Differential Effects of Uropathogenic and Non-Uropathogenic E. coli on the Mouse Urobiome and Urine NGAL Levels

Olivia Kathryn Lamanna,1,2 Rong Hu,3 Mark Khemmani,4 Alan J Wolfe,4 Suzanne L Groah,5 Catherine S Forster6,7 1Biomedical Research Institute, Rockville, MD, USA; 2Uniformed Services University, Bethesda, MD, USA; 3Department of Oncology, Georgetown University, Washington, DC, USA; 4Department of Microbiology and Immunology, Loyola University Chicago, Maywood, IL, USA; 5Department of Rehabilitative Medicine, MedStar National Rehabilitation Hospital, Washington, DC, USA; 6Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA, USA; 7University of Pittsburgh Medical Center, Children’s Hospital of Pittsburgh, Pittsburgh, PA, USACorrespondence: Catherine S Forster, University of Pittsburgh Medical Center, Children’s Hospital of Pittsburgh, 4401 Penn Ave, 3 rd Floor FP, Pittsburgh, PA, 15224, USA, Email [email protected]: To determine whether urine neutrophil gelatinase-associated lipocalin (uNGAL) or urobiome alterations can differentiate urinary tract infections (UTI) from asymptomatic bacteriuria (ASB).Methods: Female 8-week-old C57BL/6 mice were instilled with either Escherichia coli CFT073 (UTI model, n=12), E. coli 83972 (ASB model, n=12), or saline (control, n=3). uNGAL was measured daily for 3 days post-instillation. Urobiome composition was assessed pre- and post-instillation using 16S rRNA sequencing. At day 3, kidneys were harvested for culture. Comparisons were made across groups for uNGAL levels and urobiome diversity.Results: Baseline β diversity did not differ between groups. Post-instillation, β diversity significantly differed across groups (p=0.01), driven by increased relative abundance of E. coli in UTI mice compared to ASB mice. Median uNGAL levels increased significantly in both UTI and ASB groups relative to controls, but no significant difference was observed between UTI and ASB groups.Conclusion: Introduction of a uropathogenic E. coli strain reduced urobiome diversity, while a non-uropathogenic strain did not, suggesting strain-specific effects on microbial ecology. Bladder instillation itself also altered the urobiome. Elevated uNGAL levels were observed in both UTI and ASB models, indicating that while uNGAL reflects bacterial exposure, it does not distinguish between uropathogenic and non-uropathogenic E. coli. These findings highlight urobiome analysis as a potential tool for differentiating UTI from ASB, whereas uNGAL alone is insufficient.Keywords: urinary tract infection, biomarker, mice, microbiome, asymptomatic bacteriuria