Dynamic persistence of UPEC intracellular bacterial communities in a human bladder-chip model of urinary tract infection

Uropathogenic Escherichia coli (UPEC) proliferate within superficial bladder umbrella cells to form intracellular bacterial communities (IBCs) during early stages of urinary tract infections. However, the dynamic responses of IBCs to host stresses and antibiotic therapy are difficult to assess in situ. We develop a human bladder-chip model wherein umbrella cells and bladder microvascular endothelial cells are co-cultured under flow in urine and nutritive media respectively, and bladder filling and voiding mimicked mechanically by application and release of linear strain. Using time-lapse microscopy, we show that rapid recruitment of neutrophils from the vascular channel to sites of infection leads to swarm and neutrophil extracellular trap formation but does not prevent IBC formation. Subsequently, we tracked bacterial growth dynamics in individual IBCs through two cycles of antibiotic administration interspersed with recovery periods which revealed that the elimination of bacteria within IBCs by the antibiotic was delayed, and in some instances, did not occur at all. During the recovery period, rapid proliferation in a significant fraction of IBCs reseeded new foci of infection through bacterial shedding and host cell exfoliation. These insights reinforce a dynamic role for IBCs as harbours of bacterial persistence, with significant consequences for non-compliance with antibiotic regimens.

The Crohn’s disease-related bacterial strain LF82 assembles biofilm-like communities to protect itself from phagolysosomal attack

Patients with Crohn’s disease exhibit abnormal colonization of the intestine by adherent invasive E. coli (AIEC). They adhere to epithelial cells, colonize them and survive inside macrophages. It appeared recently that AIEC LF82 adaptation to phagolysosomal stress involves a long lag phase in which many LF82 cells become antibiotic tolerant. Later during infection, they proliferate in vacuoles and form colonies harboring dozens of LF82 bacteria. In the present work, we investigated the mechanism sustaining this phase of growth. We found that intracellular LF82 produced an extrabacterial matrix that acts as a biofilm and controls the formation of LF82 intracellular bacterial communities (IBCs) for several days post infection. We revealed the crucial role played by the pathogenicity island encoding the yersiniabactin iron capture system to form IBCs and for optimal LF82 survival. These results illustrate that AIECs use original strategies to establish their replicative niche within macrophages.

Uropathogenic E. coli induces DNA damage in the bladder

Urinary tract infections (UTIs) are among the most common outpatient infections, with a lifetime incidence of around 60% in women. We analysed urine samples from 223 patients with community-acquired UTIs and report the presence of the cleavage product released during the synthesis of colibactin, a bacterial genotoxin, in 55 of the samples examined. Uropathogenic Escherichia coli strains isolated from these patients, as well as the archetypal E. coli strain UTI89, were found to produce colibactin. In a murine model of UTI, the machinery producing colibactin was expressed during the early hours of the infection, when intracellular bacterial communities form. We observed extensive DNA damage both in umbrella and bladder progenitor cells. To the best of our knowledge this is the first report of colibactin production in UTIs in humans and its genotoxicity in bladder cells.

Dynamic persistence of intracellular bacterial communities of uropathogenic Escherichia coli in a human bladder-chip model of urinary tract infections

Uropathogenic Escherichia coli (UPEC) is the most common causative agent of urinary tract infections and is a major cause for antibiotic prescriptions. Previous studies have shown that infection of terminally differentiated urinary bladder cells leads to the formation of intracellular bacterial communities (IBCs). However, the precise role of IBCs in recurrence of infection and antibiotic persistence, is not completely understood in part because the in situ dynamic responses of bacteria within these structures to antibiotic stress is difficult to assess in animal models. Here, we develop and characterize a human bladder-chip model of UPEC infection wherein superficial bladder epithelial cells and bladder microvascular endothelial cells are co-cultured under flow in urine and nutritive media respectively, and the mechanics of bladder filling and voiding cycles mimicked by application and release of linear strain. Time-lapse microscopy showed that infection of epithelial cells under shear stress in diluted urine led to the rapid recruitment and diapedesis of neutrophils across the endothelial-epithelial barrier and the formation of neutrophil swarms and neutrophil extracellular traps. Subsequently, two cycles of antibiotic administration interspersed with recovery periods revealed both non-growing and rapidly proliferating IBCs. Multiple stages of IBC formation captured on-chip with single-cell resolution revealed that that bacterial killing within IBCs was substantially delayed, outcomes such as shedding of bacteria and exfoliation are not mutually exclusive and rapidly reseeded the infection, and in rare instances bacterial growth in IBCs continued for the entire period of antibiotic administration. These new insights into the early stages of pathogenesis revisit the role of IBCs as harbours of persistent bacterial populations, with significant consequences for non-compliance with antibiotic regimens.

