Authors acknowledge funding from DBT - Biotechnology Teaching Program, DBT - BUILDER Program and FIST. MR thanks fellowship received from SHODH. HP thanks fellowship received from CSIR.

All bacterial isolates of P. aeruginosa were obtained from medical microbiology laboratories from Vadodara and Jaipur, India. All selected clinical isolates were handled in Biosafety Cabinet (BSL2) and utmost care was taken while handling bacterial isolates during the experiments. The commercial details of all the reagents/solutions are provided in the Table of Materials.
1. Preparation of Chrome Azurol Sulfonate (CAS) dye and agar media
<ol>
	<li>Prepare CAS dye (100 mL) with the following composition:
	<ol>
		<li>Dissolve 60 mg of CAS in 50 mL of distilled water (Solution 1).</li>
		<li>Dissolve 2.7 mg of FeCl3&#183;6H2O in 10 mL of 10 mM HCl (Solution 2).</li>
		<li>Dissolve 73 mg of Cetrimonium bromide (HDTMA) in 40 mL of distilled water (Solution 3).</li>
		<li>Carefully mix Solution 1, Solution 2, and Solution 3. Store it in a glass bottle.</li>
	</ol>
	</li>
	<li>Prepare CAS agar following the steps below:
	<ol>
		<li>Add 100 mL of MM9 salt solution to 750 mL of distilled water.</li>
		<li>Dissolve 32.24 g of piperazine-N,N&#39;-bis(2-ethanesulfonic acid) (PIPES) free acid.</li>
		<li>Add 15 g of Agar agar. Autoclave and allow it to cool.</li>
		<li>Add 30 mL of sterile Casamino acid solution and 10 mL of sterile 20% glucose solution to the mixture.</li>
		<li>Add 100 mL of CAS dye, mix, and pour it in aseptic conditions. 
		NOTE: Preparation of MM9 media, Casamino acid solution, and 8-Hydroxyquinoline are provided in Supplementary File 1. PIPES buffer is pH-sensitive. It will not dissolve until pH 5.6 is achieved. Ensure that the pH is constantly monitored because when PIPES starts to dissolve in water, it will further lower the pH. Extract Casamino acid with the same volume of 3% 8-hydroxyquinoline dissolved in chloroform. Leave the immiscible solution at 4 &#176;C for around 20 min and carefully collect the upper phase of the solution without disturbing the lower phase.</li>
	</ol>
	</li>
</ol>
2. Qualitative analysis of siderophores production
<ol>
	<li>Set the OD600 nm to 0.2 for 24 h grown cultures of P. aeruginosa.</li>
	<li>Using a sterile wire loop, streak the bacterial culture on a CAS agar plate.</li>
	<li>Incubate at 30 &#176;C for 24 h. 
	NOTE: Peptone water media or 0.8% normal saline can be used to dilute the bacterial culture. If no bacterial growth is observed at 24 h, incubate CAS plates for 48 to 72 h.</li>
</ol>
3. Quantitative estimation of total siderophores
<ol>
	<li>Re-inoculate 24-h-grown cultures of P. aeruginosa in Peptone water media after adjusting OD600 nm to 0.25, and incubate at 30 &#176;C for 48 h.</li>
	<li>After 48 h, centrifuge the bacterial culture at 4650 x g for 10 min at room temperature.</li>
	<li>After centrifugation, add 100 &#181;L of cell-free supernatant to a 96-well microtiter plate and add 100 &#181;L of CAS dye to it.</li>
	<li>Cover the plate with aluminum foil and incubate at room temperature for 20 min.</li>
	<li>After incubation, take spectrophotometric readings at 630 nm.</li>
	<li>Calculate the results obtained for the quantification of total siderophores as Percent Siderophore Unit (PSU). 
