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

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

National University of Singapore

Urinary tract infections (UTIs) are a major infection of humans, particularly affecting women. Recurrent UTIs can cause significant discomfort and expose patients to high levels of antibiotic use, which in turn contributes to the development of higher antibiotic resistance rates. Most UTIs are caused by uropathogenic , which is able to form intracellular collections (termed intracellular bacterial communities [IBCs]) within the epithelial cells lining the bladder lumen. IBCs are seen in both infected mice and humans and are a potential cause of recurrent UTI. Genetic and molecular studies of IBCs have been hampered both by the low number of bacteria in IBCs relative to the number extracellular bacteria and by population bottlenecks that occur during IBC formation. We now report the development of a simple and rapid technique for isolating pure IBCs from experimentally infected mice. We verified the specificity and purity of the isolated IBCs via microscopy, gene expression, and culture-based methods. Our results further demonstrated that our isolation technique practically enables specific molecular studies of IBCs. In the first such direct measurement, we determined that a single epithelial cell containing an early IBC typically contains 10 viable bacteria. Our isolation technique complements recent progress in low-input, single-cell genomics to enable future genomic studies of the formation of IBCs and their activation pathways during recurrent UTI, which may lead to novel strategies to eliminate them from the bladder.

Purification of Intracellular Bacterial Communities during Experimental Urinary Tract Infection Reveals an Abundant and Viable Bacterial Reservoir.

A novel L-glutamate-gated anion channel (IscaGluCl1) has been cloned from the black-legged tick, Ixodes scapularis, which transmits multiple pathogens including the agents of Lyme disease and human granulocytic anaplasmosis. When mRNA encoding IscaGluCl1 was expressed in Xenopus laevis oocytes, we detected robust 50-400 nA currents in response to 100 μM L-glutamate. Responses to L-glutamate were concentration-dependent (pEC 3.64 ± 0.11). Ibotenate was a partial agonist on IscaGluCl1. We detected no response to 100 μM aspartate, quisqualate, kainate, AMPA or NMDA. Ivermectin at 1 μM activated IscaGluCl1, whereas picrotoxinin (pIC 6.20 ± 0.04) and the phenylpyrazole fipronil (pIC 6.90 ± 0.04) showed concentration-dependent block of the L-glutamate response. The indole alkaloid okaramine B, isolated from fermentation products of Penicillium simplicissimum (strain AK40) grown on okara pulp, activated IscaGluCl1 in a concentration-dependent manner (pEC 5.43 ± 0.43) and may serve as a candidate lead compound for the development of new acaricides.

Kristin Lees

Division of Infectious Diseases,
Department of Medicine,
Yong Loo Lin School of Medicine,
Division of Infectious Diseases, Department of Medicine, Yong Loo Lin School of Medicine

National University of Singapore

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

Kristin Lees

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Division of Infectious Diseases,Department of Medicine,Yong Loo Lin School of Medicine,Division of Infectious Diseases, Department of Medicine, Yong Loo Lin School of Medicine

National University of Singapore

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

Division of Infectious Diseases,
Department of Medicine,
Yong Loo Lin School of Medicine,
Division of Infectious Diseases, Department of Medicine, Yong Loo Lin School of Medicine