Detecting bacteria and determining their susceptibility to antibiotics by stochastic confinement in nanoliter droplets using plug-based microfluidics

Abstract

This article describes plug-based microfluidic technology that enables rapid detection and drug susceptibility screening of bacteria in samples, including complex biological matrices, without pre-incubation. Unlike conventional bacterial culture and detection methods, which rely on incubation of a sample to increase the concentration of bacteria to detectable levels, this method confines individual bacteria into droplets nanoliters in volume. When single cells are confined into plugs of small volume such that the loading is less than one bacterium per plug, the detection time is proportional to plug volume. Confinement increases cell density and allows released molecules to accumulate around the cell, eliminating the pre-incubation step and reducing the time required to detect the bacteria. We refer to this approach as ‘stochastic confinement’. Using the microfluidic hybrid method, this technology was used to determine the antibiogram — or chart of antibiotic sensitivity — of methicillin-resistant Staphylococcus aureus (MRSA) to many antibiotics in a single experiment and to measure the minimal inhibitory concentration (MIC) of the drug cefoxitin (CFX) against this strain. In addition, this technology was used to distinguish between sensitive and resistant strains of S. aureus in samples of humanblood plasma. High-throughput microfluidic techniques combined with single-cell measurements also enable multiple tests to be performed simultaneously on a single sample containing bacteria. This technology may provide a method of rapid and effective patient-specific treatment of bacterial infections and could be extended to a variety of applications that require multiple functional tests of bacterial samples on reduced timescales.

Encapsulation of single cells into monodisperse droplets by fluorescence-activated droplet formation on a microfluidic chip

“…In addition to compartmentalizing reactions, droplet-based microfluidics can also be used to encapsulate prokaryotic [14][15][16][17][18] and eukaryotic cells [19][20][21][22][23], and even the embryos of multicellular organisms [24,25], which opens up a new avenue for cell analysis. Recently, Brouzes et al [19] developed a droplet-based viability assay that permitted quantitative analysis of cell viability and growth within compartmentalized aqueous droplets.…”

Microfluidics for cell-based high throughput screening platforms—A review

“…Shortening the diagnosis time to identify specific antibiotics to treat bacterial infections could decrease the patient mortality, and reduce the cost of patient treatment. Boedicker et al [60] described a plug-based microfluidic technique that enabled to characterize the drug sensitivity of bacteria in samples and measure the minimal inhibitory concentration of antibiotics. By confining the cells in nanoliter droplets, the cell density was increased without preincubation and the time required to detect the bacteria was reduced.…”

Optofluidic ultrahigh-throughput detection of fluorescent drops

“…The optical detection of fluorescence signal is commonly performed in a serial manner, where drops are injected into a funnelshaped microchannel consisting of a narrow constriction which forces the drops to arrange in a single file, and to ensure that drops enter the detection region one at a time. [2][3][4][5][6]9,10 We have recently shown that the throughput of the serial interrogation process is limited by the rate at which droplets become unstable and undergo undesirable break-up as they flow through the constriction. 11 To achieve a droplet break-up rate of less than 1%, the maximum throughput was approximately 7000 drops s −1 for 40 pL drops.…”

“…[1][2][3][4][5][6][7] It is now possible to generate and manipulate droplets at kilohertz speeds. 8 In many biochemical assays, fluorescence is used as a read-out for the reactions occurring inside the drops, and can indicate the presence of cells or molecules of interest.…”

“…8 In many biochemical assays, fluorescence is used as a read-out for the reactions occurring inside the drops, and can indicate the presence of cells or molecules of interest. In some applications where the sample is compartmentalized at a limiting dilution such that each drop contains one or zero cell or molecule, the number of fluorescent drops directly quantifies the concentration of bacteria present, 4,5 or the amount of DNA mutation. 2 Ability to enumerate fluorescent drops in a high throughput manner is thus advantageous for the rapid detection of various diseases such as sepsis or cancer.…”