wastewater samples. To further enhance sensitivity, we synthesized an advanced two-dimensional material, MXenes@CNTs@AuNPs, which offers an extensive specific surface area that supports the enrichment and immobilization of NBs. This integration with catalase-modified magnetic probes facilitates signal generation. Subsequently, our sensor achieved a detection limit of 0.0207 pg/mL for the rotavirus VP6 antigen, significantly outperforming commercial antigen kits with a sensitivity enhancement of 3.77 × 105-fold. The exceptional sensor performance extended to specificity, repeatability, stability, and accuracy across various sample types, establishing it as a promising tool for rotavirus detection. This research outlines a viable strategy for creating a robust and ultrasensitive analytical nanoprobe, thereby addressing the critical need for efficient and reliable viral detection methods in various environments.

Managed bees commonly suffer from cross-contamination with acaricides and neonicotinoids, posing robust threats to bee population health. However, their residual characteristics and spatial distribution in beehives and surrounding environments are poorly understood. This study detected two common acaricides and five neonicotinoids in 240 beehive samples and 44 surrounding environmental samples collected from 25 Chinese provinces. The results showed that 40.0% of the honey samples contained acaricides and 83.1% contained neonicotinoids. Neonicotinoid concentrations in honey were geographically distinguished by the “Hu Huanyong line”, and concentrations of neonicotinoids in honey from eastern areas were 2.65-fold higher than those in honey from western areas. Compared to the approved acaricide amitraz, the banned acaricide coumaphos was detected more frequently in honey and was positively correlated with that quantified in the paired pollen samples. Although coumaphos was identified in only three soil samples, lower coumaphos residues in honey might be associated with persistent pollution in the surrounding environment. Conversely, neonicotinoids were detected at higher levels in honey than in the pollen and soil, demonstrating that the neonicotinoid residues in honey have a cumulative effect. This study contributes to a better understanding of the pesticide contamination scenarios that underlie the exposure risks of bees.

Antimicrobial resistance is a major threat for public health. Plasmids play a critical role in the spread of antimicrobial resistance via horizontal gene transfer between bacterial species. However, it remains unclear how plasmids originally recruit and assemble various antibiotic resistance genes (ARGs). Here, we track ARG recruitment and assembly in clinically relevant plasmids by combining a systematic analysis of 2420 complete plasmid genomes and experimental validation. Results showed that ARG transfer across plasmids is prevalent, and 87% ARGs were observed to potentially transfer among various plasmids among 8229 plasmid-borne ARGs. Interestingly, recruitment and assembly of ARGs occur mostly among compatible plasmids within the same bacterial cell, with over 88% of ARG transfers occurring between compatible plasmids. Integron and insertion sequences drive the ongoing ARG acquisition by plasmids, especially in which IS26 facilitates 63.1% of ARG transfer events among plasmids. In vitro experiment validated the important role of IS26 involved in transferring gentamicin resistance gene aacC1 between compatible plasmids. Network analysis showed four beta-lactam genes (blaTEM-1, blaNDM-4, blaKPC-2, and blaSHV-1) shuffling among 1029 plasmids and 45 clinical pathogens, suggesting that clinically alarming ARGs transferred accelerate the propagation of antibiotic resistance in clinical pathogens. ARGs in plasmids are also able to transmit across clinical and environmental boundaries, in terms of the high-sequence similarities of plasmid-borne ARGs between clinical and environmental plasmids. This study demonstrated that inter-plasmid ARG transfer is a universal mechanism for plasmid to recruit various ARGs, thus advancing our understanding of the emergence of multidrug-resistant plasmids.

