METHODS: We used the genome-wide screening tool TraDIS (Transposon Directed Insertion-site Sequencing) to identify B. pseudomallei essential genes. Transposon-flanking regions were sequenced and gene essentiality was assessed based on the frequency of transposon insertions within each gene. Transposon mutants were grown in LB and M9 minimal medium to determine conditionally essential genes required for growth under laboratory conditions. The Caenorhabditis elegans infection model was used to assess genes associated with in vivo B. pseudomallei survival. Transposon mutants were fed to the worms, recovered from worm intestines, and sequenced. Two selected mutants were constructed and evaluated for the bacteria's ability to survive and proliferate in the nematode intestinal lumen.
RESULTS: Approximately 500,000 transposon-insertion mutants of B. pseudomallei strain R15 were generated. A total of 848,811 unique transposon insertion sites were identified in the B. pseudomallei R15 genome and 492 genes carrying low insertion frequencies were predicted to be essential. A total of 96 genes specifically required to support growth under nutrient-depleted conditions were identified. Genes most likely to be involved in B. pseudomallei survival and adaptation in the C. elegans intestinal lumen, were identified. When compared to wild type B. pseudomallei, a Tn5 mutant of bpsl2988 exhibited reduced survival in the worm intestine, was attenuated in C. elegans killing and showed decreased colonization in the organs of infected mice.
DISCUSSION: The B. pseudomallei conditional essential proteins should provide further insights into the bacteria's niche adaptation, pathogenesis, and virulence.
METHODS: Data for 66 adult HPVG patients who visited the EDs of 2 research hospitals between October 1999 and April 2016 were analyzed. REMS, RAPS, and MEWS were calculated based on data in the ED, and probability of death was calculated for each patient based on these scores. The ability of REMS, RAPS, and MEWS to predict group mortality was assessed by using receiver operating characteristic (ROC) curve analysis and calibration analysis.
RESULTS: The sensitivity, specificity, and accuracy for each scoring system were 92.1%, 89.3%, and 90.9% for REMS, 86.8%, 82.1%, and 84.8% for RAPS, and 78.9%, 89.3%, and 83.3% for MEWS respectively. In the ROC curve analysis, the areas under the curve for REMS, RAPS, and MEWS were 0.929, 0.877, and 0.856 respectively.
CONCLUSION: Our study is the largest series performed in a population of adult HPVG patients in the ED. The results from this study demonstrate that REMS is superior in predicting the mortality of these patients compared to RAPS and MEWS. We therefore recommend that REMS be used for outcome prediction and risk stratification of adult HPVG in the ED.
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