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Rapid and accurate molecular diagnosis is a prerequisite for precision medicine, food safety, and environmental monitoring. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)-based detection, as a cutting-edged technique, has become an immensely effective tool for molecular diagnosis because of its outstanding advantages including attomolar level sensitivity, sequence-targeted single-base specificity, and rapid turnover time. However, the CRISPR/Cas-based detection methods typically require a pre-amplification step to elevate the concentration of the analyte, which may produce non-specific amplicons, prolong the detection time, and raise the risk of carryover contamination. Hence, various strategies for target amplification-free CRISPR/Cas-based detection have been developed, aiming to minimize the sensitivity loss due to lack of pre-amplification, enable detection for non-nucleic acid targets, and facilitate integration in portable devices. In this review, the current status and challenges of target amplification-free CRISPR/Cas-based detection are first summarized, followed by highlighting the four main strategies to promote the performance of target amplification-free CRISPR/Cas-based technology. Furthermore, we discuss future perspectives that will contribute to developing more efficient amplification-free CRISPR/Cas detection systems.

Background: Swine farming is a widespread agricultural practice that produces a substantial amount of antibiotics and genes associated with antibiotic resistance in swine manure. The presence of these antibiotic resistance genes (ARGs) poses a potential threat to the environment and human health. In order to investigate the microbiota and ARG profiles of swine manure and soil, samples from five swine farms on Chongming Island were collected and high-throughput sequencing and bioinformatics analysis were conducted. The sequencing data and MetaCompare allowed us to estimate the risk of ARG transmission to human pathogens in the sampled environments and prioritize the potential for antibiotic resistance.Results: Significant differences in microbial communities and antibiotic resistance gene (ARG) profiles were revealed both in swine manure and soil samples from five farms. Bacteroidetes was the dominant phylum in swine manure, while Proteobacteria was dominant in soil. The abundance of different drug classes varied from manure to soil. Multidrug, tetracycline, glycopeptide, peptide, fluoroquinolone, beta-lactam, rifamycin, macrolide, and aminoglycoside were the main drug classes of ARGs identified in both samples, with a large proportion of macrolide-lincosamide-streptogramin (MLS) and carbapenem resistance genes. Samples from the same farm showed a strong correlation in microbiota abundance and resistance gene diversity, indicating the effect of swine manure application and composting on the soil. However, microbial communities and ARGs varied among samples collected from different farms, influenced by factors including but not limited to feed composition, feeding density, husbandry practices, and sanitation. Beneficial bacteria in swine manure, including Bacillus spp., Eubacterium spp., Lactobacillus spp., Blautia, Dorea, and Methanobrevibacter, had symbiotic relationships with one another at the pig manure-soil interface. These genera were able to inhibit the growth of opportunistic pathogenic bacteria, such as Escherichia spp. However, some beneficial bacteria, represented by the NmcR gene, may promote the spread of antimicrobial resistance. We also observed numerous symbiotic relationships between different species of drug resistance genes, including cdeA, Bbif_ileS_MUP, macB, patB, vanSD, and tetQ, among others. Microbiota were found to determine the composition of drug resistance gene profiles. Quantifying the health risks associated with antibiotic resistance can help us identify and mitigate the risk. The mean risk value of ARGs for swine manure and soil samples from the five farms was 23.42 and 22.23 respectively, with higher values indicating greater risk.Conclusion Our study sheds light on the complex relationships between microbiota and ARG profiles in swine manure and soil, and highlights the potential impact of swine farming practices on the environment and public health.

Identifying climate change risks, vulnerability of exposed population and responses to climate change are critical to develop effective strategies to mitigate climate hazards. In this study, we have developed a climate change impacts and responses (CCIR) index that incorporates comprehensive information on climate risks, health burdens and actions that are taken in response to climate risks and damage. According to our knowledge, the CCIR index is the first of its kind that explores both climate impacts and mitigation actions. The CCIR index was positively correlated with national GDP per capita because wealthier countries can allocate more resources to mitigating climate impacts. Countries with better climate education tended to lower their carbon footprint. Furthermore, countries with higher risks of emerging infectious diseases that were more likely to consume more renewable energy. By identifying climate risks and opportunities, the CCIR index can help policymakers design, refine, and implement adaptation policies and actions to respond to the impacts of climate change.

With increasing attention paid to the concept of the microbiota–gut–brain axis, mounting evidence reveals that the gut microbiota is involved in a variety of neurological and psychiatric diseases. However, gut microbiota changes in major depressive disorder (MDD) patients and their association with disease mechanisms remain undefined. Fifty MDD patients and sixty healthy controls were recruited from the Shanghai Healthy Mental Center, China. Fecal samples were collected, and the compositional characteristics of the intestinal flora were determined in MDD patients by MiSeq sequencing. Venous blood was collected for the detection of plasma indoleamine-2,3-dioxygenase (Ido), kynurenine (Kyn) and tryptophan (Trp) levels. Stool samples of bacterial 16S sequencing was carried out. A total of 2,705,809 optimized sequences were obtained, with an average of 54,116 per sample. More unique OTUs were observed at the family, genus and species levels in the control group compared with the MDD cases. Further analysis showed significant changes in the α- and β-diversities and relative abundance levels of gut microbial entities in MDD patients, as well as elevated amounts of Ido and Kyn indicating Kyn pathway activation, KEGG bacterial 16S function prediction analysis shows a variety of amino acids and metabolic (including Ido, Trp and Kyn) changes in the body of patients with MDD. These may result in increased neurotoxic metabolites and reduced generation of serotonin in the disease process. These changed factors may potentially be utilized as biomarkers for MDD in the future, playing more important roles in the disease course.

