Gut pathobiome mediates behavioral and developmental disorders in biotoxin-exposed amphibians.

Emerging evidence suggests a link between alterations in the gut microbiome and adverse health outcomes in the hosts exposed to environmental pollutants. Yet, the causal relationships and underlying mechanisms remain largely undefined. Here we show that exposure to biotoxins can affect gut pathobiome assembly in amphibians, which in turn triggers the toxicity of exogenous pollutants. We used Xenopus laevis as a model in this study. Tadpoles exposed to tropolone demonstrated notable developmental impairments and increased locomotor activity, with a reduction in total length by 4.37%-22.48% and an increase in swimming speed by 49.96%-84.83%. Fusobacterium and Cetobacterium are predominant taxa in the gut pathobiome of tropolone-exposed tadpoles. The tropolone-induced developmental and behavioral disorders in the host were mediated by assembly of the gut pathobiome, leading to transcriptome reprogramming. This study not only advances our understanding of the intricate interactions between environmental pollutants, the gut pathobiome, and host health but also emphasizes the potential of the gut pathobiome in mediating the toxicological effects of environmental contaminants.

Post-traumatic stress, depression, and anxiety during the 2021 Myanmar conflict: a nationwide population-based survey.

Background: The UN warns that Myanmar faces the 'triple crises' of mass conflict, uncontrolled COVID-19, and economic collapse. Therefore, we aimed to assess the population mental health burden, healthcare needs, and the associated risk factors in Myanmar. Methods: We established a nationwide random sample and recruited 1038 adults via random digit dialling from July 3-Aug 9, 2021, during the ongoing conflict since Feb 1, 2021, and surge in SARS-CoV-2 infections. Probable post-traumatic stress disorder (PTSD) was assessed using the PTSD Checklist-Civilian Version. Probable depression and anxiety were assessed using the Patient Health Questionnaire-2 and the Generalized Anxiety Disorder-2. We calculated population attributable fractions for probable mental disorders using multivariable logistic regression models. Based on the mental health burden and healthcare-seeking patterns, we projected the need for mental health services. Findings: During the 'triple crises', a third of adults in Myanmar (34.9%, 95% CI 32.0-37.7) reported a probable mental disorder. Prevalence of probable PTSD, depression, and anxiety were 8.1% (6.6-9.7), 14.3% (12.0-16.6), and 22.2% (19.7-24.7), respectively. We estimated that up to 79.9% (43.8-97.9) of probable PTSD was attributable to political stress. This corresponds to 2.1 million (1.1-3.2 million) fewer adults with probable PTSD if political stress was removed from the population. The mental health burden could translate into roughly 5.9 million adults seeking mental health services. Interpretation: The mental health burden in Myanmar is substantial, and population mental health might only be restored when the three crises have ended. An accelerated peace process is critical to protecting Myanmar's population mental health. Funding: This research was supported the Research Grants Council of the Hong Kong Special Administrative Region, China (Project No. HKU 17606122) and the Michele Tansella Award.

Spatially distributed metabolites SWEETen the root for microbes.

Limited understanding exists on the spatial configuration of underground plant-microbe interactions. In this issue of Cell Host & Microbe, Loo et al. illustrate the sugar transporter-involved interdependent interaction between root metabolites and microbial spatial colonization, providing insights into metabolic-associated organization of plant-microbe interactions.

Redox mediator chlorophyll accelerates low-temperature biological denitrification with responses of extracellular polymers and changes in microbial community composition.

Low temperatures limit the denitrification wastewater in activated sludge systems, but this can be mitigated by addition of redox mediators (RMs). Here, the effects of chlorophyll (Chl), 1,2-naphthoquinone-4-sulfonic acid (NQS), humic acid (HA), and riboflavin (RF), each tested at three concentrations, were compared for denitrification performance at low temperature, by monitoring the produced extracellular polymeric substances (EPS), and characterizing microbial communities and their metabolic potential. Chl increased the denitrification rate most, namely 4.12-fold compared to the control, followed by NQS (2.62-fold increase) and HA (1.35-fold increase), but RF had an inhibitory effect. Chl promoted the secretion of tryptophan-like and tyrosine-like proteins in the EPS and aided the conversion of protein from tightly bound EPS into loosely bound EPS, which improved the material transfer efficiency. NQS, HA, and RF also altered the EPS components. The four RMs affected the microbial community structure, whereby both conditionally abundant taxa (CAT) and conditionally rare or abundant taxa (CRAT) were key taxa. Among them, CRAT members interacted most with the other taxa. Chl promoted Flavobacterium enrichment in low-temperature activated sludge systems. In addition, Chl promoted the abundance of nitrate reduction genes narGHI and napAB and of nitrite reduction genes nirKS, norBC, and nosZ. Moreover, Chl increased abundance of genes involved in acetate metabolism and in the TCA cycle, thereby improving carbon source utilization. This study increases our understanding of the enhancement of low-temperature activated sludge by RMs, and demonstrates positive effects, in particular by Chl.

