Digital Quantification of DNA Replication and Chromosome Segregation Enables Determination of Antimicrobial Susceptibility after only 15†Minutes of Antibiotic Exposure.

Rapid antimicrobial susceptibility testing (AST) would decrease misuse and overuse of antibiotics. The "holy grail" of AST is a phenotype-based test that can be performed within a doctor visit. Such a test requires the ability to determine a pathogen's susceptibility after only a short antibiotic exposure. Herein, digital PCR (dPCR) was employed to test whether measuring DNA replication of the target pathogen through digital single-molecule counting would shorten the required time of antibiotic exposure. Partitioning bacterial chromosomal DNA into many small volumes during dPCR enabled AST results after short exposure times by 1) precise quantification and 2) a measurement of how antibiotics affect the states of macromolecular assembly of bacterial chromosomes. This digital AST (dAST) determined susceptibility of clinical isolates from urinary tract infections (UTIs) after 15 min of exposure for all four antibiotic classes relevant to UTIs. This work lays the foundation to develop a rapid, point-of-care AST and strengthen global antibiotic stewardship.

Single-cell measurement of higher-order 3D genome organization with scSPRITE.

Although three-dimensional (3D) genome organization is central to many aspects of nuclear function, it has been difficult to measure at the single-cell level. To address this, we developed 'single-cell split-pool recognition of interactions by tag extension' (scSPRITE). scSPRITE uses split-and-pool barcoding to tag DNA fragments in the same nucleus and their 3D spatial arrangement. Because scSPRITE measures multiway DNA contacts, it generates higher-resolution maps within an individual cell than can be achieved by proximity ligation. We applied scSPRITE to thousands of mouse embryonic stem cells and detected known genome structures, including chromosome territories, active and inactive compartments, and topologically associating domains (TADs) as well as long-range inter-chromosomal structures organized around various nuclear bodies. We observe that these structures exhibit different levels of heterogeneity across the population, with TADs representing dynamic units of genome organization across cells. We expect that scSPRITE will be a critical tool for studying genome structure within heterogeneous populations.

Rapid pathogen-specific phenotypic antibiotic susceptibility testing using digital LAMP quantification in clinical samples.

Rapid antimicrobial susceptibility testing (AST) is urgently needed for informing treatment decisions and preventing the spread of antimicrobial resistance resulting from the misuse and overuse of antibiotics. To date, no phenotypic AST exists that can be performed within a single patient visit (30 min) directly from clinical samples. We show that AST results can be obtained by using digital nucleic acid quantification to measure the phenotypic response of Escherichia coli present within clinical urine samples exposed to an antibiotic for 15 min. We performed this rapid AST using our ultrafast (~7 min) digital real-time loop-mediated isothermal amplification (dLAMP) assay [area under the curve (AUC), 0.96] and compared the results to a commercial (~2 hours) digital polymerase chain reaction assay (AUC, 0.98). The rapid dLAMP assay can be used with SlipChip microfluidic devices to determine the phenotypic antibiotic susceptibility of E. coli directly from clinical urine samples in less than 30 min. With further development for additional pathogens, antibiotics, and sample types, rapid digital AST (dAST) could enable rapid clinical decision-making, improve management of infectious diseases, and facilitate antimicrobial stewardship.

A temporary immersion plant propagation bioreactor with decoupled gas and liquid flows for enhanced control of gas phase.

Temporary immersion bioreactors (TIBs) are being used to propagate superior plant species on a commercial scale. We demonstrate a new TIB design, a Hydrostatic-driven TIB (Hy-TIB), where periodic raising and lowering the media reservoir maintains the advantages of temporary immersion of plant tissues without requiring large amounts of gas to move the media that is a characteristic of other TIB designs. The advantage of utilizing low volumes of gas mixtures (that are more expensive than air) is shown by a doubling of the growth rate of plant root cultures under elevated (40%) oxygen in air, and with CO2 supplementation showing improved phototrophic and photomixotrophic growth of seedless watermelon meristem cultures. The development of this bioreactor system involved overcoming contamination issues associated with utilizing very low gas flow rates and included utilizing microchip pressure sensors to diagnose unexpected changes in internal bioreactor pressure (± 20 Pa ∼0.0002 atm) caused by flexing of non-rigid plastic bag vessels. The overall design seeks to achieve versatility, scalability and minimum cost such that bioreactor technology can play an increasing role in the critical need to improve plant productivity in the face of increasing demand for food, reduced resources, and environmental degradation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:337-345, 2016.