Search

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.

ABSTRACT 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.

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

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.Emerging Microbes & Infections (2017) 6, e30 doi: 10.1038/emi.2017.16; published online 24 May 2017

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.

BackgroundLeptospirosis 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 findingsJiangxi 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, pConclusions/significanceOur study demonstrated high genetic diversity of pathogenic Leptospira strains from wild small animals in Jiangxi from 2002 to 2015. A. agrarius was the most abundantly trapped animal reservoir, and serogroup Icterohaemorrhagiae and ST1 were the most dominant in Jiangxi. Significant geographic variation and host diversity in the distribution of dominant species, STs and serogroups were observed. Moreover, rat-to-human transmission might play a crucial role in the circulation of Leptospirosis in Jiangxi. Details of the serological and molecular characteristics circulating in this region will be essential in implementing prevention and intervention measures to reduce the risk of disease transmission in China. However, phylogenetic analysis of more Leptospira isolates should explore the impact of ecological change on leptospirosis transmission dynamics and investigate how such new knowledge might better impact environmental monitoring for disease control and prevention at a public health level.

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–host interactions, increasing our current understandings of anti-phage defense mechanisms in this important MDR pathogen.

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.

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.

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.

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.

Objective: Activation of nuclear factor kappaB by diverse bacteria regulates the secretion of chemokines and cytokines. Staphylococcus aureus (S. aureus)-infected osteoblasts can significantly increase the secretion of interleukin-6 and monocyte chemoattractant protein-1. The aim of this study was to investigate whether S. aureus can activate nuclear factor kappaB in human osteoblasts, and whether the activation of nuclear factor kappaB by S. aureus regulates the secretion of interleukin-6 and monocyte chemoattractant protein-1. Methods: Immunoblot and electrophoretic mobility shift assay were used to detect the degradation of IκBa and activation of nuclear factor kappaB in human osteoblasts in response to S. aureus, respectively. Enzyme-linked immunosorbent assay was used to measure the secretion of interleukin-6 and monocyte chemoattractant protein-1 in the supernatants. Lastly, carbobenzoxyl-l-leucinyl-l-leucinyl-l-leucinal, an inhibitor of the nuclear factor kappaB, was used to determine if activation of nuclear factor kappaB by S. aureus in human osteoblasts regulates the secretions of interleukin-6 and monocyte chemoattractant protein-1. Results: Our results for the first time demonstrated that S. aureus can induce the degradation of IκBa and activation of nuclear factor kappaB in human osteoblasts in a time and dose-dependent manner. In addition, inhibition of nuclear factor kappaB by carbobenzoxyl-l-leucinyl-l-leucinyl-l-leucinal suppressed the secretion of interleukin-6 and monocyte chemoattractant protein-1 in the supernatants of S. aureus-infected human osteoblasts in a dose-dependent manner. Conclusions: These findings suggest that S. aureus can activate nuclear factor kappaB in human osteoblasts, and subsequently regulate the secretion of interleukin-6 and monocyte chemoattractant protein-1. The nuclear factor kappaB transcription factor regulates a number of genes involved in a wide variety of biological processes. Further study of the effects of nuclear factor kappaB activation on S. aureus-infected human osteoblast may provide us new insights into discovery of the immune mechanisms in osteomyelitis. Keywords: Staphylococcus aureus, osteoblasts, NF-kappaB, interleukin-6, chemokine CCL2

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.

Leptospirosis, caused by pathogenic Leptospira, is a worldwide zoonotic infection. The genus Leptospira includes at least 21 species clustered into three groups--pathogens, non-pathogens, and intermediates--based on 16S rRNA phylogeny. Research on Leptospira is difficult due to slow growth and poor transformability of the pathogens. Recent identification of extrachromosomal elements besides the two chromosomes in L. interrogans has provided new insight into genome complexity of the genus Leptospira. The large size, low copy number, and high similarity of the sequence of these extrachromosomal elements with the chromosomes present challenges in isolating and detecting them without careful genome assembly. In this study, two extrachromosomal elements were identified in L. borgpetersenii serovar Ballum strain 56604 through whole genome assembly combined with S1 nuclease digestion following pulsed-field gel electrophoresis (S1-PFGE) analysis. Further, extrachromosomal elements in additional 15 Chinese epidemic strains of Leptospira, comprising L. borgpetersenii, L. weilii, and L. interrogans, were successfully separated and identified, independent of genome sequence data. Southern blot hybridization with extrachromosomal element-specific probes, designated as lcp1, lcp2 and lcp3-rep, further confirmed their occurrences as extrachromosomal elements. In total, 24 plasmids were detected in 13 out of 15 tested strains, among which 11 can hybridize with the lcp1-rep probe and 11 with the lcp2-rep probe, whereas two can hybridize with the lcp3-rep probe. None of them are likely to be species-specific. Blastp search of the lcp1, lcp2, and lcp3-rep genes with a nonredundant protein database of Leptospira species genomes showed that their homologous sequences are widely distributed among clades of pathogens but not non-pathogens or intermediates. These results suggest that the plasmids are widely distributed in Leptospira species, and further elucidation of their biological significance might contribute to our understanding of biology and infectivity of pathogenic spirochetes.