Invasion into the bladder wall generates solitary subpopulations of uropathogenic Escherichia coli that are protected from killing by antibiotics and neutrophil swarms in an organoid model

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.

Distinct Morphological Fates of Uropathogenic Escherichia coli Intracellular Bacterial Communities: Dependency on Urine Composition and pH

ABSTRACT Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections. These bacteria undertake a multistage infection cycle involving invasion of and proliferation within urinary tract epithelial cells, leading to the rupture of the host cell and dispersal of the bacteria, some of which have a highly filamentous morphology. Here, we established a microfluidics-based model of UPEC infection of immortalized human bladder epithelial cells that recapitulates the main stages of bacterial morphological changes during the acute infection cycle in vivo and allows the development and fate of individual cells to be monitored in real time by fluorescence microscopy. The UPEC-infected bladder cells remained alive and mobile in nonconfluent monolayers during the development of intracellular bacterial communities (IBCs). Switching from a flow of growth medium to human urine resulted in immobilization of both uninfected and infected bladder cells. Some IBCs continued to develop and then released many highly filamentous bacteria via an extrusion-like process, whereas other IBCs showed strong UPEC proliferation, and yet no filamentation was detected. The filamentation response was dependent on the weak acidity of human urine and required component(s) in a low molecular-mass (<3,000 Da) fraction from a mildly dehydrated donor. The developmental fate for bacteria therefore appears to be controlled by multiple factors that act at the level of the whole IBC, suggesting that variable local environments or stochastic differentiation pathways influence IBC developmental fates during infection.

Uropathogenic E. coli induces DNA damage in the bladder

Urinary tract infections (UTIs) are among the most common outpatient infections, with a lifetime incidence of around 60% in women. We analysed urine samples from 223 patients with community-acquired UTIs and report the presence of a metabolite released during the synthesis of colibactin, a bacterial genotoxin, in 50 of the samples examined. Uropathogenic Escherichia coli strains isolated from these patients, as well as the archetypal E. coli strain UTI89, were found to produce colibactin. In a murine model of UTI, the machinery producing colibactin was expressed during the early hours of the infection, when intracellular bacterial communities form. We observed extensive DNA damage both in umbrella and bladder progenitor cells. To the best of our knowledge this is the first report of colibactin production in UTIs in humans and its genotoxicity in bladder cells. This bacterial genotoxin, which is increasingly suspected to promote colorectal cancer, should also be scrutinised in the context of bladder cancer.

The Crohn’s disease-related AIEC strain LF82 assembles a biofilm-like matrix to protect intracellular microcolonies from phagolysosomal attack

Patients with Crohn’s disease exhibit abnormal colonization of the intestine by proteobacteria, and among these bacteria, the adherent invasive E. coli (AIEC) family. They are predominant in the mucus, adhere to epithelial cells, colonize them and survive inside macrophages. We recently demonstrated that the acclimation of the AIEC strain LF82 to phagolysosomal stress requires stringent and SOS responses. Such adaptation involves a long lag phase in which many LF82 cells become antibiotic tolerant. Later during infection, they proliferate in vacuoles and form colonies harboring dozens of LF82 bacteria. In the present work, we investigated the mechanism sustaining this phase of growth. We found that intracellular LF82 produced an extrabacterial matrix composed of exopolysaccharides and amyloid fibers that surrounded each individual LF82 cell. This matrix acts as a biofilm and controls the formation of LF82 intracellular bacterial communities (IBCs) inside phagolysosomes for several days post infection. Using genomics assays, we characterized the gene set involved in IBCs formation and revealed the crucial role played by a pathogenicity island presents in the genome of most AIEC strains in this process. Iron capture, by the yersiniabactin system encoded by this pathogenicity island, is essential to form IBC and LF82 survival within macrophages. These results demonstrate that AIEC have developed a sophisticated strategy to establish their replicative niche within macrophages, which might have implications for envisioning future antibacterial strategies for Crohn’s disease.