Biogenic amine tryptamine in human vaginal probiotic isolates mediates matrix inhibition and thwarts uropathogenic E. coli biofilm.

Probiotics offer a promising prophylactic approach against various pathogens and represent an alternative strategy to combat biofilm-related infections. In this study, we isolated vaginal commensal microbiota from 54 healthy Indian women to investigate their probiotic traits. We primarily explored the ability of cell-free supernatant (CFS) from Lactobacilli to prevent Uropathogenic Escherichia coli (UPEC) colonization and biofilm formation. Our findings revealed that CFS effectively reduced UPEC's swimming and swarming motility, decreased cell surface hydrophobicity, and hindered matrix production by downregulating specific genes (fimA, fimH, papG, and csgA). Subsequent GC-MS analysis identified Tryptamine, a monoamine compound, as the potent bioactive substance from Lactobacilli CFS, inhibiting UPEC biofilms with an MBIC of 4 µg/ml and an MBEC of 8 µg/ml. Tryptamine induced significant changes in E. coli colony biofilm morphology, transitioning from the Red, Dry, and Rough (RDAR) to the Smooth and White phenotype, indicating reduced extracellular matrix production. Biofilm time-kill assays demonstrated a four-log reduction in UPEC viability when treated with Tryptamine, highlighting its potent antibacterial properties, comparable to CFS treatment. Biofilm ROS assays indicated a significant elevation in ROS generation within UPEC biofilms, suggesting a potential antibacterial mechanism. Gene expression studies with Tryptamine-treated samples showed a reduction in expression of curli gene (csgA), consistent with CFS treatment. This study underscores the potential of Tryptamine from probiotic Lactobacilli CFS as a promising antibiofilm agent against UPEC biofilms.

High-throughput combination assay for studying biofilm formation of uropathogenic Escherichia coli.

Uropathogenic Escherichia coli, the most common cause for urinary tract infections, forms biofilm enhancing its antibiotic resistance. To assess the effects of compounds on biofilm formation of uropathogenic Escherichia coli UMN026 strain, a high-throughput combination assay using resazurin followed by crystal violet staining was optimized for 384-well microplate. Optimized assay parameters included, for example, resazurin and crystal violet concentrations, and incubation time for readouts. For the assay validation, quality parameters Z' factor, coefficient of variation, signal-to-noise, and signal-to-background were calculated. Microplate uniformity, signal variability, edge well effects, and fold shift were also assessed. Finally, a screening with known antibacterial compounds was conducted to evaluate the assay performance. The best conditions found were achieved by using 12 µg/mL resazurin for 150 min and 0.023% crystal violet. This assay was able to detect compounds displaying antibiofilm activity against UMN026 strain at sub-inhibitory concentrations, in terms of metabolic activity and/or biomass.

Multispecies bacterial invasion of human host cells.

Urinary tract infection (UTI), one of the most common bacterial infections worldwide, is a typical example of an infection that is often polymicrobial in nature. While the overall infection course is known on a macroscale, bacterial behavior is not fully understood at the cellular level and bacterial pathophysiology during multispecies infection is not well characterized. Here, using clinically relevant bacteria, human epithelial bladder cells and human urine, we establish co-infection models combined with high resolution imaging to compare single- and multi-species bladder cell invasion events in three common uropathogens: uropathogenic Escherichia coli (UPEC), Klebsiella pneumoniae and Enterococcus faecalis. While all three species invaded the bladder cells, under flow conditions the Gram-positive E. faecalis was significantly less invasive compared to the Gram-negative UPEC and K. pneumoniae. When introduced simultaneously during an infection experiment, all three bacterial species sometimes invaded the same bladder cell, at differing frequencies suggesting complex interactions between bacterial species and bladder cells. Inside host cells, we observed encasement of E. faecalis colonies specifically by UPEC. During subsequent dispersal from the host cells, only the Gram-negative bacteria underwent infection-related filamentation (IRF). Taken together, our data suggest that bacterial multispecies invasions of single bladder cells are frequent and support earlier studies showing intraspecies cooperation on a biochemical level during UTI.