Interaction, mindset, and order: A qualitative study on Christian fellowship.

The rapid advancement and uncertainty of societal changes have not only accelerated social risks, but also plunged individuals into anxiety and restlessness in group interactions. In this context, using in-depth interview method, this study collects data from 10 Christian members to interpret their understanding of their sacred selves that are constructed in their fellowship interactions. The study reveals that Christians eventually develop the group interaction order based on their faith in God. Incorporating both care for individual lives and the importance of establishing interaction standards, this group interaction order is crucial in advancing collaboration. Moreover, the group interaction order is also constructed with the "Self-God" as the core belief mindset. The findings not only present characteristics of Christians' individual faith, but also reveal the profound significance of group interaction norms in reality.

Disparity in risk factors of ischemic stroke in four coastal-area hospitals in China.

Background: Currently, ischemic stroke is the leading cause of death in China. To compare regional differences of ischemic stroke, we analyzed the clinical characteristics of patients with ischemic stroke in four regionally representative hospitals in China. Methods: We conducted a retrospective study at four tertiary hospitals in east China, with regionally representative patients. The associated factors include hypertension, diabetes mellitus, coronary heart disease, hyperlipidemia and a combination of these factors. The standardized ratio (SR), estimated as the observed number divided by the expected number, computed as the sum of predicted probabilities from a multivariable logistic regression model derived using data from all other cities, was used to compare to average levels. Results: A total of 34,707 patients were included. The number of patients increased with age in all four hospitals and patients were predominantly male. The number of ischemic stroke cases with related factors increased with age, except for hyperlipidemia. There was no significant gender difference when multiple related factors existed simultaneously. Coronary heart disease had a more significant impact on ischemic stroke in Qingdao Municipal Hospital and the First Hospital of Qinhuangdao, while hyperlipidemia had a significant influence on ischemic stroke in the First Hospital of Qinhuangdao. Conclusions: At four hospitals in east China, with the increase of age, the risk factors associated with ischemic stroke increased, and the distribution of ischemic stroke-related factors showed regional differences.

Epidemiology of multimorbidity associated with atherosclerotic cardiovascular disease in the United States, 1999-2018.

BACKGROUND: The multimorbidity of Atherosclerotic cardiovascular disease (ASCVD) and many other chronic conditions is becoming common. This study aimed to assess multimorbidity distribution in ASCVD among adults in the United States from 1999 to 2018. METHODS: This cross-sectional survey from the National Health and Nutrition Examination Survey (NHANES) 1999-2018 using stratified multistage probability design. Among the 53,083 survey respondents during the study period, 5,729 US adults aged ≥ 20 years with ASCVD. Joinpoint regression was used to assess the statistical significance of prevalence trends in the prevalence of ASCVD stratified by multimorbidity. The Apriori association rule mining algorithm was used to identify common multimorbidity association patterns in ASCVD patients. RESULTS: Overall, 5,729 of 53,083 individuals had ASCVD, and the prevalence showed a slow declining trend (biannual percentage change = -0.81%, p = 0.035, average 7.71%). The prevalence of ASCVD significantly decreased in populations without dyslipidemia, diabetes mellitus (DM), hypertension, asthma, chronic obstructive pulmonary disease (COPD), and arthritis (all groups, p < 0.05). Additionally, 65.6% of ASCVD patients had at least four of the 12 selected chronic conditions, with four and five being the most common numbers of conditions (17.9% and 17.7%, respectively). The five most common chronic conditions were (in order) dyslipidemia, hypertension, arthritis, chronic kidney disease, and DM. The coexistence of hypertension and dyslipidemia had the highest support in association rules (support = 0.63), while the coexistence of dyslipidemia, hypertension, metabolic syndrome, and DM had the highest lift (lift = 1.82). CONCLUSIONS: During the 20-year survey period, there was a significant decrease in the overall prevalence of ASCVD. However, this reduction was primarily observed in individuals without dyslipidemia, DM, hypertension, asthma, COPD, and arthritis. Among populations with any of the evaluated chronic conditions, the prevalence of ASCVD remained unchanged. Most of ASCVD patients had four or more concurrent chronic conditions.

