The Mobilome-Enriched Genome of the Competence-Deficient Streptococcus pneumoniae BM6001, the Original Host of Integrative Conjugative Element Tn5253, Is Phylogenetically Distinct from Historical Pneumococcal Genomes

Streptococcus pneumoniae is an important human pathogen causing both mild and severe diseases. In this work, we determined the complete genome sequence of the S. pneumoniae clinical isolate BM6001, which is the original host of the ICE Tn5253. The BM6001 genome is organized in one circular chromosome of 2,293,748 base pairs (bp) in length, with an average GC content of 39.54%; the genome harbors a type 19F capsule locus, two tandem copies of pspC, the comC1-comD1 alleles and the type I restriction modification system SpnIII. The BM6001 mobilome accounts for 15.54% (356,521 bp) of the whole genome and includes (i) the ICE Tn5253 composite; (ii) the novel IME Tn7089; (iii) the novel transposon Tn7090; (iv) 3 prophages and 2 satellite prophages; (v) 5 genomic islands (GIs); (vi) 72 insertion sequences (ISs); (vii) 69 RUPs; (viii) 153 BOX elements; and (ix) 31 SPRITEs. All MGEs, except for the GIs, produce excised circular forms and attB site restoration. Tn7089 is 9089 bp long and contains 11 ORFs, of which 6 were annotated and code for three functions: integration/excision, mobilization and adaptation. Tn7090 is 9053 bp in size, flanked by two copies of ISSpn7, and contains seven ORFs organized as a single transcriptional unit, with genes encoding for proteins likely involved in the uptake and binding of Mg2+ cations in the adhesion to host cells and intracellular survival. BM6001 GIs, except for GI-BM6001.4, are variants of the pneumococcal TIGR4 RD5 region of diversity, pathogenicity island PPI1, R6 Cluster 4 and PTS island. Overall, prophages and satellite prophages contain genes predicted to encode proteins involved in DNA replication and lysogeny, in addition to genes encoding phage structural proteins and lytic enzymes carried only by prophages. & phi;BM6001.3 has a mosaic structure that shares sequences with prophages IPP69 and MM1 and disrupts the competent comGC/cglC gene after chromosomal integration. Treatment with mitomycin C results in a 10-fold increase in the frequency of & phi;BM6001.3 excised forms and comGC/cglC coding sequence restoration but does not restore competence for genetic transformation. In addition, phylogenetic analysis showed that BM6001 clusters in a small lineage with five other historical strains, but it is distantly related to the lineage due to its unique mobilome, suggesting that BM6001 has progressively accumulated many MGEs while losing competence for genetic transformation

In vivo modulation of cervicovaginal drug transporters and tissue distribution by film-released tenofovir and darunavir for topical prevention of HIV-1

We thank Gilead Science for provision of tenofovir and Janssen R&D Ireland for provision of darunavir. We thank members of the MOTIF consortium for useful discussions and exchange of ideas during the course of this study. We thank the technical staff of IDMIT, the animal care and veterinary staff at CEA, Fontenay-aux-Roses, France. Funding: this work was supported by the European Union's Seventh Programme for research, technological development and demonstration under grant agreement No 305316 as part of the MOTIF (Microbicides Formulation Through Innovative Formulation for Vaginal and Rectal Delivery) project. It has also the support of the “Investissements d’Avenir” French government program managed by the Agence Nationale de la Recherche under ANR-11-INBS-0008 funding for the Infectious Disease Models and Innovative Therapies (IDMIT, Fontenay-aux-Roses, France) infrastructure, and ANR-10-EQPX-02-01 funding for the FlowCyTech facility (IDMIT, Fontenay-aux-Roses, France).Peer reviewedPostprin

Stimulation of Human Monocytes with the Gram-Positive Vaccine Vector Streptococcus gordonii

Streptococcus gordonii is a bacterial vaccine vector which has previously been shown to activate dendritic cells in vitro and to induce local and systemic immune responses in vivo. In the present study, human monocytes (THP-1 cell line and peripheral blood monocytes) were characterized following interaction with S. gordonii. Treatment of human monocytes with S. gordonii but not latex beads induced a clear up-regulation of CD83, CD40, CD80, and CD54 and the down-regulation of CD14. Furthermore, bacterial treatment stimulated an increased expression of Toll-like receptor 5 (TLR5), TLR6, and TLR7, production of the proinflammatory cytokines tumor necrosis factor alpha and interleukin 1 beta, and reduction of the phagocytic activity. This work shows that the immunostimulatory activity of S. gordonii is not restricted to induction of dendritic-cell maturation but also affects the differentiation process of human monocytes

Interferon-γ from Brain Leukocytes Enhances Meningitis by Type 4 Streptococcus pneumoniae

Streptococcus pneumoniae is the leading cause of bacterial meningitis. Pneumococcal meningitis is a life-threatening disease with high rates of mortality and neurological sequelae. Immune targeting of S. pneumoniae is essential for clearance of infection; however, within the brain, the induced inflammatory response contributes to pathogenesis. In this study we investigate the local inflammatory response and the role of IFN-γ in a murine model of pneumococcal meningitis induced by intracranial injection of type 4 S. pneumoniae. Lymphoid and myeloid cell populations involved in meningitis, as well as cytokine gene expression, were investigated after infection. Animals were treated with a monoclonal antibody specific for murine IFN-γ to evaluate its role in animal survival. Intracranial inoculation of 3 × 104 colony-forming units of type 4 strain TIGR4 caused 75% of mice to develop meningitis within 4 days. The amount of lymphocytes, NK cells, neutrophils, monocytes and macrophages in the brain increased 48 h post infection. IFN-γ mRNA levels were about 240-fold higher in brains of infected mice compared to controls. Pro-inflammatory cytokines such as IL-1β and TNF-α, and TLR2 were also upregulated. In vivo treatment with anti-IFN-γ antibody increased survival of infected mice. This study shows that IFN-γ produced during meningitis by type 4 S. pneumoniae enhances bacterial pathogenesis exerting a negative effect on the disease outcome

