###### Abstract  
With an antibiotic crisis upon us, we need to boost antibiotic development and improve antibiotics' efficacy. Crucial is knowing how to efficiently kill bacteria, especially in more complex in vivo conditions. Indeed, many bacteria harbor antibiotic-tolerant persisters, variants that survive exposure to our most potent antibiotics and catalyze resistance development. However, persistence is often only studied in vitro as we lack flexible in vivo models. Here, I explored the potential of using _Drosophila melanogaster_ as a model for antimicrobial research, combining methods in _Drosophila_ with microbiology techniques: assessing fly development and feeding, generating germ-free or bacteria-associated _Drosophila_ and in situ microscopy. Adult flies tolerate antibiotics at high doses, although germ-free larvae show impaired development. Orally presented _E. coli_ associates with _Drosophila_ and mostly resides in the crop. _E. coli_ shows an overall high antibiotic tolerance in vivo potentially resulting from heterogeneity in growth rates. The _hipA7_ high-persistence mutant displays an increased antibiotic survival while the expected low persistence of _arelAASepT_ and _ArpoS_ mutants cannot be confirmed in vivo. In conclusion, a _Drosophila_ model for in vivo antibiotic tolerance research shows high potential and offers a flexible system to test findings from in vitro assays in a broader, more complex condition. m 20221022022202202202202202202202202202202202202202202202202202202202202202202202202220220220220222022022022202202220220220222022022022202  
Lately, much research focus has been on studying antibiotic-tolerant persister cells. These phenotypic variants within isogenic bacterial populations are much more antibiotic-tolerant compared to their sensitive siblings (i.e., they are killed at a much lower rate) and hamper complete sterilization and prolong treatment [21; 22; 23]. Recent findings even show that, like resistance, antibiotic tolerance and persistence are under strong adaptive selection when bacteria face recurrent treatments [24; 25; 26; 27; 28; 29]. Worryingly, the increased tolerance also catalyzes the acquiring of genetic resistance [30; 31; 32]. Targeting persisters will therefore slow down resistance development and prolong the effectiveness of antibiotics. However, myriad persistence mechanisms to be targeted were identified, for example resolving around toxin-antitoxin systems [33; 34; 35; 36; 37; 38; 39; 40], the stringent stress response [41; 42], low cellular energy status [43; 44; 45; 46], intracellular protein aggregation [47; 48] or acidification [49; 50; 51]. Currently, it is still unclear how all these different mechanisms fit together: are they each specific to certain conditions or do some of them work together and are all of them relevant in clinical settings? Persistence insights have been predominantly acquired from in vitro, culture-based assays which offer a great level of control but are also artificially simple in comparison to more complex in vivo conditions. More recent studies started to examine persistence in more real-life conditions. As such, reports did not only verified the presence, relevance and evolution of persistence in vivo [30; 52; 53; 54; 55; 56; 57; 58; 59; 60] but also confirmed the in vivo efficacy of some anti-persister treatments [61; 62; 63; 64] and the relevance of in vitro identified persister genes [65; 66; 57; 36; 67; 56; 68; 69; 70; 71]. At the same time, some persistence mechanisms were also found to critically depend on host factors [68; 69; 70; 71] or co-residing microbial species and social interactions [70; 72] which additionally shows why application of such models is highly important. Unfortunately, the wide implementation is hampered by the specific nature (e.g., pathogen- and infection-specific) and the high technical, financial and ethical requirements of such in vivo models that often are based on rodent hosts.