Reactivation performance and sludge transformation after long-term storage of Partial denitrification/Anammox (PD/A) process.

This study explores the startup of innovative Partial denitrification/Anammox (PD/A) process using long-term stored sludge (>2 years at 4 °C). Results indicate a swift recovery performance, characterized by a progressive increase in the activity of functional microorganisms with improved nitrogen volumetric loading rate during operation. Stable nitrogen removal efficiency of 99.6 % was attained at 14.2 °C under influent nitrate and ammonium of 120 and 100 mg/L, respectively. A distinctive transformation was observed as the initially black seeding sludge transitioned to brownish-red, accompanied by rapid sludge granulation with size notably increased from 263.1 µm (day 4) to 1255.0 µm (day 128), significantly contributing to the rapid PD/A performance recovery. Microbial community analysis revealed substantial increases in functional bacteria, Thauera (0.09 %-10.4 %) and Candidatus Brocadia (0.003 %-1.98 %), coinciding with enhanced nitrogen removal performance. Overall, this study underscores the viability of long-term stored PD/A sludge as a seed for rapid reactor startup, offering useful technical support to advance practical PD/A process implementation.

Experience of rehabilitation specialist nurses in providing bowel care for stroke patients: A qualitative study.

AIMS: This study aims to explore the experiences of rehabilitation specialist nurses in providing bowel care to stroke patients and to identify the factors that either facilitate or hinder their practice. DESIGN: This was a descriptive qualitative design study. METHODS: Between May 2022 and October 2022, we conducted in-depth and semi-structured interviews with 12 rehabilitation specialist nurses from two tertiary hospitals in Changsha, China. Thematic analysis was employed to analyse the interview transcripts. FINDINGS: Three key themes were revealed from our analysis: (1) acceptance of bowel care as a process, (2) high level of recognition improves the experience and (3) challenges stemming from limited knowledge and rights. Acceptance of bowel care as a dynamic process, coupled with a high level of recognition, enabled nurses to prioritize the health and safety of patients over personal feelings and achieve professional accomplishments. However, they encountered challenges in terms of professional development and restricted prescribing rights for bowel care. CONCLUSION: The experiences of rehabilitation specialist nurses in providing bowel care are dynamic. These findings have important implications for healthcare improvement, including the need for collaboration with healthcare professionals and nurturing nurses' self-identity, comprehensive training plans, innovative programs and expanding the scope of rehabilitation specialist nurses' rights. IMPACT: This study enhances our understanding of the challenges faced by rehabilitation specialist nurses caring for stroke patients with neurogenic bowel dysfunction. The findings provide insights into how to enhance bowel care experience and develop further in this field. REPORTING METHOD: This study adhered to the EQUATOR guideline and utilized the COREQ checklist. PATIENT OR PUBLIC CONTRIBUTIONS: This study involved participants who were registered nurses, and there were no contributions from patients or public.

Effectiveness and safety of digital rectal stimulation and abdominal massage for neurogenic bowel dysfunction in stroke patients: a randomized controlled trial protocol.

BACKGROUND: Neurogenic bowel dysfunction (NBD) is a prevalent complication among stroke patients, significantly affecting their quality of life, duration of hospitalization, medical expenses, and even mortality. Although current guidelines suggest a conservative strategy for addressing bowel dysfunction, which includes techniques such as digital rectal stimulation (DRS) and abdominal massage, the availability of interventions remains limited in healthcare facilities. METHODS: This study follows a prospective randomized controlled parallel-group clinical trial design. The control group will receive standard care, while the intervention group will undergo a program that combines DRS and abdominal massage in addition to standard care. The duration of the intervention for both groups will be 6 weeks. The primary outcome measures will be the Wexner score. Furthermore, secondary outcomes measure will be assessed, including Bristol score, Patient Assessment of Constipation-Quality of Life (PAC-QoL), and Fecal Incontinence Quality of Life (FI-QoL). DISCUSSION: This study aims to evaluate the effectiveness and safety of a bowel rehabilitation program for stroke patients with NBD. The findings will provide information that can contribute to the formulation of bowel management strategies. TRIAL REGISTRATION: The study has been registered in the Chinese Clinical Registry under the number ChiCTR2300071709. This registration was completed on May 23, 2023. All items from the World Health Organization Trial Registration Data set are described in this manuscript.

