Metatranscriptomic data mining together with microfluidic card uncovered the potential pathogens and seasonal RNA viral ecology in a drinking water source.

AIMS: Despite metatranscriptomics becoming an emerging tool for pathogen surveillance, very little is known about the feasibility of this approach for understanding the fate of human-derived pathogens in drinking water sources. METHODS AND RESULTS: We conducted multiplexed microfluidic cards and metatranscriptomic sequencing of the drinking water source in a border city of North Korea in four seasons. Microfluidic card detected norovirus, hepatitis B virus (HBV), enterovirus, and Vibrio cholerae in the water. Phylogenetic analyses showed that environmental-derived sequences from norovirus GII.17, genotype C of HBV, and coxsackievirus A6 (CA6) were genetically related to the local clinical isolates. Meanwhile, metatranscriptomic assembly suggested that several bacterial pathogens, including Acinetobacter johnsonii and V. cholerae might be prevalent in the studied region. Metatranscriptomic analysis recovered 349 species-level groups with substantial viral diversity without detection of norovirus, HBV, and CA6. Seasonally distinct virus communities were also found. Specifically, 126, 73, 126, and 457 types of viruses were identified in spring, summer, autumn, and winter, respectively. The viromes were dominated by the Pisuviricota phylum, including members from Marnaviridae, Dicistroviridae, Luteoviridae, Potyviridae, Picornaviridae, Astroviridae, and Picobirnaviridae families. Further phylogenetic analyses of RNA (Ribonucleic Acid)-dependent RNA polymerase (RdRp) sequences showed a diverse set of picorna-like viruses associated with shellfish, of which several novel picorna-like viruses were also identified. Additionally, potential animal pathogens, including infectious bronchitis virus, Bat dicibavirus, Bat nodavirus, Bat picornavirus 2, infectious bursal disease virus, and Macrobrachium rosenbergii nodavirus were also identified. CONCLUSIONS: Our data illustrate the divergence between microfluidic cards and metatranscriptomics, highlighting that the combination of both methods facilitates the source tracking of human viruses in challenging settings without sufficient clinical surveillance.

Metavirome-assembled genome sequence of a new aquatic RNA virus expands the genus Locarnavirus.

RNA viruses in marine environments have long been recognized as major players in the virosphere. In this study, the complete genome sequence of an RNA virus from Yangshan Harbor, named marine RNA virus Yangshan-LWW (YS-LWW), was obtained based on metavirome assembly. The genome of YS-LWW is 8839 nt in length and contains two open reading frames (ORFs). Both RdRP- and whole-genome-based phylogenetic analysis showed that YS-LWW, together with 45 viral isolates with sequences in public datasets, represents a new species in the genus Locarnavirus of the family Marnaviridae. PCR and public dataset mining indicate that YS-LWW and YS-LWW-like viruses have been widely detected in coastal and freshwater environments, suggesting that they might play a significant role in aquatic ecosystems.

Genomic analysis of a new type VI secretion system in Vibrio parahaemolyticus and its implications for environmental adaptation in shrimp ponds.

The type VI secretion system (T6SS) in Vibrio spp. is often used to kill heteroclonal neighbors by direct injection of toxic effectors, but its strategies in aquacultural environments receive limited attention. In this study, we conducted genomic analysis for a T6SS-harboring plasmid in V. parahaemolyticus strain VP157. Coculture assays were further conducted to verify its antibacterial function. The results showed that strain VP157 harbored a 132-kb plasmid, pVP157-1, which consists of two fragments: an 87.8-kb fragment identical to plasmid pTJ114-1 and a 44.2-kb T6SS gene cluster with only 4% DNA identity to T6SS1 in the V. parahaemolyticus reference genome. Gene-by-gene analysis of six genes representing core T6SS components suggested that each gene has distinct evolutionary origins. In vitro experimental evolution revealed that pVP157-1 can excise from the VP157 genome with an excision rate of 4%. A coculture assay suggested that strain VP157 had significantly higher antibacterial activity against Bacillus pumilus and V. cholerae than the strain without pVP157-1(VP157∆T6SS). In contrast, a rapid decline was observed for the proportion of VP157∆ T6SS in a mock microbial community, which decreased from 10.7% to 2.1% in 5 days. The results highlighted that the acquisition of T6SS fostered the fitness of V . parahaemolyticus in a complex environment.