Acute lung injury (ALI) is a devastating human malignancy characterized by excessively uncontrolled inflammation and lung endothelial dysfunction. Non-coding RNAs play essential roles in endothelial protections during the pathological processes of ALI. The precise functions and molecular mechanisms of the lncRNA-NORAD-mediated endothelial protection remain obscure. This study reports NORAD was significantly induced in human pulmonary microvascular endothelial cells (HPMECs) under lipopolysaccharide (LPS) treatment. Silencing NORAD effectively protected HPMECs against the LPS-induced cell dysfunction. In addition, RNA pull-down and luciferase assay validated that NORAD sponged miR-30c-5p, which showed reverse functions of NORAD in the LPS-induced cell injury of HPMECs. Furthermore, the glucose metabolism of HPMECs was significantly elevated under LPS stimulation which promoted the glucose consumption and extracellular acidification rate (ECAR) of HPMECs. Inhibiting NORAD or overexpressing miR-30c-5p suppressed glucose metabolism in HPMECs, leading to protective effects on HPMECs under LPS stimulation. The glycolysis key enzyme, lactate dehydrogenase-A (LDHA), was subsequently identified as a direct target of miR-30c-5p. Finally, recovery of miR-30c-5p in NORAD-overexpressing HPMECs effectively overrode the NORAD-promoted glycolysis and impaired endothelial dysfunction under LPS stimulation by targeting LDHA. Summarily, we demonstrated a NORAD-miR-30c-5p-LDHA-glycolysis axis in the LPS-induced HPMECs dysfunction in vitro and in vivo, contributing to the development of anti-ALI therapeutic approaches.

This retrospective study explored the changes in biomarkers indicators and prognosis in COVID-19 patients with mental disorders (n = 60) from the author’ Hospital between 2/13/2020 and 4/15/2020. Significant differences before and after negative conversion were observed in lymphocytes, neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio, aspartate aminotransferase, albumin, albumin/globulin ratio, direct bilirubin, alkaline phosphatase, uric acid, high-density lipoprotein cholesterol, and ApoA1 (all P

We report a case of a 63-year-old female patient who developed a recurrent urinary tract infection (UTI) with extensively drug-resistant Klebsiella pneumoniae (ERKp). In the initial two rounds of phage therapy, phage resistant mutants developed within days. Although ERKp strains were completely resistant to sulfamethoxazole-trimethoprim, the combination of sulfamethoxazole-trimethoprim with the phage cocktail inhibited the emergence of phage resistant mutant in vitro, and the UTI of patient was successfully cured by this combination. Thus, we propose that non-active antibiotic and bacteriophage synergism (NABS) might be an alternative strategy in personalized phage therapy.

The aims of this study were to evaluate the safety and probiotic characteristics of the newly isolated Enterococcus lactis strain JDM1.Safety assessment of E. lactis JDM1 was accomplished by the combination of whole genome sequence information analysis and phenotypic assays, including antimicrobial susceptibility test, haemolysis assay, biogenic amine production assay, cytotoxicity assay. The bacteriostatic experiment and gastrointestinal tolerance experiment were also conducted to evaluate its applicability.E. lactis JDM1 possesses good gastrointestinal tolerance and can inhibit the growth of the pathogenic bacteria Clostridioides difficile and Listeria monocytogenes. The chromosome size of JDM1 was 2,570,998 bp with a GC content of 38.46%, which contained a plasmid. One intact prophage, 13 genomic islands and 19 IS elements were predicted in the JDM1 chromosome. Five resistance-related genes and seven virulence-related genes were predicted in the genome. Most resistance genes were conserved, and virulence factors were not related to functional pathogenicity. Antimicrobial susceptibility tests showed that JDM1 was sensitive to tedizolid, ciprofloxacin, levofloxacin, penicillin, ampicillin, vancomycin, linezolid, tetracycline, high-level gentamicin and high-level streptomycin. Genes encoding putative enzymes responsible for adverse metabolites were not found and JDM1 was unable to produce the six main biogenic amines. Cytotoxicity test showed that the JDM1 supernatant had no toxic effect.E. lactis JDM1 is expected to be developed as a probiotic, and its probiotic properties are worthy of further exploration.

High levels of ROS cause oxidative stress, which plays a critical role in cell death. As a ROS effector protein, PRL2 senses ROS and controls phagocyte bactericidal activity during infection. Here we report PRL2 regulates oxidative stress induced cell death. PRL2 senses oxidative stress via highly reactive cysteine residues at 46 and 101. The oxidation of PRL2 causes protein degradation and supports pro-survival PDK1/AKT signal which in turn to protect cells against oxidative stress. As a result, PRL2 levels have a high correlation with oxidative stress induced cell death. In vivo experiments showed PRL2 deficient cells survive better in inflammatory oxidative environment and resist to ionizing radiation. Our finding suggests PRL2 serves as a negative regulator in cell adaptation to oxidative stress. Therefore, PRL2 could be targeted to modulate cell viability in inflammation or irradiation associated therapy.