Exploring the effects of physical exercise on inferiority feeling in children and adolescents with disabilities: a test of chain mediated effects of self-depletion and self-efficacy.

Objective: The purpose of this study was to investigate the effects of physical exercise on inferiority feeling of children and adolescents with disabilities and its mechanism of action, as well as the mediating role of self depletion and self-efficacy. Methods: The following scales were administered to 546 children and adolescents with disabilities (mean age 15.6 years): The Feelings of Inadequacy Scale, (FIS), the Self-Regulation Fatigue Scale (S-RFS), the General Self-Efficacy Scale (GSES), and the Physical Exercise Rating Scale. Results: (1) Physical exercise can directly and negatively predict inferiority feeling, self-depletion, and can directly and positively predict self-efficacy; self-depletion can directly and negatively predict self-efficacy. Similarly, self-depletion positively predicts inferiority feeling; physical exercise and self-efficacy can also directly and negatively predict inferiority feeling. (2) The indirect effect of the path with self-depletion as the mediating variable was - 0.05, the indirect effect of the path with self-efficacy as the mediating variable was - 0.09, and the indirect effect of the path with self-depletion and self-efficacy as the mediating variables was - 0.04. (3) The sum of all indirect effects was - 0.18, and the three indirect effects accounted for 15.6%, 28.1%, and 12.5% of the total effect, with mediating effect was 56.2%. Conclusion: Physical exercise can indirectly predict inferiority feeling in children and adolescents with disabilities through the independent mediation of self-depletion and self-efficacy, as well as through the chain mediation of both. This study supports that moderate physical exercise has a positive effect on the mental health of children and adolescents with disabilities, and that reducing self-depletion and improving self-efficacy are important ways to prevent inferiority feeling among children and adolescents with disabilities. It reveals the relationship between physical exercise and inferiority feeling and its mechanism of action, and further improves the research on the effect of physical exercise on inferiority feeling of children and adolescents with disabilities.

Hydroxyapatite (HAP) formation in acetate-driven partial denitrification process: Enhancing sludge granulation and phosphorus removal.

Partial denitrification/anammox (PD/A) processes have emerged as a promising technology for efficient nitrogen removal from wastewater. However, these processes fail to remove phosphorus (P), a key pollutant that contributes to water eutrophication. To address this issue, the potential of inducing hydroxyapatite (HAP) precipitation in PD processes to achieve simultaneous P removal was investigated for the first time. Specifically, three SBRs (R1-R3) for PD were operated with adding varying concentrations of external Ca (30, 60, and 120 mg/L, respectively). Results demonstrated significant P reduction in all three SBRs, particularly in R3 with high Ca, which achieved an 80 % removal efficiency. Notably, sludge granulation was observed during operation, with the granule size in R3 with high Ca reaching 906.1 µm during the stable period, exceeding those in R2 (788.7 µm) and R1 (707.1 µm). This led to good settle ability of the PD sludge, as demonstrated by the lowest SVI5 (20 mL/g MLSS). Moreover, the decrease in the MLVSS/MLSS ratio suggested that the inorganic content accumulated, as observed by confocal laser scanning microscopy in the interior of the granules. Elemental composition analysis suggested that PD granules contained high P and Ca, while the X-ray diffraction (XRD) results confirmed the formation of HAP. Overall, this study demonstrated that PD-HAP coupled granular sludge process has potential as a robust and efficient method for nitrite production, as well as effective P removal and recovery, thereby advancing the application of anammox processes in wastewater treatment.

Oxygen-doped NiCoP derived from Ni-MOFs for high performance asymmetric supercapacitor.

Oxygen doping strategy is one of the most effective methods to improve the electrochemical properties of nickel-cobalt phosphide (NiCoP)-based capacitors by adjusting its inherent electronic structure. In this paper, O-doped NiCoP microspheres derived from porous nanostructured nickel metal-organic frameworks (Ni-MOFs) were constructed through solvothermal method followed by phosphorization treatment. The O-doping concentration has a siginificant influence on the rate performance and cycle stability. The optimized O-doped NiCoP electrode material shows a specific capacitance of 632.4 F-g-1at 1 A-g-1and a high retention rate of 56.9% at 20 A g-1. The corresponding NiCoP-based asymmetric supercapacitor exhibits a high energy density of 30.1 Wh kg-1when the power density is 800.9 W kg-1, and can still maintain 82.1% of the initial capacity after 10 000 cycles at 5 A g-1.

