Abstract
The presence of microorganisms in biological fluids like urine and blood is an indication of vulnerability to infections. Iron is one of the important micronutrients required for bacterial growth. In an iron-deficit environment, bacteria release high-affinity iron-chelating compounds called siderophores which can be used as non-invasive target molecules for the detection of such pathogens. However, only limited reagents and procedures are available to detect the presence of these organic molecules. The present study aims at detecting the presence of siderophores in the iron-depleted media, exploiting the reversible quenching of Calcein Blue and iron(III) complex. The fluorescence of Calcein Blue is known to be quenched in the presence of iron(III); if a stronger chelator removes this ion from the fluorophore, the fluorescence of the fluorophore is regained. This behaviour of the fluorophore was exploited to detect and quantify siderophores down to 50 and 800 nM equivalent of standard siderophore, deferroxamine mesylate (desferal) in Dulbecco’s PBS and siderophore quantification (SPQ) medium, respectively. The siderophores released by pathogens, equivalent to standard desferal, were in the range of 1.29 to 5.00 μM and those for non-pathogens were below 1.19 μM. The simple, sensitive and cost-effective method performed in a 96-well plate was able to detect and quantify iron chelators within 7–8 h of incubation.
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Acknowledgments
We are grateful to Mr. Suresh Lingham, M/s Trivitron Pvt Ltd. for clinical samples, Dr. Sridhar, Dept. of Microbiology, Sri Ramachandra University, for providing standard bacterial cultures and Dr. J. Saibaba, National Agro Foundation, Chennai for the analytical data. We acknowledge the financial support from Centre with Potential for Excellence in Environmental Science (CPEES) of University Grants Commission.
Conflict of interest
There is no conflict of interests for the authors for submitting this article. This work was supported by the Centre with Potential for Excellence in Environmental Science (CPEES) of University Grants Commission, India. They have no involvements in the study design, in the collection, analysis and interpretation of data, in the writing of the article and in the decision to submit the article for publication.
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Sankaranarayanan, R., Alagumaruthanayagam, A. & Sankaran, K. A new fluorimetric method for the detection and quantification of siderophores using Calcein Blue, with potential as a bacterial detection tool. Appl Microbiol Biotechnol 99, 2339–2349 (2015). https://doi.org/10.1007/s00253-015-6411-x
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DOI: https://doi.org/10.1007/s00253-015-6411-x