## 3 Results
### In Vitro Identified Persistence Mutants Show Mixed Results during In Vivo Antibiotic Treatments  
With the confirmation that _D. melanogaster_ can be treated orally with high doses of antibiotics and that _E. coli_ can reside in the gut of the fly, I set out to test the in vivo antibiotic survival of _E. coli_ and, more specifically, of in vitro identified persistence mutants to benchmark the herein developed model. To this end, I associated axenic flies with various _E. coli_ strains and switched them to the CAFE setup where the antibiotic dose during treatment can be accurately estimated via the food uptake. As is evident from the bacterial loads of _D. melanogaster_ associated with a wild-type _E. coli_ strain (Figure 3A), _E. coli_ displays high tolerance in vivo towards antibiotic treatments. Clearly, the high survival after 24 h can be explained by the low food, and thus antibiotic, uptake during this first day in the CAFE setup (Figure S4C and as shown previously in Figure 1B). However, at 48 and 72 h, flies feed regularly on antibiotic-containing liquid food (Figure S4C), with a food uptake (0.94-1.23 uL day\({}^{-1}\)) that, already for the lowest applied concentrations, results in an antibiotic dosage exceeding the advised dose in humans (Figure S4D). Despite such a high dosage, bacteria tolerate the in vivo treatment very well and at least 72 h of treatment is needed to significantly lower bacterial loads. Since ofloxacin resulted in inconsistent lower bacterial loads, I continued using ampicillin and amikacin for at least 72 h.  
Next, I confirmed that the _hipA7 E. coli_ mutant, which is widely used in persistence research and results in increased persistence levels in various conditions [21; 34], also displays increased survival towards antibiotic treatment inside the _Drosophila_ gut (Figure 3B). Both the _hipA7_ mutant and the parental wild-type strains show a similar increase in untreated flies. While antibiotic treatment inhibits this increase, the loads of the _hipA7_ mutant remain at a higher level after 96 h treatments with both AP and AM. When examining the \(\Delta\)_rpoS_ and \(\Delta\)_relA\(\Delta\)_spoT_ mutants, two mutants that are frequently implicated in a lower fraction of tolerant persisters in in vitro studies [21], it becomes apparent that not all in vitro findings can be extrapolated to the more complex in vivo condition of the fly's gut. Indeed, the \(\Delta\)_rpoS_ mutant does not show a decreased survival upon in vivo treatment. Moreover, while the \(\Delta\)_relA\(\Delta\)spoT_ mutant initially seems impaired in its in vivo antibiotic tolerance, such a conclusion is clearly hampered by the confounding factor that this mutant also associates considerably less successfully with _D. melanogaster_ in absence of any antibiotics. Figure 3: _E. coli_ shows high in vivo antibiotic tolerance inside the gut of _D. melanogaster_, with in vitro identified persistence mutants showing mixed results during in vivo antibiotic treatments. (**A**) When treating _D. melanogaster_ associated with the SX43 _E. coli_ wild-type strain with AP (red), AM (blue) and OF (yellow) at 3 different concentrations (100, 400 and 1000 for AP and AM or 10, 40 and 100 \(\upmu\)g mL\({}^{-1}\) for OF, all in different intensities) via liquid food in the CAFE assay, bacterial loads only start to decrease at 48 h and more consistently at 72 h. Bacterial loads are normalized per fly (means \(\pm\) SEMs with \(n\geq\) 10 vials as gray points across \(\geq\)2 independent runs) and an lme with post hoc test shows the significance of the difference compared to the untreated control (gray bars) per time (Dunnett test, above bars) and compared to the untreated start point (Dunnett test, in bars). Nonsignificant comparisons are not annotated as such for visual purposes. (**B**) During treatment with AP and AM (both at 100 \(\upmu\)g mL\({}^{-1}\), hatched bars on the right), the bacterial loads of _D.