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.

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.

Pseudomonas pratensis sp. nov., Isolated from Grassland Soil from Inner Mongolia, China.

A novel bacterial strain, designated MHJ-10JT, was isolated from a soil sample obtained from a grassland in Inner Mongolia, China. MHJ-10JT strain could grow at 4-37 °C (optimum: 30 °C) and pH 4-9 (optimum: pH 6), as well as in the presence of 0-6% NaCl (optimum: 1%). Cells of strain MHJ-10JT are Gram-negative, rod-shaped, and motile. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain MHJ-10JT was most closely related to Pseudomonas lutea OK2T (98.5% 16S rRNA gene sequence similarity). The values of the average nucleotide identities (ANI) and digital DNA-DNA hybridization (dDDH) between strain MHJ-10JT and its related species were all below 80.5% and 24.4%, respectively, which are significantly lower than the thresholds of 95% for ANI and 70% for DDH for species delineation. The genomic G + C content of the MHJ-10JT strain is 64.8 mol%. Based on the phenotypic, genotypic, chemotaxonomic, and phylogenetic analyses, strain MHJ-10JT can be assigned to the genus Pseudomonas. In this study, we propose that strain MHJ-10JT be classified as a novel species belonging to the genus Pseudomonas with the species name Pseudomonas pratensis sp. nov. The type strain of the proposed novel species is MHJ-10JT (= KCTC 82206T = CGMCC 17322T).

Inhibition of Virulence Factors and Biofilm Formation by Wogonin Attenuates Pathogenicity of Pseudomonas aeruginosa PAO1 via Targeting pqs Quorum-Sensing System.

Pseudomonas aeruginosa, an important opportunistic pathogen, is capable of producing various virulence factors and forming biofilm that are regulated by quorum sensing (QS). It is known that targeting virulence factor production and biofilm formation instead of exerting selective pressure on growth such as conventional antibiotics can reduce multidrug resistance in bacteria. Therefore, many quorum-sensing inhibitors (QSIs) have been developed to prevent or treat this bacterial infection. In this study, wogonin, as an active ingredient from Agrimonia pilosa, was found to be able to inhibit QS system of P. aeruginosa PAO1. Wogonin downregulated the expression of QS-related genes and reduced the production of many virulence factors, such as elastase, pyocyanin, and proteolytic enzyme. In addition, wogonin decreased the extracellular polysaccharide synthesis and inhibited twitching, swimming, and swarming motilities and biofilm formation. The attenuation of pathogenicity in P. aeruginosa PAO1 by wogonin application was further validated in vivo by cabbage infection and fruit fly and nematode survival experiments. Further molecular docking analysis, pathogenicity examination of various QS-related mutants, and PQS signal molecule detection revealed that wogonin could interfere with PQS signal molecular synthesis by affecting pqsA and pqsR. Taken together, the results indicated that wogonin might be used as an anti-QS candidate drug to attenuate the infection caused by P. aeruginosa.

Baicalin Represses Type Three Secretion System of Pseudomonas aeruginosa through PQS System.

Therapeutics that target the virulence of pathogens rather than their viability offer a promising alternative for treating infectious diseases and circumventing antibiotic resistance. In this study, we searched for anti-virulence compounds against Pseudomonas aeruginosa from Chinese herbs and investigated baicalin from Scutellariae radix as such an active anti-virulence compound. The effect of baicalin on a range of important virulence factors in P. aeruginosa was assessed using luxCDABE-based reporters and by phenotypical assays. The molecular mechanism of the virulence inhibition by baicalin was investigated using genetic approaches. The impact of baicalin on P. aeruginosa pathogenicity was evaluated by both in vitro assays and in vivo animal models. The results show that baicalin diminished a plenty of important virulence factors in P. aeruginosa, including the Type III secretion system (T3SS). Baicalin treatment reduced the cellular toxicity of P. aeruginosa on the mammalian cells and attenuated in vivo pathogenicity in a Drosophila melanogaster infection model. In a rat pulmonary infection model, baicalin significantly reduced the severity of lung pathology and accelerated lung bacterial clearance. The PqsR of the Pseudomonas quinolone signal (PQS) system was found to be required for baicalin's impact on T3SS. These findings indicate that baicalin is a promising therapeutic candidate for treating P. aeruginosa infections.

