Antimicrobial resistance and plasmid properties of Leuconostoc and group N Streptococcus

A method for rapid isolation of plasmid DNA from group N streptococci was developed. Chief advantages of the method were simplicity, the utilization of microliter quantities of reagents, and the obtainment of preparations highly enriched for covalently closed circular plasmid DNA. The method was also applied to over 15 Leuconostoc strains, and recovery of plasmids in the 1-to-76 megadalton mass range was demonstrated. Similarly to lactic streptococci, leuconostocs (other than L. oenos) contained at least one, and usually more, plasmid species. Plasmid DNA could not be demonstrated in four L. oenos strains examined. Thirty-eight strains of lactic streptococci were challenged with all major classes of antimicrobials, in the Bauer-Kirby disc test. Minimal inhibitory concentrations of some antimicrobials were determined for the less susceptible strains. Presence of high-level resistance factors could not be detected, except in the case of nisin. Streptococcus lactis ATCC 7962 was resistant to >40-fold higher concentrations of this bacteriocin (>64 μg/ml) than most other strains tested. This strain was a potent nisin producer. Accidental contamination of one S. cremoris culture with Leuconostoc led to the discovery that most leuconostocs are insensitive to the antibiotic vancomycin. Tests performed with one such strain showed that resistance did not depend on drug inactivation. Derivative strains of S. lactis NCDO 1404 (a reference nisin producer which contained seven plasmid species) were obtained by protoplast- or temperature-induced plasmid curing. Curing also occurred spontaneously, during growth in broth. Comparison of derivatives and the parental strain provided evidence for plasmid linkage of proteinase and lactose fermentation in NCDO 1404. The four possible combinations of proteinase (+/-) and lactose fermentation (+/-) phenotypes could be differentiated on buffered, milk-based agar media containing pH indicators

The myeloid immune signature of Enterotoxigenic Bacteroides fragilis-induced murine colon tumorigenesis

Enterotoxigenic Bacteroides fragilis (ETBF), a human commensal and candidate pathogen in colorectal cancer (CRC), is a potent initiator of IL-17-dependent colon tumorigenesis in Min mice. In Chapters 2 and 3, we examined the role of IL-17 and ETBF on the differentiation of myeloid cells into myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM), which are known to promote tumorigenesis. The myeloid compartment associated with ETBF-induced colon tumorigenesis in Min mice was defined using flow cytometry and gene expression profiling. Cell sorted immature myeloid cells were functionally assayed for inhibition of T cell proliferation in order to delineate MDSC populations. A comparison of ETBF infection to that with other oncogenic bacteria (Fusobacterium nucleatum or pks+ E. coli) revealed a specific, ETBF-associated colonic immune infiltrate. ETBF-triggered colon tumorigenesis is associated with an IL-17-driven myeloid signature characterized by subversion of steady-state myelopoiesis in favor of the generation of protumoral monocytic (MO)-MDSC. Combined action of the Bacteroides fragilis enterotoxin bft and IL-17 on colonic epithelial cells promoted the differentiation of MO-MDSC, which selectively upregulated Arg1 and Nos2, produced NO and suppressed T cell proliferation. Evidence of a pathogenic inflammatory signature in humans colonized with ETBF may allow for the identification of populations at risk for developing colon cancer. In Chapter 4, we describe targeting MDSC with the aim of suppressing tumorigenesis. CXCR2, an inflammatory chemokine receptor expressed on neutrophils and granulocytic (PMN)-MDSC, was a strong modifier of ETBF-Min tumorigenesis. Inhibition of CXCR2, via genetic knockout or a synthetic peptide antagonist, pepducin, significantly decreased recruitment of PMN-MDSC to the colon, increased numbers of M1 macrophages and was anti-tumoral. Specific inhibitors of MDSC targeting CXC receptors are under development and may emerge as a novel arm in immunotherapy

The microbial metabolite desaminotyrosine enhances T-cell priming and cancer immunotherapy with immune checkpoint inhibitors