With increasing attention on the concept of microbiota-gut-brain axis, increasing evidence has shown that gut microbiota is involved in a variety of neurological and psychiatric diseases. However, the change of gut microbiota in Major Depressive Disorder (MDD) patients and its association with disease mechanism remains to be further elucidated. The present study showed significant changes in ⍺- and β- diversities and relative abundance of gut microbiota in MDD patients, as well as elevation of indoleamine-2,3- dioxygenase (IDO) and kynurenine (KYN) that indicate activation of KYN pathway, which may result in increased neurotoxic metabolites and reduction in generation of serotonin in disease process. These results may potentially be utilized as biomarkers for MDD and play a potential role in the disease mechanisms. Funding Information: This work was supported by the Chinese Medicine Project of Shanghai Municipal Health and Family Planning Commission under Grant 2018LP024; The Characteristic Discipline Construction Project of Shanghai Mental Health Center under Grant 2017-TSXK-07; Shanghai Natural Science fund Project under Grant 20ZR1447700; China Public Health Union(First) Project under Grant GWLM202014; and The hospital-level project of Shanghai Mental Health Center under Grant 2020-YJ05 Declaration of Interests: No potential competing interest was reported by the authors. Ethics Approval Statement: The study was approved by the Institutional Review Board (IRB) at Shanghai Healthy Mental Center, and all participants were provided with written informed consent in line with the Declaration of Helsinki before commencement of the study.

Leptospirosis, caused by pathogenicLeptospiraspecies, has emerged as a widespread zoonotic disease worldwide. Macrophages mediate the elimination of pathogens through phagocytosis and cytokine production. Scavenger receptor A1 (SR-A1), one of the critical receptors mediating this process, plays a complicated role in innate immunity. However, the role of SR-A1 in the immune response against pathogenicLeptospirainvasion is unknown. In the present study, we found that SR-A1 is an important nonopsonic phagocytic receptor on murine macrophages forLeptospira. We also found that leptospiral LPS is the ligand of SR-A1. However, intraperitoneal injection of leptospires into WT mice presented with more severe jaundice, subcutaneous hemorrhaging, and higher bacteria burdens in blood and tissues than that of SR-A1-/-mice. Exacerbated cytokine and inflammatory mediator levels were also observed in WT mice and higher recruited macrophages in the liver than those of SR-A1-/-mice. Our findings collectively reveal that although beneficial in the uptake ofLeptospiraby macrophage, SR-A1 might be exploited byLeptospirato promote bacterial dissemination and modulate inflammatory activation, which causes a more severe infection in the host. These results provide our new insights into the innate immune response during early infection byL. interrogans.

As an antimicrobial therapy, therapeutic phages, also known as “Phage therapy” are able to inactivate multi-drug resistant bacteria such as methicillin and vancomycin resistant S. aureus and thus present a possible treatment for infections that are otherwise incurable. In this paper, we present a novel phage called JD419, which has a remarkably wide host-range. The virulent phage JD419 exhibits an elongated capsid and was able to infect and lyse 83 of all 129 tested clinical strains (64.3%) of multi-drug resistant S. aureus including MRSA. To evaluate the potential as a therapeutic phage, we tested the ability of phage JD419 to remain infectious after treatment exceeding physiological pH or temperature. The lytic activity of the phage was retained at pH values of 6.0-8.0 and at temperatures below 50°C. As phages sometimes contain virulence genes, we sequenced the complete genome of JD419. The 45509 bp genome contains a predicted 65 ORFs, none of which show homology to any known virulence or antibiotic resistance genes. Our study illustrates that Staphylococcus phage JD419 has the potential to be used for diagnostic, prophylaxic and therapeutic purposes.

Multidrug-resistant (MDR) organisms have increased worldwide, posing a major challenge for the clinical management of infection. Bacteriophage is expected as potential effective therapeutic agents for difficult-to-treat infections. When performing bacteriophage therapy, the susceptibility of lytic bacteriophage to the target bacteria is selected by laboratory isolate from patients. The presence of a subpopulation in a main population of tested cells, coupled with the rapid development of phage-resistant populations, will make bacteriophage therapy ineffective. We aimed to treat a man with multifocal urinary tract infections of MDR Klebsiella pneumoniae by phage therapy. However, the presence of polyclonal co-infectious cells in his renal pelvis and bladder led to the failure of three consecutive phage therapies. After analysis, the patient was performed with percutaneous nephrostomy (PCN). A cocktail of bacteriophages was selected for activity against all 21 heterogeneous isolates and irrigated simultaneously via the kidney and bladder to eradicate multifocal colonization, combined with antibiotic treatment. Finally, the patient recovered with an obviously improved bladder. The success of this case provides valuable treatment ideas and solutions for phage treatment of complex infections. Clinical Trial Registration www.chictr.org.cn, identifier ChiCTR1900020989.

