https://orcid.org/0000-0003-0119-8920
Figures & data
Figure 1. Kaplan-Meier survival curves of G. mellonella challenged with a range of SAMTB lux inocula over a 96 h time course.
G. mellonella larvae (n = 30/group) were infected with 1 × 107–108 CFU of SAMTB lux. Infected larvae were stored in a dark box and incubated at 37°C. Survival was recorded every 24 h for a period of 96 h. Larvae were considered dead when they failed to move in response to touch. All SAMTB lux doses were statistically different from each other (P < 0.0001, Mantle-Cox test with Holm-Sidak correction) at each timepoint. Data were pooled from three independent experiments.
Figure 2. In vivo proliferation of SAMTB lux in G. mellonella larvae over a 168 h time course.
G. mellonella larvae (n ≤ 30) were infected with 2×107 CFU of SAMTB lux and at each timepoint (0, 24, 48, 72, 96 and 168 h), four larvae were individually homogenized, and the bioluminescence (RLU/ml) of the homogenate was measured to quantify the relative abundance of SAMTB lux in vivo. Data are pooled from three independent experiments. The means of each group are plotted, and the error bars represent the standard deviation of the mean.
Figure 3. Recovery of SAMTB lux from larval homogenate over a 168 h time course.
Larvae were infected with 2×107 CFU of SAMTB lux. At 0, 24, 96 and 168 h post-infection, four larvae were homogenized and plated onto Middlebrook 7H11 agar containing additional growth supplements (leucine and pantothenate) as well as piperacillin (20 µg/ml) to prevent contamination from larval microbiota. Data are pooled from two independent experiments.
Figure 4. Histological analysis of G. mellonella-SAMTB lux interaction over time.
Histological sections of G. mellonella infected with SAMTB lux stained with Ziehl-Neelsen (ZN) or hematoxylin and eosin (H&E). (A) At 24 h post infection, large numbers of individual and densely organized bacilli were stained with ZN. (B) The H&E section of the same granuloma-like structure shows host cells occupying spaces which, in the ZN section, are filled with bacilli. Loss of eosinophilia from the H&E section gives rise to a brown pigmentation which co-localizes with clumping of bacilli in the ZN section. (C) As early as 24 h post-infection, granuloma-like structures may contain independent colonies of bacilli and compact ZN reactive amorphous material. (D) The H&E section of the same structure shown in C; loss of eosinophilia coincides with the existence of a large colony of bacilli. (E-F) At 168 h post-infection, granuloma-like structures containing compact ZN positive substances and individual bacilli or colonies of bacilli, were observed. At 48 h post-infection, (G) ZN and (H) H&E staining, show a single compact granuloma with a ZN reactive core. Such granuloma-like structures can be observed as single entities or can be included in formations that include multiple compact ZN positive structures similar to panels C-F. (J-K) Granuloma-like structure at 168 h post-infection, stained with ZN and H&E respectively, shows a single compact granuloma structure. However, the ZN affiliation of the amorphous central core has been abolished to a large extend implying breakdown of the structures that bind to the ZN dye. (L) Swarms of ZN positive bacilli form a granuloma-like structure. Part of the granuloma wall is impaired allowing access of bacteria to the surrounding hemolymph. (M) shows bacilli invading and forming colonies in internal organs of a larva. There is no evident host reaction to invading bacilli. The inset shows a larger magnification of the rectangle enclosed structure which shows abundant proliferation of bacilli. Scale bar in A, B, J, K and L represents 50 µm; in C, D, G, H and the M inset, scale bar represents 20 µm; while in E, F and M the scale bar represents 100 µm.
Figure 5. Cross sectional view of whole larvae infected with SAMTB lux.
Histological sections of whole G. mellonella larvae infected with SAMTB lux 24 h (A) and 168 h (B) post challenge showing multiple granuloma-like structures. Blue arrows indicate formations where there is an abundance of active bacilli which may be also accompanied by more consolidated structures similar to those presented in . Red arrows show formations that are similar to those presented in ). Scale bar represents 1000 µm.
Figure 6. SAMTB lux associates with hemocytes following infection.
Confocal microscopy was carried out on hemocytes extracted from G. mellonella larvae infected with SAMTB lux (2x107 CFU) at (a) 1 h and (b) 168 h post-infection. Early association between hemocyte and SAMTB lux was seen at 1 h post-infection (a). Aggregates of hemocytes in an early granuloma like structure surrounding multiple clusters of SAMTB lux was observed at 168 h post-infection (b). These images are a superimposition of DAPI (nucleus, blue), SYBR gold (SAMTB lux, green) and differential interference contrast (DIC) image. Scale bars represents 10 µm.
