Approche quantitative de la croissance microbienne avec des colonies cylindriques de levures.

Microbes can form complex structures composed of millions to billions of cells. These assemblies contrast with the classical view we have of the “unicellulars” microbes. In fact, given their environment, they likely form heterogeneous connected structures. Our understanding of these assemblies is still scarce. The problem is that the models we develop suffer from the lack of experimental tools to understand these groups of cells. In this thesis, I propose to study they yeast Saccharomyces cerevisiae colonies by defining the flux of nutrients the colony receives. I use patterned filtration membranes intercalated between colonies and nutritive gel, leading to well controlled shapes. Using the cylindrical geometry resulting from a disc pattern, I first propose a quick study of the pillar organization, and then propose a simple model for colony growth in order to explain experimental growth in different environmental conditions, with respect to glucose levels, colony diameter and oxygen availability. I then discuss the biological relevance of this model with respect to cell division and nutrient absorption. To go further in investigations, I propose a automated measure of colony volume using a laser based measure with a 10 μm height precision. A microfluidic setup that mimics a two-dimensional colony growth is also proposed, where cells can be directly observed under a microscope.Les microorganismes peuvent former des structures complexes composées de millions ou milliards de cellules. Ces assemblées de cellules contrastent avec la vue classique que nous avons des microbes « unicellulaires ». En fait, étant donné leur environnements, ces microbes forment certainement des structures hétérogènes et connectées. Notre compréhension de ces assemblées reste incomplète, et les modèles que nous développons souffrent du manque de méthodes expérimentales pour comprendre ces groupes de cellules. Dans cette thèse, je propose d’étudier les colonies de levure Saccharomyces cerevisiae en imposant le flux de nutriments que la colonie reçoit. Pour cela, j’utilise des membranes de filtration modifiées que j’intercale entre les colonies et le gel nutritif pour contrôler la forme de ces colonies. En utilisant la géométrie cylindrique que l’on obtient avec un motif en forme de disque, j’étudie d’abord rapidement l’organisation du pilier obtenu, puis je propose un modèle simple de la croissance pour expliquer les données expérimentales obtenues dans différents environnements, avec une variation de la concentration en glucose, du diamètre des colonies ou de la présence d’oxygène. Je discute ensuite de la signification biologique de ce modèle en rapport à la division cellulaire et l’absorption des nutriments. Pour aller plus loin, je propose un suivi automatisé du volume des colonies en utilisant une mesure laser avec une précision en hauteur de 10 μm. Un système microfluidique qui mime une croissance bidimensionnelle de colonies est aussi proposé pour observer les cellules directement sous un microscope

Micropatterned Porous Membranes for Combinatorial Cell-Based Assays: Chapter 11

International audienceHere, we describe a protocol for producing micropatterned porous membranes which can be used for combinatorial cell-based assays. We use contact printing to pattern the surface of a porous filter membrane with a thin layer of polydimethylsiloxane (PDMS). This allows the porosity of the filter membrane to be altered at selected locations. Cells can be grown on one side of the filter membrane, while drugs and reagents can be deposited on the porous areas of the other side of the membrane. The reagents can diffuse through the pores of the membrane to the cells. The first part of the protocol describes how to design a stamp and use it to contact print PDMS. The second part describes how to create microprinted membranes for cell-based assays. The method is simple, highly customizable, can be performed at the bench, and can be used to perform combinatorial or time-dependent cell-based assays

Prolonged bacterial lag time results in small colony variants that represent a sub-population of persisters

Persisters are a subpopulation of bacteria that are not killed by antibiotics even though they lack genetic resistance. Here we provide evidence that persisters can manifest as small colony variants (SCVs) in clinical infections. We analyze growth kinetics of Staphylococcus aureus sampled from in vivo conditions and in vitro stress conditions that mimic growth in host compartments. We report that SCVs arise as a result of a long lag time, and that this phenotype emerges de novo during the growth phase in various stress conditions including abscesses and acidic media. We further observe that long lag time correlates with antibiotic usage. These observations suggest that treatment strategies should be carefully tailored to address bacterial persisters in clinics.ISSN:2041-172

Efficient microbial colony growth dynamics quantification with ColTapp, an automated image analysis application