This study aimed to compare the short-term outcomes of esophagectomy (RAMIE) versus thoracolaparoscopic esophagectomy (TLE) for patients undergoing neoadjuvant chemoradiotherapy (nCRT) for locally advanced esophageal cancer in a propensity matched cohort. Methods Data for consecutive patients receiving nCRT plus RAMIE or TLE were collected prospectively from February 2016 to December 2019. Baseline characteristics and perioperative outcomes of the RAMIE and TLE groups were retrospectively compared. Results After propensity matching, 48 pairs were identified. The conversion rate to open thoracotomy was comparable in RAMIE and TLE (4.2% vs 6.3%, P = 1). Median operative time in RAMIE was significantly shorter than TLE (237 vs 271 min, P Conclusion Compared to traditional TLE, RAMIE can achieve more lymph nodes yield at the RLN region and shorter operative time for the patients undergoing nCRT with comparable postoperative outcomes.

Type 2 diabetes is a complex metabolic disease and has been shown to involve alteration of the gut microbiota. Previous studies have primarily focused on changes in the bacterial microbiome, while ignoring the phage community composition. Extracellular phages can lyse host bacteria and thus influence the microbiota through positive or negative interactions with bacteria. We investigated changes in the extracellular phageome and discussed its role in T2D pathogenesis. We used a sequencing-based approach to identify bacteriophage after isolation of VLPs (virus like particles) from fecal samples. We identified 330 species of phages according to the predicted host bacteria from T2D patients (N=17) and nondiabetic controls (N=29). The phageome characteristics were highly diverse among individuals. In the T2D group, the intestinal phage population was altered, and the abundance of phages specific to Enterobacteriaceae hosts increased markedly. Meanwhile, the abundance of Enterobacteriaceae in the gut was significantly increased, and systemic LPS content elevation was observed in the T2D group. Additionally, a consortia of eight phages was found to distinguish T2D patients from nondiabetic controls with good performance (AUC>0.99).

Objective Goal of this work is to assess the feasibility to perform COVID-19 RNA tests within hospitals and communities experiencing SARS-CoV-2 virus outbreaks, to ultimately provide recommendations for hospitals with so-called fever clinics. In China, these specialised clinics within a hospital, specifically receive outpatients who have fever symptoms.Methods A team with expertise in the Exposure Analysis of Critical Control Points (EACCP) framework first identified potential infection routes during the testing for SARS-CoV-2, then constructed and tested flow diagrams, which were confirmed under actual conditions, demonstrating the feasibility to be carried out in hospitals with fever clinics. The team determined critical control points to mitigate the exposure risks at each control point.Findings The sampling and inactivation steps of clinical samples in fever clinics appeared to be associated with particularly high risk levels of exposure to SARS-CoV-2. Moderate levels of exposure were associated with storage and transportation of samples for inactivation; Low risk levels were associated with the transportation, storage and detection steps after inactivation.Conclusion To minimise risks of infection for personnel, optimised processes to carry out SARS-CoV-2 RNA tests in hospitals with fever clinics in China are proposed. The high risk of SARS-CoV-2 exposure during procedures preceding testing are the sampling and biological inactivation, which can be reduced by using full personal protective equipment and the use of BSL2 facilities in fever clinics or mobile BSL2 platforms. The implementation of the Exposure Analysis of Critical Control Points framework could facilitate rapid responses to outbreaks of emerging infectious diseases.

BackgroundFundamental to viral biology is identification and annotation of viral genes and their function. Determining the level of coronavirus gene expression is inherently difficult due to the positive stranded RNA genome and the identification of sub-genomic RNAs (sgRNAs) that are required for expression of most viral genes. In the COVID-19 epidemic so far, few genomic studies have looked at viral sgRNAs and none have systematically examined the sgRNA profiles of large numbers of SARS-CoV2 datasets in conjuction with data for other coronaviruses.ResultsWe developed a bioinformatic pipeline to analyze the sgRNA profiles of coronaviruses and applied it to 588 individual samples from 20 independent studies, covering more than 10 coronavirus species. Our result showed that SARS-CoV, SARS-CoV-2 and MERS-CoV each had a core sgRNA repertoire generated via a canonical mechanism. Novel sgRNAs that encode peptides with evolutionarily conserved structures were identified in several coronaviruses and were expressed in vitro and in vivo. Two novel peptides may have direct functional relevance to disease, by alluding interferon responses and disrupting IL17E (IL25) signaling. Relevant to coronavirus infectivity and transmission, we also observed that the level of Spike sgRNAs were significantly higher in-vivo than in-vitro, while the opposite held true for the Nucleocapside protein.ConclusionsOur results greatly expanded the predicted number of coronaviruses proteins and identified potential viral peptide suggested to be involved in viral virulence. These methods and findings shed new light on coronavirus biology and provides a valuable resource for future genomic studies of coronaviruses.

