Interplay between uropathogenic Escherichia coli and bladder cells and new strategies to counteract bacterial persistence

Urinary tract infections (UTIs) are the most frequent community and hospital-acquired infections. Uropathogenic Escherichia coli (UPEC) is the major causative agent of UTIs. The high level of recurrences, in which a single bacterial strain causes two or more consecutive UTIs, strongly suggests a reservoir within host. UPEC strains can invade epithelial cells of the urinary tract and replicate to form intracellular bacterial communities (IBCs) with biofilm-like properties, and may persist acting as quiescent intracellular bacterial reservoirs (QIRs). The formation of these structures represents a key event in the development of recurrences and is associate with the failure of conventional antibiotic therapies. The most crucial factor for the persistence of UTIs is the biofilm formation ability of UPEC, which protects them against harmful conditions, antimicrobial agents, and the host's immune system. Nanotechnology has been presented as a promising approach to enhance the activity of antimicrobial agents. The use of drug-delivery systems has been hypothesized for the controlled and localized release of drugs even in the case of multi-resistant bacteria and in the fight against biofilm. In order to identify bacterial characteristics possibly associated with recurrence of the infection, in this study UPEC strains, isolated from patients with recurrent UTIs, were sequenced, phenotypically and genotypically characterized, and assayed for invasion and survival ability in cell models. To explore new strategies able to induce bacterial clearance, the activity of niosomes (Nio) loaded with gentamicin (GM) was evaluated against intracellular bacteria and Nio, loaded with ciprofloxacin (CIP), was assayed for the capacity to inhibit biofilm formation. UPEC collected from patients suffering from recurrent UTIs and reference strains were used. The whole genome was performed on Illumina platform. Profiles relative to resistance genes, virulence factors, and multilocus sequence typing were determined by querying genomes against dedicated databases. The isolates were phenotypically characterized and assayed to adhere, invade, and persist in bladder cells (T-24 ATCC HTB-4) by gentamicin protection assay. ROS accumulation and interleukins production in T24 cells after UPEC infection, were detected. Empty and antibiotics loaded Nio were prepared and were characterized in term of stability and drug entrapment efficiency. To evaluate potential antiinvasive activity, non-cytotoxic and non-bactericidal concentrations of GM-Nio were added during the UPEC infection in the bladder. Furthermore, to verify the effect of CIP-Nio on biofilm production, bacterial strains were treated with niosomal preparations for 24 h. Ultrastructural biofilm modifications were visualized by Scanning Electron Microscopy (SEM). Genotypic characterization of the strains evidenced an heterogenous virulence factor profile. UPEC strains were moderate or strong biofilm producers, non-haemolytic and belonged to phylogroup D and B2. UPEC strains were able to efficiently adhere and invade, but not survive inside T24 cells. UPEC infection promoted the production of ROS but not all strains were able to induce interleukin production in bladder cells. Nio showed nanometric dimensions and a good stability profile and when loaded with antibiotics, significantly inhibited bacteria viability. Antibiotic delivered by Nio decreased UPEC invasion into bladder cells and efficiently inhibit the biofilm formation. UPEC genome is a mosaic continuum and no single feature can be used to differentiate strains. Similarly, no virulence factor alone is sufficient to cause disease. The complex interplay between bacterial infection capabilities, treatment success and host factors are likely to determine recurrence rather than specific genetic characteristics of the E. coli bacteria itself. Further studies exploring host-pathogen relationships and impact of QIR formation with organoid models in RUTI pathogenesis should be performed to contribute to translation of these results into innovative treatments.