Methicillin Resistance, Biofilm Formation and Resistance to BenzalkoniumChloride in Staphylococcus aureus Clinical Isolates

Purpose: To examine the resistance to benzalkonium chloride (BKC) and the distribution of biocide-resistance genes in S.aureus clinical isolates and to determine whether any correlation may exist with antibiotic resistance pattern and biofilm formation. Methods: MICs to BKC were determined in a collection of S.aureus (HA-MRSA, CA-MRSA and MSSA) both in suspension and on biofilm-embedded cells. Characteristic of the isolates (qac genes and biofilm formation) were determined by PCR and a plate assay, respectively. Results: MICs to BKC were higher among MRSA than MSSA, where the CA-MRSA showed MIC levels closer to the MSSA group. qacA/B genes were found only among HA-MRSA and conferred higher resistance to the disinfectant while smr gene did not. MBC, but not MIC, were higher for biofilm embedded vs. planktonic cells, but no correlation was found with the ability to form biofilm. Conclusion: We confirmed that presence of qacA/B but not smr confers higher resistance to BKC; MICs among MRSA were more spread compared to MSSA, suggesting that factors associated to the MR phenotype may confer resistance to BKC. Interestingly, MSSA showed higher biocide tolerance in both the planktonic and biofilm form according to the MIC/MBC fold change values. Although no correlation could be observed between biofilm thickness and biocide resistance, biofilm-embedded cells responded differently to disinfectants suggesting the current practices for efficacy testing of biocides may not be relevant in the evaluation of disinfectant efficacy against biofilm-embedded microorganisms

Mobilization of healthy donors with plerixafor affects the cellular composition of T-cell receptor (TCR)-αβ/CD19-depleted haploidentical stem cell grafts

Background: HLA-haploidentical hematopoietic stem cell transplantation (HSCT) is suitable for patients lacking related or unrelated HLA-matched donors. Herein, we investigated whether plerixafor (MZ), as an adjunct to G-CSF, facilitated the collection of mega-doses of hematopoietic stem cells (HSC) for TCR-αβ/CD19-depleted haploidentical HSCT, and how this agent affects the cellular graft composition. Methods: Ninety healthy donors were evaluated. Single-dose MZ was given to 30 ‘poor mobilizers’ (PM) failing to attain ≥40 CD34+ HSCs/μL after 4 daily G-CSF doses and/or with predicted apheresis yields ≤12.0x106 CD34+ cells/kg recipient’s body weight. Results: MZ significantly increased CD34+ counts in PM. Naïve/memory T and B cells, as well as natural killer (NK) cells, myeloid/plasmacytoid dendritic cells (DCs), were unchanged compared with baseline. MZ did not further promote the G-CSF-induced mobilization of CD16+ monocytes and the down-regulation of IFN-γ production by T cells. HSC grafts harvested after G-CSF + MZ were enriched in myeloid and plasmacytoid DCs, but contained low numbers of pro-inflammatory 6-sulfo-LacNAc+ (Slan)-DCs. Finally, children transplanted with G-CSF + MZ-mobilized grafts received greater numbers of monocytes, myeloid and plasmacytoid DCs, but lower numbers of NK cells, NK-like T cells and Slan-DCs. Conclusions: MZ facilitates the collection of mega-doses of CD34+ HSCs for haploidentical HSCT, while affecting graft composition

Escherichia coli Cytotoxic Necrotizing Factor 1 Blocks Cell Cycle G(2)/M Transition in Uroepithelial Cells

Evidence is accumulating that a growing number of bacterial toxins act by modulating the eukaryotic cell cycle machinery. In this context, we provide evidence that a protein toxin named cytotoxic necrotizing factor 1 (CNF1) from uropathogenic Escherichia coli is able to block cell cycle G(2)/M transition in the uroepithelial cell line T24. CNF1 permanently activates the small GTP-binding proteins of the Rho family that, beside controlling the actin cytoskeleton organization, also play a pivotal role in a large number of other cellular processes, including cell cycle regulation. The results reported here show that CNF1 is able to induce the accumulation of cells in the G(2)/M phase by sequestering cyclin B1 in the cytoplasm and down-regulating its expression. The possible role played by the Rho GTPases in the toxin-induced cell cycle deregulation has been investigated and discussed. The activity of CNF1 on cell cycle progression can offer a novel view of E. coli pathogenicity

Cytotoxic Necrotizing Factor 1 Enhances Reactive Oxygen Species-Dependent Transcription and Secretion of Proinflammatory Cytokines in Human Uroepithelial Cells

Uropathogenic Escherichia coli strains frequently produce a Rho-activating protein toxin named cytotoxic necrotizing factor type 1 (CNF1). We herein report that CNF1 promotes transcription and release of tumor necrosis factor alpha, gamma interferon, interleukin-6 (IL-6), and IL-8 proinflammatory cytokines and increases the production of reactive oxygen species (ROS) in uroepithelial T24 cells. The antioxidant N-acetyl-l-cysteine counteracts these phenomena, a fact which suggests a role for ROS-mediated signaling in CNF1-induced proinflammatory cytokine production