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. ΦBM6001.3 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 ΦBM6001.3 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.

Complete Genome Sequence of Streptococcus pneumoniae Strain Rx1, a Hex Mismatch Repair-Deficient Standard Transformation Recipient.

The complete genome sequence of Streptococcus pneumoniae strain Rx1, a Hex mismatch repair-deficient standard transformation recipient, was obtained by combining Nanopore and Illumina sequencing technologies. The genome consists of a 2.03-Mb circular chromosome, with 2,054 open reading frames and a GC content of 39.72%.

Complete Genome Sequence of Lactobacillus crispatus Type Strain ATCC 33820.

The complete genome sequence of Lactobacillus crispatus type strain ATCC 33820 was obtained by combining Nanopore and Illumina sequencing technologies. The genome consists of a 2.2-Mb circular chromosome with 2,194 open reading frames and an average GC content of 37.0%.

In Vivo Modulation of Cervicovaginal Drug Transporters and Tissue Distribution by Film-Released Tenofovir and Darunavir for Topical Prevention of HIV-1.

Clinical trials have demonstrated partial protection against HIV-1 infection by vaginal microbicide formulations based on antiretroviral (ARV) drugs. Improved formulations that will maintain sustained drug concentrations at viral target sites in the cervicovaginal mucosa are needed. We have previously demonstrated that treatment of cervicovaginal cell lines with ARV drugs can alter gene expression of drug transporters, suggesting that the mucosal disposition of ARV drugs delivered vaginally can be modulated by drug transporters. This study aimed to investigate in vivo modulation of drug transporter expression in a nonhuman primate model by tenofovir and darunavir released from film formulations. Cervicovaginal tissues were collected from drug-naïve macaques and from macaques vaginally treated with film formulations of tenofovir or darunavir. Drug release in vaginal fluid as well as drug absorption in cervicovaginal tissues and lymph nodes were verified by mass spectrometry. The effects of exposure to drugs on the expression of transporters relevant to ARV drugs were evaluated by quantitative PCR. We showed expression in cervicovaginal tissue of drug-naïve macaques of transporters important for distribution of ARV drugs, albeit at lower levels compared to human tissue for key transporters including P-glycoprotein. Concentrations of tenofovir and darunavir well above the EC50 values determined in vitro were detected in vaginal fluid and vaginal tissues of macaques treated with drug-dissolving films over 24 h and were also comparable to those shown previously to modulate drug transporter expression. Accordingly, Multidrug Resistance associated Protein 2 (MRP2) in cervicovaginal tissue was upregulated by both tenofovir and darunavir. The two drugs also differentially induced and/or inhibited expression of key uptake transporters for reverse transcriptase inhibitors and protease inhibitors. The lower expression of key transporters in macaques may result in increased retention of ARV drugs at the simian cervicovaginal mucosa compared to the human mucosa and has implications for translation of preclinical data. Modulation of drug transporter expression by tenofovir and darunavir points to the potential benefit of MRP2 inhibition to increase ARV drug penetration through the cervicovaginal epithelium.

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 × 10(4) 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-1ß 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.

New fluoroquinolones active against fluoroquinolones-resistant Mycobacterium tuberculosis strains.

A set of 21 new fluoroquinolones bearing an aromatic or heteroaromatic moiety at C-7 and an alkyl group at N-1 were synthesized based on the lead structure of pirfloxacin and tested in vitro against Mycobacterium tuberculosis (M. tuberculosis) H37Rv by MIC determination in liquid medium. Among the synthesized compounds, 1-(tert-butyl)-6-fluoro-7-(4-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2o) and 1-(tert-butyl)-6-fluoro-7-(pyridin-3-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2n) were found to be the most active ones against M. tuberculosis H37Rv with the same MICs of reference compounds ciprofloxacin (CFX) and levofloxacin (LFX). MICs of 2o and 2n were determined for fluoroquinolone-sensitive and fluoroquinolone-resistant M. tuberculosis clinical isolates and 2o was the most active compound with up 4-fold difference of MIC with respect to CFX. The activity of 2o was also tested at the concentration of 16 µg/mL against M. tuberculosis H37Rv in infected murine macrophages. The results showed a 4-fold decrease in viable count of cell-associated mycobacteria with respect to untreated controls after 48 h of drug incubation.

Sex-based differences in susceptibility to respiratory and systemic pneumococcal disease in mice.

Systemic infection with Streptococcus pneumoniae was investigated in male and female mice in models of invasive pneumonia and sepsis. Male mice were found to be more susceptible to infection, exhibiting greater weight loss, marked decrease in body temperature, and a significantly higher mortality rate compared with female mice. For pneumonia, there were significant differences in survival rates. Female mice cleared their lung infections over time, whereas male mice, compared with female mice, had significantly increased numbers of colony-forming units in early stages of infection accompanied by higher levels of neutrophil recruitment in the first 24 hours after infection. Importantly, there were significant increases in proinflammatory cytokine levels during both sepsis and pneumonia in male compared with female mice. These cytokines were indicative of T-helper 1-type responses. The data presented here describe surprising differences in survival rates, neutrophil recruitment, and proinflammatory cytokine levels, indicating a sex-based difference in susceptibility to respiratory and systemic pneumococcal disease.

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.

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.