Drosophila melanogaster as a model host for the Burkholderia cepacia complex

Abstract

Background: Colonization with bacterial species from the Burkholderia cepacia complex (Bcc) is associated with fast health decline among individuals with cystic fibrosis. In order to investigate the virulence of the Bcc, several alternative infection models have been developed. To this end, the fruit fly is increasingly used as surrogate host, and its validity to enhance our understanding of host-pathogen relationships has been demonstrated with a variety of microorganisms. Moreover, its relevance as a suitable alternative to mammalian hosts has been confirmed with vertebrate organisms.

Methodology/principal findings: The aim of this study was to establish Drosophila melanogaster as a surrogate host for species from the Bcc. While the feeding method proved unsuccessful at killing the flies, the pricking technique did generate mortality within the populations. Results obtained with the fruit fly model are comparable with results obtained using mammalian infection models. Furthermore, validity of the Drosophila infection model was confirmed with B. cenocepacia K56-2 mutants known to be less virulent in murine hosts or in other alternative models. Competitive index (CI) analyses were also performed using the fruit fly as host. Results of CI experiments agree with those obtained with mammalian models.

Conclusions/significance: We conclude that Drosophila is a useful alternative infection model for Bcc and that fly pricking assays and competition indices are two complementary methods for virulence testing. Moreover, CI results indicate that this method is more sensitive than mortality tests.

Figure 1. Survival curves for D. melanogaster flies challenged with B. cenocepacia K56-2.

Pricking assays were performed in three independent replicates, each with a minimum of 30 flies. Statistical significance (Log-rank analysis (Mantel-Cox)) between survival curves is shown with *p<0.05 and ***p<0.0005.

Figure 2. Survival curves for D. melanogaster infected with B. cenocepacia strains.

Pricking assays were performed with a minimum of 30 flies for each strain. Statistical significance (Log-rank analysis (Mantel-Cox)) between survival curves is shown with *p<0.05 and ***p<0.0005.

Figure 3. Survival curves for D. melanogaster infected with Bcc strains.

Pricking assays were performed with a minimum of 30 flies for each strain. A: B. cepacia LMG1222, B: B. cepacia LMG18821, C: B. multivorans LMG16660, D: B. stabilis LMG18870, E: B. vietnamiensis LMG22486, F: B. vietnamiensis LMG18835, G: B. dolosa LMG21819, H: B. dolosa LMG21443, I: B. ambifaria AU0212, J: B. ambifaria CEP0996, K: B. pyrrocinia LMG21824, L: B. ubonensis LMG20358.

Figure 4. Relative bacterial load kinetic of fruit flies infected with various Bcc species.

At the indicated time points, bacterial load was quantified from living fruit flies as described in Materials and Methods. A: B. cenocepacia LMG18830, B: B. cenocepacia K56-2, C: B. cepacia LMG18821.

Figure 5. Survival curves for D. melanogaster infected with mutants of B. cenocepacia K56-2.

The killing ability of wild-type B. cenocepacia K56-2 was compared to several mutants: A: zmpA^-, zmpB^- and zmpA^-zmpB^-, B: RSF12 and RSF13. C: bscN^-, D: hldA^-, E: cepI^- and cepR^-. Pricking assays were performed with a minimum of 30 flies for each strain. Statistical significance (Log-rank analysis (Mantel-Cox)) between survival curves is shown with ***p<0.0005 and ns  =  non-significant.

Figure 6. Competitive index (CI) analysis of B. cenocepacia mutants in the D. melanogaster model.

CI is defined as the ratio between the wild-type K56-2 and the mutant in the output (bacteria recovered from the fruit fly 96 h post infection) divided by their ratio in the input (inoculum). Each empty square represents the CI value obtained for one fly. A CI of less than 1 indicates a virulence defect. The mean of the CI is shown as a solid line. A. Three independent CI analyses performed with zmpA mutant. B. CI analyses of zmpA, zmpB and zmpA zmpB mutants. C. CI analyses of hldA, bscN, BCAL2831, cepI and cepR mutants. For htrA^-, the CI was determined with strain RSF12 containing a mutation in the BCAL2831 gene.