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    Applied Microbiology andBiotechnology ISSN 0175-7598Volume 99Number 5 Appl Microbiol Biotechnol (2015)99:2339-2349DOI 10.1007/s00253-015-6411-x

    A new fluorimetric method for the detectionand quantification of siderophores usingCalcein Blue, with potential as a bacterialdetection tool

    Ranjini Sankaranarayanan,AlagiachidambaramAlagumaruthanayagam & KrishnanSankaran

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  • METHODS AND PROTOCOLS

    A new fluorimetric method for the detection and quantificationof siderophores using Calcein Blue, with potential as a bacterialdetection tool

    Ranjini Sankaranarayanan &Alagiachidambaram Alagumaruthanayagam &Krishnan Sankaran

    Received: 22 October 2014 /Revised: 12 January 2015 /Accepted: 15 January 2015 /Published online: 30 January 2015# Springer-Verlag Berlin Heidelberg 2015

    Abstract The presence of microorganisms in biologicalfluids like urine and blood is an indication of vulnerability toinfections. Iron is one of the important micronutrients requiredfor bacterial growth. In an iron-deficit environment, bacteriarelease high-affinity iron-chelating compounds calledsiderophores which can be used as non-invasive target mole-cules for the detection of such pathogens. However, only lim-ited reagents and procedures are available to detect the pres-ence of these organic molecules. The present study aims atdetecting the presence of siderophores in the iron-depletedmedia, exploiting the reversible quenching of Calcein Blueand iron(III) complex. The fluorescence of Calcein Blue isknown to be quenched in the presence of iron(III); if a strongerchelator removes this ion from the fluorophore, the fluores-cence of the fluorophore is regained. This behaviour of thefluorophore was exploited to detect and quantify siderophoresdown to 50 and 800 nM equivalent of standard siderophore,deferroxamine mesylate (desferal) in Dulbeccos PBS andsiderophore quantification (SPQ) medium, respectively. Thesiderophores released by pathogens, equivalent to standarddesferal, were in the range of 1.29 to 5.00 M and those fornon-pathogens were below 1.19 M. The simple, sensitiveand cost-effective method performed in a 96-well plate wasable to detect and quantify iron chelators within 78 h ofincubation.

    Keywords Bacteria . Calcein Blue . Fluorescence assay .

    Non-invasive . Siderophores

    Introduction

    Iron is an essential Bmicronutrient^ because it is a cofactor inmany vital metabolic processes including the respiratory elec-tron transport chain of most microbes (Husain 2008). A re-view by Meithek and Maraheil (2007) considers iron to be avital Brivet^ for the structural and functional integrity of var-ious proteins. As the availability of free iron in the environ-ment, and particularly in biological medium, is severely lim-ited due to its high reactivity with atmospheric oxygen (Joshiet al. 2006) and sequestration by chelators respectively, mi-crobes are compelled to produce small, high-affinity iron-che-lating compounds called siderophores to acquire it.

    Meithke and Maraheit classified siderophores based on thechemical nature of the oxygen donating moieties for Fe(III)coordination as catecholates (with functional catecholgroups), phenolates (with functional phenol groups),hydroxamates (with functional hydroxylamine groups) andcarboxylates (with functional -hyroxy carboxylates or keto-hydroxy carboxylates). Integration of the chemical feature ofat least two classes into one molecule, namely catecholate-hydroxamates , phenola te-hydroxamates , c i t ra te-hydroxamates and citrate-catecholates is also possible and isreferred as Bmixed type^. The association and dissociation ofiron depend on the pKa of the siderophores which is deter-mined by the protonation of its functional groups (Miethkeand Marahiel 2007).

    R. Sankaranarayanan :A. Alagumaruthanayagam :K. Sankaran (*)Centre for Biotechnology, Anna University, Sardar Patel road,Guindy, Chennai 600 025, Indiae-mail: ksankran@yahoo.com

    K. Sankarane-mail: ksankaran@annauniv.edu

    Appl Microbiol Biotechnol (2015) 99:23392349DOI 10.1007/s00253-015-6411-x

    Author's personal copy

  • A study by Vagarali et al. (2008) revealed that siderophoreproduction is more frequent (about 98%) in pathogenic strainswhen compared with non-pathogenic strains (about 2 %). It isalso evident from literature that production of the virulentsiderophore pyoverdine regulates the production of at leastthree other virulence factors (Lamont et al. 2002) initiatedby the ferri-pyoverdine complex interacting with the cell sur-face receptor protein. An important adaptation of pathogens isto induce subtle changes in the siderophore molecule (like theaddition of a glucose molecule) to circumvent the host defencestrategies (Murugappan et al. 2012). This underlies our needto detect the presence of siderophores, and thus pathogenicbacteria, using a simple and sensitive method.

