Bacterial infections are a major cause of mortality and morbidity in the world, and new strategies for improving their treatment are greatly needed. A key issue limiting treatment of bacterial infections is an inability to rapidly determine antibioti susceptibilities. Conventional microbiological diagnosis depends on culturing from bodily specimens, often requiring 1-3 day incubation times that can be further delayed by the presence of slow- growing or finicky pathogens. The objective of this proposal is to develop a flow cytometric strategy for rapidly determining MICs of antibiotics against the six most common blood stream infection (BSI)-causing bacteria. Requiring <1-hr antibiotic incubation times to see characteristic responses, this new susceptibility strategy is designed to determine antimicrobial MICs within 4 hours of blood culture positivity, portending significant improvements to patient treatment outcomes and lowered incidence of drug resistance caused by excessive antibiotic use. The flow cytometric methods proposed here rely on statistical, scattered light and selective bacterial (vs. mammalian cell) delivery and enrichment technologies recently developed in our laboratories. The central hypothesis of this proposal is that """"""""Antimicrobial susceptibility can be generated within 4 hours of positive blood culture, for the six most common BSI-causing bacteria, using a combination of flow cytometry technologies that measure antibiotic-induced ROS and scatter changes in bacteria, each coupled with rigorous multidimensional statistical distance metrics for quantification."""""""" The experiments in this proposal will develop a complementary set of flow cytometric technologies that rapidly determine antibacterial MICs of bacteria either after subculture isolation or after being recovered directly from positive blood cultures. When successful, these studies will reduce the time to antibiotic susceptibility determinations to as little as ~4 hours, post positive blood culture. Removing this treatment bottleneck will enable actionable information to be garnered at least 36 hours faster than currently implemented, directly improving patient outcomes and minimizing antibiotic resistance se- lection.
Each Aim utilizes highly innovative technologies to achieve these potential significant benefits to human health.
In Aim 1, our unique maltodextrin-based bacterial targeting,30 and our highly sensitive fluorogenic ROS dosimeters31 will be combined and applied to quantify the demonstrated ROS production42-44 upon near- MIC antibiotic exposure.
Aim 2 will demonstrate that <1-hr, near-MIC antibiotic exposure induces characteristic bacterial morphology changes that are detectable with scattered light. Multidimensional statistical distance metrics vs. paired controls are developed to quantify these changes and determine label-free MICs in the presence of biovariability.
Aim 3 will combine the innovations of Aims 1 &2 to probe clinical isolates using ROS and scatter with 3-D statistical distance metrics, while gating on fluorophore-targeted bacteria rapidly recovered from blood culture will further decrease time-to-result constraints. The proposed experiments address the great need to reduce the tremendous human and economic costs of bacterial infections.

Public Health Relevance

This proposal develops new technologies to speed antimicrobial sensitivity testing from the current ~42 hours to ~4 hours after positive blood culture using both fluorescent and label-free detection. Actionable treatment information requires both identification and antibiotic sensitivity determinations - the latter being the true bottleneck in treatment that both negatively impacts patient health and increases antibiotic resistance through empiric treatments. New flow cytometric scatter and fluorescence sensing measurements, coupled with adaptive, multidimensional statistical methods developed specifically for this purpose suggest eventual translation to the clinic to positively impact treatment and overall human health.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
Project #
Application #
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Ritchie, Alec
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Georgia Institute of Technology
Schools of Arts and Sciences
United States
Zip Code
Huang, Tzu-Hsueh; Tzeng, Yih-Ling; Dickson, Robert M (2018) FAST: Rapid determinations of antibiotic susceptibility phenotypes using label-free cytometry. Cytometry A 93:639-648
Tzeng, Yih-Ling; Berman, Zachary; Toh, Evelyn et al. (2018) Heteroresistance to the model antimicrobial peptide polymyxin B in the emerging Neisseria meningitidis lineage 11.2 urethritis clade: mutations in the pilMNOPQ operon. Mol Microbiol :
Retchless, Adam C; Kretz, C├ęcilia B; Chang, How-Yi et al. (2018) Expansion of a urethritis-associated Neisseria meningitidis clade in the United States with concurrent acquisition of N. gonorrhoeae alleles. BMC Genomics 19:176
Tzeng, Yih-Ling; Bazan, Jose A; Turner, Abigail Norris et al. (2017) Emergence of a new Neisseria meningitidis clonal complex 11 lineage 11.2 clade as an effective urogenital pathogen. Proc Natl Acad Sci U S A 114:4237-4242
Bazan, Jose A; Turner, Abigail Norris; Kirkcaldy, Robert D et al. (2017) Large Cluster of Neisseria meningitidis Urethritis in Columbus, Ohio, 2015. Clin Infect Dis 65:92-99
Maity, Santanu; Sadlowski, Corinne M; George Lin, Jung-Ming et al. (2017) Thiophene bridged aldehydes (TBAs) image ALDH activity in cells via modulation of intramolecular charge transfer. Chem Sci 8:7143-7151
Fleischer, Blake C; Petty, Jeffrey T; Hsiang, Jung-Cheng et al. (2017) Optically Activated Delayed Fluorescence. J Phys Chem Lett 8:3536-3543
Hsiang, Jung-Cheng; Fleischer, Blake C; Dickson, Robert M (2016) Dark State-Modulated Fluorescence Correlation Spectroscopy for Quantitative Signal Recovery. J Phys Chem Lett 7:2496-501
Bazan, Jose A; Peterson, Amy S; Kirkcaldy, Robert D et al. (2016) Notes from the Field: Increase in Neisseria meningitidis-Associated Urethritis Among Men at Two Sentinel Clinics - Columbus, Ohio, and Oakland County, Michigan, 2015. MMWR Morb Mortal Wkly Rep 65:550-2
Lee, Kunwoo; Rafi, Mohammad; Wang, Xiaojian et al. (2015) In vivo delivery of transcription factors with multifunctional oligonucleotides. Nat Mater 14:701-6

Showing the most recent 10 out of 13 publications