BACKGROUND:Leptospirosis is one of the most important neglected tropical infectious diseases worldwide. Icterohaemorrhagiae has been throughout recent history, and still is, the predominant serogroup of this pathogen in China. However, very little in detail is known about the serovars or genotypes of this serogroup. METHODOLOGY/PRINCIPAL FINDINGS:In this study, 120 epidemic strains from five geographically diverse regions in China collected over a 50 year period (1958~2008), and 8 international reference strains characterized by 16S rRNA sequencing and MLST analysis. 115, 11 and 2 strains were identified as L. interrogans, L. borgpetersenii, and L. kirschneri, respectively. 17 different STs were identified including 69 ST1 strains, 18 ST17, 18 ST128, 9 ST143 and 2 ST209. The remaining 12 strains belonged to 12 different STs. eBURST analysis demonstrated that, among the clonal complexes isolated (CCs), CC1 accounted for 73.3% (88/120) strains representing three STs: ST1, ST128 and ST98. ST1 was the most likely ancestral strain of this CC, followed by singleton CC17 (17/120) and CC143 (11/120). Further analysis of adding 116 serogroup Icterohaemorrhagiae strains in the MLST database and studies previously described using global eBURST analysis and MST dendrogram revealed relatively similar ST clustering patterns with five main CCs and 8 singletons among these 244 strains. CC17 was found to be the most prevalent clone of pathogenic Leptospira circulating worldwide. This is the first time, to our knowledge, that ST1 and ST17 strains were distributed among 4 distinct serovars, indicating a highly complicated relationship between serovars and STs. CONCLUSIONS/SIGNIFICANCE:Our studies demonstrated a high level of genetic diversity in the serogroup Icterohaemorrhagiae strains. Distinct from ST17 or ST37 circulating elsewhere, ST1 included in CC1, has over the past 50 years or so, proven to be the most prevalent ST of pathogenic leptospires isolated in China. Moreover, the complicated relationship between STs and serovars indicates an urgent need to develop an improved scheme for Leptospira serotyping.

Pulmonary tuberculosis (TB) has gained attention in recent decades because of its rising incidence trend; simultaneously, increasing numbers of studies have identified the relationship between microbiota and chronic infectious diseases. In our work, we enrolled 32 patients with primary TB characterised by unilateral TB lesion formation diagnosed by chest radiographic exam. Bronchoalveolar lavage fluid was taken from both lungs. Twenty-four healthy people were chosen as controls. Pyrosequencing was performed on the V3 hypervariable region of 16S rDNA in all bacterial samples and used as a culture-independent method to describe the phylogenetic composition of the microbiota. Through pyrosequencing, 271,764 amplicons were detected in samples and analysed using tools in the Ribosomal Database Project (RDP) and bioinformatics. These analyses revealed significant differences in the microbiota in the lower respiratory tract (LRT) of TB patients compared with healthy controls; in contrast, the microbiota of intra/extra-TB lesions were similar. These results showed that the dominant bacterial genus in the LRT of TB patients was Cupriavidus and not Streptococcus, which resulted in a significant change in the microbiota in TB patients. The abundance of Mycobacteria and Porphyromonas significantly increased inside TB lesions when compared with non-lesion-containing contralateral lungs. From these data, it can be concluded that Cupriavidus plays an important role in TB's secondary infection and that in addition to Mycobacteria, Porphyromonas may also be a co-factor in lesion formation. The mechanisms underlying this connection warrant further research.

Neutrophils are known to play a major role in the egg granulomatous lesions caused by Schistosoma japonicum, but the precise mechanism by which eggs recruit or active neutrophil is unknown. Here we report S. japonicum egg specific EF-hand protein-SjE16.7 is a potent neutrophil recruiter and initiates the egg associated inflammatory granuloma in schistosomiasis. We show that the expression of SjE16.7 at level of both mRNA and protein is restricted to the egg stage. It locates in the miracidium and subshell area of the egg and can be secreted by the egg. The antigenic properties of SjE16.7 strongly suggest a role for SjE16.7 as an egg-derived molecule involved in host-parasite interactions. To study SjE16.7 functions in vivo, we challenged murine air pouch with recombinant SjE16.7. The results showed SjE16.7 trigged more inflammatory cell infiltration than vehicle or control protein. Using peritoneal exudate neutrophils from mice, we found that SjE16.7 significantly induced neutrophil chemotaxis in vitro, and the observed phenotypes were associated with enhanced Rac GTPase activation in SjE16.7 treated cells. Finally, in vivo hepatic granuloma formation model showed SjE16.7 coupled beads recruited more inflammatory cell infiltration than control beads. Our findings suggest SjE16.7 is an important pathogenic factor derived from egg. By recruiting neutrophils and inducing local inflammation, SjE16.7 facilitates eggs to be excreted through gut tissues and also initiates pathology in the liver; therefore SjE16.7 is a possible target for the prevention and treatment of schistosomiasis.