    Among the colorimetric assays available to detectsiderophores (chrome azurole sulphonate (CAS) (Schwynand Neilands 1987); Csaky (Csaky 1948); Arnow (Arnow1937)), the standard and most commonly used technique isthe CAS assay. It is based on the colour change that the dyeundergoes when iron is removed from it, indicating the pres-ence of iron-chelating molecules (Schwyn and Neilands1987). However, these assays have limitations; the CAS assaycan detect the presence of siderophores qualitatively or in asemi-quantitative manner and utilizes numerous chemicals,some of which are toxic, for the detection of siderophores;Csaky and Arnow assays are specific for hydroxamatesiderophores and catecholate siderophores, respectively. Thisprompted us to develop a generic method for better sensitivityand quantitative analysis of these compounds.

    Experimentally, fluorescence-based detection assays arepreferred over colorimetric methods owing to their high sen-sitivity, rapid response rate and relative low cost (Marencoet al. 2012). In this regard, 4-methylumbelliferone-8-methyliminodiacetic acid, commonly referred to as CalceinBlue, was chosen for this study. The fluorophore is knownto be quenched by iron (Glickstein et al. 2005; Huitink et al.1974), and siderophores can remove the iron bound by the dyeand make it fluoresce. Here, this property has been exploitedfor siderophore detection and quantitation.

    Materials and methods

    Collection and identification of strains

    & Standard strains:The standard strains used in the study, Shigella flexneri

    (2), Salmonella paratyphi (1), Salmonella entericasubspp. (1), Escherichia coli (4), Staphylococcus aureus(3), Klebsiella pneumonia (1), Proteus mirabilis (2),Staphylococcus haemolyticus (1), Klebsiella oxytoca (1),Pseudomonas aerugenosa (2), Streptococcus pneumonia(1), Lactobacillus acidophilus (1), Lactobacillus casei (1),Lactobacillus fermentum (1) and Lactobacillus lactus (1)

    were obtained from American Type Culture Collection(ATCC), USA, National Collection of Type Cultures(NCTC), UK, National Culture Diary Collection (NCDC),India, and Microbial Type Culture Collection (MTCC),Chandigarh, India.

    Laboratory-engineered strains of E. coliDH5 andBL21 (DE3)were obtained from Invitrogen, San Diego,USA, and GJ1158 was procured from Genei, Bangalore,India.

    & Clinical isolates:The clinical isolates of E. coli (10), Klebsiella spp. (3),

    P. mirabilis (3), Pseudomonas spp. (1) and Pseudomonasaerugenosa (1) were collected fromM/s Lister MetropolisLaboratory, Chennai, Tamil Nadu, India. The strains wereconfirmed by standard microbiological (selective mediaand motility tests), biochemical tests (methyl red/Voges-Proskauer (MR/VP) test, urease, catalase, triple sugar ironand indole tests) and 16S rRNA sequencing.

    Details of the standard and clinical isolates are provided inTable 2.

    Preparation of CB dye

    A stock concentration of 5 mM 4-methylumbelliferone-8-methyliminodiacetic acid, commonly known as Calcein Blue(CB; Sigma-Aldrich, USA) was prepared in 0.1 M potassiumhydroxide (Merck, India), and the pH was neutralized using0.1 N hydrochloric acid (HCl; Merck, India). A 0.1-M solu-tion of Dulbeccos phosphate-buffered saline (DPBS) wasprepared by adding 2.7 mM potassium chloride (KCl; Merck,India), 1.5 mM dihydrogen potassium phosphate (KH2PO4;Merck, India), 136.9 mM sodium chloride (NaCl; Merck, In-dia) and 8.9 mM disodium hydrogen phosphate (Na2HPO47H2O; Merck, India) to 1 L of distilled water, and the pH wasadjusted to 7.2 (Seto et al. 2012). The CB stock prepared wasdiluted to 200 M (working stock) in DPBS and stored forfurther use.

    Preparation of CB reagent for siderophore detection

    A concentration of 10 MCBwas prepared from the workingstock by diluting in DPBS. From this, 200 L was added to a96-well plate and the excitation/emission (Ex./Em.) maximumwas determined using the micro-plate reader (PerkinElmer,Enspire multimode plate reader, USA).

    From a stock of 10 mM ferric chloride (FeCl3; Merck,India), the quenching was studied with various concentrationsof FeCl3 in CB