Presentation Title

Avoiding spectral interference from antibiotic controls and autofluorescence in antimycobacterial assays

Faculty Mentor

Jacquline Trischman

Start Date

23-11-2019 8:45 AM

End Date

23-11-2019 9:00 AM

Location

Markstein 203

Session

oral 1

Type of Presentation

Oral Talk

Subject Area

physical_mathematical_sciences

Abstract

One of the top ten leading causes of death, tuberculosis (TB) remains an infectious epidemic of great concern worldwide. Although many useful drugs have been established and the mechanism of pathogenesis has been extensively studied, TB is capable of tolerating many hostile conditions and rapidly adapting to become resistant to current antibiotics. Multidrug-resistant TB has reached crisis levels in some parts of the world. The causative agent of TB, Mycobacterium tuberculosis, exhibits the unique characteristics of all mycobacteria, such as the presence of mycolic acids in the cell wall and the use of biofilms in the formation of granulomas. In developing assays to investigate potential new treatments for TB, our lab sought to target biofilm formation and control using a related Mycobacterial strain, M. marinum. We continue to work on these growth inhibition, biofilm formation, and quorum sensing assays. The assay procedures rely heavily on the principles of absorbance and fluorescence, and many such assays in the literature do not account for intrinsic fluorescence of the antibiotic controls used or potential autofluorescence of the study organisms. Though the most common TB antibiotics, rifampicin, isoniazid, and pyrazinamide, have well established spectroscopic characteristics, information on newly FDA approved drugs for TB such as bedaquiline or linezolid and on autofluorescence of Mycobacteria is not readily available. In this project, absorbance and emission spectra were established for thirteen TB antibiotics and a dense culture of M. marinum (strain U69D1). Each antibiotic was run initially at a high concentration to establish that it contained a chromophore. Those that demonstrated absorbance were then analyzed at a concentration exhibiting an absorbance between 0.2 and 1.0. Fluorescence spectra were subsequently established using the wavelength of maximum absorption as the excitation wavelength. Resulting spectra and their relevance to overall assay development will be discussed.

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Nov 23rd, 8:45 AM Nov 23rd, 9:00 AM

Avoiding spectral interference from antibiotic controls and autofluorescence in antimycobacterial assays

Markstein 203

One of the top ten leading causes of death, tuberculosis (TB) remains an infectious epidemic of great concern worldwide. Although many useful drugs have been established and the mechanism of pathogenesis has been extensively studied, TB is capable of tolerating many hostile conditions and rapidly adapting to become resistant to current antibiotics. Multidrug-resistant TB has reached crisis levels in some parts of the world. The causative agent of TB, Mycobacterium tuberculosis, exhibits the unique characteristics of all mycobacteria, such as the presence of mycolic acids in the cell wall and the use of biofilms in the formation of granulomas. In developing assays to investigate potential new treatments for TB, our lab sought to target biofilm formation and control using a related Mycobacterial strain, M. marinum. We continue to work on these growth inhibition, biofilm formation, and quorum sensing assays. The assay procedures rely heavily on the principles of absorbance and fluorescence, and many such assays in the literature do not account for intrinsic fluorescence of the antibiotic controls used or potential autofluorescence of the study organisms. Though the most common TB antibiotics, rifampicin, isoniazid, and pyrazinamide, have well established spectroscopic characteristics, information on newly FDA approved drugs for TB such as bedaquiline or linezolid and on autofluorescence of Mycobacteria is not readily available. In this project, absorbance and emission spectra were established for thirteen TB antibiotics and a dense culture of M. marinum (strain U69D1). Each antibiotic was run initially at a high concentration to establish that it contained a chromophore. Those that demonstrated absorbance were then analyzed at a concentration exhibiting an absorbance between 0.2 and 1.0. Fluorescence spectra were subsequently established using the wavelength of maximum absorption as the excitation wavelength. Resulting spectra and their relevance to overall assay development will be discussed.