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Leptospirosis, caused by pathogenic Leptospira species, 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 pathogenic Leptospira invasion is unknown. In the present study, we found that SR-A1 is an important nonopsonic phagocytic receptor on murine macrophages for Leptospira. However, intraperitoneal injection of leptospires into WT mice presented with more apparent 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 of Leptospira by macrophage, SR-A1 might be exploited by Leptospira to modulate inflammatory activation and increase the susceptibility of infection in the host. These results provide our new insights into the innate immune response during early infection by L. interrogans.

Phage therapy is recognized as a promising alternative to antibiotics in treating pulmonary bacterial infections, however, its use has not been reported for treating secondary bacterial infections during virus pandemics such as coronavirus disease 2019 (COVID-19). We enrolled 4 patients hospitalized with critical COVID-19 and pulmonary carbapenem-resistant Acinetobacter baumannii (CRAB) infections to compassionate phage therapy (at 2 successive doses of 109 plaque-forming unit phages). All patients in our COVID-19-specific intensive care unit (ICU) with CRAB positive in bronchoalveolar lavage fluid or sputum samples were eligible for study inclusion if antibiotic treatment failed to eradicate their CRAB infections. While phage susceptibility testing revealed an identical profile of CRAB strains from these patients, treatment with a pre-optimized 2-phage cocktail was associated with reduced CRAB burdens. Our results suggest the potential of phages on rapid responses to secondary CRAB outbreak in COVID-19 patients.

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.

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.

Background: There are ever increasing researches implying that noncoded RNAs (ncRNAs) specifically circular RNAs (circRNAs) and microRNAs (miRNAs) in exosomes play vital roles in respiratory disease. However, the detailed mechanisms persist to be unclear in mycobacterial infection. Methods: In order to detect circRNAs and miRNAs expression pattern and potential biological function in tuberculosis, we performed immense parallel sequencing for exosomal ncRNAs from THP-1-derived macrophages infected by Mycobacterium tuberculosis H37Ra, Mycobacterium bovis BCG and control Streptococcus pneumonia, respectively and uninfected normal cells. Besides, THP-1-derived macrophages were used to verify the validation of differential miRNAs, and monocytes from PBMCs and clinical plasma samples were used to further validate differentially expressed miR-185-5p. Results: Many exosomal circRNAs and miRNAs associated with tuberculosis infection were recognized. Extensive enrichment analyses were performed to illustrate the major effects of altered ncRNAs expression. Moreover, the miRNA–mRNA and circRNA–miRNA networks were created and expected to reveal their interrelationship. Further, significant differentially expressed miRNAs based on Exo-BCG, Exo-Ra and Exo-Control, were evaluated, and the potential target mRNAs and function were analyzed. Eventually, miR-185-5p was collected as a promising potential biomarker for tuberculosis. Conclusion: Our findings provide a new vision for exploring biological functions of ncRNAs in mycobacterial infection and screening novel potential biomarkers. To sum up, exosomal ncRNAs might represent useful functional biomarkers in tuberculosis pathogenesis and diagnosis.

Phage therapy represents a possible treatment option to cure infections caused by multidrug-resistant bacteria, including methicillin and vancomycin-resistant Staphylococcus aureus, to which most antibiotics have become ineffective. In the present study, we report the isolation and complete characterization of a novel phage named JD219 exhibiting a broad host range able to infect 61 of 138 clinical strains of S. aureus tested, which included MRSA strains as well. The phage JD419 exhibits a unique morphology with an elongated capsid and a flexible tail. To evaluate the potential of JD419 to be used as a therapeutic phage, we tested the ability of the phage particles to remain infectious after treatment exceeding physiological pH or temperature. The activity was retained at pH values of 6.0–8.0 and below 50°C. As phages can contain virulence genes, JD419’s complete genome was sequenced. The 45509 bp genome is predicted to contain 65 ORFs, none of which show homology to any known virulence or antibiotic resistance genes. Genome analysis indicates that JD419 is a temperate phage, despite observing rapid replication and lysis of host strains. Following the recent advances in synthetic biology, JD419 can be modified by gene engineering to remove prophage-related genes, preventing potential lysogeny, in order to be deployed as a therapeutic phage.

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 Registrationwww.chictr.org.cn, identifier ChiCTR1900020989.

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.

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.

