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Global Health Technologies

Global Health Technologies - Poster Session C & D

R3 - Development of Rapid Antimicrobial Susceptibility Testing Platform Using Hollow Core-Shell Microparticles

Friday, October 10, 2025
10:00 AM - 11:00 AM PDT
Location: Exhibit Hall F, G & H

Introduction: : Sepsis is a highly lethal condition responsible for a third of hospital deaths [1], and its treatment is further complicated by antimicrobial resistance (AMR), a global health crisis linked to 4.95 million deaths in 2019 and projected to cause 10 million deaths annually by 2050 [2,3]. AMR renders standard antimicrobial therapies ineffective, emphasizing the urgent need for swift de-escalation from empiric broad-spectrum antibiotics to targeted disease-specific antibiotics, improving patient outcomes and halting the global rise in AMR. In the clinic, this decision is typically informed using antimicrobial susceptibility testing (AST); however, existing AST methods have long turnaround times and low throughput, which delays timely interventions. We propose a novel hollow core-shell microparticle (PicoShell) AST platform that enables single-bacterium encapsulation and microscopy-based viability assessment to characterize resistance profiles in as few as 16 hours. Our results demonstrate successful bacterial encapsulation after a 12-hour preculture and detection of minimum inhibitory concentration (MIC) within 3-6 hours. Using an ampicillin dose panel (0-50 µg/mL), we determined a MIC of 10 µg/mL for wild-type E. coli, consistent with other reports [4], and observed uninhibited growth for strains resistant to ampicillin. Additionally, we leverage flow cytometry to quantify bacterial load in PicoShells, providing a label-free method for evaluating antimicrobial response and high-throughput screening based on microparticle granularity and size. This PicoShell-based rapid AST workflow offers faster results (16-18 hours) for informing early clinical decision-making, aiding antimicrobial stewardship efforts, and facilitating isolation of rare hyper-resistant pathogens for downstream investigation, all critical in addressing the global AMR crisis.

Materials and

Methods: : We present a proof-of-concept for a rapid, phenotypic AST workflow leveraging PicoShells, a hollow core-shell microparticle platform developed by the Di Carlo Lab that enables high-throughput encapsulation and phenotyping of cells using standard laboratory equipment [5]. To emulate clinically relevant scenarios, whole blood samples are spiked with E. coli to simulate infection and enriched via a 12-hour pre-culture step. The blood sample containing pathogen samples is separated from blood cells and platelets using centrifugation to isolate the pathogens in the plasma layer (Fig. 1C). Then, the bacteria are encapsulated into core-shell PicoShells using a microfluidic polydimethylsiloxane (PDMS) device that flows polyethylene glycol (PEG), dithiothreitol (DTT) crosslinker, pathogen-spiked dextran, and fluorinated oil with PicoSurf surfactant (Fig. 2A). The porous hydrogel shell facilitates small molecule diffusion and nutrient exchange, allowing encapsulated pathogens to proliferate into colonies and respond to antibiotics added in the culture medium (Fig. 2C). Single-bacterium PicoShells are then incubated in varying concentrations of antibiotics and monitored using brightfield microscopy. Finally, a fluorescence-activated cell sorting (FACS) based readout using a Sony Cell Sorter SH800 quantifies bacterial growth, providing a rapid assessment of pathogen load and bacterial susceptibility via the MIC in a clinically actionable workflow. The entire process delivers MIC results in as few as 16 hours, significantly faster than current gold-standard AST methods, which require 40 to 72 hours [6].