Air pollutants of PM2.5 can alter the composition of gut microbiota and lead to inflammation in the lung and gastrointestinal tract. The aim of this study was to evaluate the protective effect of a novel herbal extract blend, FC, composed of Lonicera japonica extract, Momordica grosvenori extract, and broccoli seed extract, on PM2.5-induced inflammation in the respiratory and intestinal tract. A549 cells and THP-1 cells, as well as C57BL/6 mice, were stimulated with PM2.5 to establish in vitro and in vivo exposure models. The models were treated with or without FC. The expression of inflammatory cytokines and tight junction proteins were studied. Proteomic analysis was performed to elucidate mechanisms. Mouse feces were collected for gut microbiota analysis. FC was shown to modulate the upregulation of pro-inflammatory cytokines mRNA expression in A549 and THP-1 cells and downregulated tight junction proteins mRNA expression in A549 cells due to PM2.5 stimulation. In animal models, the decreased expression of the anti-inflammatory factor il-10, tight junction protein ZO-1, and the elevated expression of COX-2 induced by PM2.5 were improved by FC intervention, which may be associated with zo-1 and cox-2 signaling pathways. In addition, FC was shown to improve the gut microbiota by increasing the abundance of beneficial bacteria.

Fundamental to viral biology is identification and annotation of viral genes and their function. Determining the level of coronavirus gene expression is inherently difficult due to the positive stranded RNA genome and the identification of subgenomic RNAs (sgRNAs) that are required for expression of most viral genes. We developed a bioinformatic pipeline to analyze metatranscriptomic data from 20 independent studies encompassing 588 individual samples and 10 coronavirus species. This comparative analysis defined a core sgRNA repertoire for SARS-CoV-2 and found novel sgRNAs that could encode functional short peptides. Relevant to coronavirus infectivity and transmission, we also observed that the ratio of Spike sgRNA to Nucleocapsid one is highest in SARS-CoV-2, among the β-coronaviruses examined. Furthermore, the adjustment of this ratio can be made by modifications to the viral RNA replication machinery, representing a form of viral gene regulation that may be involved in host adaption.

Colitis induced by C. difficile is one of the most common and costly healthcare-related infections for humans. Probiotics are one of the most promising approaches for controlling CDI. Here, we presented the isolation, safety, and probiotic property evaluation of a novel E. thailandicus strain, d5B, with effective antimicrobial activity against C. difficile. Strain d5B showed strong bactericidal effects on at least 54C. difficile strains. Safety tests showed that strain d5B was sensitive to clinically important antibiotics, and had no haemolytic and cytotoxic activities. Whole genomic analysis showed strain d5B only contained one aminoglycoside resistance gene located in the chromosome. Moreover, d5B was devoid of functional virulence genes. Finally, strain d5B exhibited probiotic properties, such as tolerance to the gastrointestinal tract, and adhered well to HT-29 cells. In conclusion, the E. thailandicus strain d5B should be investigated further for useful properties as a novel candidate probiotic for controlling CDI.

Dear Editors,We report the first outbreak and spread of one Coxsackievirus A16 (CV-A16) novel Genogroup D recombinant strain in Shanghai, China, in 2016, 2 years after an initial report in France1....

Background Type 2 diabetes (T2D) is a complex metabolic disease and has been proved to involve in the alternation of the gut microbiota. The previous studies primarily focused on the changes in bacteriome while ignoring the phage community composition. The extracellular phages could lyse the host bacteria, and thus influence the microbiota through the positive or negative interactions with bacteria. We investigated the change of extracellular phageome and explored its role in T2D pathogenesis.Results We used a sequencing-based approach to identify the bacteriophage after isolation of VLPs from the fecal samples. We identified 330 phages according to the predicted host bacteria. The phageome characteristics were highly diverse among individuals. In the T2D group, the intestinal phage population is altered and the abundance of 7 identified phages specific to Enterobacteriaceae hosts were found increased markedly. Additionally, the abundance of Enterobacteriaceae bacteria in gut was significantly increased and the systemic LPS elevation was observed in T2D group. Several phage consortia were found to have significant correlations with T2D disease indicators.Conclusions The alteration of bacteriophages predicted to infect Enterobacteriaceae in the gut was observed in this study, which was expected to be a new source of systemic LPS in T2D patients, and may contribute to the pathogenesis of the disease. The data present in this study revealed the similar variation trend in enteric bacteriome and the correlated bacteriophages, which is likely to shed considerable light on overall understanding the interactions between microbiome and metabolic diseases.

