## 3 Biofilms Can Promote Antibiotic Persistence in Clinical Settings
As an environment subject to ecological drivers, biofilms promote intraspecies variation that encourages persister development. [PERSON] et al. have suggested that heterogeneity promotes antibiotic tolerance through the altruistic behavior of a few variant subpopulations within the biofilm [32]. This \"bacterial charity\" is analogous to kin selection, where a subset of cells obtains resistance- or persistence-conferring capacity and provides protection to others. [PERSON] found that mutations in indole production were directly correlated to charity events in polymicrobial biofilms. By challenging _E. coli_ strains to increasing concentrations of fluoroquinolones, they found that a highly resistant and high indole-producing subpopulation triggered overall biofilm tolerance via indole signaling [32]. Biofilms are ultimately important to the study of persister formation because they represent the endogenous ecological structures that many bacteria will adopt within a host [33]. Persister cells are implicated as a causative agent in a multitude of biofilm-related recurrent infections including urinary tract infections, where sub-inhibitory antibiotic concentrations promote persister development and multi-drug tolerance [26; 34; 35]. In otitis media (OM), a commonly chronic or recurrent infection, the formation of a polymicrobial biofilm is initiated by opportunistic members of the nasopharyngeal microbiota that migrate towards the inner ear and trigger infection [27]. In OM, the role of cooperative intraspecies interactions has been well documented. In polymicrobial biofilms, cooperation promotes multi-drug tolerance by persister cells [27]. Specifically, _Moraxella catarrhalis_ provides passive \(\beta\)-lactam protection to _Streptococcus pneumoniae_ and non-typable _Haemophilus influenzae_ (NTHi), and in turn they provide tolerance towards fluoroquinolones by promoting _M. catarrhalis_ persister cell formation [27]. Here, as in periprosthetic joint implant infections, biofilm formation functions as a vehicle for persister cell development [36]. In _Staphylococcal_ infections, recurrent SSTIs have been associated with biofilms in response to prolonged antibiotic exposure, including last-line therapies such as vancomycin [37]. In catheter-related bloodstream infections, there is an effective relapse rate of approximately 20 percent, due to surviving persister populations within catheter-adhered biofilms [28]. Even extended antibiotic therapy at 1000-fold inhibitory concentrations is insufficient to eliminate the biofilm [28]. Thus, the theme of a biofilm functioning as an environment that promotes tolerant infections and persister cell development is prominent in clinical settings. The question then becomes how can we study persister formation in host-related biofilms? As with persister gene evolution, Tn-seq is an attractive option in which a host-related biofilm infection model can be established with a high-density transposon insertion library (Figure 1A). Alternatively, persister-specific Fluorescence In-Situ Hybridization (FISH) could be used to isolate biofilms from in vivo contexts and identify persisters in their native environment by quantifying expression of persister elements such as toxin-antitoxin systems [38; 39]. Laser-capture microdissection (LCMD) could then be coupled with transcriptomic analysis to isolate specific, persister-containing fragments of the biofilm, assess their transcriptional activity, and decouple it from culture-specific or in vitro-specific variation (Figure 1C). The great strength of LCMD coupled with FISH is that it allows the transcriptional analysis of populations enriched for persisters. A similar approach could and has been taken to analyze transcriptional response of persisters in liquid culture through the use of flow cytometry sorting [40; 41]. While these approaches enrich for persisters, clear challenges related to intrinsically low abundance of persisters still remain.