A novel culture-enriched metagenomic sequencing strategy effectively guarantee the microbial safety of drinking water by uncovering the low abundance pathogens.

Assessing the presence of waterborne pathogens and antibiotic resistance genes (ARGs) is crucial for managing the environmental quality of drinking water sources. However, detecting low abundance pathogens in such settings is challenging. In this study, a workflow was developed to enrich for broad spectrum pathogens from drinking water samples. A mock community was used to evaluate the effectiveness of various enrichment broths in detecting low-abundance pathogens. Monthly metagenomic surveillance was conducted in a drinking water source from May to September 2021, and water samples were subjected to five enrichment procedures for 6 h to recover the majority of waterborne bacterial pathogens. Oxford Nanopore Technology (ONT) was used for metagenomic sequencing of enriched samples to obtain high-quality pathogen genomes. The results showed that selective enrichment significantly increased the proportions of targeted bacterial pathogens. Compared to direct metagenomic sequencing of untreated water samples, targeted enrichment followed by ONT sequencing significantly improved the detection of waterborne pathogens and the quality of metagenome-assembled genomes (MAGs). Eighty-six high-quality MAGs, including 70 pathogen MAGs, were obtained from ONT sequencing, while only 12 MAGs representing 10 species were obtained from direct metagenomic sequencing of untreated water samples. In addition, ONT sequencing improved the recovery of mobile genetic elements and the accuracy of phylogenetic analysis. This study highlights the urgent need for efficient methodologies to detect and manage microbial risks in drinking water sources. The developed workflow provides a cost-effective approach for environmental management of drinking water sources with microbial risks. The study also uncovered pathogens that were not detected by traditional methods, thereby advancing microbial risk management of drinking water sources.

Distinct dynamics of Vibrio parahaemolyticus populations in two farming models.

AIMS: Despite the recent prosperity of shrimp cultivation in China, very little is known about how different shrimp farming models influence the dynamics of Vibrio parahaemolyticus populations and the antibiotic resistance of this bacterium. METHODS AND RESULTS: To this end, we conducted continuous surveillance of V. parahaemolyticus on four farms over 3 years: two traditional shrimp farms with daily water exchange and two farms operated in the recirculating aquaculture systems (RAS). No antibiotics were used in these farms to exclude the potential impacts of antibiotics on the emergence of antibacterial resistance. Multilocus sequence typing was utilized to characterize the dynamics of V. parahaemolyticus populations. Whole-genome sequencing (WGS) was conducted to determine the representative sequence types (STs) at each farm. Results revealed that the population structure of V. parahaemolyticus remained stable over time in both RAS farms, with only nine and four STs observed at each. In contrast, annual replacement of V. parahaemolyticus populations was observed in traditional farms with 26 and 28 STs identified in rearing water. WGS of 50 isolates divided them into five clusters, of which ST917a isolates harboured a genomic island that disrupted the gene recA. Pair-wised genomic comparison of isolates from the same STs showed that they were genetically related but belonged to different clones associated with geographical distribution. CONCLUSIONS: These results suggested that RAS presented a specific ecological niche by minimizing the water exchanges with the external environment. In contrast, traditional farming might pose a food safety issue by introducing new V. parahaemolyticus populations with antibiotic resistance genes. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results expose the potential food safety issue associated with conventional agriculture and should encourage the development of preventive strategies to reduce the emergence of resistant V. parahaemolyticus populations.

Delineating the key virulence factors and intraspecies divergence of Vibrio harveyi via whole-genome sequencing.

