## 3 Results
### In Vitro Identified Persistence Mutants Show Mixed Results during In Vivo Antibiotic Treatments
melanogaster_ associated with the _hipA7_ mutant (gold) remain higher compared to the loads of those associated with the MG1655 wild-type strain (gray). In contrast, bacterial loads are highly similar in absence of antibiotic treatment  
(full bars, left). Bacterial loads are normalized per fly (means \(\pm\) SEMs with \(n\geq 6\) vials as gray points across \(\geq\)2 independent runs) and a linear model with post hoc test shows the significance of the difference compared to the untreated start point (Dunnett test, in bars) and between the strains per time and treatment (above bars). (**C**) The in vitro identified low persistence mutants lacking both _relA_ and _spotT_ (gold) or _rpoS_ (blue) do not show a decreased in vivo antibiotic survival when associated with _D. melanogaster_ and treated with AP (100 \(\upmu\)g mL\({}^{-1}\)). While the \(\Delta rpoS\) mutant is not different from the BW25113 _E. coli_ wild-type strain, the loads of \(\Delta rd\Delta\Delta spT\) are significantly lower during treatment but also in the untreated controls. Bacterial loads are normalized per fly (means \(\pm\) SEMs with \(n\geq 8\) vials as gray points across 2 independent runs) and an lme with post hoc test shows the significance of the difference compared to the untreated start point (Dunnett test, in bars) and to the wild-type per time per treatment (Dunnett test, above bars). In (**A**-**C**), significance levels are ', ns, nonsignificant; \(p<0.1\); ', \(p<0.05\); ', \(p<0.01\); ', \(p<0.001\); and ', \(p<0.0001\). (**D**) The MG1655 wild-type strain and (**E**) the _hipA7_ mutant show heterogeneous in vivo growth rates as deduced from the Timer sensor. A representative confocal micrograph is shown of a section of the crop of a fly associated with both strains expressing Timer, a dsRed variant that matures quickly to a green fluorescent intermediate and slowly to a red fluorescent end product. The underlying widefield image is shown as a reference, while the fluorescence image is a composite of DAPI (staining the _D. melanogaster_ nuclei), GFP and dsRed (labeling _E. coli_ cells) images, and a gradient growth scale from green to deep orange was added. The \(z\) stack of images at 63\(\times\) magnification was projected onto one plane along with other, image-wide adjustments for visual representation with images of both strains treated similarly (see Section 2). In an initial attempt to explain the increased tolerance of the _hipA7_ mutant in vivo, I made use of a biosensor for growth status as it was shown in vitro that the _hipA7_ mutation causes increased persister levels by increased activation of the growth-inhibiting HipA toxin. The plasmid pTimer expresses a dsRed variant that quickly matures to a green fluorescent intermediate fluorophore which then slowly matures to its red fluorescent end product [58]. As such, cells that have not divided for a long time will contain both red and green products, while quickly dividing cells will dilute the red signal and only display green fluorescence. From the confocal images, it is striking that there is quite some diversity in growth rates among individual cells as deduced from the Timer fluorescence, with some green cells that are actively growing and (many) orange cells that show a slower growth (Figure 3D,E). However, while _hipA7_ cells seem to display a slower growth rate, in-depth quantitative comparison is far from trivial when doing in situ imaging and working with \(z\)-stacked images. Therefore, a formal proof of the link between slow growth rate and tolerance is the subject for future experiments. Taken together, my data do indicate that some findings on bacterial persistence discovered making use of culture-based in vitro assays can be extrapolated to the more complex conditions of the _Drosophila_ gut system while others cannot and that, inherently due to the complexity of an in vivo model, drawing strong conclusions about subtle effects affecting antibiotic tolerance can be much harder.