Figure 7. Interaction between SAMTB lux and phagocytic hemocytes.
Transmission electron microscopy (TEM) was carried out on hemocytes extracted from G. mellonella larvae infected with SAMTB lux (2x107 CFU) at: (a, b) 1 h (c) 24 h and (d) 168 h post infection. (e) shows healthy non-infected hemocytes. (f, g) show SAMTB lux bacilli in various geometric orientations. Internalization of SAMTB lux was observed as early as 1 h post-infection and presence of the bacilli persisted 24 h post-infection (A-C). By 168 h post-infection, aggregates of SAMTB lux bacilli, surrounded by hemocytes in an early granuloma-like structure, could be observed under TEM (D). H = hemocytes, N = nucleus, inverted triangles indicate SAMTB lux. Scale bars represent (a, c-e): 2 µm, (b, f, g): 500 nm.
Figure 8. Changes in hemocyte counts in SAMTB lux infected larvae.
Total number of circulating hemocytes of naïve, PBS-T injected and SAMTB lux infected (2x107 CFU) larvae were determined at 2, 4, 24 and 48 h post-injection. Five larvae were bled (approximately 60 µl of hemolymph/larva) and diluted in PBS containing 0.37% of mercaptoethanol. Hemocytes were stained 1:1 with trypan blue (0.4%) and counted in a hemocytometer. Data are pooled from three independent experiments, each with two technical repeats. Plotted are the means of each group and the error bars represent the standard deviation of the mean. PBS and SAMTB lux infected larvae were compared to the naïve control using one-way ANOVA with Dunnett’s multiple comparison test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
Figure 9. The effect of first-line and second-line antibiotics on the reduction of SAMTB lux bioluminescence within G. mellonella over a 96 h time course.
Larvae (n = 30/group) were infected with 2×107 CFU of SAMTB lux. At 1 h post-infection, larvae were treated with a single dose of INH (5 mg/kg), RIF (10 mg/kg), and BDQ (5.7 mg/kg) at the recommended clinical dosage relative to the larval body weigh; while PZA (250 mg/kg), ETH (150 mg/kg), MOX (67 mg/kg) were used at concentrations ten-folds higher than the recommended dosage. Control groups were injected larvae mock treated with PBS-T. At 0, 24, 48, 72, and 96 h post-infection, four larvae from each group were individually homogenized and the bioluminescence (RLU/ml) of the homogenates was measured to determine the relative drug efficacies. Data are pooled from three independent experiments. Plotted are the mean of each group and the error bars represent the standard deviation of the mean. Non-parametric Kruskal-Wallis test with Dunn’s multiple comparison was carried out against the PBS-T injected control for all treatment groups. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
Figure 10. The effect of INH and RIF concentration on the reduction of SAMTB lux bioluminescence within G. mellonella.
Larvae (n = 30/group) were infected with 2 × 107 CFU of SAMTB lux. At 1 h post-infection, larvae were treated with a single dose of INH (5 mg/kg) or RIF (10 mg/kg). Control groups were injected larvae mock treated with PBS-T. At (A) 24 h and (B) 96 h post-infection, four larvae from each group were individually homogenized and the bioluminescence (RLU/ml) of the homogenate was measured to determine relative drug efficacy. Data are pooled from two independent experiments. Plotted are the mean for each group and the error bars represent the standard deviation of the mean. Non-parametric Kruskal-Wallis test with Dunn’s multiple comparison was carried out against the PBS-T control for all treatment groups. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
Figure 11. The effect of RIF multiple dosing and RIF combination drug therapy on the reduction of SAMTB lux bioluminescence within G. mellonella.
Larvae (n = 30/group) were infected with 2×107 CFU of SAMTB lux. (a) At 1 h post-infection, larvae were treated with a single dose of RIF (10 mg/kg) and for dual dosing an additional dose was given 24 h post-infection. (b) At 1 h post-infection, larvae were treated with a single dose of RIF (10 mg/kg), ETH (15 mg/kg), MOX (6.7 mg/kg) or in combination as RIF/ETH or RIF/ETH/MOX. At 96 h post-infection, four larvae from each group were individually homogenized and the bioluminescence (RLU/ml) of the homogenate was measured to determine relative drug efficacy. The efficacy of dual RIF dosing was compared to single dosing using the Mann-Whitney test. As for combination drug therapy, the efficacy of each drug combination was compared to their respective single drug type efficacy using a non-parametric Kruskal-Wallis test with Dunn’s multiple comparison. For combination drug therapy, the color of * correlates to the color of the single drug type. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
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