Vibrio harveyi is one of the major pathogens in aquaculture. To identify the key virulence factors affecting pathogenesis of V. harveyi towards fish, we conducted a field investigation for three representative fish farms infected with V. harveyi. Multilocus sequence typing (MLST) and whole-genome sequencing were conducted to delineate the phylogenetic relationship and genetic divergence of V. harveyi. A total of 25 V. harveyi strains were isolated from the diseased fish and groundwater and were subtyped into 12 sequence types by MLST. Five virulence genes, mshB, pilA, hutR, ureB, and ureG, were variably presented in the sequenced strains. The virulence gene profiles strongly correlated with the distinct pathogenicity of V. harveyi strains, with a strain harboring all five genes exhibiting the highest virulence towards fish. Phenotype assay confirmed that reduced virulence correlated with decreased motility and biofilm formation ability. Additionally, three types of type VI secretion system, namely T6SS1, T6SS2, and T6SS3, were identified in V. harveyi strains, which can be classified into six, four, and 12 subtypes, respectively. In conclusion, the results indicated that the virulence level of V. harveyi is mainly determined by the above virulence genes, which may play vital roles in environmental adaptation for V. harveyi.

National Safety Survey of Animal-use Commercial Probiotics and Their Spillover Effects From Farm to Humans: An Emerging Threat to Public Health.

BACKGROUND: Human-use probiotics have recently been associated with clinical infections and antibiotic resistance transfer, raising public concern over their safety. However, despite their extensive application in aquaculture and animal husbandry, the safety of animal-use probiotics remains poorly described. METHODS: We evaluated the safety of 92 animal-use probiotics from China. The pattern of spread of pathogens from probiotics and the consequent public health implications were also examined by conducting in-field genomic surveillance at 2 farms. RESULTS: A total of 123 probiotic Bacillus species isolates were obtained from 92 brands of probiotics, of which 45 isolates were resistant to antibiotics. Notably, 33.7% of probiotic products were contaminated with life-threatening pathogens such as Klebsiella pneumoniae. Genomic surveillance at a chicken farm identified an anthrax toxin-positive Bacillus cereus strain in a probiotic product used as a feed supplement, which was transferred into the groundwater and to a nearby fish farm. Following up retrospective analysis of the surveillance data during 2015-2018 in 3 provinces retrieved 2 B. cereus strains from human with intestinal anthrax symptoms and confirmed the transmission of B. cereus from farm to human. Surveillance of anthrax toxin revealed that cya was detected in 8 of 31 farms. CONCLUSIONS: This study provides the first national safety survey of animal-use probiotics in China and confirms the spillover effects of probiotics from the farms to human. These results suggest that the large-scale application of pathogen-containing probiotics leads to the transfer of pathogens, with worrisome implications for public health. Good Manufacturing Practice should be implemented during the production of all probiotics.Animal-use probiotic products are frequently contaminated with viable pathogenic bacteria. This study revealed that virulent probiotic organisms and contaminating pathogens were colonized with farm animals and shed into the environment, which facilitated the transfer of pathogens to humans.

Comparative genomic analysis unravels the transmission pattern and intra-species divergence of acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus strains.

Acute hepatopancreatic necrosis disease (AHPND) is a recently discovered shrimp disease that has become a severe threat to global shrimp-farming industry. The causing agents of AHPND were identified as Vibrio parahaemolyticus and other vibrios harboring a plasmid encoding binary toxins PirAvp/PirBvp. However, the epidemiological involvement of environmental vibrios in AHPND is poorly understood. In this study, with an aim to reveal the possible transmission route of AHPND-causing V. parahaemolyticus, we sequenced and analyzed the genomes of four pairs of V. parahaemolyticus strains from four representative regions of shrimp farming in China, each including one strain isolated from diseased shrimp during an AHPND outbreak and one strain isolated from sediment before AHPND outbreaks. Our results showed that all the four shrimp-isolated and three of the sediment-isolated strains encode and secret PirAvp/PirBvp toxins and, therefore, are AHPND-causing strains. In silico multilocus sequence typing and high-resolution phylogenomic analysis based on single-nucleotide polymorphisms, as well as comparison of genomic loci in association with prophages and capsular polysaccharides (CPSs) consistently pointed to a close genetic relationship between the shrimp- and sediment-isolated strains obtained from the same region. In addition, our analyses revealed that the sequences associated with prophages, CPSs, and type VI secretion system-1 are highly divergent among strains from different regions, implying that these genes may play vital roles in environmental adaptation for AHPND-causing V. parahaemolyticus and thereby be potential targets for AHPND control. Summing up, this study provides the first direct evidence regarding the transmission route of AHPND-causing V. parahaemolyticus and underscores that V. parahaemolyticus in shrimp are most likely originated from local environment. The importance of environmental disinfection measures in shrimp farming was highlighted.

