Figure 1.
PAO1 (pCHAP6656) infection of the Drosophila crop.
(A) Plating of bacteria recovered from infected flies on Pseudomonas isolation agar (PIA) +/− Gm 30 (µg/ml) indicated that pCHAP6656 was not lost up to 48 h postinfection. (B) Merged image of brightfield and red fluorescence images from PAO1-infected crops (40x). Red fluorescence images of infected crops at (C) 63x and (D) 100x objectives. White arrows indicate the presence of large bacterial aggregates. Scale bars indicate 200 µM. At least three infected crops were examined from three separate infections and representative images are shown.
Figure 2.
In vivo P. aeruginosa biofilms stain positively for EPS and DNA.
(A) Red autofluorescence, (B) green autofluorescence, (C) DAPI-stained nuclei (all indicated by grey arrow) and (D) merge image of uninfected Drosophila crop. (E) Aggregative red fluorescent PAO1pCHAP6656 (white arrow) along with red autofluorescence (grey arrow), (F) green fluorescent EPS (white arrow) staining and autofluorescence (grey arrow), (G) DAPI staining of bacteria (white arrow) and Drosophila nuclei (grey arrow) and (H) merge image of PAO1-infected crops. DNAse and cellulase treatment of P. aeruginosa-infected crops. (I) Non-aggregative red fluorescent PAO1pCHAP6656 (white arrow) along with red autofluorescence (grey arrow), (J) autofluorescence (grey arrow) and lack of EPS staining with FITC-labeled HHA lectin. (K) DAPI staining of Drosophila nuclei (grey arrow) and absence of bacterial DNA staining and (L) merge image of DNAse and cellulase treated PAO1 in infected crops (white arrow). Scale bars in indicate 100 µM. At least three infected crops were examined from two separate infections and representative images are shown.
Figure 3.
Visualization and staining of in vivo microcolonies in the Drosophila crop.
(A) DAPI-stained bacterial cells (100X) and (B) digitally zoomed images (4.4X) of DAPI stained microcolonies in the crop, demonstrating a honeycomb-like structure (white arrow). Scale bars in A indicate 200 µM. Scale bars in B indicate 45.4 µM. At least three infected crops were examined from three separate infections and representative images are shown. Honeycomb-like structures were visualized in 2 out of every three PAO1-infected crops examined.
Figure 4.
Crop integrity in response to P. aeruginosa infection.
(A) The macroscopic structure of (A), uninfected and (B), PAO1pCHAP6656-infected Drosophila crops (Olympus OV100 intravital observation system). Merged fluorescent image of phallodin 488-stained actin (green) and DAPI-stained nuclei (blue) in uninfected crops using (C) 10x and (D) 63x objectives. PAO1pCHAP6656-infected crops (red) at (E) low and high (F) magnification. Scale bars in C and E indicate 400 µM; scale bars in D and E indicate 100 µM. White arrows in C and E indicate the area of the crop where higher magnification images were taken. At least five infected crops were examined from two separate infections and representative images are shown.
Figure 5.
The role of Pel EPS during in vivo biofilm formation in Drosophila.
Representative images of P. aeruginosa pCHAP6656-infected crops. (A) PAO1pCHAP6656-infected crops contain individual bacterial cells, a number of small microcolonies (grey arrows) and two large microcolonies (white arrows). (B) PelB::luxpCHAP6656-infected crops contain individual bacterial cells and no small or large microcolonies. (C) PAZH13pCHAP6656-infected crops contain some individual bacterial cells, and five large microcolonies (white arrows). At least 3 infected crops were examined for each strain. Scale bar equals 100 µM. (D) Quantitative analysis of microcolony formation in response to infection with PAO1 and relevant mutant strains. At least 3 infected crops were examined for each strain. Data presented is the frequency of individual bacterial cells, small or large bacterial microcolonies in a total of 12 fields of view. (E) Expression of pel and psl EPS genes during oral infection relative to acute infection. Values are mean +/− SEM from triplicate qRT-PCR experiments on RNA isolated from two independent Drosophila infections.