Schistosome infection contributes to cancer development, but the mechanisms are still not well-understood. SjE16.7 is an EF-hand calcium-binding protein secreted from Schistosoma japonicum eggs. It is a neutrophil attractant and macrophage activator and, as such, plays an important role in the inflammatory granuloma response in schistosomiasis. Here, we show that SjE16.7 binds to host cells by interacting with receptors for advanced glycation end products (RAGE). This ligation leads to activation of the NF-κB signaling pathway, an increase in the generation of reactive oxygen species, and production of the pro-inflammatory cytokines IL-6 and TNF-α. Using a mouse model of colorectal cancer, we demonstrate that intraperitoneal injection of SjE16.7 promotes colorectal cancer progression along with systemic myeloid cell accumulation. Thus, our results identify a new helminth antigen contributing to tumor development in the mammalian host.

C. difficile is one of the most clinically significant intestinal pathogens. Although phages have been shown to effectively control C. difficile infection, the host responses to phage predation have not been fully studied. In this study, we reported the isolation and characterization of a new phage, JD032, and analyzed the global transcriptomic changes in the hypervirulent RT078 C. difficile strain, TW11, during phage JD032 infection. We found that bacterial host mRNA was progressively replaced with phage transcripts, three temporal categories of JD032 gene expression, the extensive interplay between phage-bacterium, antiphage-like responses of the host and phage evasion, and decreased expression of sporulation- and virulence-related genes of the host after phage infection. These findings confirmed the complexity of interactions between C. difficile and phages and suggest that phages undergoing a lytic cycle may also cause different phenotypes in hosts, similar to prophages, which may inspire phage therapy for the control of C. difficile., Insights into the interaction between phages and their bacterial hosts are crucial for the development of phage therapy. However, only one study has investigated global gene expression of Clostridioides (formerly Clostridium) difficile carrying prophage, and transcriptional reprogramming during lytic infection has not been studied. Here, we presented the isolation, propagation, and characterization of a newly discovered 35,109-bp phage, JD032, and investigated the global transcriptomes of both JD032 and C. difficile ribotype 078 (RT078) strain TW11 during JD032 infection. Transcriptome sequencing (RNA-seq) revealed the progressive replacement of bacterial host mRNA with phage transcripts. The expressed genes of JD032 were clustered into early, middle, and late temporal categories that were functionally similar. Specifically, a gene (JD032_orf016) involved in the lysis-lysogeny decision was identified as an early expression gene. Only 17.7% (668/3,781) of the host genes were differentially expressed, and more genes were downregulated than upregulated. The expression of genes involved in host macromolecular synthesis (DNA/RNA/proteins) was altered by JD032 at the level of transcription. In particular, the expression of the ropA operon was downregulated. Most noteworthy is that the gene expression of some antiphage systems, including CRISPR-Cas, restriction-modification, and toxin-antitoxin systems, was suppressed by JD032 during infection. In addition, bacterial sporulation, adhesion, and virulence factor genes were significantly downregulated. This study provides the first description of the interaction between anaerobic spore-forming bacteria and phages during lytic infection and highlights new aspects of C. difficile phage-host interactions. IMPORTANCE C. difficile is one of the most clinically significant intestinal pathogens. Although phages have been shown to effectively control C. difficile infection, the host responses to phage predation have not been fully studied. In this study, we reported the isolation and characterization of a new phage, JD032, and analyzed the global transcriptomic changes in the hypervirulent RT078 C. difficile strain, TW11, during phage JD032 infection. We found that bacterial host mRNA was progressively replaced with phage transcripts, three temporal categories of JD032 gene expression, the extensive interplay between phage-bacterium, antiphage-like responses of the host and phage evasion, and decreased expression of sporulation- and virulence-related genes of the host after phage infection. These findings confirmed the complexity of interactions between C. difficile and phages and suggest that phages undergoing a lytic cycle may also cause different phenotypes in hosts, similar to prophages, which may inspire phage therapy for the control of C. difficile.

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.