Delineating the origins of the multidrug-resistant pathogens in ornamental fish farms by multilocus sequence typing and identification of a novel multidrug-resistant plasmid.

To evaluate the overuse of antibiotics and to identify the origin of pathogens in the ornamental fish industry, we conducted a field investigation of three representative fish farms in Liaoning province, China. Drug-resistant pathogens in the fishponds and groundwater were isolated and subtyped by multilocus sequence typing (MLST). In total, 33 pathogenic strains, including Aeromonas veronii and five other pathogens, were isolated from diseased fish and from groundwater. MLST revealed that A. veronii obtained from diseased fish in three fish farms can be subtyped into four sequence types, which were also identified in the corresponding groundwater. All of the isolates obtained from diseased fish showed resistance to at least four antibiotics. Notably, Citrobacter freundii JY-17 exhibited resistance to the majority of the antibiotics and was a carrier of a megaplasmid with 15 drug resistance genes. PCR assays targeting ß-lactam, kanamycin, macrolide, phenicol, sulfonamide, and trimethoprim resistance genes in the pathogens from the diseased fish and groundwater were also conducted. The results revealed strong correlations between antibiotic treatment and increased antimicrobial resistance in fish pathogens. The results suggested that groundwater is the origin of the pathogens in ornamental fish. Antibiotic treatment of ornamental fish promoted the emergence of resistant pathogens.

Characterization of Microbial Communities in Pilot-Scale Constructed Wetlands with Salicornia for Treatment of Marine Aquaculture Effluents.

Microorganisms play an essential role in the performance of constructed wetlands (CWs) for wastewater treatment. However, there has been limited discussion on the characteristics of microbial communities in CWs for treatment of effluents from marine recirculating aquaculture systems (RAS). This study is aimed at characterizing the microbial communities of pilot-scale CWs with Salicornia bigelovii for treatment of saline wastewater from a land-based Atlantic salmon RAS plant located in Northern China. Illumina high-throughput sequencing was employed to identify the profile of microbial communities of three CWs receiving wastewater under different total ammonia nitrogen (TAN) concentrations. Results of this study showed remarkable spatial variations in diversity and composition of microbial communities between roots and substrates in three CWs, with distinct response to different TAN concentrations. In particular, Proteobacteria, Firmicutes, Cyanobacteria, and Bacteroidetes were predominant in roots, while Cyanobacteria, Proteobacteria, Firmicutes, Verrucomicrobia, and Bacteroidetes were prevalent in substrates. Moreover, redundancy analysis indicated that specific functional genera, such as Nitrosopumilus, Vibrio, Pseudoalteromonas, Nitrospina, and Planctomyces, played key roles in the removal of nitrogen/phosphorus pollutants and growth of wetland plants. From a microorganism perspective, the findings of this study could contribute to better understanding of contaminants' removal mechanism and improved management of CWs for treatment of effluents from land-based marine aquaculture.

Defining the Core Genome of Salmonella enterica Serovar Typhimurium for Genomic Surveillance and Epidemiological Typing.