	NOTE: PSU can be calculated as: [(Ar&#160;- As)/Ar] x 100 
	where, Ar = absorbance of the reference at 630 nm, As = absorbance of the cell-free supernatant of the sample. For the reference, CAS dye should be added to uninoculated peptone water media. Fill all glassware, such as test tubes, flasks, etc., with 6 M HCl for 2 h and rinse them twice with distilled water to remove any trace of iron on them.</li>
</ol>
4. Quantitative estimation of pyoverdine
<ol>
	<li>Re-inoculate 24-h-grown cultures of P. aeruginosa in Peptone water media after adjusting OD600 nm to 0.25 and incubate at 30 &#176;C for 48 h.</li>
	<li>After 48 h, measure the OD600 nm of the bacterial growth before proceeding further.</li>
	<li>Centrifuge the bacterial culture at 4650 x g for 10 min at room temperature.</li>
	<li>After centrifugation, add 100 &#181;L of cell-free supernatant to a 96-well microtiter plate and add 100 &#181;L of 50 mM Tris-HCl (pH 8.0) to it.</li>
	<li>Take spectrophotometric readings at OD405 nm.</li>
</ol>
5. Pyochelin extraction and spectrophotometry
<ol>
	<li>Re-inoculate 24-h-grown cultures of P. aeruginosa in King&#39;s B media (Supplementary File 1) after adjusting OD600 nm to 0.25 and incubate at 30 &#176;C for 24 h.</li>
	<li>After 24 h, take 100 mL of culture and centrifuge at 4650 x g for 10 min at room temperature.</li>
	<li>After centrifugation, add 5 mL of 1 M citric acid to the supernatant. Extract twice with 50 mL of Ethyl acetate.</li>
	<li>Filter the organic phase with magnesium sulfate through a syringe filter. Store the filtered organic phase at -20 &#176;C.</li>
	<li>Take spectrophotometric readings at 320 nm. 
	NOTE: As pyochelin is a highly unstable compound at room temperature, perform the extraction process on ice. Use a 50 mL sterile syringe for filter separation. Place cotton at the tip of the syringe and add 1 gm of magnesium sulfate on it. Fix a sterile syringe filter at the tip of the syringe and collect the filtrate in a sterile tube.</li>
</ol>

Assessment of Siderophore Production from Pseudomonas aeruginosa Isolates

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+chemical+assay+for+the+detection+and+determination+of+siderophores.">Universal chemical assay for the detection and determination of siderophores.</a> Analytical Biochemistry. 160 (1), 47-56 (1987).</li><li>Louden, B. C., Haarmann, D., Lynne, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+blue+agar+cas+assay+for+siderophore+detection.">Use of blue agar cas assay for siderophore detection.</a> Journal of Microbiology &amp; Biology Education. 12 (1), 51-53 (2011).</li><li>Arora, N. K., Verma, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+microplate+method+for+rapid+and+efficient+estimation+of+siderophore+produced+by+bacteria.">Modified microplate method for rapid and efficient estimation of siderophore produced by bacteria.</a> 3 Biotech. 7 (381), 1-9 (2017).</li><li>Frac, M., Gryta, A., Oszust, K., Kotowicz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+accurate+microplate+method+(biolog+mt2)+for+detection+of+fusarium+fungicides+resistance/sensitivity.">Fast and accurate microplate method (biolog mt2) for detection of fusarium fungicides resistance/sensitivity.</a> Frontiers in Microbiology. 7 (4), 1-16 (2016).</li><li>Cezard, C., Farvacques, N., Sonnet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+and+biology+of+pyoverdines,+pseudomonas+primary+siderophores.">Chemistry and biology of pyoverdines, pseudomonas primary siderophores.</a> Current Medicinal Chemistry. 22 (2), 165-186 (2015).</li><li>Braud, A., Hoegy, F., Jezequel, K., Lebeau, T., Schalk, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+metal+specificity+of+the+pseudomonas+aeruginosa+pyoverdine-iron+uptake+pathway.">New insights into the metal specificity of the pseudomonas aeruginosa pyoverdine-iron uptake pathway.</a> Environmental Microbiology. 11 (5), 1079-1091 (2009).</li><li>Brandel, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+siderophore+of+pseudomonas+aeruginosa:+Physicochemical+characterization+of+the+iron(iii),+copper+(ii)+and+zinc+(ii)+complexes.">a siderophore of pseudomonas aeruginosa: Physicochemical characterization of the iron(iii), copper (ii) and zinc (ii) complexes.</a> Dalton Transactions. 41 (9), 2820-2834 (2012).</li><li>Hoegy, F., Mislin, G. L. A., Schalk, I. 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The authors have nothing to disclose.

This protocol enables researchers to quantitate total siderophores and two different siderophores of P. aeruginosa, namely pyoverdine and pyochelin, from the bacterial cell-free supernatant. In the CAS agar plates assay, CAS dye and Fe3+ ions form a complex. When bacteria produce siderophores, they quench Fe3+ ions from the CAS-Fe3+ complex, leading to a color change around the bacterial growth. This change results in a clear orange halo around the bacterial growth14,15. While bacteria can be directly streaked onto CAS agar plates, the overall results may be affected. Streaking a high number of bacterial cells might create a wide orange halo around the bacterial growth, while using fewer bacterial cells might result in a narrow clear zone. To address this, we streaked a uniform number of bacterial cells by adjusting the OD600. Fastidious bacteria may show results in 24 h, while slow-growing bacteria might take more than 72 hours to demonstrate siderophore production.
