Determining the phenotype and genotype of single cells is central to understand microbial evolution. DNA sequencing technologies allow the detection of mutants at high resolution, but similar approaches for phenotypic analyses are still lacking. We show that a drop-based millifluidic system enables the detection of heritable phenotypic changes in evolving bacterial populations. At time intervals, cells were sampled and individually compartmentalized in 100 nL drops. Growth through 15 generations was monitored using a fluorescent protein reporter. Amplification of heritable changes–via growth–over multiple generations yields phenotypically distinct clusters reflecting variation relevant for evolution. To demonstrate the utility of this approach, we follow the evolution of Escherichia coli populations during 30 days of starvation. Phenotypic diversity was observed to rapidly increase upon starvation with the emergence of heritable phenotypes. Mutations corresponding to each phenotypic class were identified by DNA sequencing. This scalable lineage-tracking technology opens the door to large-scale phenotyping methods with special utility for microbiology and microbial population biology.

http://dx.doi.org/10.1371/journal.pone.0152395

Microbiology has continuously pushed efforts towards understanding microbial diversity. Technologies and methods have also evolved, from plating, and use of mi- croscopes and cytometers, towards micro-well handling robots and, finally, fluidic- based devices. The aim of this review was to bring microbiologists attention to the outstanding analytical and handling power of millifluidic droplet technologies for analysing and sorting phenotypic diversity in the microbial world. This new format overcomes many limitations of previous approaches. It provides outstanding repro- ducible growth conditions over droplet reservoirs allowing unprecedented sensitive read-out over thousands of colonies over time. The confinement of the millifluidic train within tubes and the implementation of a three phases format excludes any con- tamination issues. The automation and handling of reservoir droplets is inherently facilitated. We show as a proof of principle the efficiency of capturing phenotypic diversity within a bacterial sample submitted to a sub-minimum inhibitory con- centration of antibiotic. The precision offered by the millifluidic format allows the detection of a variety of resistance strategies that compete and coexist. The review finally explores the potential of this approach to address new challenges such as community-based growth of multiple-strain systems.

To address possible cell-to-cell heterogeneity in growth dynamics of isogenic cell popula- tions of Chlamydomonas reinhardtii, we developed a millifluidic drop-based device that not only allows the analysis of populations grown from single cells over periods of a week, but is also able to sort and collect drops of interest, containing viable and healthy cells, which can be used for further experimentation. In this study, we used isogenic algal cells that were first synchronized in mixotrophic growth conditions. We show that these synchronized cells, when placed in droplets and kept in mixotrophic growth conditions, exhibit mostly homoge- neous growth statistics, but with two distinct subpopulations: a major population with a short doubling-time (fast-growers) and a significant subpopulation of slowly dividing cells (slow- growers). These observations suggest that algal cells from an isogenic population may be present in either of two states, a state of restricted division and a state of active division. When isogenic cells were allowed to propagate for about 1000 generations on solid agar plates, they displayed an increased heterogeneity in their growth dynamics. Although we could still identify the original populations of slow- and fast-growers, drops inoculated with a single progenitor cell now displayed a wider diversity of doubling-times. Moreover, popula- tions dividing with the same growth-rate often reached different cell numbers in stationary phase, suggesting that the progenitor cells differed in the number of cell divisions they could undertake. We discuss possible explanations for these cell-to-cell heterogeneities in growth dynamics, such as mutations, differential aging or stochastic variations in metabolites and macromolecules yielding molecular switches, in the light of single-cell heterogeneities that have been reported among isogenic populations of other eu- and prokaryotes.

