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Detection and Discrimination of Bacteria and Pathological Analytes using Photoacoustic Flow Cytometry

Edgar, Robert H (2022) Detection and Discrimination of Bacteria and Pathological Analytes using Photoacoustic Flow Cytometry. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Infections caused by antibiotic-resistant bacteria, such as Clodostrium difficile, Acinetobacter baumannii, carbapenem-resistant Enterobacteriaceae, and methicillin-resistant Staphylococcus aureus (MRSA), are increasing worldwide and cause significant strain on the healthcare system. Without early intervention, bacteremia can progress to sepsis, a potentially lethal condition. Rising prevalence of resistant bacteria have necessitated a move towards more rapid and quantifiable diagnostic tools, as time is of the essence in effective treatment. Current treatment relies on prescribing broad-spectrum antibiotics until blood cultures can be completed, a process which usually takes days to complete. Furthermore, sub-populations of seemingly isogenic bacteria may exhibit a range of antibiotic susceptibilities, often called heterogeneous resistance. These heterogeneous antibiotic-resistant infections are often misdiagnosed as hospital-acquired secondary infections because there are no clinically used tests that can differentiate between homogeneous and heterogeneous antibiotic resistance.
To address the need to improve rapid diagnostics for antibiotic-resistant “superbugs,” we describe the development and proof of concept of rapid bacterial identification using photoacoustic flow cytometry and labeled bacteriophages with the characterization and differentiation of heterogeneous antibiotic resistant bacterial infections. Bacteriophages are viruses that infect bacteria, using specialized attachment proteins called tailspikes, to specifically bind to their target bacterial cell-surface proteins. We exploit this bacteriophage-host interaction specificity conferred by tailspikes, along with other specific proteins such as antibodies and bacteriocins, by attaching these proteins to streptavidin-coated chromophores and passing them through the photoacoustic flow cytometer. In this setup, pulsed laser light is delivered to a sample containing tagged bacteria flowing past a focused transducer, with tagged particles absorbing laser light to create a photoacoustic response. We illustrate the ability of this technique to discriminate and enumerate any bacterial population using a bacteriophage-chromophore tag through a proof-of-principle experiment detecting and differentiating Salmonella from Escherichia coli. This research presents an innovative way of identifying and differentiating bacterial strains and sub-populations of antibiotic sensitivity. This method can be further developed for use with other bacterial pathogens in blood cultures representing a major step forward in clinical practice, speeding up delivery of effective treatment to patients by eliminating the need to culture samples.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Edgar, Robert Hrhe5@pitt.eduRHE50000-0002-3906-1930
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairRoy, Parthapar19@pitt.edupar19
Committee MemberFederspiel, WilliamWFEDERSP@Pitt.eduWFEDERSP
Committee MemberKellum, JohnKellum@Pitt.eduKellum
Committee MemberViator,
Committee MemberHempel,
Date: 10 June 2022
Date Type: Publication
Defense Date: 13 December 2021
Approval Date: 10 June 2022
Submission Date: 3 March 2022
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 170
Institution: University of Pittsburgh
Schools and Programs: Swanson School of Engineering > Bioengineering
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Bacteriophage, Photoacoustics, Optoacoustics, bacterial infection, rapid diagnostics
Date Deposited: 10 Jun 2022 19:40
Last Modified: 10 Jun 2022 19:40


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