Phage therapy is a potential and promising avenue for controlling the emergence and spread of multidrug-resistant (MDR) Klebsiella pneumoniae, however, the rapid development of anti-phage resistance has been identified as an obstacle to the development of phage therapy. Little is known about the mechanism employed by MDR K. pneumoniae strains and how they protect themselves from lytic phage predation in vitro and in vivo. In this study, comparative genomic analysis shows undecaprenyl-phosphate glucose-1-phosphate transferase (WcaJ), the initial enzyme catalyzing the biosynthesis of colanic acid, is necessary for the adsorption of phage 117 (Podoviridae) to the host strain Kp36 to complete its lytic life cycle. In-frame deletion of wcaJ alone was sufficient to provide phage 117 resistance in the Kp36 wild-type strain. Complementation assays demonstrated the susceptibility of phage 117, and the mucoid phenotype could be restored in the resistant strain Kp36-117R by expressing the wild-type version of wcaJ. Remarkably, we found that bacterial mobile genetic elements (insA and insB) block phage 117 infections by disrupting the coding region of wcaJ, thus preventing phage adsorption to its phage receptor. Further, we revealed that the wcaJ mutation likely occurred spontaneously rather than adapted by phage 117 predation under unfavorable environments. Taken together, our results address a crucial evolutionary question around the mechanisms of phage&ndash, host interactions, increasing our current understandings of anti-phage defense mechanisms in this important MDR pathogen.

The increasing incidence of carbapenem-resistant Enterobacteriaceae has become a challenge for clinical therapy. In our study, we analysed the molecular characteristics of imipenem-hydrolyzing β-lactamase (IMI) in Enterobacteriaceae isolates. Two reported clinical isolates, the IMI-3-producing Raoultella ornithinolytica RJ46C and the IMI-2-producing Escherichia coli RJ18 were identified in our retrospective review of isolates collected from June 2010 to June 2013, both isolates were resistant to carbapenem but sensitive to expanded-spectrum cephalosporins. The blaIMI gene was located on different â¼170-kb plasmids in both isolates. The blaIMI-3 gene was carried by the plasmid pRJ46C, which was extracted from the transconjugant and identified to be a 166,620-bp conjugative IncFIIY plasmid that contained 193 open reading frames, including replication-, plasmid conjugal transfer-, partitioning-, and mobilization-associated structures. The blaIMI-3 gene was located on a 15-kb region with a completely inverted sequence relative to that of plasmid pGA45, two ISEcl1-like elements containing two 33-bp complete inverted repeats were in an inverted orientation on both sides of the 15-kb region. We identified this typical structure as a novel composite transposon named Tn6306, indicating the occurrence of transposition. In addition, the blaIMI-2-carrying pRJ18 was an IncFIB plasmid, and a similar ISEcl1-like element was identified in an inverted direction upstream of IMI-2 in pRJ18. The identification of blaIMI in R. ornithinolytica and E. coli highlights the diversity of spreading carbapenemases in Enterobacteriaceae between hospitals and the environment in China. The novel transposon Tn6306, and other insert sequences, may play important roles in blaIMI mobilization. Keywords: blaIMI in Enterobacteriaceae, Genetic environment, Plasmids, Novel transposon structure Tn6306, ISEcl1-like element

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.

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.

Little is known regarding differences in the gut microbiomes of rheumatoid arthritis (RA) patients and healthy cohorts in China. This study aimed to identify differences in the fecal microbiomes of 66 Chinese patients with RA and 60 healthy Chinese controls. The V3-V4 variable regions of bacterial 16S rRNA genes were sequenced with the Illumina system to define the bacterial composition. The alpha-diversity index of the microbiome of the RA patients was significantly lower than that of the control group. The bacterial genera Bacteroides (p = 0.02202) and Escherichia-Shigella (p = 0.03137) were more abundant in RA patients. In contrast, Lactobacillus (p = 0.000014), Alloprevotella (p = 0.0000008615), Enterobacter (p = 0.000005759), and Odoribacter (p = 0.0000166) were less abundant in the RA group than in the control group. Spearman correlation analysis of blood physiological measures of RA showed that bacterial genera such as Dorea and Ruminococcus were positively correlated with RF-IgA and anti-CCP antibodies. Furthermore, Alloprevotella and Parabacteroides were positively correlated with the erythrocyte sedimentation rate, and Prevotella-2 and Alloprevotella were positively correlated with C-reactive protein, both biomarkers of inflammation. These findings suggest that the gut microbiota may contribute to RA development via interactions with the host immune system.

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.