Application of epicutaneo-cava catheters with 24G indwelling needles in very low birth weight infants: a safe and simple innovative technique.

Background: Epicutaneo-cava catheter (ECC) is an ideal venous access for very low birth weight (VLBW) infants. However, because veins of VLBW infants are thin, ECC catheter is difficult to insert, and the success rate of puncture is low. This study aimed to use ECC with 24G indwelling needles to improve the outcomes of VLBW infants. Methods: This retrospective study enrolled 121 VLBW infants (birthweight <1,500 g) who required ECC catheterization and were admitted to the Neonatal Intensive Care Unit of the Children's Hospital of Zhejiang University School of Medicine between January 2021 and December 2021. The patients were divided into the indwelling needle group and the conventional technique group according to the technique of ECC. The demographic and treatment data of the two groups were collected, and the success rate of first attempt cannulation of ECC and catheter-related complications of the two groups were analyzed and compared. Results: There were no significant differences in gender, age, and body weight between the two groups on the day of ECC insertion and venipuncture site. It can be seen through model analysis that the success rate of first-attempt cannulation of ECC in the indwelling needle group was significantly higher than in the conventional technique group. In contrast, average catheterization time and catheterization-related bleeding risk in the indwelling needle group were significantly lower than in the conventional technique group (p = 0.00,and 0.00, respectively). Infection during catheter placement, indwelling catheter duration and catheter-related infection between the two groups (p > 0.05). Conclusion: Application of ECC with 24G indwelling needles in VLBW infants can improve the success rate of first attempt cannulation of ECC, reduce the time of catheterization and the risk of bleeding, which may be popularized for widespread application.

A Transparent Additive-Free Fibrous Facial Mask for Skin Moisturizing.

Most sheet facial masks for skincare are made of nonwovens and loaded with liquid active ingredients, which are usually opaque and require additives for long-term preservation. Herein, a Transparent Additive-Free Fibrous (TAFF) facial mask is reported for skin moisturizing. The TAFF facial mask consists of a bilayer fibrous membrane. The inner layer is fabricated by electrospinning functional components of gelatin (GE) and hyaluronic acid (HA) into a solid fibrous membrane to get rid of additives, the outer layer is an ultrathin PA6 fibrous membrane that is highly transparent, especially after absorbing water. The results indicate that the GE-HA membrane can quickly absorb water and become a transparent hydrogel film. By employing the hydrophobic PA6 membrane as the outer layer, directional water transport is achieved, which enables TAFF facial mask with excellent skin moisturizing effect. The skin moisture content is up to 84% ± 7% after placing the TAFF facial mask on the skin for 10 min. In addition, the relative transparency of the TAFF facial mask on the skin reaches 97.0% ± 1.9% when ultrathin PA6 membrane is used as the outer layer. The design of the transparent additive-free facial mask may serve as a guideline for developing new functional facial masks.

Construction of a telomere-related gene signature to predict prognosis and immune landscape for glioma.

Background: Glioma is one of the commonest malignant tumors of the brain. However, glioma present with a poor clinical prognosis. Therefore, specific detection markers and therapeutic targets need to be explored as a way to promote the survival rate of BC patients. Therefore, we need to search for quality immune checkpoints to support the efficacy of immunotherapy for glioma. Methods: We first recognized differentially expressed telomere-related genes (TRGs) and accordingly developed a risk model by univariate and multivariate Cox analysis. The accuracy of the model is then verified. We evaluated the variations in immune function and looked at the expression levels of immune checkpoint genes. Finally, to assess the anti-tumor medications often used in the clinical treatment of glioma, we computed the half inhibitory concentration of pharmaceuticals. Results: We finally identified nine TRGs and built a risk model. Through the validation of the model, we found good agreement between the predicted and observed values. Then, we found 633 differentially expressed genes between various risk groups to identify the various molecular pathways between different groups. The enrichment of CD4+ T cells, CD8+ T cells, fibroblasts, endothelial cells, macrophages M0, M1, and M2, mast cells, myeloid dendritic cells, and neutrophils was favorably correlated with the risk score, but the enrichment of B cells and NK cells was negatively correlated with the risk score. The expression of several immune checkpoint-related genes differed significantly across the risk groups. Finally, in order to create individualized treatment plans for diverse individuals, we searched for numerous chemotherapeutic medications for patients in various groups. Conclusion: The findings of this research provide evidence that TRGs may predict a patient's prognosis for glioma, assist in identifying efficient targets for glioma immunotherapy, and provide a foundation for an efficient, customized approach to treating glioma patients.