PA0335, a Gene Encoding Histidinol Phosphate Phosphatase, Mediates Histidine Auxotrophy in Pseudomonas aeruginosa.

The biosynthesis of histidine, a proteinogenic amino acid, has been extensively studied due to its importance in bacterial growth and survival. Histidinol-phosphate phosphatase (Hol-Pase), which is responsible for the penultimate step of histidine biosynthesis, is generally the last enzyme to be characterized in many bacteria because its origin and evolution are more complex compared to other enzymes in histidine biosynthesis. However, none of the enzymes in histidine biosynthesis, including Hol-Pase, have been characterized in Pseudomonas aeruginosa, which is an important opportunistic Gram-negative pathogen that can cause serious human infections. In our previous work, a transposon mutant of P. aeruginosa was found to display a growth defect on glucose-containing minimal solid medium. In this study, we found that the growth defect was due to incomplete histidine auxotrophy caused by PA0335 inactivation. Subsequently, PA0335 was shown to encode Hol-Pase, and its function and enzymatic activity were investigated using genetic and biochemical methods. In addition to PA0335, the roles of 12 other predicted genes involved in histidine biosynthesis in P. aeruginosa were examined. Among them, hisC2 (PA3165), hisH2 (PA3152), and hisF2 (PA3151) were found to be dispensable for histidine synthesis, whereas hisG (PA4449), hisE (PA5067), hisF1 (PA5140), hisB (PA5143), hisI (PA5066), hisC1 (PA4447), and hisA (PA5141) were essential because deletion of each resulted in complete histidine auxotrophy; similar to the case for PA0335, hisH1 (PA5142) or hisD (PA4448) deletion caused incomplete histidine auxotrophy. Taken together, our results outline the histidine synthesis pathway of P. aeruginosaIMPORTANCE Histidine is a common amino acid in proteins. Because it plays critical roles in bacterial metabolism, its biosynthetic pathway in many bacteria has been elucidated. However, the pathway remains unclear in Pseudomonas aeruginosa, an important opportunistic pathogen in clinical settings; in particular, there is scant knowledge about histidinol-phosphate phosphatase (Hol-Pase), which has a complex origin and evolution. In this study, P. aeruginosa Hol-Pase was identified and characterized. Furthermore, the roles of all other predicted genes involved in histidine biosynthesis were examined. Our results illustrate the histidine synthesis pathway of P. aeruginosa The knowledge obtained from this study may help in developing strategies to control P. aeruginosa-related infections. In addition, some enzymes of the histidine synthesis pathway from P. aeruginosa might be used as elements of histidine synthetic biology in other industrial microorganisms.

Regulatory Effect of DNA Topoisomerase I on T3SS Activity, Antibiotic Susceptibility and Quorum- Sensing-Independent Pyocyanin Synthesis in Pseudomonas aeruginosa.

Topoisomerases are required for alleviating supercoiling of DNA during transcription and replication. Recent evidence suggests that supercoiling of bacterial DNA can affect bacterial pathogenicity. To understand the potential regulatory role of a topoisomerase I (TopA) in Pseudomonas aeruginosa, we investigated a previously isolated topA mutation using genetic approaches. We here report the effects of the altered topoisomerase in P. aeruginosa on type III secretion system, antibiotic susceptibility, biofilm initiation, and pyocyanin production. We found that topA was essential in P. aeruginosa, but a transposon mutant lacking the 13 amino acid residues at the C-terminal of the TopA and a mutant, named topA-RM, in which topA was split into three fragments were viable. The reduced T3SS expression in topA-RM seemed to be directly related to TopA functionality, but not to DNA supercoiling. The drastically increased pyocyanin production in the mutant was a result of up-regulation of the pyocyanin related genes, and the regulation was mediated through the transcriptional regulator PrtN, which is known to regulate bacteriocin. The well-established regulatory pathway, quorum sensing, was unexpectedly not involved in the increased pyocyanin synthesis. Our results demonstrated the unique roles of TopA in T3SS activity, antibiotic susceptibility, initial biofilm formation, and secondary metabolite production, and revealed previously unknown regulatory pathways.