For the quantitative determination of total siderophores, classical spectroscopy was initially employed, which necessitates a significant volume of supernatant for siderophore detection, making it impractical. The quantification of total siderophores is now conducted using a 96-well plate with a modified CAS assay. This approach has resulted in a reduction in the usage of reagents, bacterial supernatant, time, and increased accuracy compared to traditional spectroscopic methods16. Other studies have also reported the use of microtiter plates as a time-saving and more efficient method17. The quantification of total siderophores using a microtiter plate has proven to be an efficient approach. In this study, 48-h-old bacterial cultures grown in iron-free media were selected for examination. A comparative study was also conducted to assess siderophore production in different growth media, such as Luria Broth, Luria Broth (extracted with 3% 8-hydroxyquinoline), King&#39;s B media (extracted with 3% 8-hydroxyquinoline), and Peptone water media (extracted with 3% 8-hydroxyquinoline). It was observed that Peptone water media exhibited the highest amount of siderophore production, and thus, it was selected for further experiments. A graph depicting siderophore production in different growth media is included in Supplementary Figure 1.
Pyoverdine can be detected and quantified using spectrofluorimetric readings, but only pyoverdine is known to exhibit fluorescence. Pyoverdine in complex with Fe3+ ions does not exhibit fluorescence18,19. For pyoverdine detection and quantification, 48-h-old bacterial cultures were selected for study. P. aeruginosa is known to produce pyochelin under initial iron-starving conditions and switches to pyoverdine production after exposure to severe iron-stress conditions. It has been demonstrated that pyoverdine can chelate other metallic cations, such as aluminum, gallium, manganese, and chromium, but only iron is successfully transported to the cell membrane19.
Similar to total siderophores and pyoverdine, pyochelin cannot be directly quantified from cell-free supernatant and must be extracted using dichloromethane20. Dichloromethane is immiscible with the cell-free supernatant and forms a separate layer at the bottom. In this study, various nutrient media were tested, including Luria broth, Peptone water, and Casamino acid media, but a significant amount of pyochelin was obtained from King&#39;s B media. While Hoegy et al. had used CAA media in their protocol, we modified it with King&#39;s B media, as CAA media did not support bacterial growth21. The method was further modified by extracting nutrient media with 8-hydroxyquinoline to remove any iron residues from the media, creating an iron-deficient medium. Additionally, incubation time was found to significantly affect pyochelin production. Dumas et al. demonstrated that P. aeruginosa switches from pyochelin to pyoverdine production under severe iron-limiting conditions12. In this study, pyochelin extraction and detection were performed from 24-h-old bacterial cultures, as pyochelin was not detected in 48-h cultures. The volume of the supernatant also affects pyochelin detection, and thus, 100 mL of cell-free supernatant was used for extraction. Furthermore, pyochelin production was significantly higher in MR1, TL7, and J3 isolates, possibly due to bacterial strains that are unable to produce pyoverdine despite having potent iron chelators22.
Ji et al. introduced a novel and sensitive approach for pyochelin quantification, validating it using LC/MS/MS. However, this method only applied to P. aeruginosa isolates from the sputum of cystic fibrosis patients. LC/MS/MS, being a costly setup, may not be affordable for most laboratories23. Visaggio et al. developed a bioluminescent whole cell-based biosensor, which enables rapid, sensitive, and single-step pyochelin quantification. Nevertheless, this method cannot provide quantification for total siderophores and pyoverdine24.
Siderophore iron uptake systems could have an antimicrobial application in drug delivery for multidrug-resistant bacteria, as they play a crucial role in bacterial survival and virulence. This approach may offer a new way to combat multidrug-resistant bacteria25. Drugs that cannot penetrate the bacterial cell membrane can be linked to a siderophore, and the resulting Fe-siderophore complex can be transported inside the bacterial membrane26.