Figure 6.
In vivo localization and antibiotic resistance profiling of biofilm and non-biofilm infections.
(A) Localization of bacteria in the fly 5 days postinfection. The GI tract including the crop, was dissected out, crushed and plated on PIA agar to determine CFU per GI tract/fly. The remainder of the fly body, including the head, was crushed separately and plated on PIA to determine CFU/rest of body per fly. (B) The number of CFU recovered from Drosophila hemolymph 2- and 5-days postinfection with PAO1, PAZH13 or pelB::lux. Two biological replicate experiments were performed, each containing 20 Drosophila, and values represented are mean +/−SEM. (C) Antibiotic resistance profiling of biofilm and non-biofilm infections. Increase in antibiotic resistance, as measured by zone of inhibition in disk diffusion assay, in P. aeruginosa strains recovered for Drosophila after oral infection relative to planktonic cultures. Antibiotic concentration indicated in µg/ml. Two biological replicate experiments were performed in triplicate and mean +/− SEM is shown. * p<0.05, ** p<0.01, ***p<0.001.
Figure 7.
Kaplan-Meier survival curves post P. aeruginosa infection.
Survival curves of (A) oral and (B) acute infection with PAO1, pelB::lux, PAZH13 or 5% sucrose control. Experiments were performed at least 3 times each with a minimum of 80 flies and representative curves (mean +/− standard deviation) are shown. *** p<0.001.
Figure 8.
Biofilm infections induce antimicrobial peptide gene expression in Drosophila.
Real time RT-PCR analysis of (A) cecropin A1 (B) diptericin and (C) drosomycin following oral infection with PAO1, pelB::lux, PAZH13 or following oral co-infection with a 1∶1 ratio of PAO1-PAO1p16Slux, PAO1p16Slux-pelB::lux or PAO1p16Slux-PAZH13. For co-infection experiments (last 3 bars) the strains used for each infection are listed, separated by a hyphen. The levels of AMP gene expression was represented as fold change relative to uninfected flies. Values are mean +/− SEM from triplicate qPCR experiments on RNA isolated from two independent Drosophila infections. a, significant fold change (p<0.05, ANOVA) relative to uninfected flies; b, significant fold change (p<0.05, ANOVA) relative to PAO1-infected flies. (D) Kaplan-Meier survival curves of Drosophila following oral co-infection with a 1∶1 ratio of PAO1-PAO1p16Slux, PAO1p16Slux-pelB::lux, PAO1p16Slux-PAZH13 and relevant controls. Experiments were performed at least 3 times each with a minimum of 80 flies and representative curves (mean +/− standard deviated) are shown. a, significant difference (p<0.05, ANOVA) relative to PAO1-PAO1p16Slux-infected flies (green); b, significant difference (p<0.05, ANOVA) relative to pelB::lux-infected flies.
Figure 9.
Kaplan-Meier survival curves of Drosophila orally infected (feeding) for 24h followed by subsequent acute (nicking) or secondary oral infection.
Survival following oral infection with (A) PAO1, (B) pelB::lux or (C) PAZH13 and relevant controls followed by acute infection with PAO1 or relevant controls. (D) Drosophila survival following oral infection with PAO1, pelB::lux, PAZH13 or uninfected (sucrose control) followed by oral infection with pelB::lux or uninfected (sucrose control). Strain name preceding the forward slash "/" indicates the strain or uninfected sucrose control used for oral infection of Drosophila; the strain name following the forward slash "/" indicates the strain or nicked (LB) or uninfected sucrose controls used for subsequent acute or secondary oral infection of Drosophila. Experiments were performed at least twice, each with a minimum of 100 flies and representative curves (mean +/- standard deviated) are shown. * p<0.05, ***p<0.001.