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

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.

Integration of microfluidics and multimodal optics for translational bioanalytics

Daniel Migliozzi

Numerous analytical techniques for life sciences have been at the basis of the improvement of medical diagnostics and treatments. Current challenges in bioanalytics are related to the conception of methods enabling large amount of information to be extracted from biological samples, in order to be translated into concrete solutions for the patient. From this point of view, microfluidics constitutes a key element to automate, accelerate and standardize bioanalytical tests. At the same time, obtaining precise information from the analyte of interest is crucial to specifically address diagnoses and therapies. Since the high flexibility of optical readouts can give access to a multitude of diverse aspects of biological matter in multiplexed fashion, in this thesis we have chosen to integrate microfluidics and multimodal optics to address specific problems in translational bioanalytics: drug screening and tumor diagnostics. In the first part of the work, we approached the problem of drug screening on living organisms, and especially the possibility to provide as much information as possible on potential mechanisms of action and clinical targets. For this, we aimed at developing a method to study the model organism Caenorhabditis elegans, i.e. a small soil worm that is widely used by researchers to study physiopathology at whole-organism scale. In fact, while offering the systemic information that is missing with cellular models, this small organism is much easier to handle and grow in large amounts compared to rodents, for which the costs and the ethical concerns are substantial. Moreover, its large genetic homology with humans supports translational analysis and its genetic manipulation is well mastered to model human diseases. Thus, we developed a microfluidic array to select small separated populations of C. elegans specimens, and combined fluorescence and bright-field imaging along with high-throughput feature recognition and signal detection to identify the mode-of-action of an antibiotic. Also, we demonstrated real-time, very large field-of-view capability on multiplexed motility assays for the assessment of the dose-response relation of an anesthetic. Furthermore, to enable on-site antibiotic testing, we explored a method to empower low-magnification optical systems with dielectric micro- and macro-spheres for the detection of pathogens in liquid media. Such dielectric objects offer potential solutions to increase the resolution and the sensitivity of imaging systems in a cost-effective fashion. We first characterized the parameters that govern the resolution increase in the case of microsphere-assisted incoherent microscopy and demonstrated it by imaging silicon microstructures and fluorescent bacteria. Then, we developed a method to easily fabricate microfluidic chips that integrate custom designs and precise location of highly-diffractive macro-spheres, and used it for on-chip detection of bacteria in a flow. In the second part of the work, we aimed at developing a technique to assess the status of individual tumor and immune cells in the conventional tissue sections used in the clinics. The first step was to combine fluorescence-based image segmentation with automated microfluidics to quantify, at the single-cell level, the overexpression of two proteins used as biomarkers for breast cancer diagnostics. We first assessed our method on biological controls such as breast cancer cell lines, and then combined it with ...

EPFL2019

Microfluidic culture platforms for C. elegans bacterial interaction and digestion studies at single-organism resolution

Vittorio Viri

Increasing interest in understanding fundamental biological processes, among which aging and nutrition, has led to the study of simple and powerful model organisms, as the round-worm Caenorhabditis elegans (C. elegans). The nematode, selected by Sydney Brenner , shares slightly over one-third of its genetic base code with human beings, making it an optimal model organism for many human related studies. It has been established that bacteria, besides being the principal nutrition source for these nematodes, play an active role in their aging process. In later life stage, i.e. after the larval development, the bacterial grinder of the worms loses efficiency permitting viable bacteria to reach and colonize the gut of the nematodes . Intestinal persistency of bacteria is highly related both to the strain of the hosted organism and of the host; in the first case the pathogenicity of bacteria determines colonization whereas in the second case the speed of the ingestion and digestion mechanism adopted by the host is one of the causes that can determine the occurrence of a condition of illness. Conventional methods commonly employed by biologists for C. elegans observation, widely applied in studies of interaction between worms and bacteria, food intake and processing analysis, result in some cases to be time consuming and difficult to operate. In order to solve these study related issues, such as observations of digestion related pheno-types on individual worms during the entire developmental time and reproducible food stimulation, microfluidics techniques, based on control of microliters of fluid in a laminar flow regime conditions, revealed to be useful for a consistent increase of the overall accuracy. Technical improvement in research and new analytical tools allowed single worm reversible immobilization and imaging and high throughput analysis, but gathering phenotypic information at single-cell or individual organism level, with higher throughput and better precision than conventional methods, still remains challenging. Combining the multiple technical advantages brought by microfluidics to the study of C. elegans, we focus our attention in developing new devices and protocols aiming to investigate how microbiota interact with the host organism. In particular, we intend to develop a novel microfluidic approach for performing long-term live imaging, bacterial processing and standardized food intake assays in an automated way. Worms sorting and immobilization by passive PDMS structures and accurate food concentration dispens-ing will be accomplished. We intend to obtain quantitative measurement of C. elegans food digestion, intake and development during the adult stage first and then in all stages, showing how digestion process, food intake and bacterial hosting is highly correlated with multiple characterizing phenotypes. To obtain food absorption measurement at single worm resolution, we apply an optical method of quantification using green fluorescent protein (GFP) labeled E. coli bacteria together with fluorescent microscopy techniques. Feeding behaviour and physiological digestion response under different exposure conditions will be tested in order to investigate how these factors impact on the development of the organism.

EPFL2021

Microfluidic culture platforms for C. elegans bacterial interaction and digestion studies at single-organism resolution

Vittorio Viri

EPFL2021

Integration of microfluidics and multimodal optics for translational bioanalytics

Daniel Migliozzi

EPFL2019