© 2020, The Author(s). Populations of genetically identical bacteria are phenotypically heterogeneous, giving rise to population functionalities that would not be possible in homogeneous populations. For instance, a proportion of non-dividing bacteria could persist through antibiotic challenges and secure population survival. This heterogeneity can be studied in complex environmental or clinical samples by spreading the bacteria on agar plates and monitoring time to growth resumption in order to infer their metabolic state distribution. We present ColTapp, the Colony Time-lapse application for bacterial colony growth quantification. Its intuitive graphical user interface allows users to analyze time-lapse images of agar plates to monitor size, color and morphology of colonies. Additionally, images at isolated timepoints can be used to estimate lag time. Using ColTapp, we analyze a dataset of Staphylococcus aureus time-lapse images including populations with heterogeneous lag time. Colonies on dense plates reach saturation early, leading to overestimation of lag time from isolated images. We show that this bias can be corrected by taking into account the area available to each colony on the plate. We envision that in clinical settings, improved analysis of colony growth dynamics may help treatment decisions oriented towards personalized antibiotic therapies.ISSN:2045-232

Antibacterial Neutrophil Effector Response: Ex Vivo Quantification of Regulated Cell Death Associated with Extracellular Trap Release

Regulated cell death (RCD) and the concomitant release of extracellular traps by neutrophils (NETs) constitute an important antibacterial effector response. Usually, the dynamic processes of RCD and NETs release are assessed independently of each other by either unspecific or time-consuming methods. Here, we describe a flow cytometry-based high-throughput analysis method incorporating neutrophil RCD and NETs release with visual live-imaging conformation upon ex vivo bacterial challenge. This combined approach allows to quantify and closely follow the kinetics of the dynamic neutrophil effector response towards bacterial infection

A microfluidic device for inferring metabolic landscapes in yeast monolayer colonies

International audienc

C-di-AMP levels modulate Staphylococcus aureus cell wall thickness, response to oxidative stress, and antibiotic resistance and tolerance

Antibiotic resistance and tolerance are substantial healthcare-related problems, hampering effective treatment of bacterial infections. Mutations in the phosphodiesterase GdpP, which degrades cyclic di-3', 5'-adenosine monophosphate (c-di-AMP), have recently been associated with resistance to beta-lactam antibiotics in clinical Staphylococcus aureus isolates. In this study, we show that high c-di-AMP levels decreased the cell size and increased the cell wall thickness in S. aureus mutant strains. As a consequence, an increase in resistance to cell wall targeting antibiotics, such as oxacillin and fosfomycin as well as in tolerance to ceftaroline, a cephalosporine used to treat methicillin-resistant S. aureus infections, was observed. These findings underline the importance of investigating the role of c-di-AMP in the development of tolerance and resistance to antibiotics in order to optimize treatment in the clinical setting

Identification of individual cells from z-stacks of bright-field microscopy images

Obtaining single cell data from time-lapse microscopy images is critical for quantitative biology, but bottlenecks in cell identification and segmentation must be overcome. We propose a novel, versatile method that uses machine learning classifiers to identify cell morphologies from z-stack bright-field microscopy images. We show that axial information is enough to successfully classify the pixels of an image, without the need to consider in focus morphological features. This fast, robust method can be used to identify different cell morphologies, including the features of E. coli, S. cerevisiae and epithelial cells, even in mixed cultures. Our method demonstrates the potential of acquiring and processing Z-stacks for single-layer, single-cell imaging and segmentation.ISSN:2045-232

Wide lag time distributions break a trade-off between reproduction and survival in bacteria

Many microorganisms face a fundamental trade-off between reproduction and survival: Rapid growth boosts population size but makes microorganisms sensitive to external stressors. Here, we show that starved bacteria encountering new resources can break this trade-off by evolving phenotypic heterogeneity in lag time. We quantify the distribution of single-cell lag times of populations of starved Escherichia coli and show that population growth after starvation is primarily determined by the cells with shortest lag due to the exponential nature of bacterial population dynamics. As a consequence, cells with long lag times have no substantial effect on population growth resumption. However, we observe that these cells provide tolerance to stressors such as antibiotics. This allows an isogenic population to break the trade-off between reproduction and survival. We support this argument with an evolutionary model which shows that bacteria evolve wide lag time distributions when both rapid growth resumption and survival under stressful conditions are under selection. Our results can explain the prevalence of antibiotic tolerance by lag and demonstrate that the benefits of phenotypic heterogeneity in fluctuating environments are particularly high when minorities with extreme phenotypes dominate population dynamics.ISSN:0027-8424ISSN:1091-649