Leptospirosis is an emerging worldwide zoonosis caused by pathogenic Leptospira spp. Our understanding of leptospirosis pathogenesis and host immune response remains limited, while mechanistic studies are hindered by a lack of proper animal models and immunological reagents. Here we established a murine model of acute and self-resolving leptospirosis by infecting 10-week-old C57BL/6 mice with Leptospira interrogans serovar Autumnalis strain 56606v, with characteristic manifestations including jaundice as well as subcutaneous and pulmonary bleeding, but no kidney lesions. We also verified that the lipopolysaccharide (LPS) of strain 56606v signaled through a TLR4-dependent pathway in murine bone marrow-derived macrophages (BMDMs), rather than the previously reported TLR2. In addition, upon infection with Leptospira strain 56606v, TLR4−/− C57BL/6 mice presented more severe jaundice and liver injury as well as higher bacterial loads than WT mice but milder pulmonary hemorrhaging. Molecular studies showed that leptospirosis-related bleeding coincides with the temporal kinetics of iNOS production, while jaundice and liver injury are probably due to insufficiently controlled bacterial loads in the liver. These results suggested that TLR4 is essential in mediating host leptospiral clearance and, to some extent, is associated with pulmonary and subcutaneous hemorrhage, probably through downstream inflammatory mediators, iNOS in particular. Overall, our murine model using immunocompetent mice might facilitate future studies into the pathogenesis of jaundice and bleeding in leptospirosis. Meanwhile, our study suggests the prospect of combining antibiotics and immunosuppressants in the treatment of severe leptospirosis presenting with pulmonary hemorrhage.

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.

Although it is well-recognized that inflammation enhances leukocyte bactericidal activity, the underlying mechanisms are not clear. Here we report that PRL2 is sensitive to oxidative stress at inflamed sites. Reduced PRL2 in phagocytes causes increased respiratory burst activity and enhances phagocyte bactericidal activity. PRL2 (Phosphatase Regenerating Liver 2) is highly expressed in resting immune cells, but is markedly downregulated by inflammation. in vitro experiments showed that PRL2 was sensitive to hydrogen peroxide (H2O2), a common damage signal at inflamed sites. In response to infection, PRL2 knockout (KO) phagocytes were hyper activated, produced more reactive oxygen species (ROS) and exhibited enhanced bactericidal activity. Mice with PRL2 deficiency in the myeloid cell compartment were resistant to lethal listeria infection and cleared the bacteria more rapidly and effectively. Moreover, in vitro experiments demonstrated that PRL2 binds to GTPase Rac and regulates ROS production. Rac GTPases were more active in PRL2 (KO) phagocytes than in wild type cells after bacterium infection. Our findings indicate that PRL2 senses ROS at inflamed sites and regulates ROS production in phagocytes. This positive feedback mechanism promotes bactericidal activity of phagocytes and may play an important role in innate anti-bacterial immunity.

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.

Aim: In this study, Staphylococcus phage JD007 bactericidal activity and induced immune responses during treatment were assessed in a dermal abscess model. Materials and Methods: Dermal abscesses in nude mice were established by injecting a clinical isolate of S. aureus SA325 isolated from the back under-dermal abscess of an in-patient. Results: Phage JD007 was able to inhibit the growth of S. aureus SA325 at MOI = 1 or 10, significantly preventing the formation of dermal abscesses. Moderate immune responses were observed in the prevention group through detection of cytokines. Conclusion: Phage JD007 inhibits the formation of dermal abscesses caused by a clinical S. aureus strain in nude mice without robust immune responses.

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.

Carbapenem-resistant Acinetobacter baumannii (CRAB) which is noted as a major pathogen associated with healthcare-associated infections has steadily developed beyond antibiotic control. Lytic bacteriophages with the characteristics of infecting and lysing specific bacteria have been used as a potential alternative to traditional antibiotics to solve multidrug-resistant bacterial infections. Here, we isolated A. baumannii-specific lytic phages and evaluated their potential therapeutic effect against lung infection caused by CRAB clinical strains. The combined lysis spectrum of four lytic phages' ranges was 87.5% (42 of 48) against CRAB clinical isolates. Genome sequence and analysis indicated that phage SH-Ab15519 is a novel phage which does not contain the virulence or antibiotic resistance genes. In vivo study indicated that phage SH-Ab15519 administered intranasally can effectively rescue mice from lethal A. baumannii lung infection without deleterious side effects. Our work explores the potential use of phages as an alternative therapeutic agent against the lung infection caused by CRAB strains.