The Two-Operon-Coded ABC Transporter Complex FpvWXYZCDEF is Required for Pseudomonas aeruginosa Growth and Virulence Under Iron-Limiting Conditions.

Iron is essential for all organisms. Bacteria have devolved sophisticated systems to maintain intracellular iron homeostasis. FpvCDEF(PA2407-2410) has been reported as an ABC transporter involved in pyoverdine-Fe uptake which does not affect growth under iron-limiting condition, when it is deleted in PAO1. In this study, we proved that fpvCDEF and fpvWXYZ(PA2403-2406) constituted an ABC transporter complex containing two operons: fpvWXYZCDE and fpvF. The operon fpvWXYZCDE was regulated by iron negatively and the single gene operon fpvF was constitutively expressed. Inactivation of any one of the components, fpvW, fpvC, fpvD, fpvE, and fpvF, led to increased expression of fpvWXYZCDE suggesting that each component of fpvWXYZCDEF could be involved in iron uptake. The ABC transporter complex encoded by fpvWXYZCDEF plays important roles in growth, oxidative stress resistance, and virulence, since the deletion of fpvWXYZCDEF resulted in defective growth, increased sensitivity to H2O2, and decreased virulence compared with PAO1(ΔfpvCDEF) and the wild type PAO1 under iron-limiting condition.

Microbial Community Dynamics and Assembly Follow Trajectories of an Early-Spring Diatom Bloom in a Semienclosed Bay.

Harmful algal blooms (HABs) are serious ecological disasters in coastal areas, significantly influencing biogeochemical cycles driven by bacteria. The shifts in microbial communities during HABs have been widely investigated, but the assembly mechanisms of microbial communities during HABs are poorly understood. Here, using 16S rRNA gene amplicon sequencing, we analyzed the microbial communities during an early-spring diatom bloom, in order to investigate the dynamics of microbial assembly processes. Rhodobacteraceae, Flavobacteriaceae, and Microbacteriaceae were the main bacterial families during the bloom. The 30 most abundant operational taxonomic units (OTUs) segregated into 4 clusters according to specific bloom stages, exhibiting clear successional patterns during the bloom process. The succession of microbial communities correlated with changes in the dynamics of algal species. Based on the ß-nearest taxon distance, we constructed a simulation model, which demonstrated that the assembly of microbial communities shifted from strong heterogenous selection in the early stage of the bloom to stochasticity in the middle stage and then to strong homogeneous selection in the late and after-bloom stages. These successions were driven mainly by chlorophyll a contents, which were affected mainly by Skeletonema costatum Moreover, functional prediction of microbial communities showed that microbial metabolic functions were significantly related to nitrogen metabolism. In summary, our results clearly suggested a dominant role of determinacy in microbial community assembly in HABs and will facilitate deeper understanding of the ecological processes shaping microbial communities during the algal bloom process.IMPORTANCE Harmful algal blooms (HABs) significantly influence biogeochemical cycles driven by bacteria. The shifts in microbial communities during HABs have been studied intensively, but the assembly mechanisms of microbial communities during HABs are poorly understood, with limited investigation of the balance of deterministic and stochastic processes in shaping microbial communities in HABs. In this study, the dynamics and assembly of microbial communities in an early-spring diatom bloom process were investigated. Our data both confirm previously observed general microbial successional patterns and show new detailed mechanisms for microbial assembly in HABs. These results will facilitate deeper understanding of the ecological processes shaping microbial communities in HABs. In addition, predictions of metabolic potential in this study will facilitate understanding of the influence of HABs on nitrogen metabolism in marine environments.

