Fig. 4
From: Novel probiotic approach to counter Paenibacillus larvae infection in honey bees
In vitro growth and cell viability of Paenibacillus larvae is reduced by Lp39. a Growth curves of P. larvae in MY media supplemented with cell-free supernatant from lactobacilli strains of interest. b Percent maximal growth was determined from growth curve data (OD600) at 48 h using the area under the curve for P. larvae grown in MY media supplemented with CFS from the specified lactobacilli. Data are depicted as means ± standard deviation (one-way ANOVA with Dunnett’s multiple comparisons) of n = 3 biological replicates performed with duplicate technical repeats. c Zone of inhibition measurements represent the mean ± standard deviation radius clearance (minus the disk) on a P. larvae lawn grown on MY agar. Experiments were performed in biological triplicate (n = 3 for each group) with technical duplicates. Statistical analysis is shown for one-way ANOVA with Dunnett’s multiple comparisons made against 30 µg of oxytetracycline. Enterobacter hormaechei B0003, Paenibacillus illinoisensis B0004, Hafnia paralvei B0008, and Lactobacillus apis B0011 represent isolates previously obtained from a healthy hive. d Lp39 (short rod-shaped) and P. larvae (long rod-shaped) were incubated in nutrient-limited media for 60 min and subsequently stained with cell-permeable (4′,6-diamidino-2-phenylindole; DAPI) and non-permeable (SYTOX Green) nucleic acid markers, as well as Texas Red-WGA that selectively binds to the surface of gram-positive bacteria. Cells were visualized using a Nikon Eclipse Ti2 confocal microscope. Increased uptake of SYTOX Green indicates reduced cell viability based on plasma membrane integrity. Yellow arrow points to P. larvae, white arrow points to Lp39. Bacterial cells that were incubated with 70% ethanol (EtOH) served as a positive control to validate the assay. Scale bar = 20 µM. ns = not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001
