088 - Advancing Group B Streptococcus Research: Human Intestinal Organoids as a New Model for Neonatal Infections

Publication Number: 88.3964

Nichol John-Lewis Edwards, University of South Florida, Port Richey, FL, United States; Alexia N. Pearah, USF Health Morsani College of Medicine, Tampa, FL, United States; April Lindon, University of south florida, Tampa, FL, United States; Sophie Darch, University of South Florida, Tampa, FL, United States; Tara M. Randis, University of South Florida, Tampa, FL, United States

Background: Late-onset Group B Streptococcus (GBS) disease is a significant cause of infant morbidity and mortality. GBS intestinal colonization is a critical precursor to late-onset disease, but understanding the transition from colonization to invasive infection is challenging due to the lack of models that accurately reflect the neonatal intestinal environment. We have previously described the use of human tissue derived enteroids, however, this model lacks important stromal features relevant to GBS pathogenesis. To address this gap, we have developed a human intestinal organoid (HIO) model derived from induced pluripotent stem cells (iPSCs) that mimic the structural and functional characteristics of the neonatal intestine.

Objective: Our aim was to establish and optimize a human intestinal organoid model that closely replicates the neonatal intestinal environment to investigate the mechanisms of GBS colonization and progression to invasive infection.

Design/Methods: HIOs were derived from iPSCs and embedded in Matrigel to promote self-assembly. Apical-out HIOs were generated by reversing epithelial polarity using EDTA. On day three, the organoids were infected with GBS strain COH-1 at optical densities of 0.4 and 0.1, followed by a two-hour incubation at 37°C. Following GBS exposure, organoids were washed three times with PBS and fixed with paraformaldehyde. Antibodies against MUC2, villin, and GBS were applied for confocal scanning laser microscopy visualization. Images were analyzed using Imaris software.

Results: We captured the first images of GBS interacting with the apical surface of human intestinal organoids. Confocal microscopy revealed direct adherence of GBS to the epithelial cells' apical membrane, providing visual confirmation of colonization on the organoid surface. GBS was also observed within the cytoplasm of epithelial cells, suggesting GBS intracellular survival.

Conclusion(s): This novel organotypic model has significant potential to advance our understanding of GBS pathogenicity, particularly concerning neonatal intestinal colonization and progression to late-onset infection. We anticipate this model will enable us to delineate the pathways by which GBS traverses intestinal epithelial barriers (i.e. transcellular vs paracellular). Future work will focus on optimizing this model by incorporating important components, such as microfold cells, and developing an HIO monolayer co-culture system that includes innate immune cells to better recapitulate the complexity of the neonatal intestinal barrier.

GBS Adherence to Human Intestinal Organoids
Figure 1. Laser confocal scanning microscopy image of GBS adherence to apical-out epithelial border of human intestinal organoids A) at 63x mag B) 63x (zoomed in) C) 100x. Blue = nuclei, DAPI; magenta = GBS, anti-GBS IgG Alexa Fluor 568.