## 4 Discussion and Conclusions  
Here, I explored the use of _D. melanogaster_ as a practical model to study bacterial antibiotic-tolerant persisters in the in vivo and more complex condition that is the fly gut. In building the model, I verified that, while axenic larvae suffer from antibiotics, adult _D. melanogaster_ flies are very tolerant towards high-dose antibiotic treatments. In addition, when presenting axenic individuals orally with a bacterial inoculum, _Drosophila_ flies can host a substantial amount of _E. coli_ cells in their gut, specifically in the crop, at a number that increases over time. Furthermore, I showed that the in vivo conditions induce an overall increased antibiotic tolerance in gut-residing bacteria, as 3-4 days of treatment with high antibiotic doses resulted in only limited decreases of bacterial loads. For the in vitro widely used _hipA7_ high-persistence mutant, in vivo antibiotic survival was even higher, but the changed tolerance levels of other in vitro persistence mutants could not be extrapolated to the complex gut environment. Studying _E. coli_ in _D. melanogaster_ might seem highly irrelevant. Indeed, microbiome compositions of Drosophila never reported the presence of _E. coli_[111; 113]. Furthermore, the physiology of a fly gut is, at points, quite different from that of the human gut, e.g., showing more oxygenation [77; 115]. Indeed, a Drosophila-based model will never have the relevance of pathogen-specific infection models with large eukaryotic hosts. In fact, it is not at all possible to \"humanize\" Drosophila models [77]. Nevertheless, they allow testing, with throughput and power and devoid of ethical issues, the effect of various aspects of environmental complexity and unpredictability on antibacterial tolerance, e.g., the structural and temporal heterogeneity in food and antibiotics or the interaction with host cells. In addition, many aspects of the immune response are conserved between Drosophila and humans, and as _E. coli_ and _D. melanogaster_ are model organisms, a plethora of knowledge and tools are available for both (e.g., mutant libraries, sensors or defined fly food [116]). Furthermore, _D. melanogaster_ is very promiscuous towards its bacterial partners, being a capable host for many nonpathogenic bacteria [117] while sensitive to a wide array of important human pathogens [118; 119], making it widely applicable in in vivo research on antibiotic tolerance. The herein found, specific localization of _E. coli_ in the crop nicely agrees with some previous findings on other microbes that more naturally occur in the _Drosophila_ gut. The [PERSON] group showed that a gut symbiont of _Drosophila_, _Aectobacter thailandicus_, remains associated in a very stable fashion in the specific niche that is the crop and foregut [108]. In addition, _Lactiplantiobacillus plantarum_, another member of the natural microbiome, was also shown to be strongly enriched in the crop and cardio region of the foregut [120]. Even in a _Drosophila_-based model for oral infection with the opportunistic pathogen _Pseudomonas aeruginosa_, bacteria were found specifically in the crop [121]. The crop is an organ in most Diptera insects that stores food (and fulfills a range of other functions) and is diverticulated from the linear digestive tract [122]. As such, the crop can offer bacteria protection from physical clearance by the food flux due to peristaltic movements of the gut. Furthermore, antimicrobial peptides are only to a lesser extent produced in the crop [81; 122]. Nevertheless, the crop does contract at times to funnel food into the midgut and produces digestive enzymes. The absence of a mucus layer and the peritrophic matrix that protect the epithelial cells in the midgut and confines bacteria to the gut lumen [86] could aid in a better adherence of bacteria and resilience against evacuation due to crop contractions. Similar to what was found for natural symbionts or opportunistic pathogens, I also observe patch-like clusters of _E.