The bacterial pathogen Klebsiella pneumoniae causes urinary tract infections in immunocompromised patients. Generally, the overuse of antibiotics contributes to the potential development and the spread of antibiotic resistance. In fact, certain strains of K. pneumoniae are becoming increasingly resistant to antibiotics, making infection by these strains more difficult to treat. The use of bacteriophages to control pathogens may offer a non-antibiotic-based approach to treat multidrug-resistant (MDR) infections. However, a detailed understanding of phage–host interactions is crucial in order to explore the potential success of phage-therapy for treatment. In this study, we investigated the molecular epidemiology of nine carbapenemase-producing K. pneumoniae isolates from a local hospital in Shanghai, China. All strain isolates belong to sequence type 11 (ST11) and harbor the blaKPC-2 gene. The S1-PFGE (S1 nuclease pulsed field gel electrophoresis) pattern of the isolates did not show any relationship to the multilocus sequence typing (MLST) profiles. In addition, we characterized phage 117 and phage 31 and assessed the potential application of phage therapy in treating K. pneumoniae infections in vitro. The results of morphological and genomic analyses suggested that both phages are affiliated to the T7 virus genus of the Podoviridae family. We also explored phage–host interactions during growth in both planktonic cells and biofilms. The phages’ heterogeneous lytic capacities against K. pneumoniae strains were demonstrated experimentally. Subsequent culture and urine experiments with phage 117 and host Kp36 initially demonstrated a strong lytic activity of the phages. However, rapid regrowth was observed following the initial lysis which suggests that phage resistant mutants were selected in the host populations. Additionally, a phage cocktail (117 + 31) was prepared and investigated for antimicrobial activity. In Luria Broth (LB) cultures, we observed that the cocktail showed significantly higher antimicrobial activity than phage 117 alone, but this was not observed in urine samples. Together, the results demonstrate the potential therapeutic value of phages in treating K. pneumoniae urinary tract infections.

Background The diagnosis of major depression disorder (MDD) and other mental disorders were depended on some subjective survey scales. There are confirmed relationship between the gut flora and the mental states of MDD patients. Methods The V3–V4 region of the 16S rRNA gene was extracted from the fecal microbial communities in MDD patients, PCR amplified and sequenced on the Illumina Miseq platform. Results More phylum Firmicutes, less Bacteroidetes, and more genus Prevotella, Klebsiella, Streptococcus and Clostridium XI were found in MDD patients. The changes of the proportion of Prevotella and Klebsiella were consistent with Hamilton depression rating scale. Limitations The conclusion was limited by small sample sizes and potential uncontrollable influence factors on fecal microbiota. Discussion Prevotella and Klebsiella proportion in fecal microbial communities should be concerned in the diagnosis and therapeutic monitoring of MDD in future.

Staphylococcus aureus can adhere to most foreign materials and form biofilm on the surface of medical devices. Biofilm infections are difficult to resolve. The goal of this in vitro study was to explore the use of chitosan-coated nanoparticles to prevent biofilm formation. For this purpose, S. aureus was seeded in 96-well plates to incubate with chitosan-coated iron oxide nanoparticles in order to study the efficiency of biofilm formation inhibition. The biofilm bacteria count was determined using the spread plate method; biomass formation was measured using the crystal violet staining method. Confocal laser scanning microscopy and scanning electron microscopy were used to study the biofilm formation. The results showed decreased viable bacteria numbers and biomass formation when incubated with chitosan-coated iron oxide nanoparticles at all test concentrations. Confocal laser scanning microscopy showed increased dead bacteria and thinner biofilm when incubated with nanoparticles at a concentration of 500 µg/mL. Scanning electron microscopy revealed that chitosan-coated iron oxide nanoparticles inhibited biofilm formation in polystyrene plates. Future studies should be performed to study these nanoparticles for anti-infective use.

Toxin-antitoxin (TA) systems are widespread in bacteria and archaea. However, the roles of chromosomally encoded TA systems in bacterial physiology are still open to debate. In this study, a TA module-relBE in Bifidobacterium longum JDM301 (relBE(Bif)) was identified and its function in stress response was evaluated. Bioinformatics analysis of the whole genome sequences of JDM301 revealed a pair of linked genes encoding a RelBE-like TA system (RelBE(Bif)). Our results revealed a bicistronic operon formed by relBE(Bif) in JDM301. Over-expression of RelE(Bif) had a toxic effect on Escherichia coli, which could be neutralized by co-expression of its cognate antitoxin, RelB(Bif) Our data also demonstrated that RelE(Bif) is an mRNA interferase and that the activity of RelE(Bif) can be inhibited by RelB(Bif) These results suggest that RelE(Bif) is a toxic nuclease which arrests cell growth through mRNA degradation, and that the activity of RelE(Bif) can be abolished by co-expression of RelB(Bif) In addition, we also found that the expression of RelBE(Bif) is increased during osmotic stress, suggesting that RelBE(Bif) is activated under this adverse condition. Our results imply that the RelBE(Bif) TA module may represent a cell growth modulator which helps B. longum to deal with osmotic stress.

Introduction Staphylococcus aureus is a common human pathogen that causes various diseases including infections on the skin, in the bloodstream and the lower respiratory tracts. The emergence of methicillin-resistant S. aureus (MRSA) made the treatment of the bacterial infection more difficult, calling for development of new therapeutics. Compared with conventional antibiotic therapy, phage therapy offers a promising alternative to combat infections caused by MRSA. Results Here we showed that phage VB_SauS_SH-St 15644 isolated from sewage inhibited MRSA isolates in vitro and in the murine skin infection model. Phage VB_SauS_SH-St 15644 belongs to Siphoviridae. The genome of the phage is a linear, 45,111 bp double-stranded DNA with GC content of 33.35%. Among the 37 clinical MRSA isolates tested, 12 (32%) were lysed by the phage in vitro. The phage was relatively stable at temperatures up to 40 °C or between pH 6 and 9. However, the phage was sensitive to UV light. 80% of the phage was approximately adsorbed to the host MRSA isolate in 4 min. The one-step growth curve showed that the latent period was about 12 min followed by the growth period (about 9 min). The burst size was estimated at 13 PFU per infected cell. Furthermore, in a murine skin infection model, the phage significantly inhibited MRSA infection. Conclusions Our study suggested that phage VB_SauS_SH-St 15644 has a potential to inhibit MRSA skin infection.