Background Inter-individual differences in response to immune checkpoint inhibitors (ICI) remain a major challenge in cancer treatment. The composition of the gut microbiome has been associated with differential ICI outcome, but the underlying molecular mechanisms remain unclear, and therapeutic modulation challenging. Methods We established an in vivo model to treat C57Bl/6j mice with the type-I interferon (IFN-I)-modulating, bacterial-derived metabolite desaminotyrosine (DAT) to improve ICI therapy. Broad spectrum antibiotics were used to mimic gut microbial dysbiosis and associated ICI resistance. We utilized genetic mouse models to address the role of host IFN-I in DAT-modulated antitumour immunity. Changes in gut microbiota were assessed using 16S-rRNA sequencing analyses. Findings We found that oral supplementation of mice with the microbial metabolite DAT delays tumour growth and promotes ICI immunotherapy with anti-CTLA-4 or anti-PD-1. DAT-enhanced antitumour immunity was associated with more activated T cells and natural killer cells in the tumour microenvironment and was dependent on host IFN-I signalling. Consistent with this, DAT potently enhanced expansion of antigen-specific T cells following vaccination with an IFN-I-inducing adjuvant. DAT supplementation in mice compensated for the negative effects of broad-spectrum antibiotic-induced dysbiosis on anti-CTLA-4-mediated antitumour immunity. Oral administration of DAT altered the gut microbial composition in mice with increased abundance of bacterial taxa that are associated with beneficial response to ICI immunotherapy. Interpretation We introduce the therapeutic use of an IFN-I-modulating bacterial-derived metabolite to overcome resistance to ICI. This approach is a promising strategy particularly for patients with a history of broad-spectrum antibiotic use and associated loss of gut microbial diversity

Participação no processo civil: repensando litisconsórcio, intervenção de terceiros e outras formas de atuação

- Divulgação dos SUMÁRIOS das obras recentemente incorporadas ao acervo da Biblioteca Ministro Oscar Saraiva do STJ. Em respeito à Lei de Direitos Autorais, não disponibilizamos a obra na íntegra.- Localização na estante: 347.921(81) T279

The myeloid immune signature of Enterotoxigenic Bacteroides fragilis-induced murine colon tumorigenesis

Enterotoxigenic Bacteroides fragilis (ETBF), a human commensal and candidate pathogen in colorectal cancer (CRC), is a potent initiator of IL-17-dependent colon tumorigenesis in Min mice. In Chapters 2 and 3, we examined the role of IL-17 and ETBF on the differentiation of myeloid cells into myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM), which are known to promote tumorigenesis. The myeloid compartment associated with ETBF-induced colon tumorigenesis in Min mice was defined using flow cytometry and gene expression profiling. Cell sorted immature myeloid cells were functionally assayed for inhibition of T cell proliferation in order to delineate MDSC populations. A comparison of ETBF infection to that with other oncogenic bacteria (Fusobacterium nucleatum or pks+ E. coli) revealed a specific, ETBF-associated colonic immune infiltrate. ETBF-triggered colon tumorigenesis is associated with an IL-17-driven myeloid signature characterized by subversion of steady-state myelopoiesis in favor of the generation of protumoral monocytic (MO)-MDSC. Combined action of the Bacteroides fragilis enterotoxin bft and IL-17 on colonic epithelial cells promoted the differentiation of MO-MDSC, which selectively upregulated Arg1 and Nos2, produced NO and suppressed T cell proliferation. Evidence of a pathogenic inflammatory signature in humans colonized with ETBF may allow for the identification of populations at risk for developing colon cancer. In Chapter 4, we describe targeting MDSC with the aim of suppressing tumorigenesis. CXCR2, an inflammatory chemokine receptor expressed on neutrophils and granulocytic (PMN)-MDSC, was a strong modifier of ETBF-Min tumorigenesis. Inhibition of CXCR2, via genetic knockout or a synthetic peptide antagonist, pepducin, significantly decreased recruitment of PMN-MDSC to the colon, increased numbers of M1 macrophages and was anti-tumoral. Specific inhibitors of MDSC targeting CXC receptors are under development and may emerge as a novel arm in immunotherapy

Group 7: Experimental Social Psychology

We are interested in learning the impact of music genre and tempo on stress