[Study on the Migration Resistance of Additives in Disposable Photophobic Infusion Set].

The disposable photophobic infusion was used to simulate clinical infusion under different conditions. The simulated liquid was collected every 30 min (total 4 h),and detected the additives (Fe3+, MDA and antioxidant 1076) in simulated liquid by spectroscopic method and chromatography method. The method is simple and stable, and can be used for the technical monitoring of the disposable photophobic infusion set in the future.

Pleiotropic effects of temperature-regulated 2-OH-lauroytransferase (PA0011) on Pseudomonas aeruginosa antibiotic resistance, virulence and type III secretion system.

Pseudomonas aeruginosa is an important human pathogen which adapts to changing environment, such as temperature variations and entering host by regulating their gene expression. Here, we report that gene PA0011 in P. aeruginosa PAO1, which encodes a 2-OH-lauroytransferase participating in lipid A biosynthesis, is involved in carbapenem resistance and virulence in a temperature-regulated manner in PAO1. The expression of PA0011 was higher at an environment temperature (21 °C) than that at a body temperature (37 °C). The inactivation of PA0011 rendered increased antibiotic susceptibility and decreased virulence both in vivo and in vitro. The impaired integrity and the decreased stability of the outer membrane were the cause of the increased susceptibility of PAO1(Δ0011) to carbapenem and many other common antibiotics. The reduced endotoxic activity of lipopolysaccharide (LPS) contributed to the decreased virulence both at 21 °C and 37 °C in PAO1 (Δ0011). In addition, we have found that PA0011 repressed the expression of TTSS virulence factors both at transcriptional and translational levels, similar to the effect of O antigen of LPS but unlike any effect of its homologue reported in other bacteria. The effect of PA0011 on resistance to many antibiotics including carbapenem and virulence in P. aeruginosa makes it a target for novel antimicrobial therapies.

The role of the temperature-regulated acyltransferase (PA3242) on growth, antibiotic resistance and virulence in Pseudomonas aeruginosa.

Pseudomonas aeruginosa (PAO1) is an important opportunistic pathogen that thrives in various environments. It is known that the structural variations of the lipopolysaccharide (LPS), including lipid A moiety play an important role in encountering environmental changes. Genes PA3242 and PA0011 have recently been reported to be responsible for secondary-acylation of lipid A in P. aeruginosa. In this study, we confirmed that the PA3242-dependant secondary acylation affects the growth, antibiotic resistance and virulence of PAO1 and functions as a more predominant acyltransferase than PA0011. PA3242 mutant showed inhibited growth at 37 °C and inviability at 28 °C in rich medium LB. The inactivation of PA3242 leads to more sensitivity to a wide range of antibiotics than PAO1(ΔPA0011). Moreover, the virulence of PAO1(ΔPA3242) was attenuated more significantly than that of PAO1 and PAO1(ΔPA0011). The outer membrane integrity and stability of PAO1(ΔPA3242) were seriously compromised. Furthermore, PAO1(ΔPA3242) lost most of pilus and exhibited severely damaged cell envelope, which is probably responsible for the deficiency of swimming, swarming and twitching. These results partially explained the decreased antibiotic resistance and attenuated virulence of PAO1(ΔPA3242) compared to PAO1(ΔPA0011) and PAO1. Our study demonstrated that PA3242-dependent secondary acylation of lipid A plays a predominant role in growth, antibiotic resistance and virulence of PAO1 than PA0011.

Hybrid sensor kinase PA1611 in Pseudomonas aeruginosa regulates transitions between acute and chronic infection through direct interaction with RetS.

