## 3 Biofilms Can Promote Antibiotic Persistence in Clinical Settings  
While persister development is an adaptive strategy at an individual level, bacteria can also exhibit community-wide adaptations to survive antibiotic challenge. Committing to a biofilm community structure can aid in bacterial fitness and promote persister development, particularly if the biofilm is slow-growing in nature [21]. Biofilms are an amalgam of one or more bacterial species that colonize and adhere to physical surfaces in a density-dependent manner. The biofilm creates heterogeneous gradients in signaling molecules, nutrients, and environmental exposures that generate diverse micro-niches [1]. Biofilm formation is found ubiquitously across microbial phyla, and facilitates colonization of both abiotic and biotic surfaces with relative ease [22]. Figure 1: Utilization of next-generation technologies for studying persister cells. (**A**) Both human patients and murine models provide an opportunity to study in vivo persister formation via 16S rRNA profiling, community metagenomics, and RNA-Seq of the intestinal flora following antibiotic exposure. Single-organism persister formation can be studied in vivo through animal infection with high-density transposon insertion libraries and Tn-Seq. (**B**) Metagenomic and RNA-Seq data can be used to study selective pressure on persister genes in either closely related or divergent taxa. This can be done in retrospective clinical cohort groupings or in animal model infections over the course of antibiotic therapy, identifying how certain therapeutic regimens can select for the expression of specific (red) or ubiquitous (blue) persister elements. (**C**) Persisters can be studied from either in vitro-generated biofilms or patient biopsy-derived biofilms. Persister-specific Fluorescence In-Situ Hybridization (FISH) labeling can allow for visualization and study of persisters within the 3D context of the biofilm, and laser-capture microdissection (LCMD) sectioning can facilitate labeled cell extraction for downstream transcriptomic analyses. Biofilms are characteristically stress-resilient, and they are a great example of how population size and fitness are positively correlated through the Allee effect [23]. The Allee effect describes scenarios in which biological characteristics correlate the population density of a given ecosystem with the fitness of individual species or the population within that ecosystem [23]. In microbial ecology, biofilms increase the total population density irrespective of whether they are mono- or polymicrobial. As the population becomes stabilized by density, intraspecies variation and thus fitness drastically increase due to cooperative interactions and reduced genetic drift [24; 25]. The biofilm as a total population exhibits antibiotic tolerance, and increased cooperative interactions within the population might generate conditions that increase persister cell formation. Clinically, biofilms are associated with antibiotic recalcitrance, infection recurrence, and persister formation [1]. Biofilm formation has been documented in both Gram-positive and Gram-negative pathogens and is clinically significant in various infection types, ranging from skin and soft tissue infections (SSTI), implanted device infections, urinary tract infections, endocarditis, otitis media, and more [12; 26; 27; 28; 22]. Approximately 50 percent of all nosocomial infections originate from implanted medical devices such as prosthetic joints, catheters, and prosthetic heart valves, all of which provide abiotic surfaces for the development of biofilms [12]. In patients, tissue location and biofilm progression can result in varied antibiotic exposure even in the presence of clinically appropriate dosing [16]. Ultimately, this results in bacterial exposure to sub-inhibitory antibiotic concentrations, which can promote persister development [16; 29; 30; 31]. Biofilm formation appears to confer significant fitness advantages to pathogenic bacteria.