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.

ICESag37, a novel integrative and conjugative element carrying multidrug resistance and potential virulence factors, was characterized in a clinical isolate of Streptococcus agalactiae. Two clinical strains of S. agalactiae, Sag37 and Sag158, were isolated from blood samples of new-borns with bacteremia. Sag37 was highly resistant to erythromycin and tetracycline, and susceptible to levofloxacin and penicillin, while Sag158 was resistant to tetracycline and levofloxacin, and susceptible to erythromycin. Transfer experiments were performed and selection was carried out with suitable antibiotic concentrations. Through mating experiments, the erythromycin resistance gene was found to be transferable from Sag37 to Sag158. SmaI-PFGE revealed a new SmaI fragment, confirming the transfer of the fragment containing the erythromycin resistance gene. Whole genome sequencing and sequence analysis revealed a mobile element, ICESag37, which was characterized using several molecular methods and in silico analyses. ICESag37 was excised to generate a covalent circular intermediate, which was transferable to S. agalactiae. Inverse PCR was performed to detect the circular form. A serine family integrase mediated its chromosomal integration into rumA, which is a known hotspot for the integration of streptococcal ICEs. The integration site was confirmed using PCR. ICESag37 carried genes for resistance to multiple antibiotics, including erythromycin [erm(B)], tetracycline [tet(O)], and aminoglycosides [aadE, aphA, and ant(6)]. Potential virulence factors, including a two-component signal transduction system (nisK/nisR), were also observed in ICESag37. S1-PFGE analysis ruled out the existence of plasmids. ICESag37 is the first ICESa2603 family-like element identified in S. agalactiae carrying both resistance and potential virulence determinants. It might act as a vehicle for the dissemination of multidrug resistance and pathogenicity among S. agalactiae.

Staphylococcus aureus or methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen causing pneumonia among children. To estimate the prevalence and molecular properties of S. aureus in children pneumonia in Shanghai, China, One hundred and seven hospitalized children with S. aureus pneumonia from two children’s hospitals from January 2014 through June 2015 were studied. S. aureus isolates from the respiratory specimens were characterized by antimicrobial susceptibility, agr typing, toxin genes, multilocus sequence typing (MLST), spa and SCCmec typing. Fifty-eight (54.2%, 58/107) were MSSA (methicillin-susceptible Staphylococcus aureus) and 49 (45.8%, 49/107) were MRSA. No isolates were found resistant to teicoplanin, sulfamethoxazole/trimethoprim, rifampicin, quinupristin/dalfopristin, linezolid, or vancomycin. However，these isolates showed high resistant rates to erythromycin, fosfomycin-trometamol and clindamycin. The agrⅠ (87/107, 81.3%) was the most common agr allele, followed by agrⅢ(10/107, 9.3%), agrⅡ(9/107, 8.4%) and agrⅣ(1/107, 0.9%). Six pvl-positive isolates (3 MRSA and 3 MSSA) and 7 isolates of livestock associated clone ST398 (4 MRSA, 3 MSSA) were identified. CC59 was found in 35 isolates (33 MRSA and 2 MSSA), constituting majority of MRSA (33/49, 67.35%). The dominant CC were CC59 (32.7%), CC188 (13.1%), CC7 (12.1%) and CC398 (9.3%) while t172 (16.8%), t189 (12.1%), t437 (9.3%) and t091 (9.3%) were the most common spa types. In conclusion, more particular concern should appeal to ST59-SCCmecIV-t172/t437 as it is the most common epidemic clone causing pneumonia among children in Shanghai.

Leptospirosis, which is caused by pathogenic species of the genus Leptospira, has emerged as one of the most widespread zoonotic diseases in the world. The exact mechanism of pathogenesis remains unknown, and the interaction between Leptospira and macrophages is not well understood. In this study, we report that carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) can efficiently label different Leptospira interrogans strains without affecting bacterial motility, viability, or virulence. Following co-incubation, CFDA-SE-labeled leptospires associated with macrophages were quantified by flow cytometry or confocal microscopy. In addition, we showed that trypan blue efficiently quenched the extracellular fluorescence from the adherent leptospires, which enabled intracellular and extracellular bacteria to be distinguished.