Pseudomonas aeruginosa causes serious acute and chronic infections in humans. Major differences exist in disease pathogenesis, clinical treatment and outcomes between acute and chronic infections. P. aeruginosa acute infection characteristically involves the type III secretion systems (T3SS) while chronic infection is often associated with the formation of biofilms, a major cause of difficulties to eradicate chronic infections. The choice between acute and chronic infection or the switch between them by P. aeruginosa is controlled by regulatory pathways that control major virulence factors and genes associated with biofilm formation. In this study, we characterized a hybrid sensor kinase PA1611 that controls the expression of genes associated with acute and chronic infections in P. aeruginosa PAO1. Expression of PA1611 completely repressed T3SS and swarming motility while it promoted biofilm formation. The protein PA1611 regulates two small RNAs (sRNAs), rsmY and rsmZ which in turn control RsmA. Independent of phosphate relay, PA1611 interacts directly with RetS in vivo. The positive effect of RetS on factors associated with acute infection could presumably be restrained by PA1611 when chronic infection conditions are present. This RetS-PA1611 interaction, together with the known RetS-GacS interaction, may control disease progression and the lifestyle choice of P. aeruginosa.

[PA2580 is involved in Pseudomonas aeruginosa resistance to environmental stress].

OBJECTIVE: To investigate the function of gene PA2580 in Pseudomonas aeruginosa PAO1. METHODS: We constructed a PA2580 knockout mutant of PAO1 and a complemented strain of the mutant. We studied the function of the gene PA2580 using both genetic and biochemical methods, including antibiotic minimum inhibition concentration (MIC) comparison, measurement of gene expression levels in different conditions, protein expression and purification in vitro and enzymatic activity detection. RESULTS: PA2580 mutant was more sensitive to carbenicillin, chloramphenicol and ciprofloxacin. PA2580 expression was regulated by sub-inhibitory concentrations of antibiotics. PA2580 protein reduced various quinones efficiently using NADPH as the electron donor. PA2580 mutant was more sensitive to hydrogen peroxide and the mutant showed decreased expression of catalase. These results indicate that PA2580 is involved in the tolerance of oxidative stress in P. aeruginosa. CONCLUSION: The P. aeruginosa PA2580 protein physiologically functions as an NADPH quinone reductase which plays an important role in dealing with environment stress.

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.

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.

Construction of a tightly-controlled expression system for use in Pseudomonas.

Transcriptional analysis of czcCBA gene cluster in Pseudomonas aeruginosa PAO1 revealed that the promoter of this operon is tightly controlled by Zn. The promoter activity is undetectable in the uninduced condition but reaches high levels when induced. We used the czcCBA promoter to construct a tightly-controlled expression vector series, pLY vectors (pLY-A, pLY-B and pLY-C). These differed by just one base pair at the multiple cloning sites, allowing convenient in-frame cloning of any genes for expression. Using the luxCDABE reporter, this expression system was shown to be controlled by Zn in a dose-dependent manner in Pseudomonas; cloning the sacB gene encoding levansucrase onto the pLY-C vector enabled Zn-controlled growth of P. aeruginosa PAO1. These pLY vectors provide a useful tool for functional characterization of genes and controlled production of difficult or toxic proteins in Pseudomonas sp.

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.

Advance on Engineering of Bacteriophages by Synthetic Biology.

Since bacteriophages (phages) were firstly reported at the beginning of the 20th century, the study on them experiences booming-fading-emerging with discovery and overuse of antibiotics. Although they are the hotspots for therapy of antibiotic-resistant strains nowadays, natural phage applications encounter some challenges such as limited host range and bacterial resistance to phages. Synthetic biology, one of the most dramatic directions in the recent 20-years study of microbiology, has generated numerous methods and tools and has contributed a lot to understanding phage evolution, engineering modification, and controlling phage-bacteria interactions. In order to better modify and apply phages by using synthetic biology techniques in the future, in this review, we comprehensively introduce various strategies on engineering or modification of phage genome and rebooting of recombinant phages, summarize the recent researches and potential directions of phage synthetic biology, and outline the current application of engineered phages in practice.