The stability of Staphylococcal bacteriophage in presence of local vancomycin concentrations used in clinical practice.

PURPOSE: To evaluate the stability of a clinically used Staphylococcal bacteriophage with doses of vancomycin that are encountered with local administration of vancomycin for musculoskeletal infections. METHODS: A Staphylococcal bacteriophage was evaluated for stability in different pH ranges. Then that same bacteriophage was evaluated for stability with different concentrations of vancomycin and with vancomycin biodegradable antibiotic beads. RESULTS: The bacteriophage had stability within a pH range of 4-10. There was a statistically significant (P < 0.05) decrease in the amount of bacteriophage over 24 h for vancomycin concentrations of 10 mg/mL and 100 mg/mL compared to lower vancomycin concentrations (1 mg/mL, 0.1 mg/mL and normal saline). However, no statistically significant decrease in the amount of bacteriophage was seen with biodegradable vancomycin beads over 24 h. CONCLUSION: These findings have important clinical ramifications in that they show local administration of bacteriophages with concomitant local vancomycin powder therapy should be avoided. Moreover, these findings should spearhead further research into bacteriophage stability in in vivo environments.

Functional Identification of Corynespora cassiicola-Responsive miRNAs and Their Targets in Cucumber.

Target leaf spot (TLS), which is caused by Corynespora cassiicola (C. cassiicola), is one of the most important diseases in cucumber (Cucumis sativus L.). Our previous research identified several C. cassiicola-responsive miRNAs in cucumber by high-throughput sequencing, including two known miRNAs and two novel miRNAs. The target genes of these miRNAs were related to secondary metabolism. In this study, we verified the interaction between these miRNAs and target genes by histochemical staining and fluorescence quantitative assays of GUS. We transiently expressed the candidate miRNAs and target genes in cucumber cotyledons to investigate the resistance to C. cassiicola. Transient expression of miR164d, miR396b, Novel-miR1, and Novel-miR7 in cucumber resulted in decreased resistance to C. cassiicola, while transient expression of NAC (inhibited by miR164d), APE (inhibited by miR396b), 4CL (inhibited by Novel-miR1), and PAL (inhibited by Novel-miR7) led to enhanced resistance to C. cassiicola. In addition, overexpression of 4CL and PAL downregulated lignin synthesis, and overexpression of Novel-miR1 and Novel-miR7 also downregulated lignin synthesis, indicating that the regulation of 4CL and PAL by Novel-miR1 and Novel-miR7 could affect lignin content. The tobacco rattle virus (TRV) induced short tandem target mimic (STTM)-miRNA silencing vector was successfully constructed, and target miRNAs were successfully silenced. The identification of disease resistance and lignin content showed that silencing candidate miRNAs could improve cucumber resistance to C. cassiicola.

Cucumber Mildew Resistance Locus O Interacts with Calmodulin and Regulates Plant Cell Death Associated with Plant Immunity.

Pathogen-induced cell death is closely related to plant disease susceptibility and resistance. The cucumber (Cucumis sativus L.) mildew resistance locus O (CsMLO1) and calmodulin (CsCaM3) genes, as molecular components, are linked to nonhost resistance and hypersensitive cell death. In this study, we demonstrate that CsMLO1 interacts with CsCaM3 via yeast two-hybrid, firefly luciferase (LUC) complementation and bimolecular fluorescence complementation (BiFC) experiments. A subcellular localization analysis of green fluorescent protein (GFP) fusion reveals that CsCaM3 is transferred from the cytoplasm to the plasma membrane in Nicotiana benthamiana, and CsCaM3 green fluorescence is significantly attenuated via the coexpression of CsMLO1 and CsCaM3. CsMLO1 negatively regulates CsCaM3 expression in transiently transformed cucumbers, and hypersensitive cell death is disrupted by CsCaM3 and/or CsMLO1 expression under Corynespora cassiicola infection. Additionally, CsMLO1 silencing significantly enhances the expression of reactive oxygen species (ROS)-related genes (CsPO1, CsRbohD, and CsRbohF), defense marker genes (CsPR1 and CsPR3) and callose deposition-related gene (CsGSL) in infected cucumbers. These results suggest that the interaction of CsMLO1 with CsCaM3 may act as a cell death regulator associated with plant immunity and disease.

Loop-mediated isothermal amplification assays for screening of bacterial integrons

BACKGROUND: The occurrence and prevalence of integrons in clinical microorganisms and their role played in antimicrobial resistance have been well studied recently. As screening and detection of integrons are concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for integrons detection. RESULTS: In this study, three loop-mediated isothermal amplification (LAMP) assays targeting on class 1, 2 and 3 integrons were implemented and evaluated. Optimization of these detection assays were performed, including studing on the reaction temperature, volume, time, sensitivity and specificity (both primers and targets). Application of the established LAMP assays were further verified on a total of 1082 isolates (previously identified to be 397 integron-positive and 685 integron-negative strains). According to the results, the indispensability of each primer had been confirmed and the optimal reaction temperature, volume and time were found to be 65°C, 45 min and 25 µL, respectively. As application was concerned, 361, 28 and 8 isolates carrying intI1, intI2 and intI3 yielded positive amplicons, respectively. Other 685 integron-negative bacteria were negative for the integron-screening LAMP assays, totaling the detection rate and specificity to be 100%. CONCLUSIONS: The intI1-, intI2- and intI3-LAMP assays established in this study were demonstrated to be the valid and rapid detection methodologies for the screening of bacterial integrons.