Organisms require iron to perform various vital functions, such as electron transport and DNA replication1. Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is known to possess a variety of virulence factors to establish infection in the host, among which one mechanism is siderophore formation2. During iron-depleting conditions, P. aeruginosa releases specialized molecules called siderophores, which quench iron from the surrounding environment. Siderophores chelate iron extracellularly, and the resulting ferric-siderophore complex is actively transported back to the cell3.
P. aeruginosa is known to produce two siderophores, pyoverdine and pyochelin. Pyoverdine is known to have a higher iron chelating affinity (1:1), whereas pyochelin is known to have a lesser iron chelating affinity (2:1)4. Pyochelin is also called a secondary siderophore because it has a lower iron chelating affinity5. The production and regulation of siderophores are actively controlled by Quorum Sensing (QS) systems in P. aeruginosa6.
Besides iron quenching, siderophores are also involved in regulating virulence factors and play an active role in biofilm formation7. Siderophores serve additional crucial roles, including involvement in cell signaling, defense against oxidative stress, and facilitation of interactions between microbial communities8. Siderophores are typically categorized based on the specific functional groups through which they chelate iron. The three primary bidentate ligands in this classification are catecholate, hydroxamate, and &#945;-hydroxycarboxylate3. Pyoverdines are hallmarks of fluorescent Pseudomonas species such as P. aeruginosa and P. fluorescens5. They consist of a mixed green fluorescent chromophore coupled to an oligopeptide containing 6-12 amino acids. Several non-ribosomal peptide synthetases (NRPs) are involved in their synthesis9. Four genes involved in pyoverdine production and regulation are pvdL, pvdI, pvdJ, and pvdD10. Pyoverdine is also responsible for infection and virulence in mammals11. P. aeruginosa is noted to produce pyochelin in moderate iron-limiting conditions, while pyoverdine is produced during severe iron-limiting environments12. Two operons involved in pyochelin production are pchDCBA and pchEFGHI13. It is noted that in the presence of pyocyanin, pyochelin (catecholate) induces oxidative damage and inflammation and generates hydroxyl radicals, which are harmful to host tissues11.
The Chrome Azurol Sulfonate (CAS) assay is widely adopted due to its comprehensiveness, high sensitivity, and greater convenience compared to microbiological assays, which, although sensitive, can be overly specific14. The CAS assay can be conducted on agar surfaces or in a solution. It relies on the color change that occurs when the ferric ion transitions from its intense blue complex to orange. The CAS colorimetric assay quantifies the depletion of iron from a Fe-CAS-surfactant ternary complex. This particular complex, consisting of metal, organic dye, and surfactant, has a blue color and exhibits an absorption peak at 630 nm.
This report presents a method for the qualitative detection of siderophore production, where one can detect the production of siderophores on an agar plate. A method for the quantitative estimation of total siderophore production in a microtiter plate and the detection and quantitative analysis of two siderophores, pyoverdine and pyochelin, from P. aeruginosa, is also provided.

<table><tbody><tr><td>Agar Agar, Type I</td><td>HIMEDIA</td><td>GRM666</td><td></td></tr><tr><td>8-Hydroxyquinoline</td><td>Loba Chemie</td><td>4151</td><td></td></tr><tr><td>Casamino Acid</td><td>SRL Chemicals</td><td>68806</td><td></td></tr><tr><td>Cetyltrimethyl Ammonium Bromide (CTAB)</td><td>HIMEDIA</td><td>RM4867-100G</td><td></td></tr><tr><td>Chloroform</td><td>Merck</td><td>1070242521</td><td></td></tr><tr><td>Chrome azurol sulfonate</td><td>HIMEDIA</td><td>RM336-10G</td><td></td></tr><tr><td>Citric acid</td><td>Merck</td><td>100241</td><td></td></tr><tr><td>Dextrose monohydrate</td><td>Merck</td><td>108342</td><td></td></tr><tr><td>Dichloromethane</td><td>Merck</td><td>107020</td><td></td></tr><tr><td>Ferric chloride hexahydrate</td><td>HIMEDIA</td><td>GRM6353</td><td></td></tr><tr><td>Glass Flasks</td><td>Borosil</td><td>5100021</td><td></td></tr><tr><td>Glass Test-tubes</td><td>Borosil</td><td>9820U05</td><td></td></tr><tr><td>Hydrochloric acid</td><td>SDFCL</td><td>20125</td><td></td></tr><tr><td>King&#039;s medium B base</td><td>HIMEDIA</td><td>M1544-500G</td><td></td></tr><tr><td>M9 Minimal Medium Salts</td><td>HIMEDIA</td><td>G013-500G</td><td></td></tr><tr><td>Magnesium