Effects of temperature on the virulence of <i>M</i>. <i>abscessus</i> subsp. <i>abscessus</i> ATCC19977 in silkworms.

Silkworms were injected with saline (50 μl) or M. abscessus subsp. abscessus ATCC19977 cell suspension (1.4 x 107 cells per 50 μl) and incubated at (A) 27˚C and (B) 37˚C. The curves were drawn by the Kaplan-Meier method. Seven silkworms were used per group. (C) Silkworms were injected with M. abscessus subsp. abscessus ATCC19977 cell suspension (2.9 x 108 cells per 50 μl) and incubated at 27˚C and 37˚C. Silkworm hemolymph was harvested at 18 h post-infection. Statistically significant differences between groups were evaluated using the Student t-test. Three silkworms were used per group.</p

Effects of autoclaved cells and antibacterial treatment in silkworms infected with <i>M</i>. <i>abscessus</i> subsp. <i>abscessus</i>.

(A) Silkworms were injected with either saline (50 μl), M. abscessus subsp. abscessus ATCC19977 cell suspension (1.1 x 107 cells per 50 μl), or autoclaved M. abscessus subsp. abscessus ATCC19977 cell suspension and incubated at 37˚C. Ten silkworms were used per group. (B) Silkworms were injected with either saline (50 μl) or M. abscessus subsp. abscessus ATCC19977 cell suspension (6.3 x 107 cells per 50 μl) followed by clarithromycin (25 μg g-1 larva). The number of surviving silkworms following incubation at 37˚C was measured for 66 h. Statistically significant differences between groups were evaluated using a log-rank test based on the curves by the Kaplan-Meier method. Ten silkworms were used per group.</p

Datasets included in this study.

(XLSX)</p

Attached-cell counts of human THP-1 macrophages after infection with <i>M</i>. <i>abscessus subsp</i>. <i>abscessus</i> Mb-10 and Mb-17 isolates.

Attached-cell counts of human THP-1 macrophages at 48 h after infection with M. abscessus subsp. abscessus Mb-10 or Mb-17 cells at a multiplicity of infection of 50. The number of nuclei of macrophages attached to the well was calculated using High-Content Imaging System Operetta CLS with Harmony software. Three independent samples were used per group. Statistically significant differences between groups were evaluated using the Turkey test with one-way ANOVA.</p

Viable cell counts of <i>M</i>. <i>abscessus</i> subsp. <i>abscessus</i> Mb-10 and Mb-17 isolates in silkworms.

Silkworms were injected with saline (50 μl) or M. abscessus subsp. abscessus Mb-10 cell suspension (3.6 x 108 cells per 50 μl) or Mb-17 cell suspension (3.8 x 108 cells per 50 μl) and incubated at 37˚C. Silkworm hemolymph was harvested at 18 h post-infection. Five silkworms were used per group. Statistically significant differences between groups were evaluated using the Student t-test.</p

Histopathological features of hind footpads.

A-B) Untreated control mouse at day 33 post-infection. A) Erosion, edema, and fibrinous exudates are observed (H&E x290). B) Aggregated acid-fast bacilli are observed primarily in the stroma, with some present in monocytes (arrowhead) (Fite-Faraco Staining x1750). C) Rifalazil (5mg/kg)-treated mouse at one-week of treatment. Some bacterial cells are fragmented (arrowheads) (Fite-Faraco staining x1750). D-E) Rifalazil (5mg/kg)-treated mouse at 15-weeks of treatment. D) Epithelioid cell granuloma with mild infiltration of lymphocytes is noted. Epidermal erosion, edema, fibrin, and neutrophil aggregation are not observed (H&E x120). E) Granularly degenerated acid-fast bacilli are observed in monocytes (arrowheads) (Fite-Faraco staining x1750). F-G) Rifalazil (5mg/kg)-treated mouse 15 weeks following termination of treatment. F) Epithelioid cell granuloma with mild infiltration of lymphocytes is noted. Epidermal erosion, edema, fibrin, and neutrophil aggregation are not observed (H&E x290). G) Completely degenerated acid-fast bacilli are observed in an epithelioid cell granuloma (Fite-Faraco staining x1750).</p

Gross skin erosion of hind footpads in untreated and rifalazil-treated <i>M</i>. <i>ulcerans</i> infected mice.

Gross skin erosion of hind footpads in untreated and rifalazil-treated M. ulcerans infected mice.</p

Comparative rifamycin efficacy against intracellular <i>M</i>. <i>ulcerans</i> with THP-1 human macrophage infection model.

The sum of fluorescence intensity (Alexa Flour 488) of intracellular bacterium after 72 hours incubation with RFP10μg/mL, RFP10μg/mL+SM10μg/mL, RLZ 10μg/mL and RLZ 10μg/mL+SM10μg/mL. Data are shown as mean ±SD. *p< 0.05 by unpaired t-test.</p

Successive measurement of hind footpad thickness.

Measurements were started 33 days post-infection. † All of the mice in the untreated group reached to the endpoint at 4 weeks after the measurement was started.</p

Workflow of Harmony software image analysis for intracellular <i>M</i>. <i>ulcerans</i>.

THP-1 cell cytoplasm, nuclei, and bacterial immunostaining input image were shown in Fig 4A. Cytoplasm with nucleus were detected by HCS Cell Mask Deep Red and Hoechst 33258 channel respectively (Fig 4B). Bacterial cells were identified as Alexa 488-staining objects within cytoplasm were specifically detected, and calculated the intensity of them (Fig 4C).</p