Droplet microfluidics has rapidly emerged as one of the key technologies opening up new experimental possibilities in microbiology.

Since antibiotic resistance is a major threat to global health, recent observations that the traditional test of minimum inhibitory concentration (MIC) is not informative enough to guide effective antibiotic treatment are alarming. Bacterial heteroresistance, in which seemingly susceptible isogenic bacterial populations contain resistant sub-populations, underlies much of this challenge. To close this gap, here we developed a droplet-based digital MIC screen that constitutes a practical analytical platform for quantifying the single-cell distribution of phenotypic responses to antibiotics, as well as for measuring inoculum effect with high accuracy. We found that antibiotic efficacy is determined by the amount of antibiotic used per bacterial colony forming unit (CFU), not by the absolute antibiotic concentration, as shown by the treatment of beta-lactamase-carrying Escherichia coli with cefotaxime. We also noted that cells exhibited a pronounced clustering phenotype when exposed to near-inhibitory amounts of cefotaxime. Overall, our method facilitates research into the interplay between heteroresistance and antibiotic efficacy, as well as research into the origin and stimulation of heterogeneity by exposure to antibiotics. Due to the absolute bacteria quantification in this digital assay, our method provides a platform for developing reference MIC assays that are robust against inoculum-density variations.

There is a growing need for quick and reliable methods for microorganism detection and identification worldwide. Although traditional culture-based technologies are trustworthy and accurate at a relatively low cost, they are also time- and labor-consuming and are limited to culturable bacteria. Those weaknesses have created a necessity for alternative technologies that are capable for faster and more precise bacterial identification from medical, food or environmental samples. The most common current approach is to analyze the nucleic acid component of analyte solution and determine the bacterial composition according to the specific nucleic acid profiles that are present. This review aims to give an up-to-date overview of different nucleic acid target sequences and respective analytical technologies.

Measurement of antibiotic susceptibility at the level of single cells is important as it reveals the concentration of an antibiotic that leads to drug resistance in bacterial strains. To date, no solution for large-scale studies of antibiotic susceptibility at the single-cell level has been shown. Here, we present a method for production and separation of emulsions consisting of subnanoliter droplets that allows us to identify each emulsion by their spatial position in the train of emulsions without chemical barcoding. The emulsions of droplets are separated by a third immiscible phase, thus forming large compartments—tankers—each filled with an emulsion of droplet reactors. Each tanker in a train can be set under different reaction conditions for hundreds or thousands of replications of the same reaction. The tankers allow for long term incubation – needed to check for growth of bacteria under a screen of conditions. We use microfluidic tankers to analyze susceptibility to cefotaxime in ca. 1900 replications for each concentration of the antibiotic in one experiment. We test cefotaxime susceptibility for different initial concentrations of bacteria, showing the inoculum effect down to the level of single cells for more than a hundred single-cell events per tanker. Lastly, we use tankers to observe the formation of aggregates of bacteria in the presence of cefotaxime in the increasing concentration of the antibiotic. The microfluidic tankers allow for facile studies of the inoculum effect and antibiotic susceptibility, and constitute an attractive, label-free screening method for a variety of other experiments in chemistry and biology.