## 10 Future Directions  
The past decade has brought many advancements in the study of bacterial persistence. Despite these advances, persistent infections remain a major public health burden and work is needed to translate new discoveries to improved clinical outcomes. One potential area of research that could help bridge this gap is determining the role that host metabolism plays in bacterial persistence. To date, the vast majority of persistence research has been conducted in vitro under nutrient conditions that differ considerably from what is found in vivo. Human metabolism is a complex phenotypic trait that is dependent on a multitude of factors such as genetics, diet, and microbiome composition [109; 110; 111; 112]. Further complicating the role of human metabolism is the fact that the human host comprises of a multitude of micro-niches that harbor vastly different nutrient conditions. A breadth of research has demonstrated that metabolic activity is a key factor in the development of bacterial persistence. Therefore, it is likely that nutrient availability in these micro-niches may act as a determinant of bacterial metabolism and thus persister formation. If so, understanding the impact that human metabolism plays in bacterial persistence and treatment efficacy is crucial to improving patient outcomes. Furthermore, uncovering the links between host metabolism and bacterial persistence could open the door to new therapeutic strategies that improve the efficacy of treatment by modulating host metabolism. Such strategies could lay the foundation for personalized medicine by allowing medical professionals to tailor treatment based on infection site and the patient's overall metabolic state. Another untapped area of research is the potential link between persister formation and the microbiome. Most, if not all, pre-existing persister research has been conducted in vitro using human pathogens. However, it is unknown if persister formation occurs within the complex polymicrobial communities that comprise the microbiome. Though persistence is typically viewed negatively in the context of recurrent clinical infections, it is possible that it may serve a beneficial role in the context of the microbiome. Antibiotic treatment is known to decrease the diversity and count of bacteria in a number of niches within these communities, which, in turn are associated with dysbiosis and other negative health outcomes [113; 114; 115; 116; 117; 118]. However, a form of persistence may enable beneficial microbes to survive perturbations such as antibiotic treatment, infection, or dietary shifts, thus allowing them to replenish a healthy microbiota. Conversely, these strategies could also explain the bloom of opportunistic pathogens following antibiotic therapy. In either case, it is crucial to understand if and how persistence mechanisms are utilized in the context of the microbiome. Addressing these questions would have been logistically daunting in past decades due to the diversity of the microbiome and the inability to culture many of its resident microbes. However, the advance of next-generation sequencing technologies in the past decade has enabled new insights into the development of persistence. For example, experiments utilizing RNA-Seq demonstrated that persisters overexpress the TolC efflux pump, indicating a previously unknown role of drug efflux in this phenomenon [90]. Additionally, [PERSON] et al. recently developed a platform that integrated fluorescence-activated cell sorting (FACS), traditional antibiotic susceptibility assays, and next-generation sequencing to assay persister physiology [41]. The extreme rarity of persisters within polymicrobial communities makes many in vivo analyses logistically difficult. The development of single-cell sequencing technologies, combined with persister enrichment protocols such as FACS and LCMD, present robust avenues for analysis in vitro and in vivo [42; 43; 44; 45; 46; 47; 48]. Critically, many of the methods that utilize these technologies are culture-independent.