Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

Abstract

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

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

“…UPEC invasion of the urinary bladder generates substantial changes to bladder morphology and a robust immune response. Much of our current understanding of early stages of UTI and the intracellular lifestyle is derived from studies in the mouse model ( Anderson, 2003 ; Duraiswamy et al, 2018 ; Hannan et al, 2012 ; Hung et al, 2009 ; Justice et al, 2004 ; Schwartz et al, 2011 ; Yang et al, 2019 ). Examinations of mouse bladder explants via microscopy have revealed the formation of intracellular bacterial communities (IBCs) composed of thousands of bacteria within individual superficial bladder cells ( Justice et al, 2004 ).…”

Dynamic persistence of intracellular bacterial communities of uropathogenicEscherichia coliin a human bladder-chip model of urinary tract infections

“…UPEC invasion of the urinary bladder generates substantial changes to bladder morphology and a robust immune response. Much of our current understanding of early stages of UTI and the intracellular lifestyle is derived from studies in the mouse model (Anderson et al, 2003; Duraiswamy et al, 2018; Hannan et al, 2012; Hung et al, 2009; Justice et al, 2004; Schwartz et al, 2011; Yang et al, 2019). Examinations of mouse bladder explants via microscopy have revealed the formation of intracellular bacterial communities (IBCs) composed of thousands of bacteria within individual superficial bladder cells (Justice et al, 2004).…”