Pseudomonas aeruginosa cystic fibrosis isolates from individual patients demonstrate a range of levels of lethality in two Drosophila melanogaster infection models

Abstract

Recently, two Drosophila melanogaster models of infection, fly feeding and fly nicking, have been developed that allow a determination of pathogenic potential of Pseudomonas aeruginosa isolates. In this study, control strains, isolates from burn wounds, and isolates from the sputa of cystic fibrosis (CF) patients were used to compare the two infection models to determine whether any of the isolates might be better adapted to either of the models. In addition, our goal was to determine the variability of isolates from individual CF patients. Three of four control strains (PAO1, PAK, and PA14) caused significant mortality in the flies in both models of infection. The remaining control strain, PA103, was lethal to flies in the nicking model but lacked significant lethality in the feeding model. The burn wound isolates had a high level of lethality in both models. Interestingly, the CF isolates had the largest diversity of lethality in both models of infection. The range of pathogenic potentials of the CF isolates occurred across a cohort of patients, both at the patient level and down to the level of individual sputum samples. The majority of all isolates had similar levels of lethality in both fly infection models. However, two CF isolates were significantly more lethal in the nicking model, and three CF isolates were significantly more lethal in the feeding model. In conclusion, the two Drosophila infection models were useful for the analysis of the diversity of pathogenic potentials of P. aeruginosa isolates.

FIG. 1.

Survival curves for D. melanogaster flies challenged with Pseudomonas isolates. D. melanogaster fly feeding is shown in panels A, C, and E, and fly nicking is shown in panels B, D, and F. The percent survival of PAO1 (*), PAK (▪), PA14 (▾), PA103 (▴), and medium control (⋄) was graphed over time in days (A) and hours (B). The Pseudomonas strains from acute infections graphed in panels C and D are Utah4 (▴), Utah3 (⧫), and ATCC 27853 (•). In panels E and F, the percent survival values for the CF isolates are indicated as follows: 14649 (*), 14673 (⧫), 14671 (⋄), 7307 (▪), 14684 (▴), 14670 (•), 14716 (□), 14683 (▵), 5588 (▾), 5552 (-⧫-), 14715 (▾), 5585 (○), 14661 (-×-), 14690 (-+-), 14703 (▪), 14651 (-•-), 14656 (-□-), 14655 (-▿-), 14672 (-▾-), 14685 (-▴-), 4384 (×), 14660 (-⋄-), 14717 (-*-), 14650 (|), 5166 (+), 5154 (-○-), and 6106 (-|-). Symbols without dashes indicate symbols connected by a solid line on the graph, and symbols with dashes indicate symbols connected by a dashed line on the graph. Fly mortality was measured hourly in the fly nicking assays and daily in the fly feeding assays using sets of 30 flies. The data are representative of the means of three or more replicate experiments performed in triplicate.

FIG. 2.

Cluster analysis of fly lethality. Survival curves of Drosophila infected with Pseudomonas are compared by using Cluster 3.0 and visualized by using Java Treeview 1.1.1. The average fly survival of all strains, in the present study, was used as the baseline for comparison. Green boxes indicate where fewer flies were alive compared to the average, red boxes indicate where more flies were alive than the average, and black boxes indicate that the number of flies alive were equal to the average. Four groups were identified in clustering; isolates that had above-average lethality in both models (group 1), above-average lethality in fly feeding and less-than-average lethality in fly nicking (group 2), and below-average lethality in fly feeding and above-average lethality in fly nicking (group 3) and isolates that had less-than-average lethality in both models (group 4).

FIG. 3.

Pseudomonas CF isolates in cluster group 2 exhibit above-average lethality in the fly feeding model. D. melanogaster survival curves plotted against percentage of time in the experiment for challenge with CF isolates 5154 (A), 14671 (B), and 14650 (C). Results for fly feeding experiments (▪) and fly nicking experiments (▴) are shown. The data are representative of assays performed three times in triplicate, with error bars representing the standard error of the mean.

FIG. 4.

Pseudomonas CF isolates in cluster group 3 exhibit above-average lethality in the fly nicking model. Survival curves are shown for D. melanogaster when challenged with isolates 14684 (A), 14672 (B), and PA103 (C). Results for fly feeding experiments (▪) and fly nicking experiments (▴) are shown. Survival curves were plotted as a percentage of time since the duration of each experiment was different. The data are representative of assays performed three times in triplicate, with error bars representing the standard error of the mean.

FIG. 5.

Pseudomonas survival on sucrose filters in fly feeding assays. Viable bacterial counts per filter were determined for bacterial filters on sucrose agar every second day. Strains: PAO1 (A), Utah4 (B), PA103 (C), 14649 (D), 14672 (E), 6106 (F), 14650 (G). The data are representative of the assay performed twice in triplicate, with the standard error of the mean graphed as error bars.

FIG. 6.

Pseudomonas viability in Drosophila during fly feeding assays. Viable bacterial counts were determined for fly homogenates every second day. Strains: PAO1 (A), Utah4 (B), PA103 (C), 14649 (D), 14672 (E), 6106 (F), 14650 (G). The data are representative of the assay performed twice in triplicate, with the standard error of the mean graphed as error bars.

FIG. 7.

Colonization of D. melanogaster during P. aeruginosa infection. (A) Fruit flies were infected with PAO1 (▪) and transferred to sucrose at 2 (•), 3 (□), and 4 (○) days postinfection. (B) Bacterial CFU per fly was determined on day 8 for PAO1. The days of exposure to Pseudomonas are shown on the x axis. NT, flies maintained on filters inoculated with PAO1 for the entire duration of the experiment.

FIG. 8.

Lethality of strains from CF patients simultaneously infected with multiple Pseudomonas strains. (A and B) The lethality of Pseudomonas CF isolates from patient 57 (A) and patient 89 (B) was graphed. The pathogenic potential of strains was determined by fly nicking. The strains shown in panel A are 14660 (▴) and 14661 (▪), and the strains shown in panel B are 14649 (○), 14651 (⧫), and 14650 (•). The data are representative of the assay performed twice in triplicate, with error bars representing the standard error of the mean.