Neutrophil elastase inhibition during <i>ΔaroA Salmonella</i> infection eliminates the effect of infection on gut microbiota composition.

<p>Six- to eight-week-old female mice were treated with 450 mg/L of streptomycin for 2 days in their drinking water. After antibiotic withdrawal, mice were infected with 2.7×10⁸ CFUs of the indicated strain. Neutrophil elastase was inhibited by injection of Sivelestat sodium hydrate (Sivel.) intraperitoneally twice a day on days -1, 0, 1, 2, 3, and 4 post infection. A) Ceca were harvested at five days post infection and single cell suspensions were obtained. Staining and flow cytometry analyses were used to confirm that neutrophils were not decreased in mice treated with the neutrophil elastase inhibitor. T cells (CD45+CD3+NK1.1-), neutrophils (CD45+GR1+MPO+) and macrophages (CD45+CD11c+CD11b+) were analyzed. B) <i>Salmonella</i> colonization levels were determined five days post infection by plating serial dilutions of cecum homogenates on LB plates supplemented with 100 µg/ml of streptomycin. Outliers were detected using the Grubbs’ test and removed from the dataset. C) and D) Bacterial 16s rRNA genes were amplified from fecal samples at day 5 post infection for TRFLP analysis. C) graph showing percent similarity of microbiota between treatment groups D) dendrogram showing the separation of treatment groups based on changes in microbial composition. <i>ΔaroA-</i>infected mice receiving neutrophil elastase inhibitor showed a significantly different gut microbial composition compared to untreated mice but similar to <i>ΔaroAΔinvA</i>-infected mice. Four mice per group were used, and experiments were repeated at least three times. Outliers were removed from data sets using Grubbs’ test. * indicates <i>p</i><0.05, **p<0.01.