Salmonella enterica serovar Typhimurium is the most common Salmonella serovar causing foodborne infections in Australia and many other countries. Twenty-one S. Typhimurium strains from Salmonella reference collection A (SARA) were analyzed using Illumina high-throughput genome sequencing. Single nucleotide polymorphisms (SNPs) in 21 SARA strains ranged from 46 to 11,916 SNPs, with an average of 1,577 SNPs per strain. Together with 47 strains selected from publicly available S. Typhimurium genomes, the S. Typhimurium core genes (STCG) were determined. The STCG consist of 3,846 genes, a set that is much larger than that of the 2,882 Salmonella core genes (SCG) found previously. The STCG together with 1,576 core intergenic regions (IGRs) were defined as the S. Typhimurium core genome. Using 93 S. Typhimurium genomes from 13 epidemiologically confirmed community outbreaks, we demonstrated that typing based on the S. Typhimurium core genome (STCG plus core IGRs) provides superior resolution and higher discriminatory power than that based on SCG for outbreak investigation and molecular epidemiology of S. Typhimurium. STCG and STCG plus core IGR typing achieved 100% separation of all outbreaks compared to that of SCG typing, which failed to separate isolates from two outbreaks from background isolates. Defining the S. Typhimurium core genome allows standardization of genes/regions to be used for high-resolution epidemiological typing and genomic surveillance of S. Typhimurium.

Veratryl Alcohol Attenuates the Virulence and Pathogenicity of Pseudomonas aeruginosa Mainly via Targeting las Quorum-Sensing System.

Pseudomonas aeruginosa is an opportunistic pathogen that usually causes chronic infections and even death in patients. The treatment of P. aeruginosa infection has become more challenging due to the prevalence of antibiotic resistance and the slow pace of new antibiotic development. Therefore, it is essential to explore non-antibiotic methods. A new strategy involves screening for drugs that target the quorum-sensing (QS) system. The QS system regulates the infection and drug resistance in P. aeruginosa. In this study, veratryl alcohol (VA) was found as an effective QS inhibitor (QSI). It effectively suppressed the expression of QS-related genes and the subsequent production of virulence factors under the control of QS including elastase, protease, pyocyanin and rhamnolipid at sub-inhibitory concentrations. In addition, motility activity and biofilm formation, which were correlated with the infection of P. aeruginosa, were also suppressed by VA. In vivo experiments demonstrated that VA could weaken the pathogenicity of P. aeruginosa in Chinese cabbage, Drosophila melanogaster, and Caenorhabditis elegans infection models. Molecular docking, combined with QS quintuple mutant infection analysis, identified that the mechanism of VA could target the LasR protein of the las system mainly. Moreover, VA increased the susceptibility of P. aeruginosa to conventional antibiotics of tobramycin, kanamycin and gentamicin. The results firstly demonstrate that VA is a promising QSI to treat infections caused by P. aeruginosa.

Targeted amplicon sequencing facilitated a novel risk assessment framework for assessing the prevalence of broad spectrum bacterial and coronavirus diseases.

How to effectively leverage wastewater data to estimate the risk of various infectious diseases remains a great challenge. To address this issue, we conducted continuous wastewater surveillance in Dalian city during the summer-autumn seasons of 2022, targeting coronavirus and bacterial diseases. The surveillance included daily sampling at a wastewater treatment plant (WWTP) and weekly sampling in three sewersheds. Targeting the bacteria's 16S rRNA gene and the coronavirus's RNA-dependent RNA polymerase (RdRp) gene, we first employed RT-PCR and amplicon sequencing techniques to analyze the presence and phylogenetic relationship of detected coronavirus and bacterial pathogens. Next, qPCR was used to quantify the abundances of detected coronavirus and bacterial species. Based on the daily shedding dynamics of SARS-CoV-2, a novel model was developed to predict daily new cases. Based on the medium shedding density of 12 pathogens, two thresholds of sewage pathogen load (indicating 0.1 % and 1 % infection rates) were proposed. Our PanCoV RT-PCR detected coronavirus on 12th August and from 26th August to 12th September 2022. Targeted amplicon sequencing further identified human coronavirus OC43 (hCoV-OC43) on 12th August and the SARS-CoV-2 Omicron variant since 26th August in samples from WWTPs and sewersheds. Phylogenetic analysis revealed that hCoV-OC43 from this study belonged to genotype K and suggested a close relationship between the amplified coronavirus sequences from wastewater and clinical samples in a local COVID-19 outbreak on 26th August. Amplicon sequencing targeting the bacterial 16S rRNA gene also revealed the presence of several bacterial pathogens. Finally, we assessed the microbial risk of specific pathogens in sewersheds and identified a number of pathogens that reached high (>1 % prevalence) and medium risk levels (>0.1 % prevalence) at sewershed B. Our findings underline wastewater surveillance as a valuable early warning system for coronavirus and other waterborne bacterial diseases, complementing public health response measures.

