Files and links (1)
Rapid detection of bacterial infections with nuclease-activatable molecular imaging probes Final4.36 MB
Free to read and download, Open Access
Dissertation
Rapid detection of bacterial infections with nuclease-activatable molecular imaging probes
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Autumn 2019
DOI: 10.17077/etd.006304
Abstract
Currently, bacterial infections are diagnosed symptomatically or by the screening of a sample (tissue, urine, blood, etc.) collected in both hospital and clinical settings. Samples are used to generate a microbial culture of the invading microbe followed by secondary culture-based techniques including microscopy. Cultures can also be used to run biochemical tests, such as the Gram stain or the acid-fast stain for identification. Additionally, collected samples can be used for molecular diagnostics such as PCR. Although these methods have been the gold standard for infectious disease diagnostics, they still have some significant drawbacks. When a patient presents with a focal infection, a rapid diagnosis may be essential to survival. However, current diagnostic methods can take days to weeks or more if there is a fastidious organism involved in the infection. Current methods are also subject to false-negative test results, which can further delay correct diagnosis, losing valuable time. To address these issues, this study investigates a new method of identifying Staphylococcus aureus (S. aureus) which is the leading cause of various types of focal infections in humans. Our group has carried out a previous study using a molecular imaging technology that rapidly detects S. aureus infections in a mouse pyomyositis infection model. This was accomplished with an activatable probe fluorescently labeled in the near infrared (NIR) wavelength, to which a fluorophore and a quencher were attached on opposite ends to a synthetic modified RNA oligonucleotide which is selectively cleaved by the S. aureus micrococcal nuclease (MN). With this probe, we were able to rapidly detect and localize the pathogen (via specific MN digestion of the oligonucleotide sequence) in mouse thigh tissue. Despite the promising results from this study, a critical need for rapid clinically relevant pathogenic infections remains. In this dissertation, we investigated whether a second-generation probe could be used to rapidly detect a skin and catheter-associated biofilm infection by S. aureus in a noninvasive manner. We discovered that the DyLight800- P2 & P3 NIR probe could be used to detect a catheter-associated biofilm infection by S. aureus within 20 minutes of intravenous administration. However, the DyLight800 probe was not activated (with significance) in the presence of a S. epidermidis, a nuclease-knockout S. aureus (mutant), or a sham-infected catheter-associated biofilm infection. We discovered that the second-generation probe labeled with fluorescein could be used to rapidly detect a skin infection by S. aureus. Additionally, we report development and use of first-generation probe to rapidly detect a skin infection by S. pyogenes (2nd leading cause of skin and soft tissue infections worldwide). Using the two probes, we have also developed a multiplex imaging approach to detect a skin infection by either S. aureus or S. pyogenes, the two most common pathogens in North America. The findings from this study provide a novel approach to rapidly diagnosing two common classes of S. aureus infections seen worldwide. Additionally, we believe this technology has great potential to serve as a platform for other relevant classes of pathogenic infections.
Details
- Title: Subtitle
- Rapid detection of bacterial infections with nuclease-activatable molecular imaging probes
- Creators
- Franklin Oluwole Bright-Smith
- Contributors
- James O McNamara (Advisor)Lee-Ann H Allen (Committee Member)Paloma H Giangrande (Committee Member)Alexander R Horswill (Committee Member)Aloysius J Klingelhutz (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Molecular and Cellular Biology
- Date degree season
- Autumn 2019
- Publisher
- University of Iowa
- DOI
- 10.17077/etd.006304
- Number of pages
- xii, 82 pages
- Copyright
- Copyright 2019 Franklin Bright
- Language
- English
- Description illustrations
- color illustrations
- Description bibliographic
- Includes bibliographical references (pages 75-82).
- Academic Unit
- Interdisciplinary Graduate Program in Molecular Medicine
- Record Identifier
- 9984210642702771
Metrics
17 File views/ downloads
26 Record Views