Aside from enterovirus 71 (EV71) and coxsackie virus A16 (CV-A16), viruses that are known to cause hand-foot-and-mouth disease (HFMD), epidemiological profiles of other enteroviruses that induce HFMD are limited. We collected 9949 laboratory surveillance HFMD cases and 1230 serum samples from infants and children in Shanghai from 2012-2016. Since 2013, CV-A6 has displaced EV71 and CV-A16 to become the predominant serotype. Interestingly, novel epidemiological patterns in EV71 and CV-A16 infections were observed, with one large peak in both 2012 and 2014, followed by two smaller peaks in the respective following years (2013 and 2015). Through sequencing, we found that C4a, B1b, D-Cluster-1 and B constituted the major subgenotypes of EV71, CV-A16, CV-A6 and CV-A10, respectively. Among healthy individuals, 50.49% and 54.23% had positive neutralising antibodies (NtAbs) against EV71 and CV-A16, respectively, indicating that EV71 and CV-A16 silent infections were common. These populations may be an important potential source of infection. The overall seropositive rate of EV71 NtAbs showed a fluctuating, markedly downward trend, indicating the potential risk of a future EV71 epidemic. High CV-A16 NtAb seroprevalence corroborated a documented CV-A16 'silent' epidemic. Children aged 1-5 years had the lowest EV71 NtAb seropositive rate, whereas those aged 1-2 years exhibited the lowest CV-A16 NtAb seropositive rate. This is the first comprehensive investigation of the epidemiology and aetiology, as well as the seroprevalence, of HFMD in Shanghai between 2012 and 2016. This study provides the latest insights into developing a more efficient HMFD vaccination programme.

A novel Gram-stain-positive, strictly aerobic strain, NEAU-SA1

The idea that healthy uterine cavity is sterile is challenged nowadays. It is still debatable whether the bacteria present in the uterine cavity during pregnancy are residents or invaders. To reveal microbiome composition and its characteristics in the womb of pregnant women, 41 decidual tissue samples and 64 amniotic fluid samples were taken from pregnant Chinese women. DNA extraction was followed by pyrosequencing of the hypervariable V4 region of the 16S rDNA gene to characterize womb microbiome. Both types of samples had low diversity microbiome with Enterobacteriaceae being the dominant phylotypes at family level. To characterize the nature of colonization during pregnancy, the presence of endogenous biomass was confirmed by cultivation. Surprisingly, all of the 50 amniotic fluid samples studied were culture-negative, whereas 379 out of 1,832 placenta samples were culture-positive. Our results suggested that womb contained microbiome with low diversity. Culture-based investigation of amniotic fluid and placenta samples confirmed the presence of cultivable microorganisms in the placenta but not in amniotic fluid. Thus it suggests that bacterial colonization does occur during healthy pregnancy.

Probiotics might offer an attractive alternative to prevent and control Clostridium difficile (C. difficile) infection (CDI). Limited information is available on the ability of commercially used bifidobacterial strains to inhibit C. difficile. This study examined the anti-clostridial effects of Bifidobacterium longum JDM301, a widely used commercial probiotic strain in China, in vitro and in vivo. In vitro evaluation revealed a significant reduction in C. difficile counts when JDM301 was co-cultured with C. difficile, which was correlated with the significant decrease in clostridial toxin titres (TcdA and TcdB). Furthermore, the cell-free culture supernatants (CFS) of JDM301 inhibited C. difficile growth and degraded TcdA and TcdB. Notably, the results showed that acid pH promoted the degradation of TcdA by CFS from JDM301. Furthermore, comparative studies among 10 B. longum strains were performed, which showed that the inhibitory effect of CFS from JDM301 was similar with the other 8 B. longum strains and higher than strain BLY1. However, when it was neutralized, the significant different was lost. When present together, it was suggested that the acid pH induced by probiotics not only played important roles in the growth inhibition against C. difficile resulting in the reduction of toxins titres, but also directly promoted the degradation of clostridial toxin. In vivo studies proved that JDM301 partially relieved damage to tissues caused by C. difficile and also decreased the number of C. difficile and toxin levels. In summary, our results demonstrated that the commercial strain, JDM301 could be considered a probiotic able to exert anti-toxin capability and most of the CFS from Bifidobacterium were able to inhibit the growth of C. difficile, depending on acid pH. These results highlighted a potential that JDM301 could be helpful in preventing CDI and that most of the bifidobacterial strains could (at least partially) exert protective effects by reducing toxin titres through growth inhibition against toxigenic C. difficile.