Environmental viruses in wastewater and sludge are widely recognized for their roles in waterborne diseases. However, previous studies mainly focused on RNA viruses, little is known about the diversity of DNA viral communities and their driving factors in municipal wastewater treatment environments. Herein, we conducted a pilot study to explore DNA virus profiles in municipal wastewater and recycled sludge by metagenomics method, as well as track their temporal changes in northern China. Results showed that 467 viral species were co-shared among all the samples. We identified 13 families of human viruses with a prevalence of 0.1%, which were rare but relatively stable in wastewater and sludge for six months. Adenoviridae, Parvoviridae, Herpersviridae were the most dominant human viral families in municipal wastewater and recycled sludge. A time series of samples revealed that the dynamic changes of human DNA viruses were stable based on qPCR results, particularly for high risk fecal-oral transmission viruses of adenovirus, bocavavirus, and polyomavirus. Concentrations of Adenovirus (5.39-7.48log10 copies/mL) and bocavirus (4.36-7.48 log10 copies/mL) were observed to be the highest in these samples compared to other viruses. Our findings demonstrated the high prevalence and persistence of DNA viruses in municipal wastewater treatment environments, also highlighting the value of enhancing public health responses based on wastewater-based viral epidemiology.

Antibiotic resistance is propagating worldwide, but the predominant dissemination mechanisms are not fully understood. Here, we report that antibiotic resistance gene (ARG) abundance in conjugative plasmids that are recorded in the National Center for Biotechnology Information (NCBI) RefSeq plasmid database is increasing globally, which is likely a key factor in the propagation of resistance. ARG abundance in plasmids increased by 10-fold on a global scale from the year 2000 to the year 2020 (from 0.25 to 2.93 ARG copies/plasmid), with a more pronounced increase being observed in low-to-middle income countries. This increasing trend of plasmid-borne ARGs was corroborated by bootstrap resampling from each year of the NCBI RefSeq plasmid database. The results of a correlation analysis imply that if antibiotic consumption keeps growing at the current rates, a 2.7-fold global increase in the ARG abundance of clinically relevant plasmids may be reached by 2030. High sequence similarities of clinically relevant, conjugative plasmids that are isolated both from clinics and from the environment raise concerns about the environmental resistome serving as a potential ARG maintenance reservoir that facilitates transmission across these ecological boundaries. IMPORTANCE Antibiotic resistance propagation is a significant concern due to its projected impacts on both global health and the economy. However, global propagation mechanisms are not fully understood, including regional and temporal trends in the abundance of resistance plasmids that facilitate antibiotic resistance gene (ARG) dissemination. This unprecedented study reports that ARG abundance in the conjugative plasmids that are recorded in the National Center for Biotechnology Information (NCBI) database and harbor ARGs is increasing globally with antibiotic consumption, especially in low-to-medium income countries. Through network and comparative genomic analyses, we also found high sequence similarities of clinically relevant conjugative resistance plasmids that were isolated from clinical and environmental sources, suggesting transmission between these ecological boundaries. Therefore, this study informs the One Health perspective to develop effective strategies by which to curtail the propagation of plasmid-borne antibiotic resistance.

The emergence of antimicrobial resistance (AMR) is a growing public health threat worldwide and antibiotic consumption is being increasingly recognized as the main selective pressure driving this resistance. However, global trend in antibiotic resistance in response to antibiotic consumption is not fully understood. In this study, we collected national resistance data on specific resistant pathogens considered by the World Health Organization (WHO) as priority and antibiotic consumption data for 61 countries to assess the global trends in antibiotic resistance of those common bacterial pathogens and their association with antibiotic consumption. The low- and middle-income countries (LMICs) represented the largest hotspots of resistance, which presented relatively higher resistance rates in common bacterial pathogens but lower antibiotic consumption rates compared to high-income countries (HICs). Specifically, we developed the Normalized Antibiotic Resistance/Consumption Index (NARCI) and produced global maps of NARCI to roughly assess the appropriateness of antibiotic consumption across countries and to indicate the potentially inappropriate antibiotic consumption in LMICs compared with HICs. Additionally, we linked antibiotic consumption rates and resistance rates of target pathogens, in conjunction with NARCI and the correlation analysis between antibiotic use and resistance, to inform strategies to alleviate the threat of antibiotic resistance worldwide.