Identification and validation of a novel prognostic circadian rhythm-related gene signature for stomach adenocarcinoma.

Circadian rhythm genes were reported to be strongly associated with the development and prognosis of circadian rhythm disorders related to stomach adenocarcinoma (STAD), which is one of the most prevalent cancers. This study aimed to identify a circadian rhythm-related gene signature that could help predict STAD outcome. Using bioinformatics analysis approaches, 105 genes were examined in 350 patients with STAD. Overall, six hub-type circadian rhythm-associated genes (GNA11, PER1, SOX14, EZH2, MAGED1, and NR1D1) were identified using univariate and multivariate Cox regression analyses. These genes were then used to build a genetic predictive model, which was further validated using a publicly available dataset (GSE26899). Overall, genes associated with the circadian rhythm were found to be substantially correlated with the characteristics of the STAD patients (grade, sex, and M stage). In addition, the circadian rhythm-related gene signature was significantly associated with the MAPK and Notch signaling pathways, which are known risk factors for poorer STAD outcome. Taken together, these findings suggest that the herein proposed prognostic model based on six circadian rhythm-associated genes may have predictive value and potential application for clinical decision-making and for personalized treatment of STAD.

Phyllosphere microbiome induces host metabolic defence against rice false-smut disease.

Mutualistic interactions between host plants and their microbiota have the potential to provide disease resistance. Most research has focused on the rhizosphere, but it is unclear how the microbiome associated with the aerial surface of plants protects against infection. Here we identify a metabolic defence underlying the mutualistic interaction between the panicle and the resident microbiota in rice to defend against a globally prevalent phytopathogen, Ustilaginoidea virens, which causes false-smut disease. Analysis of the 16S ribosomal RNA gene and internal transcribed spacer sequencing data identified keystone microbial taxa enriched in the disease-suppressive panicle, in particular Lactobacillus spp. and Aspergillus spp. Integration of these data with primary metabolism profiling, host genome editing and microbial isolate transplantation experiments revealed that plants with these taxa could resist U. virens infection in a host branched-chain amino acid (BCAA)-dependent manner. Leucine, a predominant BCAA, suppressed U. virens pathogenicity by inducing apoptosis-like cell death through H2O2 overproduction. Additionally, preliminary field experiments showed that leucine could be used in combination with chemical fungicides with a 50% reduction in dose but similar efficacy to higher fungicide concentrations. These findings may facilitate protection of crops from panicle diseases prevalent at a global scale.

Effects of combined antibiotics on nitrification, bacteria and antibiotic resistance genes in activated sludge: Insights from legacy effect of antibiotics.

The effect of combined antibiotics exposure on nitrogen removal, microbial community assembly and proliferation of antibiotics resistance genes (ARGs) is a hotspot in activated sludge system. However, it is unclear that how the historical antibiotic stress affects the subsequent responses of microbes and ARGs to combined antibiotics. In this study, the effects of combined sulfamethoxazole (SMX) and trimethoprim (TMP) pollution on activated sludge under legacy of SMX or TMP stress with different doses (0.005-30 mg/L) were investigated to clarify antibiotic legacy effects. Nitrification activity was inhibited under higher level of combined exposure but a high total nitrogen removal (∼70%) occurred. Based on the full-scale classification, the legacy effect of past antibiotic stress had a marked effect on community composition of conditionally abundant taxa (CAT) and conditionally rare or abundant taxa (CRAT). Rare taxa (RT) were the keystone taxa in the microbial network, and the responses of hub genera were also affected by the legacy of antibiotic stress. Nitrifying bacteria and genes were inhibited by the antibiotics and aerobic denitrifying bacteria (Pseudomonas, Thaurea and Hydrogenophaga) were enriched under legacy of high dose, as were the key denitrifying genes (napA, nirK and norB). Furthermore, the occurrences and co-selection relationship of 94 ARGs were affected by legacy effect. While, some shared hosts (eg., Citrobacter) and hub ARGs (eg., mdtD, mdtE and acrD) were identified. Overall, antibiotic legacy could affect responses of activated sludge to combined antibiotic and the legacy effect was stronger at higher exposure levels.