Background: Staphylococcus aureus is one of the predominant causes of skin and soft tissue infections (SSTIs), but limited data were available regarding the characterization of S. aureus from SSTIs patients in Jiangsu Province in China. We aimed to investigate the molecular epidemiology of S. aureus among SSTIs patients in two hospitals of Jiangsu Province. Methods: Sixty-two patients with SSTIs from two Chinese hospitals in Jiangsu Province were enrolled in this study, and 62 S. aureus isolates were collected from February 2014 to January 2015. S. aureus isolates were characterized by antimicrobial susceptibility testing, toxin gene detection, and molecular typing with sequence type, Staphylococcus protein A gene type, accessory gene regulator ( agr ) group, and Staphylococcal cassette chromosome mec t ype. Results: Sixteen (25.8%) methicillin-resistant S. aureus (MRSA) isolates were detected, and there was no isolate found resistant to vancomycin, teicoplanin, sulfamethoxazole-trimethoprim, and linezolid. The sei was the toxin gene most frequently found, and no lukS/F-PV -positive isolates were detected among the SSTIs' patients. Molecular analysis revealed that ST398 (10/62, 16.1%; 2 MRSA and 8 methicillin-susceptible S. aureus ) to be the dominant clone, followed by ST5 (8/62, 12.9%) and ST7 (8/62, 12.9%). Conclusions: The livestock ST398 was the most common clone among patients with S. aureus SSTIs in Jiangsu Province, China. Surveillance and further studies on the important livestock ST398 clone in human infections are necessarily requested.

SjE16.7 is an egg-specific protein from Schistosoma japonicum that recruits neutrophils and initiates an inflammatory granuloma response in host tissue. However, since macrophages are known to be important regulators of egg granuloma formation we investigated the effect of SjE16.7 on this cell type. Here we report that SjE16.7 is a potent macrophage activator, inducing macrophage chemotaxis and stimulating cytokine production. Treatment of murine primary macrophages with SjE16.7 resulted in upregulation of both pro- and anti-inflammatory cytokines (IL-10, IL-12, IL-6 and TNF-α), as well as phosphorylation of mitogen-activated protein kinases (MAPKs). Moreover, SjE16.7 treatment increased MHC Class II expression on the surface of macrophages. Importantly, in vivo blockade of SjE16.7 significantly reduced egg-induced pathology, as a result of decreased leucocyte infiltration and reduced granuloma size. Our results suggest that SjE16.7 is an important pathogenic factor and a potential treatment target for this disease.

Probiotics might offer an attractive alternative to prevent and control Clostridium difficile (C. difficile) infection (CDI). Limited information is available on the ability of commercially used bifidobacterial strains to inhibit C. difficile. This study examined the anti-clostridial effects of Bifidobacterium longum JDM301, a widely used commercial probiotic strain in China, in vitro and in vivo. In vitro evaluation revealed a significant reduction in C. difficile counts when JDM301 was co-cultured with C. difficile, which was correlated with the significant decrease in clostridial toxin titres (TcdA and TcdB). Furthermore, the cell-free culture supernatants (CFS) of JDM301 inhibited C. difficile growth and degraded TcdA and TcdB. Notably, the results showed that acid pH promoted the degradation of TcdA by CFS from JDM301. Furthermore, comparative studies among 10 B. longum strains were performed, which showed that the inhibitory effect of CFS from JDM301 was similar with the other 8 B. longum strains and higher than strain BLY1. However, when it was neutralized, the significant different was lost. When present together, it was suggested that the acid pH induced by probiotics not only played important roles in the growth inhibition against C. difficile resulting in the reduction of toxins titres, but also directly promoted the degradation of clostridial toxin. In vivo studies proved that JDM301 partially relieved damage to tissues caused by C. difficile and also decreased the number of C. difficile and toxin levels. In summary, our results demonstrated that the commercial strain, JDM301 could be considered a probiotic able to exert anti-toxin capability and most of the CFS from Bifidobacterium were able to inhibit the growth of C. difficile, depending on acid pH. These results highlighted a potential that JDM301 could be helpful in preventing CDI and that most of the bifidobacterial strains could (at least partially) exert protective effects by reducing toxin titres through growth inhibition against toxigenic C. difficile.

Irritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder yet it still lacks effective prevention therapies. The aim of this study is to determine whether a novel prebiotic blend (PB) composed of fructo-oligosaccharide (FOS), galactooligosaccharide (GOS), inulin and anthocyanins could be effective in preventing the development of IBS. We explored the possible mechanisms both in animal and in cells. Post-infectious IBS models in C57BL/6 mice were established and were pretreated with the PB, PB and probiotic strains 8 weeks in advance of infection. Eight weeks after infection, intestinal tissues were collected for assessing histomorphology, visceral sensitivity, barrier function, pro-inflammatory cytokines expression and proteomics analysis. Fecal samples were also collected for microbiota analysis. The pro-inflammatory cytokines expression in Caco-2 cells were evaluated after co-incubation with PB and Salmonella typhimurium 14028. The results showed that PB significantly decreased the pro-inflammatory cytokines both in infected Caco-2 cells and PI-IBS models. The loss of body weight, decreased expression of tight junction protein Occludin (OCLN), and changes of the microbiota composition induced by infections could be greatly improved by PB intervention (p < 0.05). The proteomics analysis revealed that this function was associated with Peroxisome proliferator-activated receptor (PPAR)γ pathway.