A high prevalence of the rtI187V polymerase substitution of hepatitis B virus (HBV) was detected in nucleoside/nucleotide-analogue-naive and -treated chronic hepatitis B (CHB) patients. We aimed at assessing the replicative capacity and susceptibility to lamivudine (LAM) and adefovir (ADV) in vitro of HBV harbouring rtI187V alone or in conjunction with LAM- or ADV-resistant mutations. The reverse transcriptase region of HBV isolates was directly sequenced from a cohort of 300 CHB patients from China. Replication-competent HBV constructs containing rtI187V and combined with LAM-resistant (rtM204I, rtL180M/rtM204V) mutations were generated, and compared with WT, LAM-resistant single (rtM204I) or double (rtL180M/rtM204V) and ADV-resistant (rtN236T) clones. In a Chinese cohort of 300 CHB patients, 8.7 % (26/300) showed substitution of rtI187 with V. Of note, the rtI187V prevalence in HBV genotype B was significantly higher than that in HBV genotype C (95.2 vs 4.8 %). In vitro phenotypic assays showed that the viruses bearing the rtI187V substitution had impaired replication efficacy when compared with the WT and the virus carrying rtI187V combined with LAM-resistant single or double mutations showed even more significantly impaired replicative capacities. Furthermore, rtI187V HBV remained susceptible towards treatment with LAM or ADV in vitro whereas the combination of the rtI187V substitution with LAM-resistant mutations rendered HBV resistant to LAM but still sensitive to ADV. Our study revealed that the rtI187V substitution in the HBV polymerase frequently occurred in CHB patients, particularly those with HBV genotype B. However, the emergence of the rtI187V substitution significantly impaired viral replication but without affecting drug sensitivity in vitro.

Introduction Viral infection is a significant cause of chronic obstructive pulmonary disease (COPD) and acute exacerbation of COPD. Retinoic acid inducible gene I (RIG-I)-like receptors (RLRs), including RIG-I and melanoma differentiation associated gene 5 (MDA-5), are important pattern recognition receptors for viral elimination. Objective The study aims to investigate the role of RIG-I and MDA-5 in COPD pathogenesis. Methods We examined the expression of RIG-I and MDA-5 by immunohistochemistry, real-time PCR and Western blots in COPD patients and control subjects. Results Our results showed that MDA-5 expression was upregulated in lung tissues and peripheral blood mononuclear cells of COPD patients and there was a negative correlation between MDA-5 mRNA levels and forced expiratory volume in 1 s %pred. COPD patients had higher interleukin (IL)-1 and IL-8 mRNA expression levels, and these inflammatory cytokines positively correlate with MDA-5 levels. However, there was no difference in the expression of RIG-I between COPD patients and control subjects. Conclusion Our results suggested that MDA-5, but not RIG-I, may play a critical role in airway inflammation in COPD.