Results, Conclusions, and Discussions:: To demonstrate the clinical utility of our proposed rapid AST workflow, wild-type (WT) and ampicillin-resistant (AmpR) strains of E. coli were inoculated in blood culture, encapsulated in PicoShells, and treated with an ampicillin panel with concentrations ranging from 0 to 50 µg/mL. Encapsulated colonies were then incubated and imaged hourly to assess growth. The experimental endpoint was determined as the time at which multiple confluent PicoShells were observed (T=3 hours for the WT, T=6 hours for the AmpR), and representative PicoShells from each condition at this endpoint are pictured in Fig. 2E. Based on these results, an MIC of 10 µg/mL was estimated for the wild-type strain, as no PicoShells were showing observable growth at this concentration. No distinguishable growth inhibition was detected for the ampicillin-resistant strain. These results are in agreement with values reported in the literature, providing compelling evidence to differentiate between susceptible and inhibited growth [4].
Using FACS, we demonstrate a quantitative readout of the susceptibility of wild-type E. coli encapsulated in PicoShells. Bacterial growth inside PicoShells causes increased radius and granularity, which correlate with high forward and side scatter, respectively, on FACS plots. Minimal growth is observed above 10 µg/mL of ampicillin, aligning with the microscopy and literature MIC values (Fig. 3D) [4]. The number of PicoShells with viable bacterial colonies can be quantified based on their position in high-scatter regions. Specifically, we observed 60% bacteria-positive colonies in PicoShells in the 0 µg/mL control condition, and progressively lower percentages were observed at increasing ampicillin concentrations, confirming a dose-dependent reduction in growth.
Overall, we have developed a PicoShell-based rAST workflow that enables high-throughput, single-cell AMR characterization with FACS-based growth analysis. This platform provides MIC results approximately 30 hours faster than gold-standard AST methods, demonstrating the clinical potential of the platform to facilitate early, targeted antimicrobial therapy and prevent the overuse of broad-spectrum antibiotics. Furthermore, encapsulated single cells from whole blood samples can be cultured into distinct monoclonal populations, allowing rare resistant pathogens or pathogens exhibiting multidrug resistance to be more easily identified and isolated for downstream genotypic testing or research.

Acknowledgements and/or References (Optional): : [1] Rhee, C., Dantes, R., Epstein, L., Murphy, D. J., Seymour, C. W., Iwashyna, T. J., Kadri, S. S., Angus, D. C., Danner, R. L., Fiore, A. E., Jernigan, J. A., Martin, G. S., Septimus, E., Warren, D. K., Karcz, A., Chan, C., Menchaca, J. T., Wang, R., Gruber, S., & Klompas, M. (2017). Incidence and trends of sepsis in US hospitals using clinical vs claims data, 2009-2014. JAMA, 318(13), 1241. https://doi.org/10.1001/jama.2017.13836
[2] Murray, C. J., Ikuta, K. S., Sharara, F., Swetschinski, L., Robles Aguilar, G., Gray, A., Han, C., Bisignano, C., Rao, P., Wool, E., Johnson, S. C., Browne, A. J., Chipeta, M. G., Fell, F., Hackett, S., Haines-Woodhouse, G., Kashef Hamadani, B. H., Kumaran, E. A., McManigal, B., … Naghavi, M. (2022). Global burden of bacterial antimicrobial resistance in 2019: A systematic analysis. The Lancet, 399(10325), 629–655. https://doi.org/10.1016/s0140-6736(21)02724-0
[3] O’Neill, J. (2016). Tackling drug-resistant infections globally: final report and recommendations. Government of the United Kingdom. https://apo.org.au/node/63983
[4] Fino, C., Vestergaard, M., Ingmer, H., Pierrel, F., Gerdes, K., & Harms, A. (2020). PasT of Escherichia coli sustains antibiotic tolerance and aerobic respiration as a bacterial homolog of mitochondrial Coq10. MicrobiologyOpen, 9(8), e1064. https://doi.org/10.1002/mbo3.1064
[5] van Zee, M., de Rutte, J., Rumyan, R., Williamson, C., Burnes, T., Radakovits, R., Sonico Eugenio, A., Badih, S., Lee, S., Lee, D.-H., Archang, M., & Di Carlo, D. (2022a). High-throughput selection of cells based on accumulated growth and division using PicoShell particles. Proceedings of the National Academy of Sciences, 119(4). https://doi.org/10.1073/pnas.2109430119
[6] Waites, K. B. (2011). Methods for antimicrobial susceptibility testing for human mycoplasmas: Approved guideline. Clinical and Laboratory Standards Institute. (CLSI publication / Clinical and Laboratory Standards Institute, No. 31.19.) Available from: https://www.ncbi.nlm.nih.gov/books/NBK544375/