| Enterohemorrhagic Escherichia coli serotype O157:H7 (EHEC O157:H7) is a major food-borne pathogen causing hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS). Although most clinical isolates of E. coli O157:H7 have an F-like 92-kb plasmid, pO157, the role of pO157 in the pathogenesis of EHEC O157:H7 infections remains unknown. Here, we compared wild-type (WT) EHEC O157:H7 EDL933 and an isogenic plasmid elimination mutant (ΔpO157) for adhesive properties, cytotoxicity to pheripheral blood mononuclear cell (PBMC) and human macrophages, cytokines release from PBMC and human macrophages. In vitro adherence assay, ApO157 exhibited a dramatic adherence defect compared with the WT.ΔpO157 was less cytotoxic to PBMC and human macrophages than the WT measured by releasing of the cytoplasmic enzyme lactate dehydrogenase (LDH) and cell detachment assay. The level of cytokines interleukin-6 (IL-6), tumor necrosis factor-a (TNF-a) and monocyte chemotactic protein-1 (MCP-1) released from PBMC with the WT infection were significantly higher than with ApO157 infection. Cytokines IL-6, IL-1β, IL-8, IL-10, TNF-a, MCP-1, chemokine CC motif ligand 5 (CCL5/RANETS) production were examined from human macrophages with the WT and ApO157 infection. Almost no production of IL-6, IL-10 was observed with the WT and ApO157 infection. The WT induced more production of IL-1βbut not TNF-a, IL-8, MCP-1, RANETS, from human macrophages. The results suggested that plasmid pO157 played a very important role in adherence, cytotoxicity to PBMC and human macrophages and cytokines production from PBMC and human macrophages.To further investigate which gene on plasmid contributed to the significant difference in macrophages cytotoxicity and IL-1βproduction between the WT andΔpO157, we constructed gene hlyA and espP deletion of the WT:ΔhlyA andΔespP. No significant difference of cytotxocity and IL-1βproduction between the WT and AespP were observed. We found that cytotoxicity caused byΔhlyA was significantly reduced compared with caused by the WT, IL-1βsecretion from macrophage stimulated byΔhlyA mutant was significantly lower than stimulated by the WT, but higher than stimulated byΔpO157. The data suggest that EHEC O157:H7 hemolysin caused human macrophages cytotoxicity and induced IL-1βproduction from human macrophage. Our finding may provide a clue to reveal the possible role played by hemolysin in pathogenicity of EHEC O157:H7 infection, which has not been well understood. The Aim of this study was to establish an appropriate pulsed-field gel electrophoresis (PFGE) protocol for subtyping of Citrobacter isolates. We standardized the plug preparation procedure focused on the cell suspension concentration (turbidity of 2.5 to 3.5), SDS addition (no SDS needed) and lysis time (1 h), selected the appropriate digestion enzyme (Xbal and Blnl) and optimized the electrophoretic parameters (1s-20s for 19h). We evaluated the discriminatory power, typeability and reproducibility of this PFGE method for Citrobacter subtyping. BlnI PFGE had a higher discriminatory power than XbaI PFGE in this study, so we recommend the use of Blnl as the primary enzyme with Xbal used when further differentiation is needed. The results also showed this method gave good typeability and is highly reproducible. A rapid PFGE protocol was established here, which could be used for genotyping and other research of Citrobacter. This study aimed to optimize the PFGE procedure of Citrobacter and an appropriate protocol was developed. This protocol is anticipated to be used widely for Citrobacter genotyping for epidemiological surveys and other researchs.
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