Background Leptospirosis is one of the most important neglected tropical bacterial diseases worldwide. However, there is limited information on the genetic diversity and host selectivity of pathogenic Leptospira in wild small mammal populations. Methodology/Principal findings Jiangxi Province, located in southern China, is a region highly endemic for leptospirosis. In this study, among a total of 3,531 trapped rodents dominated by Apodemus agrarius (59.7%), 330 Leptospira strains were successfully isolated from six different sites in Jiangxi between 2002 and 2015. Adding 71 local strains from humans, various kinds of livestock and wild animals in Jiangxi, a total of 401 epidemic strains were characterized using 16S rRNA gene senquencing, multilocus sequence typing (MLST) and the microscopic agglutination test (MAT). Among them, the most prevalent serogroup was Icterohaemorrhagiae (61.10%), followed by Javanica (19.20%) and Australis (9.73%); the remaining five serogroups, Canicola, Autumnalis, Grippotyphosa, Hebdomadis and Pomona, accounted for 9.97%. Species identification revealed that 325 were L. interrogans and 76 were L. borgpetersenii. Moreover, L. interrogans was the only pathogenic species in Fuliang and Shanggao and was predominant in Shangrao (95.0%); L. borgpetersenii was the most common in the remaining three sites. Twenty-one sequence types (STs) were identified. Similarly, ST1 and serogroup Icterohaemorrhagiae were most prevalent in Shangrao (86.0% and 86.4%) and Fuliang (90.4% and 90.4%), ST143 and serogroup Javanica in Shangyou (88.5% and 90.4%) and Longnan (73.1% and 73.1%), and ST105 and serogroup Australis in Shanggao (46.3% and 56.1%). Serogroup Icterohaemorhagiae primarily linked to A. agrarius (86.9%), serogroup Canicola to dogs (83.3%). There were significant differences in the distribution of leptospiral species/serogroups/STs prevalence across host species/collected locations among the 394 animal-associated strains (Fisher’s exact test, p, Author summary Leptospirosis, caused by pathogenic Leptospira spp, is one of the most widespread zoonoses. In recent years, human leptospirosis with occasionally fatal infections has been frequently reported in Jiangxi Province, a highly endemic region located in the south of China. However, there is a lack of information on circulating Leptospira strains in this province. To identify the etiological characteristics, 401 Leptospira from Jiangxi were characterized using serological and molecular typing methods. Serological typing revealed that 61.10% of the isolates belonged to serogroup Icterohaemorrhagiae. Two species, L. interrogans and L. borgpetersenii, were identified using 16S rRNA gene sequencing. A. agrarius may be the main carrier of leptospirosis in this endemic region. Furthermore, the diversity of leptospiral isolates was demonstrated using MLST analysis. ST1, as the most prevalent ST of pathogenic leptospires, was widely dispersed in China. Significant geographic variation and host diversity in the distribution of dominant species, serogroups and STs were found in Jiangxi. This study is the first to demonstrate the distribution of Leptospira in domestic and wildlife animals in Jiangxi. This retrospective study represents the longest and largest field epidemiological investigation on the etiological characteristics and genetic diversity of pathogenic Leptospira among large wild animal reservoirs and human populations in Jiangxi. A better understanding of the circulating etiological agents and epidemiology of leptospirosis will provide a good starting point for efforts to control and prevent this disease.

Alarmone Guanosine 5'-diphosphate (or 5'-triphosphate) 3'-diphosphate [(p)ppGpp] is the key component that globally regulates stringent control in bacteria. There are two homologous enzymes, RelA and SpoT in Escherichia coli, which are responsible for fluctuations in (p)ppGpp concentration inside the cell, whereas there exists only a single RelA/SpoT enzyme in Gram-positive bacteria. We have identified a bifunctional enzyme with (p)ppGpp-hydrolase/synthase activity in Leptospira interrogans. We show that the relLin gene (LA_3085) encodes a protein that fully complements the relA/spoT double mutants in E. coli. The protein functions as a (p)ppGpp degradase as well as a (p)ppGpp synthase when the cells encounter amino acid stress and deprivation of carbon sources. N-terminus HD and RSD domains of relLin (relLinN ) were observed to restore growth of double mutants of E. coli. Finally, We demonstrate that purified RelLin and RelLinN show high (p)ppGpp synthesis activity in vitro. Taken together, our results suggest that L. interrogans contain a single Rel-like bifunctional protein, RelLin , which plays an important role in maintaining the basal level of (p)ppGpp in the cell potentially contributing to the regulation of bacterial stress response.

The Leptospira interrogans genome encodes two copies of cheR genes and each of them is able to complement for the swarming defective phenotype of Escherichia coli cheR null mutant RP1254 to certain extent, while over-expression of either of them inhibits the swarming of the chemotactic wild-type E. coli strain, RP437. Therefore, both CheR1 and CheR2 ought to bear the methyltransferase activities, although CheR1 has only one instead of two conserved basic amino acid residues located on the positively charged face of α2-helix. When this residue as well as the Lys48 and Arg55 of CheR2 was mutated, none of the CheRs was able to maintain aforementioned complementation functions, suggesting their critical roles in recognition of methyl-accepting chemotaxis proteins similar to that of E. coli. Demonstrated by microarray assay, the expression of cheR1 in L. interrogans cultured at 28°C in Ellinghausen-McCullough-Johnson-Harris medium was significantly lower than the average transcription level of all other genes, while the transcription of cheR2 was significantly higher than that of cheR1 in accordance with real-time reverse transcriptase-polymerase chain reaction assay. Tandem MS-MS data mining for the proteome of the same culture detected 16 peptides derived from CheR2 but none from CheR1. Therefore, although both genes were shown to be functional in E. coli, the structurally more conserved CheR2 rather than CheR1 might be the major functional component of L. interrogans chemotaxis adaptation system under our laboratory culture conditions.