Interferon-γ from brain leukocytes enhances meningitis by type 4 Streptococcus pneumoniae

Streptococcus pneumoniae is the leading cause of bacterial meningitis. Pneumococcal meningitis is a life-threatening disease with high rates of mortality and neurological sequelae. Immune targeting of S. pneumoniae is essential for clearance of infection; however, within the brain, the induced inflammatory response contributes to pathogenesis. In this study we investigate the local inflammatory response and the role of IFN-γ in a murine model of pneumococcal meningitis induced by intracranial injection of type 4 S. pneumoniae. Lymphoid and myeloid cell populations involved in meningitis, as well as cytokine gene expression, were investigated after infection. Animals were treated with a monoclonal antibody specific for murine IFN-γ to evaluate its role in animal survival. Intracranial inoculation of 3×104 colony-forming units of type 4 strain TIGR4 caused 75% of mice to develop meningitis within 4 days. The amount of lymphocytes, NK cells, neutrophils, monocytes and macrophages in the brain increased 48 hours post infection. IFN-γ mRNA levels were about 240-fold higher in brains of infected mice compared to controls. Pro-inflammatory cytokines such as IL-1β and TNF-α, and TLR2 were also upregulated. In vivo treatment with anti-IFN-γ antibody increased survival of infected mice. This study shows that IFN-γ produced during meningitis by type 4 S. pneumoniae enhances bacterial pathogenesis exerting a negative effect on the disease outcome

Interferon-γ from Brain Leukocytes Enhances Meningitis by Type 4 Streptococcus pneumoniae

Streptococcus pneumoniae is the leading cause of bacterial meningitis. Pneumococcal meningitis is a life-threatening disease with high rates of mortality and neurological sequelae. Immune targeting of S. pneumoniae is essential for clearance of infection; however, within the brain, the induced inflammatory response contributes to pathogenesis. In this study we investigate the local inflammatory response and the role of IFN-γ in a murine model of pneumococcal meningitis induced by intracranial injection of type 4 S. pneumoniae. Lymphoid and myeloid cell populations involved in meningitis, as well as cytokine gene expression, were investigated after infection. Animals were treated with a monoclonal antibody specific for murine IFN-γ to evaluate its role in animal survival. Intracranial inoculation of 3 × 104 colony-forming units of type 4 strain TIGR4 caused 75% of mice to develop meningitis within 4 days. The amount of lymphocytes, NK cells, neutrophils, monocytes and macrophages in the brain increased 48 h post infection. IFN-γ mRNA levels were about 240-fold higher in brains of infected mice compared to controls. Pro-inflammatory cytokines such as IL-1β and TNF-α, and TLR2 were also upregulated. In vivo treatment with anti-IFN-γ antibody increased survival of infected mice. This study shows that IFN-γ produced during meningitis by type 4 S. pneumoniae enhances bacterial pathogenesis exerting a negative effect on the disease outcome

In Vivo Activation of Naive CD4(+) T Cells in Nasal Mucosa-Associated Lymphoid Tissue following Intranasal Immunization with Recombinant Streptococcus gordonii

The antigen-specific primary activation of CD4(+) T cells was studied in vivo by adoptive transfer of ovalbumin-specific transgenic T cells (KJ1-26(+) CD4(+)) following intranasal immunization with recombinant Streptococcus gordonii. A strain of S. gordonii expressing on its surface a model vaccine antigen fused to the ovalbumin (OVA) peptide from position 323 to 339 was constructed and used to study the OVA-specific T-cell activation in nasal mucosa-associated lymphoid tissue (NALT), lymph nodes, and spleens of mice immunized by the intranasal route. The recombinant strain, but not the wild type, activated the OVA-specific CD4(+) T-cell population in the NALT (89% of KJ1-26(+) CD4(+) T cells) just 3 days following immunization. In the cervical lymph nodes and in the spleen, the percentage of proliferating cells was initially low, but it reached the peak of activation at day 5 (90%). This antigen-specific clonal expansion of KJ1-26(+) CD4(+) T cells after intranasal immunization was obtained with live and inactivated recombinant bacteria, and it indicates that the NALT is the site of antigen-specific T-cell priming

Risk Management in healthcare: results from a national-level survey and scientometric analysis in Italy

Risk management in healthcare, intended as all processes employed to detect, monitor, assess, mitigate, and prevent risks in healthcare facilities and safeguard patient safety, is a crucial component of Italy' National Health Service. Aim of the current study is to assess the role and progress of  research and training, in the field of Risk Management. We carried out a scientometric analysis to quantify and describe scientific outputs on Risk Management at the global and national level, over the last forty years; in addiction, we conducted a national-level cross-sectional survey to systematically retrieve and assess research and training activities within Italian postgraduate medical programmes in Hygiene and Preventive Medicine.   We report increasing scientific production on Risk Management-related topics from 1980 to 2017 at the global level (12% annual increase rate). Clinical Trials and Systematic reviews/meta-analysis make up for respectively 5% and 6% of global scientific output. Italy ranks 4th for scientific production, after USA, UK and Germany. 88% of Italian postgraduate medical programmes in Hygiene and Preventive medicine research on Risk Management, 42% through international collaborations. The main research themes are Healthcare-Associated Infections (HAIs) (97%), analysis of organizational models for safety in healthcare (62%), while training is focused on internships (87%) and academic lectures (73%). While research provides the evidence required to plan, implement and monitor effective interventions in healthcare risk management, training allows its dissemination in a synergic action to promote the value of patient safety and quality of care