An In Vivo Microfluidic Study of Bacterial Load Dynamics and Absorption in the C. elegans Intestine

Abstract

Caenorhabditiselegans (C. elegans) has gained importance as a model for studying host-microbiota interactions and bacterial infections related to human pathogens. Assessing the fate of ingested bacteria in the worm’s intestine is therefore of great interest, in particular with respect to normal bacterial digestion or intestinal colonization by pathogens. Here, we report an in vivo study of bacteria in the gut of C. elegans. We take advantage of a polydimethylsiloxane (PDMS) microfluidic device enabling passive immobilization of adult worms under physiological conditions. Non-pathogenic Escherichia coli (E. coli) bacteria expressing either pH-sensitive or pH-insensitive fluorescence reporters as well as fluorescently marked indigestible microbeads were used for the different assays. Dynamic fluorescence patterns of the bacterial load in the worm gut were conveniently monitored by time-lapse imaging. Cyclic motion of the bacterial load due to peristaltic activity of the gut was observed and biochemical digestion of E. coli was characterized by high-resolution fluorescence imaging of the worm’s intestine. We could discriminate between individual intact bacteria and diffuse signals related to disrupted bacteria that can be digested. From the decay of the diffuse fluorescent signal, we determined a digestion time constant of 14 ± 4 s. In order to evaluate the possibility to perform infection assays with our platform, immobilized C. elegans worms were fed pathogenic Mycobacterium marinum (M. marinum) bacteria. We analyzed bacterial fate and accumulation in the gut of N2 worms and mitochondrial stress response in a hsp-6::gfp mutant.

SICKO: Systematic Imaging ofCaenorhabditisKilling Organisms

“…The single-worm culture method isolates individual C. elegans on small NGM pads, allowing a more direct comparison to the majority of previous studies that use C. elegans to examine host-microbe interactions on solid media, and distinguishing this system from related methods that use microfluidic systems to monitor bacterial infection. 13 The enclosed chamber is humidified with water absorbing beads, allowing for long-term cultivation of animals for repeated sampling of infection data and monitoring of longevity ( Fig. 1c,d ).…”

High-resolution imaging and analysis of the intestinal bacterial load ofCaenorhabditis elegansduring early adulthood

“…Both of these phenotypes can be tracked in vivo using microuidic solutions. 21,28 Moreover, infection with bacteria can also damage the intestinal cells. For example, it has been reported that S. marcescens is a pathogen of the worm that over time causes vacuolation of the intestinal cells and reduces the volume of the intestinal epithelium.…”

“…In the more recent works by Viri et al , 27,28 the dynamics of bacterial transition, load and absorption in the gut of the worms are studied in an automated fashion through fluorescence imaging. The first microfluidic device 27 contained 4 channels, each of which comprised 5 consecutive chambers, separated by filter structures.…”

“…[22][23][24] However, only a few microuidic devices have been reported for studying the interactions between bacteria and C. elegans. [25][26][27][28] Yang et al, 25 fabricated a microuidic chip featuring 32 chambers arranged radially and 4 concentration gradient generators for evaluation of in vivo antimicrobial activity of several compounds. Each concentration generator supplies 8 unique chambers with 8 different concentrations of a given antibiotic.…”

Reciprocating intestinal flows enhance glucose uptake in C. elegans

“…The mean gut residence time is reportedly < 2 min; approximately 43 ± 10% of the maximum volume of the intestinal lumen is expelled through the anus during each activation of the DMP 22 . The estimated time constant needed for digestion of bacteria is 14 ± 4 s 24 . We previously reported that a C. elegans worm must eat at least three bacterial cells per second to survive for 3 days, which implies the consumption of hundreds of thousands of bacteria daily 39 .…”