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Water-in-oil droplet microfluidics promises capacity for high-throughput single-cell antimicrobial susceptibility assays and investigation of drug resistance mechanisms. Every droplet must serve as an isolated environment with a controlled antibiotic concentration in such assays. While technologies for generation, incubation, screening, and sorting droplets mature, predictable retention of active molecules inside droplets remains a major outstanding challenge. Here, we analyzed 36 descriptors of the antibiotic molecules against experimental results on the cross-talk of antibiotics in droplets. We show that partition coefficient and fractional polar surface area are the key physicochemical properties that predict antibiotic retention. We verified the prediction by monitoring growth inhibition by antibiotic-loaded neighboring droplets. Our experiments also demonstrate that transfer of antibiotics between droplets is concentration- and distance-dependent. Our findings immediately apply to designing droplet antibiotic assays and give deeper insight into the retention of small molecules in water-in-oil emulsions.

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.

Application of droplet-based methods enables (i) faster detection, (ii) increased sensitivity, (iii) characterization of the level of heterogeneity in response to antibiotics by bacterial populations, and (iv) expanded screening of the effectiveness of antibiotic combinations. Hereby, we discuss the key steps and parameters of droplet-based experiments to investigate antimicrobial resistance. We also review recent findings accomplished with these methods and highlight their advantages and capacity to yield new insights into the problem of antimicrobial resistance.

We demonstrate the utility of non-contact printing to fabricate the mAST&mdash, an easy-to-operate, microwell-based microfluidic device for combinatorial antibiotic susceptibility testing (AST) in a point-of-care format. The wells are prefilled with antibiotics in any desired concentration and combination by non-contact printing (spotting). For the execution of the AST, the only requirements are the mAST device, the sample, and the incubation chamber. Bacteria proliferation can be continuously monitored by using an absorbance reader. We investigate the profile of resistance of two reference Escherichia coli strains, report the minimum inhibitory concentration (MIC) for single antibiotics, and assess drug&ndash, drug interactions in cocktails by using the Bliss independence model.

A method to monitor the level of oxygen in microdroplets is presented. Optical sensor nanoparticles are dispersed in the aqueous phase of the microfluidic droplets for culturing bacteria. The oxygen sensor nanoparticles consist of phosphorescent indicator dye embedded in poly(styrene-block-vinylpyrrolidone) nanobeads. The nanoparticles are excitable by red light and emit in the near-infrared spectra region which minimizes background fluorescence from biological matter. The biocompatibility of the nanoparticles was proven. Nanoparticles sensors were read out by adapted miniaturized oxygen meters. The instruments can be easily integrated into the microfluidic system by placing it next to the tubing and measuring through the tubing wall. The phosphorescence lifetime-based measurement circumvents the drawbacks of intensity-based measurements and enables the determination of the absolute oxygen concentration in individual moving droplets. The technique can also be used for monitoring the growth of bacteria in microdroplets. We demonstrate simultaneous measurement of concentration of oxygen and optical density (OD) from micro cultures of E. coli and M. smegmatis.

This paper proves that dodecylresorufin (C12R) outperforms resorufin (the conventional form of this dye) in droplet microfluidic bacterial assays. Resorufin is a marker dye that is widely used in different fields of microbiology and has increasingly been applied in droplet microfluidic assays and experiments. The main concern associated with resorufin in droplet-based systems is dye leakage into the oil phase and neighboring droplets. The leakage decreases the performance of assays because it causes averaging of the signal between the positive (bacteria-containing) and negative (empty) droplets. Here we show that C12R is a promising alternative to conventional resorufin because it maintains higher sensitivity, specificity, and signal-to-noise ratio over time. These characteristics make C12R a suitable reagent for droplet digital assays and for monitoring of microbial growth in droplets.

Heteroresistance is a phenomenon where isogenic bacteria population exhibits a diverse antibiotic resistance pattern at sub-population or single cell level. The sub-populations with higher resistance can remain undetected with conventional diagnostics which makes them subsequently harder to treat. Such surviving phenotypically heterogeneous sub-populations are also a potential hotbed for novel mutations, thus increasing the resistance permanently in bacteria. Droplet microfluidics gives tools for high-throughput analysis of bacteria and their response to antibiotics at single cell level, which is difficult to obtain with traditional agar plate technologies. In here we show for the first time the precise digital quantification of drug resistance profile in isogenic population at single cell level. We also see that the inhibiting amount of drug per bacteria remains quite stable regardless of bacteria density. Interestingly, the bacteria clump together preferably near these sub-inhibitory conditions. The technology and findings we describe here provide novel quantitative insight into the heteroresistance which is a key step in understanding the pathways leading to drug resistance. This knowledge is crucial in the context of global drug resistance threat as it can help us to find tools to prevent further escalation of drug resistance.

Standard digital assays need a large number of compartments for precise quantification of a sample over a broad dynamic range. We address this issue with an optimized droplet digital approach that uses a drastically reduced number of compartments for quantification. We generate serial logarithmic dilutions of an initial bacterial sample as an array of microliter-sized droplet plugs. In a subsequent step, these droplets are split into libraries of nanoliter droplets and pooled together for incubation and analysis. We show that our technology is at par with traditional dilution plate count for quantification of bacteria, but has the advantage of simplifying the experimental setup and reducing the manual workload. The method also has the potential to reduce the assay time significantly.

The in vitro antifungal potency of six series of 4-arylthiosemicarbazides was evaluated. Two isoquinoline derivatives with an ortho-methoxy or ortho-methyl group at the phenyl ring were the most potent antifungal agents. Molecular modeling studies and docking of all 4-arylthiosemicarbazides into the active sites of sterol 14α-demethylase (CYP51), topoisomerase II (topo II), l-glutamine: d-fructose-6-phosphate amidotransferase (GlcN-6-P), secreted aspartic proteinase (SAP), N-myristoyltransferase (NMT), and UDP-N-acetylmuramoyl-l-alanine:d-glutamate ligase (MurD) indicated the importance of both structural and electronic factors in ligand recognition and thus for the antifungal effectiveness of 4-arylthiosemicarbazides. A possible antifungal target was identified (NMT) and isoquinoline-thiosemicarbazides showed more favorable affinity than the native ligand. Figure Electrostatic potential surface of isoquiniline derivative compound 6o with antifungal activity Electronic supplementary material The online version of this article (doi:10.1007/s00894-012-1420-5) contains supplementary material, which is available to authorized users.

The alarming dynamics of antibiotic-resistant infections calls for the development of rapid and point-of-care (POC) antibiotic susceptibility testing (AST) methods. Here, we demonstrated the first completely stand-alone microfluidic system that allowed the execution of digital enumeration of bacteria and digital antibiograms without any specialized microfluidic instrumentation. A four-chamber gravity-driven step emulsification device generated ∼2000 monodisperse 2 nanoliter droplets with a coefficient of variation of 8.9% of volumes for 95% of droplets within less than 10 minutes. The manual workload required for droplet generation was limited to the sample preparation, the deposition into the sample inlet of the chip and subsequent orientation of the chip vertically without an additional pumping system. The use of shallow chambers imposing a 2D droplet arrangement provided superior stability of the droplets against coalescence and minimized the leakage of the reporter viability dye between adjacent droplets during long-term culture. By using resazurin as an indicator of the growth of bacteria, we were also able to reduce the assay time to ∼5 hours compared to 20 hours using the standard culture-based test.