The global increase in multidrug-resistant (MDR) bacteria has led to phage therapy being refocused upon. A novel endolysin, LysPA26, containing a lysozyme-like domain, was screened against Pseudomonas aeruginosa in this study. It had activity against MDR P. aeruginosa without pretreatment with an outer-membrane permeabilizer. LysPA26 could kill up to 4 log units P. aeruginosa in 30 min. In addition, temperature and pH effect assays revealed that LysPA26 had good stability over a broad range of pH and temperatures. Moreover, LysPA26 could kill other Gram-negative bacteria, such as Klebsiella pneumonia, Acinetobacter baumannii and Escherichia coli, but not Gram-positive bacteria. Furthermore, LysPA26 could eliminate P. aeruginosa in biofilm formation. Our current results show that LysPA26 is a new and promising antimicrobial agent for the combat of Gram-negative pathogens.

Objectives: To describe the genetic environment, transferability, and antibiotic susceptibility of one clinical Klebsiella pneumoniae isolate harboring both blaOXA-48 and blaNDM-1 on different plasmids from a Chinese hospital. Methods: The isolate was subjected to antimicrobial susceptibility testing and multilocus sequence typing using Etest and PCR. The plasmids harboring blaOXA-48 and blaNDM-1 were analyzed through conjugation experiments, S1-nuclease pulsed-field gel electrophoresis, and hybridization with specific probes. Plasmid DNA was sequenced using Pacbio RS II and annotated using RAST. Results: K. pneumoniae RJ119, carrying both blaOXA-48 and blaNDM-1, was resistant to almost all carbapenems, cephalosporins, fluoroquinolone, and aminoglycosides and belonged to ST307. blaOXA-48 was located on a 61,748-bp IncL/M conjugative plasmid, which displayed overall nucleotide identity (99%) to pKPN-E1-Nr.7. blaNDM-1 was located on a 335,317-bp conjugative plasmid, which was a fusion of a blaNDM-1-harboring InA/C plasmid pNDM-US (140,825 bp, 99% identity) and an IncFIB plasmid pKPN-c22 (178,563 bp, 99% identity). The transconjugant RJ119-1 harboring blaNDM-1 was susceptible to carbapenem, and there was an insertion of IS10 into the blaNDM-1 gene. Conclusions: This is the first report of the coexistence of blaOXA-48 and blaNDM-1 in one K. pneumoniae clinical isolate in China. OXA-48 in RJ119 contributed to the majority to its high resistance to carbapenems, whereas NDM-1 remained unexpressed, most likely due to the insertion of IS10. Our results provide new insight for the relationship between genetic diagnosis and clinical treatment. They also indicate that increased surveillance of blaOXA-48 is urgently needed in China.

Klebsiella pneumoniae (K.pneumoniae) is a common nosocomial pathogen causing bloodstream infections. Antibiotic susceptibility surveillance and molecular characterization will facilitate prevention and management of K. pneumoniae bloodstream infections. K. pneumoniae isolates causing bloodstream infections were consecutively collected between January 2012 and December 2015 in Shanghai. Eighty isolates (20 per year) were randomly selected and enrolled in this study. Drug susceptibility were determined by the disk diffusion method. Polymerase chain reaction (PCR) was employed to detect extended-spectrum β-lactamases (ESBLs), carbapenemases, and seven housekeeping genes of K. pneumoniae. eBURST was used for multi-locus sequence typing (MLST). More than 50% isolates were resistant to cefuroxime, ampicillin-sulbactam, and piperacillin, while carbapenems had lower resistant rates than other antibiotics. Of the 80 isolates, 22 produced ESBLs, and 14 were carbapenemase producers. In the ESBL-producing K. pneumoniae isolates, the most common ESBL genes were blaSHV and blaCTX−M. Thirteen carbapenemase producers harbored blaKPC−2 and one other carried blaNDM−5. ST11 (14/80) was the most frequent sequence type (ST), followed by ST15 (7/80) and ST29 (4/80). Our data revealed high prevalence of antibiotic resistant K. pneumoniae isolates from bloodstream infections but their genetic diversity suggested no clonal dissemination in the region. Also, one K. pneumoniae isolate harbored blaNDM−5 in this study, which was firstly reported in Shanghai.