Early invasion of uropathogenicEscherichia coliinto the bladder wall by solitary bacteria that are protected from antibiotics and neutrophil swarms in an organoid model

Abstract

Uropathogenic Escherichia coli (UPEC) is the most common cause of urinary tract infections (UTIs) and require antibiotic therapy. Recurrent infection, which occurs in 25% of treated individuals, is thought to be caused by the release of bacteria from persistent intracellular bacterial communities (IBCs) in the outermost layer of umbrella cells in the bladder wall. Here, we present a bladder organoid model of UPEC infection that recapitulates the bladder architecture within a volume that is suitable for long-term time-lapse imaging of host-pathogen dynamics with high spatiotemporal resolution. We show that bacteria injected into the organoid lumen rapidly invade the bladder epithelium and proliferate intracellularly to form IBCs that are refractory to clearance by antibiotics or neutrophils. Unexpectedly, we find that individual 'solitary' bacteria within deeper layers of the organoid wall also resist elimination by antibiotics and neutrophil swarms. Volumetric serial block face scanning electron microscopy of infected organoids reveals that solitary bacteria may be intracellular or pericellular (located between uroepithelial cells). Solitary bacteria are enriched after neutrophil attack and continue to express flagellin (unlike bacteria within the lumen or IBCs, which are flagellin-deficient), suggesting a mechanism for invasion of solitary bacteria into deeper layers of tissue. Through live-cell imaging, we show that solitary bacteria in the organoid wall, that might resemble quiescent reservoirs, are spatially distinct from IBCs but have temporal overlap, indicating that bacterial shedding from IBCs is not the only mechanism for the establishment of solitary bacterial populations in the bladder wall.