High-throughput screening of antibiotic-resistant bacteria in picodroplets

Abstract

The prevalence of clinically-relevant bacterial strains resistant to current antibiotic therapies is increasing and has been recognized as a major health threat. For example, multidrug-resistant tuberculosis and methicillin-resistant Staphylococcus aureus are of global concern. Novel methodologies are needed to identify new targets or novel compounds unaffected by pre-existing resistance mechanisms. Recently, water-in-oil picodroplets have been used as an alternative to conventional high-throughput methods, especially for phenotypic screening. Here we demonstrate a novel microfluidic-based picodroplet platform which enables high-throughput assessment and isolation of antibiotic-resistant bacteria in a label-free manner. As a proof-of-concept, the system was used to isolate fusidic acid-resistant mutants and estimate the frequency of resistance among a population of Escherichia coli (strain HS151). This approach can be used for rapid screening of rare antibiotic-resistant mutants to help identify novel compound/target pairs.

Emerging Microtechnologies and Automated Systems for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

“…149–154 In particular, combining bacterial culture enrichment in small-volume compartments with downstream genetic analysis, including PCR and sequencing, represents a great approach to obtain both phenotypic and molecular information. 61,72,155 …”

Application of genomic technologies to measure and monitor antibiotic resistance in animals

“…As one example, an agarose droplet–based microfluidic emulsion PCR was established with the ability to detect a single pathogenic E. coli O157:H7 cell in a high background of 100,000 excess normal K12 cells . As another example, a novel microfluidic‐based picodroplet platform was used to screen and isolate fusidic acid–resistant strains in a high‐throughput manner at an estimated frequency of resistance at 10 −8 ; further sequence analysis of fus A detected novel single‐nucleotide polymorphisms in isolated mutants . Single‐cell genomics can also help reveal the genomic mechanisms of heteroresistance and lend insight into the changes of bacterial metabolism under selection pressure …”

“…185 As another example, a novel microfluidic-based picodroplet platform was used to screen and isolate fusidic acid-resistant strains in a high-throughput manner at an estimated frequency of resistance at 10 −8 ; further sequence analysis of fusA detected novel single-nucleotide polymorphisms in isolated mutants. 196 Single-cell genomics can also help reveal the genomic mechanisms of heteroresistance and lend insight into the changes of bacterial metabolism under selection pressure. 172,197,198 Single-cell genomic methods have advanced rapidly in recent years and resulted in new discoveries that bulk genomic methodologies have failed to achieve.…”

Accelerating bacterial growth detection and antimicrobial susceptibility assessment in integrated picoliter droplet platform

“…The relatively large droplet volume of 4 nL in this work necessitated 7 h incubation for reliable growth detection. In a later work, Liu et al used smaller droplets of 330 pL for detecting a mutant resistant to fusidic acid, but required 8 h incubation due to a less-sensitive non-fluorescent detection approach based on light scattering (Liu et al, 2016). Notably, these techniques were demonstrated on experimental workflows requiring a separate device for droplet generation, an incubator for off-chip droplet incubation, and an additional device for droplet detection (Boedicker et al, 2008; Liu et al, 2009, 2016).…”

“…In a later work, Liu et al used smaller droplets of 330 pL for detecting a mutant resistant to fusidic acid, but required 8 h incubation due to a less-sensitive non-fluorescent detection approach based on light scattering (Liu et al, 2016). Notably, these techniques were demonstrated on experimental workflows requiring a separate device for droplet generation, an incubator for off-chip droplet incubation, and an additional device for droplet detection (Boedicker et al, 2008; Liu et al, 2009, 2016). To accelerate the analysis time for assessing antimicrobial susceptibility to the timescale of bacterial replication, an integrated workflow that ensures precise temporal control from single-bacteria confinement to detection must also be implemented.…”