## 4 Discussion and Conclusions
coli_ cells that seem to be adhered as microcolonies to the crop epithelial cells [108; 120; 101], although in-depth research needs to further confirm these findings. The bacterial loads that I obtained after association by feeding strongly resemble those that were previously found in a setup where associations were already started in the larval stage [117]. However, compared to the latter setup, if restricting the use of the model to the adult phase, the association-by-feeding strategy offers higher flexibility and additional checkpoints. The obtained numbers furthermore remained stable over time. While I switched flies back to sterile solid food after feeding on the bacterial inoculum, it is hard to speculate what these stable bacterial loads mean in terms of stability of the association between _E. coli_ and _D. melanogaster_. I cannot formally exclude that bacteria cycle from food to gut, gut to food and vice versa and that associations are quite unstable. Indeed, it is generally believed that associations, even between naturally occurring microbial gut residents and _D. melanogaster_, are highly flexible and that bacteria need to be replenished orally to maintain stable in vivo densities [123], although two genuinely stable symbionts have been described [108; 120]. Very frequent (\(\geq\)daily) replacement of the sterile food would offer a formal examination in the case of _E. coli_ and likely result in declining bacterial numbers over time. However, several of my findings do support some form of stability. To start, a similar bacterial load was observed when flies were maintained in the empty tubes of the CAFE assays where the capillaries with sterile food were replaced daily. Whilecoprophagy could still occur in these vials as the vials themselves were not replaced, it is then striking that antibiotic treatments imposed during CAFE do not result in a stronger decrease (as antibiotics would have excluded further ex vivo proliferation needed to maintain stable bacterial loads). Furthermore, the number of bacteria even increases, which matches with the in vivo detection of microcolony growth patterns and the fact that the Timer growth sensor shows cells that are actively growing. Clearly, a more detailed examination is needed to examine the stability of the association between _E. coli_ and the fly. Bacterial antibiotic tolerance was very high despite the high applied antibiotic dosage. While perhaps surprising at first, antibiotics are known to be less efficient/officious in vivo, although the reasons are not always clear and multiple explanations exist [56]. Here, the heterogeneous in vivo conditions might be an important contributor. Indeed, a heterogeneous in vivo growth status was found among individual bacteria which previously resulted in a delayed eradication of _Salmonella_ by antibiotics in mice [58]. A slightly lower growth rate in the more antibiotic-tolerant _hipA7_ mutant was apparent from confocal microscopy; however, differences were small and future detailed quantification is needed. In addition, in vivo exposure to stress (e.g., limited nutrient availability, digestive enzymes, immune system, acidity) can further induce antibiotic tolerance [20]. To end, while unlikely given the high dosage and the fact that the increase in bacterial loads was effectively inhibited by treatment, the host and its microbial partners (here single _E. coli_ strains) can modulate drugs, leading to lower effective concentrations [124]. The nontransferability of the generally in vitro observed low persistence of the \(\Delta\)_relAASpoT_ and \(\Delta\)_rpoS_ mutants to the in vivo setting could also be understood in the context of the more complex in vivo conditions. For _rpoS_, even in vitro, some exceptions exist where its deletion in fact can cause an increase in persister levels [125; 126], which seems to highly depend on the exact environment and used antibiotic. My results for the fly model further confirm that the environment is extremely important for the precise role of RpoS in antibiotic tolerance.