Tailored wastewater surveillance framework uncovered the epidemics of key pathogens in a Northwestern city of China.

Wastewater surveillance enables rapid pathogen monitoring and community prevalence estimation. However, how to design an integrated and tailored wastewater surveillance framework to monitor major health threats in metropolises remains a major challenge. In this study, we first analyzed the historical clinical data of Xi'an city and designed a wastewater surveillance framework covering five key endemic viruses, namely, SARS-CoV-2, norovirus, influenza A virus (IAV), influenza B virus (IBV), respiratory syncytial virus (RSV), and hantavirus. Amplicon sequencing of SARS-CoV-2, norovirus and hantavirus was conducted biweekly to determine the prevalent community genotypes circulating in this region. The results showed that from April 2023 to August 2023, Xi'an experienced two waves of SARS-CoV-2 infection, which peaked in the middle of May-2023 and late August-2023. The sewage concentrations of IAV and RSV peaked in early March and early May 2023, respectively, while the sewage concentrations of norovirus fluctuated throughout the study period and peaked in late August. The dynamics of the sewage concentrations of SARS-CoV-2, norovirus, IAV, RSV, and hantavirus were in line with the trends in the sentinel hospital percent positivity data, indicating the role of wastewater surveillance in enhancing the understanding of epidemic trends. Amplicon sequencing of SARS-CoV-2 revealed a transition in the predominant genotype, which changed from DY.1 and FR.1.4 to the XBB and EG.5 subvariants. Amplicon sequencing also revealed that there was only one predominant hantavirus genotype in the local population, while highly diverse genotypes of norovirus GI and GII were found in the wastewater. In conclusion, this study provided valuable insights into the dynamics of infection trends and predominant genotypes of key pathogens in a city without sufficient clinical surveillance, highlighting the role of a tailored wastewater surveillance framework in addressing public health priorities. More importantly, our study provides the first evidence demonstrating the applicability of wastewater surveillance for hantavirus, which is a major health threat locally.

Wastewater surveillance together with metaviromic data revealed the unusual resurgence of infectious diseases after the first wave of the COVID-19 outbreak.

How to address public health priorities after COVID-19 is becoming a critical task. To this end, we conducted wastewater surveillance for six leading pathogens, namely, SARS-CoV-2, norovirus, rotavirus, influenza A virus (IAV), enteroviruses and respiratory syncytial virus (RSV), in Nanchang city from January to April 2023. Metaviromic sequencing was conducted at the 1st, 4th, 7th, 9th, 12th and 14th weeks to reveal the dynamics of viral pathogens that were not covered by qPCR. Amplicon sequencing of the conserved region of norovirus GI and GII and the rotavirus and region encoding nonstructural protein of RSV was also conducted weekly. The results showed that after a rapid decrease in SARS-CoV-2 sewage concentrations occurred in January 2023, surges of norovirus, rotavirus, IAV and RSV started at the 6th, 7th, 8th and 11th weeks, respectively. The dynamics of the sewage concentrations of norovirus, rotavirus, IAV and RSV were consistent with the off-season resurgence of the above infectious diseases. Notably, peak sewage concentrations of norovirus GI, GII, rotavirus, IAV and RSV were found at the 6th, 3rd, 7th, 7th and 8th weeks, respectively. Astroviruses also resurge after the 7th week, as revealed by metaviromic data, suggesting that wastewater surveillance together with metaviromic data provides an essential early warning tool for revealing patterns of infectious disease resurgence.

