Genes mcr improve the intestinal fitness of pathogenic E. coli and balance their lifestyle to commensalism.

BACKGROUND: The plasmid-mediated resistance gene mcr-1 confers colistin resistance in Escherichia coli and paves the way for the evolution to pan-drug resistance. We investigated the impact of mcr-1 in gut colonization in the absence of antibiotics using isogenic E. coli strains transformed with a plasmid encoding or devoid of mcr-1. RESULTS: In gnotobiotic and conventional mice, mcr-1 significantly enhanced intestinal anchoring of E. coli but impaired their lethal effect. This improvement of intestinal fitness was associated with a downregulation of intestinal inflammatory markers and the preservation of intestinal microbiota composition. The mcr-1 gene mediated a cross-resistance to antimicrobial peptides secreted by the microbiota and intestinal epithelial cells (IECs), enhanced E. coli adhesion to IECs, and decreased the proinflammatory activity of both E. coli and its lipopolysaccharides. CONCLUSION: Overall, mcr-1 changed multiple facets of bacterial behaviour and appeared as a factor enhancing commensal lifestyle and persistence in the gut even in the absence of antibiotics. Video Abstract.

Mesenchymal content of fresh bone marrow: a proposed quality control method for cell therapy.

The scarcity of mesenchymal stem cells (MSC) in bone marrow (BM) has justified their ex vivo expansion before therapeutic use, but a method to evaluate the quality of initial mesenchymal content and track the modifications induced by graft processing has not yet been proposed. The aim of this study was to establish such a procedure. Flow cytometric and functional assay methods were modified to count CD45(-) CD14(-)/CD73(+) subsets containing all MSC and used them to study BM from spongy bone (SB) and iliac crest aspirate (ICA). These methods detected the target subsets in all BM suspensions derived from SB (n = 154) and ICA, (n = 44) with a satisfactory correlation between immuno-phenotyping and functional tests by low-density plating. We noted a higher overall MSC frequency in SB cell suspensions but a lower plating efficiency of CD45(-) CD14(-)/CD73(+) SB cells under standard culture conditions. We propose a cell quality control on un-manipulated BM cell suspensions to quantify the mesenchymal compartment with regard to varying donor factors, such as age and sampling site, that influence expansion and define a therapeutic threshold value. Furthermore, we were able to confirm differences in plating efficiency and proliferative capacity between two BM origins.

Characterization of nonexpanded mesenchymal progenitor cells from normal adult human bone marrow.

OBJECTIVE: Adult bone marrow (BM) mesenchymal stem/progenitor cells (MS/PC) are a potentially useful tool for cell therapy and tissue repair. However, the identification of cell subsets rich in MS/PC from fresh BM has not been described. We have developed a means of identifying such subsets from untouched bone marrow. MATERIAL AND METHODS: First, MS/PC were enriched by short-time adherence (D(1-3)) before any cell division to evaluate the efficiency of CD73, CD105, CDw90, and CD49a antigens to select highly purified CD45(-)CD14(-) fluorescence-activated sorted subsets enriched in clonogenic mesenchymal cells. Then, we adapted this method to unmanipulated BM mononuclear cells (MNC). RESULTS: Short-time (D(1-3)) adherent CD45(-)CD14(-) cells expressing CD73 or CD49a antigens contained all the CFU-F, even though the CD105(+) and CDw90(+) subsets comprised less than half the total. In fresh unmanipulated BM MNC, CD73 and CD49a were also highly discriminative and allowed up to a 3 log enrichment of CFU-F when compared to BM MNC. Normal culture conditions upregulated most of the tested antigens. CONCLUSION: The CD45(-)CD14(-)/CD73(+) and CD45(-)CD14(-)/CD49a(+) phenotypes identified subsets containing all the CFU-F and sufficiently enriched to detect them in fresh BM, enabling evaluation of mesenchymal content of BM collections for cell therapy.

CD34+CDw90(Thy-1)+ subset colocated with mesenchymal progenitors in human normal bone marrow hematon units is enriched in colony-forming unit megakaryocytes and long-term culture-initiating cells.

OBJECTIVE: The progress made in the supportive care of allografts and the identification of mesenchymal stem cells in adult human bone marrow (BM) has prompted renewed interest in the use of BM as a form of cell therapy. With the aim of optimizing the collection of BM cells, we evaluated the hematopoietic and mesenchymal immature cell contents of BM hematon units (HUs), which usually are eliminated during graft processing. MATERIALS AND METHODS: Hematopoietic CD34+ progenitors from HU and buffy coat (BC) compartments were characterized in short-term culture. The sorted CD34+CDw90(Thy-1)+ primitive subset was assessed in colony-forming cell (CFC) and long-term culture-initiating cell (LTC-IC) assays, then further characterized by the expression of additional antigens. In parallel, we evaluated the colony-forming unit fibroblast (CFU-F) number and phenotyped the fresh adherent (D1-3) cells. RESULTS: The plating efficiencies of CD34+ cells derived from HU and BC were identical. However, the HU CD34+CDw90(Thy-1)+ subset was enriched in colony-forming unit megakaryocyte (2.3x), LTC-IC (4.6x), and cells coexpressing CD105 (5x). We found a higher frequency of CFU-F (4.7x), considered to be the mesenchymal stem cell-containing population, correlated with an enrichment in fresh adherent (CD45/GPA)-CD14- cells. CONCLUSIONS: We show for the first time that functional properties of the CD34+CDw90+ subset are related to its in vivo location in HU, which may represent the BM mesenchymal reserve compartment. The location in HU of 35.6%, 59.1%, and 58.7% of CD34+ cells, CD34+CDw90+ LTC-IC, and CFU-F, respectively, justifies the development of a procedure to collect them in order to reduce the therapeutic BM volume.