Microfluidic droplet systems are popular analytical methods used in antimicrobial susceptibility testing (AST) to help study antimicrobial resistance (AMR). Droplets are unique systems that make it possible to analyze individual bacterial cells. Droplet methods have many advantages over conventional AST methods: They enable faster detection, they can be used to look at factors like heterogeneity, and they have higher sensitivity. In addition, you can analyze droplets in parallel, for a high-throughput way to test different antibiotic concentrations and combinations against various different bacterial strains. In essence, each droplet acts like a mini microreactor, so it’s a completely controlled environment. You can also overcome the inoculum effect (this is where the initial concentration of the bacteria can influence the measured minimum inhibitory concentration (MIC) value.
Droplet systems must meet certain criteria to be used in biological research. The aqueous phase (the sample) is mixed with oil containing a surfactant. The role of the surfactant is to protect against droplet coalescence. Additionally, the droplets must present a barrier to the transport of chemical ingredients. In fact, the transport of small molecules between droplets highly depends on the characteristics of these small molecules within the droplets.
Investigators at the Institute of Physical Chemistry in Poland used a cell-based reporter system and found a creative way to tag droplets using dyes so they could tell them apart. Droplets containing bacteria were prepared and encapsulated. The type of bacteria used was a fluorescent strain of E. coli and these droplets were blue due to blue fluorescent dye (Cascade Blue). Droplets containing antibiotics were red due to red fluorescent dye (Alexa Fluor). They mixed them in the same test tube and incubated overnight, essentially developing a color-coded method for the indirect analysis of antibiotic leakage in droplets. An inverted fluorescence microscope was used to image droplets in three channels: red, green, and blue (RGB). Thus, the droplets were essentially ‘barcoded’. They used microfluidic chips for first generating the droplets, and then detection. They imaged droplets at 3 different wavelengths: 405 nm for blue, 488 nm for green, and 635 nm for red.
If antibiotics migrating from neighbor droplets inhibited the bacterial replication in these encapsulated drops, the droplets themselves remained blue. If the transfer of antibiotics not occur in the emulsion, then these bacteria can happily grow and divide and produce a strong green signal (due to green fluorescent protein ie GFP).
Authors observed different responses of bacteria co-incubated with antibiotic-loaded droplets. For certain antibiotics, there was no ‘leakage’ ie no transfer of antibiotics between the droplet and thus the droplets remained blue. The other group of antibiotics did not decrease the fluorescence of bacteria in accompanying emulsions and thus resulted in no leakage and these droplets had an intensive green signal. There was also a weak green signal which they associated with the cross-talk of antibiotics between droplets.
Microfluidics is a powerful tool in antibiotic resistance research. Each droplet must not leak or take up the components of the assay. In this study, authors found a novel way to analyze the chemical factors that accelerate the escape of nonfluorescent reagents from droplets. They also looked at the physicochemical parameters of antimicrobial molecules themselves. They discovered that two main properties: a) partition coefficient (how a compound distributes itself between two different solvents like oil and water) and b) fractional polar surface area (a measure of the surface area that is polar) are good predictors for retention inside droplets.
Several antibiotics from TOKU-E were used in this research including:
Amikacin Sulfate, Chloramphenicol, Ciprofloxacin, Fosfomycin, Gentamicin Sulfate, Imipenem, Levofloxacin, Meropenem, Nitrofurantoin, Norfloxacin and Tobramycin Sulfate.
Authors found aminoglycosides to be nonleaky, while all quinolones, tetracyclines, and macrolides were leaky. Beta-lactams and glycopeptides had both leaky and nonleaky antibiotics present such that carbapenems and most of the cephems were nonleaky, while most of the penicillins showed potentially leaky parameters. Authors hope their study will open a discussion on two-phase microfluidic systems for susceptibility testing applications.
References
Ruszczak A, Jankowski P, Vasantham S, Scheler O and Garstiecki P (2023) Physicochemical properties predict retention of antibiotics in water-in-oil droplets. Anal. Chem. 95(2): 1574−1581 Link
Ruszczak A, Bartkova S, Zapotoczna M, Scheler O, and Garstecki P (2022) Droplet-based methods for tackling antimicrobial resistance. Curr. Opinion Biotech. 76: 102755 Link.