Temporal variability of microbiome in the different plankton hosts revealed distinct environmental persistence of Vibrio parahaemolyticus in shrimp farms.

Plankton-bacteria interactions may play essential roles in maintaining the persistence of pathogenic Vibrio spp. However, the actual plankton host of the toxigenic Vibrio parahaemolyticus that harbors thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) remains unclear. To answer this question, we measured the abundance of toxigenic and nontoxigenic Vibrio parahaemolyticus in different types of plankton by most probable number (MPN)-PCR. We next investigated the dynamics of the microbiomes of rearing water, copepods, Chlorella, four predominant diatom genera (Nitzschia, Melosira, Skeletonema and Chaetoceros) and the gut of the shrimp in two recirculated shrimp farming systems from April to September using high-throughput 16 S rRNA amplicon sequencing. The survival of trh-positive and trh-negative V. parahaemolyticus in seawater and different plankton hosts was examined under low temperature and starvation conditions. The results showed that copepods harbored the highest proportion of trh-positive V. parahaemolyticus, followed by diatoms. Chitinous diatoms (CD) harbored a high proportion of Vibrionaceae, of which a high abundance of V. parahaemolyticus was found in summer. In contrast, Vibrio spp. is rarely found in Chlorella and nonchitinous diatoms. Taxon-specific associations were also observed, including a relatively high abundance of Vibrio and Halodesulfovibrio on copepods and covariation of Aeromonas and Bacillus inside the Chlorella. The survival assays showed that, in comparison to trh-negative V. parahaemolyticus, trh-positive V. parahaemolyticus showed better survival in copepods and CD under starvation conditions and maintained high persistence in the above hosts at low temperature. In conclusion, the results herein suggested that chitinous plankton might provide protection for V. parahaemolyticus, especially trh-positive V. parahaemolyticus, and improve their persistence under harsh conditions. Our study provided in-depth insights into the persistence of V. parahaemolyticus in the environment, which would promote targeted disease prevention measures.

Development of quantitative wastewater surveillance models facilitated the precise epidemic management of COVID-19.

Wastewater surveillance serves as a promising approach to elucidate the silent transmission of SARS-CoV-2 in communities. To understand the decay of the coronavirus in sewage pipes, the decay of the coronavirus traveling over 20 km distance of pipeline was analyzed. Based on the decay model, a WWTP and a community model were then proposed for predicting COVID-19 cases in Xi'an and Nanchang city during the COVID-19 outbreak in 2021 and 2022. The results suggested that Monte Carol simulations estimated 23.3, 50.1, 127.3 and 524.2 infected persons in the Yanta district of Xi'an city on December 14th, 18th, 22nd and 26th of 2021, respectively, which is largely consistent with the clinical reports. Next, we further conducted wastewater surveillance in two WWTPs that covered the whole metropolitan region in Nanchang to validate the robustness of the WWTP model from December 2021 to April 2022. SARS-CoV-2 signals were detected in two WWTPs from March 15th to April 5th. Predicted infection numbers were in agreement with the actual infection cases, which promoted precise epidemic control. Finally, community wastewater surveillance was conducted for 40 communities that were not 100 % covered by massive nucleic acid testing in Nanchang city, which accurately identified the SARS-CoV-2 carriers not detected by massive nucleic acid testing. In conclusion, accurate prediction of COVID-19 cases based on WWTP and community models promoted precise epidemic control. This work highlights the viability of wastewater surveillance for outbreak evaluation and identification of hidden cases, which provides an extraordinary example for implementing precise epidemic control of COVID-19.

Longitudinal wastewater surveillance of four key pathogens during an unprecedented large-scale COVID-19 outbreak in China facilitated a novel strategy for addressing public health priorities-A proof of concept study.