Bacteria play a major role in our environment. Their ubiquity comes from their amazing ability to adapt to environmental conditions. Understand their adaptability can help to cure in fections or to use bacteria for biotechnology. The diversity of phenotypes within a population - different observable characters - and the diversification mechanisms contribute to the bacterial adaptability. In order to study the adaptation of bacteria, we followed the temporal evolution of diversity within populations of Escherichia coli through environmental transition. We get a picture of the diversity with a phenotyping tool in millifluidic allowing parallel acquisition of one thousand the bacteria growth curves. Through three examples, we show the potential of this approach to read the phenotypes within a population and we explore the roles of diversification and Darwinian selection on the dynamics of adaptation observed. The phase variation of the ag43 gene and the exposure to a non lethal concentration of antibiotic illustrate a first phase of adaptation governed by the competition between the phenotypes with the highest apparition probabilities. Finally we study the adaptation of a population in a depleted environment for thirty days. After fifteen days we observe a convergence towards a single phenotype. This determinism is common to all three examples. However, the end of the experiment is unpredictable: the observation is different for each repetition. It corresponds to a second phase of adaptation which is dominated by the rare appearance of beneficial mutations.

En 10 ans le coût du séquençage d’un génome a été divisé par 10 000. Alors qu’il fallait trois ans pour séquencer le génome humain il ne faut plus que 10 jours avec les séquenceurs de dernière génération. Ces progrès technologiques sur la « lecture » du code génétique ont des répercussions sur de nombreux domaines de la biologie.

L'utilisation des antibiotiques est un procédé curatif très courant (parfois trop), utilisé contre les infections microbiennes. La désinfection d'agents pathogènes est précédée d'études portant sur l'action des antibiotiques à l'égard des bactéries. Les micro-organismes possèdent naturellement une résistance à un sous dosage en antibiotique. Il existe une concentration minimale en antibiotique, relative à chaque espèce microbienne, à partir de laquelle il est possible d'inhiber les organismes. Cette concentration seuil est appelée la Concentration Minimale Inhibitrice (CMI), c'est un critère essentiel à la détermination de la posologie d'un antibiotique.En ce qui concerne la détermination de la CMI d'un antibiotique sur un organisme, les méthodes de mesures se révèlent fastidieuses à mettre en place, et possèdent une faible résolution. Ces méthodes ont très peu évolué depuis leur création, seules des machineries automatisées ont pu faciliter les manipulations. L'utilisation de ces outils révèle que la CMI d'un antibiotique sur un organisme, dépend de la densité initiale de la population bactérienne. Mais surtout que cette dépendance n'est pas linéaire.

Au cours de ma thèse, nous avons développé un procédé expérimental permettant de mesurer la CMI d'un antibiotique. Le dispositif est fondé sur l'utilisation de la milli-fluidique pour concevoir des gouttes d'émulsion, qui sont les échantillons de mesures gérés de manière automatisée. L'utilisation de cette méthodologie a été réservée à la conception d'un gradient de concentration en antibiotique. Chaque échantillon, d'un train d'un millier de gouttes, contenant une quantité en antibiotique différente de sa voisine.Ce procédé donne la faculté de simplifier l'échantillonnage par rapport aux méthodes usuelles. Il permet de contrôler de manière précise l'environnement direct des cellules, et également d'identifier chaque échantillon. Donnant ainsi la capacité d'observer la dynamique de croissance dans les gouttes, et l'influence des paramètres environnementaux. La machine ainsi utilisée a permis d'affirmer et de compléter les informations de dépendance non linéaire entre la CMI et la densité initiale de cellules pour un couple antibiotique/bactérie.

We present a novel millifluidic droplet analyser (MDA) for precisely monitoring the dynamics of microbial populations over multiple generations in numerous (>10³) aqueous emulsion droplets (~100 nL). As a first application, we measure the growth rate of a bacterial strain and determine the minimal inhibitory concentration (MIC) for the antibiotic cefotaxime by incubating bacteria in a fine gradient of antibiotic concentrations. The detection of cell activity is based on the automated detection of an epifluorescent signal that allows the monitoring of microbial populations up to a size of ~10⁶ cells. We believe that this device is helpful for the study of population dynamic consequences of microbe-environment interactions and of individual cell differences. Moreover, the fluidic machine may improve clinical tests, as it simplifies, automates and miniaturizes the screening of numerous microbial populations that grow and evolve in compartments with a finely tuned composition.