Droplet-based digital antibiotic susceptibility screen reveals single-cell clonal heteroresistance in an isogenic bacterial population.

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.

Simultaneous Measurement of Viscosity and Optical Density of Bacterial Growth and Death in a Microdroplet.

Herein, we describe a novel method for the assessment of droplet viscosity moving inside microfluidic channels. The method allows for the monitoring of the rate of the continuous growth of bacterial culture. It is based on the analysis of the hydrodynamic resistance of a droplet that is present in a microfluidic channel, which affects its motion. As a result, we were able to observe and quantify the change in the viscosity of the dispersed phase that is caused by the increasing population of interacting bacteria inside a size-limited system. The technique allows for finding the correlation between the viscosity of the medium with a bacterial culture and its optical density. These features, together with the high precision of the measurement, make our viscometer a promising tool for various experiments in the field of analytical chemistry and microbiology, where the rigorous control of the conditions of the reaction and the monitoring of the size of bacterial culture are vital.

Continuous Recirculation of Microdroplets in a Closed Loop Tailored for Screening of Bacteria Cultures.

Emerging microfluidic technology has introduced new precision controls over reaction conditions. Owing to the small amount of reagents, microfluidics significantly lowers the cost of carrying a single reaction. Moreover, in two-phase systems, each part of a dispersed fluid can be treated as an independent chemical reactor with a volume from femtoliters to microliters, increasing the throughput. In this work, we propose a microfluidic device that provides continuous recirculation of droplets in a closed loop, maintaining low consumption of oil phase, no cross-contamination, stabilized temperature, a constant condition of gas exchange, dynamic feedback control on droplet volume, and a real-time optical characterization of bacterial growth in a droplet. The channels (tubing) and junction cubes are made of Teflon fluorinated ethylene propylene (FEP) to ensure non-wetting conditions and to prevent the formation of biofilm, which is particularly crucial for biological experiments. We show the design and operation of a novel microfluidic loop with the circular motion of microdroplet reactors monitored with optical sensors and precision temperature controls. We have employed the proposed system for long term monitoring of bacterial growth during the antibiotic chloramphenicol treatment. The proposed system can find applications in a broad field of biomedical diagnostics and therapy.

Calibration-free assays on standard real-time PCR devices.

Quantitative Polymerase Chain Reaction (qPCR) is one of central techniques in molecular biology and important tool in medical diagnostics. While being a golden standard qPCR techniques depend on reference measurements and are susceptible to large errors caused by even small changes of reaction efficiency or conditions that are typically not marked by decreased precision. Digital PCR (dPCR) technologies should alleviate the need for calibration by providing absolute quantitation using binary (yes/no) signals from partitions provided that the basic assumption of amplification a single target molecule into a positive signal is met. Still, the access to digital techniques is limited because they require new instruments. We show an analog-digital method that can be executed on standard (real-time) qPCR devices. It benefits from real-time readout, providing calibration-free assessment. The method combines advantages of qPCR and dPCR and bypasses their drawbacks. The protocols provide for small simplified partitioning that can be fitted within standard well plate format. We demonstrate that with the use of synergistic assay design standard qPCR devices are capable of absolute quantitation when normal qPCR protocols fail to provide accurate estimates. We list practical recipes how to design assays for required parameters, and how to analyze signals to estimate concentration.

Designing and interpretation of digital assays: Concentration of target in the sample and in the source of sample.

We explain how to design classic digital assays, comprising identical partitions, in order to obtain the required precision of the estimate within a defined range of concentrations. The design, including the number and volume of partitions, depends significantly on whether the assay is to assess the concentration of the target analyte in the sample or in the source of the sample (e.g. a patient body) with a given precision. We also show how to translate the result referring to the concentration in the sample into the concentration in the source of the sample, including the significant change in the breath of the confidence intervals.

Rational design of digital assays.

Optimum algorithm for digital assays treats chemical compartments as bits of probabilistic information and arranges these bits in a fractional positional system. Maximization of information gain reduces, by orders of magnitude, the number of partitions required to achieve the requested dynamic range and precision of the assay. The method simplifies the execution of digital analytical methods providing for more accessible use of absolute quantization in research and in diagnostics.