## 4 Discussion and Conclusions
For the \(\Delta\)_relAASpoT_ mutant, a similar explanatory statement could be made. In addition, such a double mutant lacking ppGpp production is known to be highly crippled and, as shown by my results, clearly has a severely impaired fitness in the fly's gut even in absence of antibiotics, which acts as a strong confounding effect for any observed differences in in vivo antibiotic survival. In the future, the use of this model could be expanded to test some of the many other persistence mutants identified in vitro in _E. coli_[127], examine other physiological traits linked to antibiotic tolerance (e.g., ATP levels) or study antibiotic tolerance in a wide array of bacterial strains or species. Furthermore, to examine the effect of interactions between drugs, host and coresiding bacterial communities on antibiotic tolerance, the Drosophila model could be extremely powerful [124]. Indeed, in vitro, it was already shown that the tolerance of L. plantarum towards rifampicin is affected by other important members of the Drosophila gut microbiome, namely Acetobacter species [128]. To end, the model would be extremely suitable to perform in vivo experimental evolution under antibiotic pressure. A growing number of evolution experiments selecting for increased persistence have been performed in monocultures in the lab; however, it is unclear how evolutionary trajectories are dependent on the environmental conditions. A recent evolution experiment with L. plantarum in association with Drosophila shows that the host diet is a dominant factor for evolution, but it remains to be seen whether this is also true when including antibiotics. In conclusion, my results presented here show a high potential for the wide application of a Drosophila-based model to examine antibiotic tolerance in vivo. **Supplementary Materials:** The following are available online at [[https://www.mdpi.com/article/10.3390/microorganisms10010119/s1](https://www.mdpi.com/article/10.3390/microorganisms10010119/s1)]([https://www.mdpi.com/article/10.3390/microorganisms10010119/s1](https://www.mdpi.com/article/10.3390/microorganisms10010119/s1)), Figure S1: Direct effects of antibiotics on _Drosophila melanogaster_ are limited to lethality in the axenic, larval stage, Figure S2: Ontobiotic flies associated with _E. coli_ display similar feeding and mortality compared to an axenic control group, Figure S3: _E. coli_ is taken up orally by axenic _D. melanogaster_ flies and is specifically enriched in the crop, Figure S4: Ontobiotic flies maintained under the CAFE assay show microcolonies of _E. coli_ in the crop and show a regular feeding rate leading to an antibiotic dosage similar to in humans. **Funding:** This research was funded by the Research Foundation-Flanders (FWO), grant numbers 12O1917N, V428917N and 1513120N; the Belgian American Educational Foundation (BAEF), grant number 2016-E083; and the European Molecular Biology Organization (EMBO; ALTF 344-2017). The APC was funded by the Research Foundation-Flanders (postdoctoral fellowship 12O1922N). **Institutional Review Board Statement:** Not applicable. **Informed Consent Statement:** Not applicable. **Data Availability Statement:** The data presented in this study will be made openly available upon publication in Mendeley Data at doi:10.17632/n6 hx7p9 trk.1. **Acknowledgments:** I want to thank [PERSON] (retired from Cornell University) for her support during my stay in her lab, her kindness and intellectual brain I could pick, the opportunities she offered me and her vision on a life in academia in general. I will never forget this period of my life which shaped me as a person. Next, I want to thank various [PERSON] lab members for their valuable discussions, introductions in advanced statistics and practical support during my stay, more specifically [PERSON], [PERSON], [PERSON], [PERSON], [PERSON] and [PERSON].