Platelet-activating factor (PAF) plays an important role in the pathogenesis of sepsis, and the level of plasma PAF acetylhydrolase (pPAF-AH), which inactivates PAF, decreases in sepsis patients except for the sepsis caused by severe leptospirosis. Usually, increase of pPAF-AH activity was observed in lipopolysaccharide (LPS)-induced Syrian hamster and rat sepsis models, while contradictory effects were reported for mouse model in different studies. Here, we demonstrated the in vivo effects of LPS upon the change of pPAF-AH activity in C57BL/6 mice and Mongolian gerbils. After LPS-treatment, the clinical manifestations of Mongolian gerbil model were apparently similar to that of C57BL/6 mouse sepsis model. The pPAF-AH activity increased in C57BL/6 mice after LPS induction, but decreased in Mongolian gerbils, which was similar to that of the human sepsis. It thus suggests that among the LPS-induced rodent sepsis models, only Mongolian gerbil could be used for the study of pPAF-AH related to the pathogenesis of human sepsis. Proper application of this model might enable people to clarify the underline mechanism accounted for the contradictory results between the phase II and phase III clinical trials for the administration of recombinant human pPAF-AH in the sepsis therapy.

Pulmonary hemorrhage has been recognized as a major, often lethal, manifestation of severe leptospirosis albeit the pathogenesis remains unclear. The Leptospira interrogans virulent serogroup Icterohaemorrhagiae serovar Lai encodes a protein (LA2144), which exhibited the platelet-activating factor acetylhydrolase (PAF-AH) activity in vitro similar to that of human serum with respect to its substrate affinity and specificity and thus designated L-PAF-AH. On the other hand, the primary amino acid sequence of L-PAF-AH is homologous to the alpha1-subunit of the bovine brain PAF-AH isoform I. The L-PAF-AH was proven to be an intracellular protein, which was encoded unanimously and expressed similarly in either pathogenic or saprophytic leptospires. Mongolian gerbil is an appropriate experimental model to study the PAF-AH level in serum with its basal activity level comparable to that of human while elevated directly associated with the course of pulmonary hemorrhage during severe leptospirosis. Mortality occurred around the peak of pulmonary hemorrhage, along with the transition of the PAF-AH activity level in serum, from the increasing phase to the final decreasing phase. Limited clinical data indicated that the serum activity of PAF-AH was likely to be elevated in the patients infected by L. interrogans serogroup Icterohaemorrhagiae, but not in those infected by other less severe serogroups. Although L-PAF-AH might be released into the micro-environment via cell lysis, its PAF-AH activity apparently contributed little to this elevation. Therefore, the change of PAF-AH in serum not only may be influential for pulmonary hemorrhage, but also seems suitable for disease monitoring to ensure prompt clinical treatment, which is critical for reducing the mortality of severe leptospirosis.

OBJECTIVE: Activation of nuclear factor kappaB by diverse bacteria regulates the secretion of chemokines and cytokines. Staphylococcus aureus (S. aureus)-infected osteoblasts can significantly increase the secretion of interleukin-6 and monocyte chemoattractant protein-1. The aim of this study was to investigate whether S. aureus can activate nuclear factor kappaB in human osteoblasts, and whether the activation of nuclear factor kappaB by S. aureus regulates the secretion of interleukin-6 and monocyte chemoattractant protein-1. METHODS: Immunoblot and electrophoretic mobility shift assay were used to detect the degradation of IκBa and activation of nuclear factor kappaB in human osteoblasts in response to S. aureus, respectively. Enzyme-linked immunosorbent assay was used to measure the secretion of interleukin-6 and monocyte chemoattractant protein-1 in the supernatants. Lastly, carbobenzoxyl-l-leucinyl-l-leucinyl-l-leucinal, an inhibitor of the nuclear factor kappaB, was used to determine if activation of nuclear factor kappaB by S. aureus in human osteoblasts regulates the secretions of interleukin-6 and monocyte chemoattractant protein-1. RESULTS: Our results for the first time demonstrated that S. aureus can induce the degradation of IκBa and activation of nuclear factor kappaB in human osteoblasts in a time and dose-dependent manner. In addition, inhibition of nuclear factor kappaB by carbobenzoxyl-l-leucinyl-l-leucinyl-l-leucinal suppressed the secretion of interleukin-6 and monocyte chemoattractant protein-1 in the supernatants of S. aureus-infected human osteoblasts in a dose-dependent manner. CONCLUSION: These findings suggest that S. aureus can activate nuclear factor kappaB in human osteoblasts, and subsequently regulate the secretion of interleukin-6 and monocyte chemoattractant protein-1. The nuclear factor kappaB transcription factor regulates a number of genes involved in a wide variety of biological processes. Further study of the effects of nuclear factor kappaB activation on S. aureus-infected human osteoblast may provide us new insights into discovery of the immune mechanisms in osteomyelitis.