Wastewater-based epidemiology (WBE) is a promising tool for monitoring the spread of SARS-CoV-2 and other pathogens, providing a novel public health strategy to combat disease. In this study, we first analysed nationwide reports of infectious diseases and selected Salmonella, norovirus, and influenza A virus (IAV) as prioritized targets apart from SARS-CoV-2 for wastewater surveillance. Next, the decay rates of Salmonella, norovirus, and IAV in wastewater at various temperatures were established to obtain corrected pathogen concentrations in sewage. We then monitored the concentrations of these pathogens in wastewater treatment plant (WWTP) influents in three cities, establishing a prediction model to estimate the number of infected individuals based on the mass balance between total viral load in sewage and individual viral shedding. From October 2022 to March 2023, we conducted multipathogen wastewater surveillance (MPWS) in a WWTP serving one million people in Xi'an City, monitoring the concentration dynamics of SARS-CoV-2, Salmonella, norovirus, and IAV in sewage. The infection peaks of each pathogen were different, with Salmonella cases and sewage concentration declining from October to December 2022 and only occasionally detected thereafter. The SARS-CoV-2 concentration rapidly increased from December 5th, peaked on December 26th, and then quickly decreased until the end of the study. Norovirus and IAV were detected in wastewater from January to March 2023, peaking in February and March, respectively. We used the prediction models to estimate the rate of SARS-CoV-2 infection in Xi'an city, with nearly 90 % of the population infected in urban regions. There was no significant difference between the predicted and actual number of hospital admissions for IAV. We also accurately predicted the number of norovirus cases relative to the reported cases. Our findings highlight the importance of wastewater surveillance in addressing public health priorities, underscoring the need for a novel workflow that links the prediction results of populations with public health interventions and allocation of medical resources at the community level. This approach would prevent medical resource panic squeezes, reduce the severity and mortality of patients, and enhance overall public health outcomes.

Metagenomic sequencing combined with flow cytometry facilitated a novel microbial risk assessment framework for bacterial pathogens in municipal wastewater without cultivation.

A workflow that combined metagenomic sequencing with flow cytometry was developed. The absolute abundance of pathogens was accurately estimated in mock communities and real samples. Metagenome-assembled genomes binned from metagenomic data set is robust in phylogenetic analysis and virulence profiling.

Genomic Characteristion of Opportunistic Pathogen Kluyvera Reveals a Novel CTX-M Subgroup.

A rising incidence of clinical infections has been caused by Kluyvera, a significant opportunistic pathogen. Meanwhile, Kluyvera acts as an important reservoir of blaCTX-Ms, which are the dominant genes of class A extended-spectrum ß-lactamases (ESBLs). In this work, 60 strains of Kluyvera were subjected to phylogenetic relationship reconstruction, antimicrobial susceptibility testing, and antibiotic resistance genes prediction. All mature blaCTX-Ms were gathered to perform subgroup reclassification. The findings demonstrate that Kluyvera has a large gene pool with significant genetic flexibility. Notably, 25% of strains showed simultaneous detection of ESBLs and carbapenem resistance genes. The genotypes of fourteen novel blaCTX-Ms were identified. A new subgroup classification approach for blaCTX-Ms was defined by using 20 amino acid site variants, which could split blaCTX-Ms into 10 subgroups. The results of the subgroup division were consistent with the phylogenetic clustering. More significantly, we proposed a novel blaCTX-M subgroup, KLUS, that is chromosomally encoded in K. sichuanensis and the new species put forward in this study, showing amino acid differences from the currently known sequences. Cloning and transformation tests demonstrated that the recipient bacteria had a robust phenotype of cefotaxime resistance. Closely related Kluyvera species had blaCTX-Ms in the same subgroup. Our research lays the groundwork for a deeper comprehension of Kluyvera and emphasizes how important a blaCTX-M reservoir it is. We provide an update on blaCTX-M subgroups reclassification from the aspects of phylogenetic relationship, amino acid differences, and the new subgroup KLUS, which needs to be strengthen monitored due to its strong resistance phenotype to cefotaxime.