Abstract
A variety of direct and indirect methods have been used to quantify planktonic and biofilm bacterial cells. Direct counting methods to determine the total number of cells include plate counts, microscopic cell counts, Coulter cell counting, flow cytometry, and fluorescence microscopy. However, indirect methods are often used to supplement direct cell counting, as they are often more convenient, less time-consuming, and require less material, while providing a number that can be related to the direct cell count. Herein, an indirect method is presented that uses fluorescence emission intensity as a proxy marker for studying bacterial accumulation. A clinical strain of Pseudomonas aeruginosa was genetically modified to express a green fluorescent protein (PA14/EGFP). The fluorescence intensity of EGFP in live cells was used as an indirect measure of live cell density, and was compared with the traditional cell counting methods of optical density (OD600) and plate counting (colony-forming units (CFUs)). While both OD600 and CFUs are well-established methods, the use of fluorescence spectroscopy to quantify bacteria is less common. This study demonstrates that EGFP intensity is a convenient reporter for bacterial quantification. In addition, we demonstrate the potential for fluorescence spectroscopy to be used to measure the quantity of PA14/EGFP biofilms, which have important human health implications due to their antimicrobial resistance. Therefore, fluorescence spectroscopy could serve as an alternative or complementary quick assay to quantify bacteria in planktonic cultures and biofilms.
Highlights
The goal of this study is to investigate the potential of fluorescence spectroscopy to: (1) quantify genetically engineered Pseudomonas aeruginosa (PA14/EGFP) through the detection of the enhanced green fluorescent protein (EGFP) and to (2) correlate fluorescence to plate counting colony-forming units (CFUs), which is the gold standard of bacterial quantification.Pseudomonas aeruginosa (PA) is a common Gram-negative bacterium that is responsible for more than 10% of all hospital acquired infections [1]
PA14/EGFP living cells are fluorescent and display an emission profile characterized by a living cells are fluorescent and display an emission characterized by a maximum at 514 nm when excited with visible light
This study demonstrated the use of fluorescence spectroscopy for the quantification of PA14 tagged with EGFP in suspension and biofilm growth
Summary
The goal of this study is to investigate the potential of fluorescence spectroscopy to: (1) quantify genetically engineered Pseudomonas aeruginosa (PA14/EGFP) through the detection of the enhanced green fluorescent protein (EGFP) and to (2) correlate fluorescence to plate counting colony-forming units (CFUs), which is the gold standard of bacterial quantification.Pseudomonas aeruginosa (PA) is a common Gram-negative bacterium that is responsible for more than 10% of all hospital acquired infections [1]. Chemosensors 2018, 6, 21 flora, and is virtually impossible to prevent exposure in natural environments [2,3] While this bacterium is typically found on the surface of the body, it is an opportunistic pathogen that can form antibiotic-resistant biofilms, causing a wide array of infections [2,4,5,6,7,8], such as skin ( in burn patients), urinary tract, kidney, and surgical site infections, as well as pneumonia and sepsis [5,9,10]. These infections are persistent, they are severe when compared with many other bacterial infections [1]
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