Urinary tract infections (UTIs) have complex dynamics, with uropathogenic Escherichia coli (UPEC), the major causative agent, capable of colonization from the urethra to the kidneys in both extracellular and intracellular niches while also producing chronic persistent infections and frequent recurrent disease. In mouse and human bladders, UPEC invades the superficial epithelium, and some bacteria enter the cytoplasm to rapidly replicate into intracellular bacterial communities (IBCs) comprised of ϳ10 4 bacteria each. Through IBC formation, UPEC expands in numbers while subverting aspects of the innate immune response. Within 12 h of murine bladder infection, half of the bacteria are intracellular, with 3 to 700 IBCs formed. Using mixed infections with green fluorescent protein (GFP) and wild-type (WT) UPEC, we discovered that each IBC is clonally derived from a single bacterium. Genetically tagged UPEC and a multiplex PCR assay were employed to investigate the distribution of UPEC throughout urinary tract niches over time. In the first 24 h postinfection (hpi), the fraction of tags dramatically decreased in the bladder and kidney, while the number of CFU increased. The percentage of tags detected at 6 hpi correlated to the number of IBCs produced, which closely matched a calculated multinomial distribution based on IBC clonality. The fraction of tags remaining thereafter depended on UTI outcome, which ranged from resolution of infection with or without quiescent intracellular reservoirs (QIRs) to the development of chronic cystitis as defined by persistent bacteriuria. Significantly more tags remained in mice that developed chronic cystitis, arguing that during the acute stages of infection, a higher number of IBCs precedes chronic cystitis than precedes QIR formation.Population bottlenecks exist for many infections and are particularly well documented during transmission between hosts for RNA viruses and parasites (1,3,27,28,35,41). Localizing bottlenecks in time and space during an infection can identify steps in pathogenesis where an organism encounters the strongest barriers to establishing a foothold within a host. Bottlenecks may also represent important steps in host colonization and pathogenesis to target with therapeutics. Similar studies have been undertaken to identify genes important for tissue colonization and transit between tissues for bacterial pathogens (7,36,37). Several potential bottlenecks limiting the progression of uropathogenic Escherichia coli (UPEC) to later stages of infection exist in the pathogenic cascade of urinary tract infections (UTIs): (i) invasion of the superficial bladder epithelium, (ii) avoidance of Toll-like receptor 4 (TLR4)-mediated expulsion (46), (iii) persistence in the face of superficial facet cell exfoliation, (iv) the maturation process of intracellular bacterial communities (IBCs), (v) ascension from bladder to the kidneys, and (vi) possible descent from kidneys to the bladder. These population dynamics all occur in the face of clearance mechanisms, including mi...