Sulphate&amp;nbsp;</td><td>Qualigens</td><td>10034</td><td></td></tr><tr><td>MultiskanGO UV Spectrophotometer</td><td>Thermo Scientific</td><td>51119200</td><td></td></tr><tr><td>Peptone Type I, Bacteriological</td><td>HIMEDIA</td><td>RM667-500G</td><td></td></tr><tr><td>PIPES free acid</td><td>MP Biomedicals</td><td>190257</td><td></td></tr><tr><td>Potassium dihydrogen phosphate</td><td>Merck</td><td>1048731000</td><td></td></tr><tr><td>Proteose peptone</td><td>HIMEDIA</td><td>RM005-500G</td><td></td></tr><tr><td>Shimadzu UV-Vis Spectrophotometer</td><td>Shimadzu</td><td>2072310058</td><td></td></tr><tr><td>Sigma Laborzentrifuge</td><td>Sigma-Aldrich</td><td>3-18K</td><td></td></tr><tr><td>Sodium chloride</td><td>Qualigens</td><td>15915</td><td></td></tr></tbody></table>

qualitative and quantitative analysis of siderophore production from pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is known for its production of a diverse range of virulence factors to establish infections in the host. One such mechanism is the scavenging of iron through siderophore production. P. aeruginosa produces two different siderophores: pyochelin, which has lower iron-chelating affinity, and pyoverdine, which has higher iron-chelating affinity. This report demonstrates that pyoverdine can be directly quantified from bacterial supernatants, while pyochelin needs to be extracted from supernatants before quantification.
The primary method for qualitatively analyzing siderophore production is the Chrome Azurol Sulfonate (CAS) agar plate assay. In this assay, the release of CAS dye from the Fe3+-Dye complex leads to a color change from blue to orange, indicating siderophore production. For the quantification of total siderophores, bacterial supernatants were mixed in equal proportions with CAS dye in a microtiter plate, followed by spectrophotometric analysis at 630 nm. Pyoverdine was directly quantified from the bacterial supernatant by mixing it in equal proportions with 50 mM Tris-HCl, followed by spectrophotometric analysis. A peak at 380 nm confirmed the presence of pyoverdine. As for Pyochelin, direct quantification from the bacterial supernatant was not possible, so it had to be extracted first. Subsequent spectrophotometric analysis revealed the presence of pyochelin, with a peak at 313 nm.

Before quantification of siderophores from clinical isolates, a qualitative screening for siderophore production was carried out to ensure siderophores production. Qualitative detection of siderophores from clinical isolates was observed by streaking bacteria on CAS agar plates. Three clinical isolates, namely MR1, TL7, J3, along with PAO1 (the reference strain), were selected for the study. All three clinical isolates and PAO1 showed positive results for siderophore production, where a clear orange halo around the bacterial growth on the blue agar surface indicated positive siderophore production (Figure 1). The halo formation on the CAS agar (Figure 1) gave a rough estimation of the siderophore production by clinical isolates. Hence, quantitative estimation of siderophore production using CAS reagent and liquid media was performed.
Total siderophore quantification was performed directly from the cell-free supernatant. Post-incubation, a color change was observed where yellow color indicated the removal of CAS dye from the Fe-complex. Here, CAS dye with uninoculated growth media was used as control, which was taken for siderophores calculation. There was no significant difference in total siderophore production in isolates MR1 and TL7, while there was a significant total siderophore production in J3 compared to PAO1 (Figure 2).
Next, pyoverdine quantification was performed directly from cell-free supernatants. Pyoverdines are released in cell environments, so the cell-free supernatant was used for pyoverdine quantification. A UV range scan by the spectrophotometer was also performed, where the peak at 380 nm confirmed the presence of pyoverdine (Figure 3A). Spectrophotometric readings were taken at 405&#160;nm, and results were interpreted as OD405/OD600 nm. All isolates showed pyoverdine production, where isolates MR1 and J3 showed significantly lower pyoverdine compared to PAO1, while no significant difference was observed for TL7 (Figure 3B).
Pyochelin cannot be directly quantified from the cell-free supernatant. It was not possible to extract pyochelin from 1 mL of cell-free supernatant, so it was extracted from 100 mL of cell-free supernatant. It was acidified with 1 M citric acid. As pyochelin is a highly unstable compound, the extraction process was performed on ice. Pyochelin detection was performed by running a UV range scan from a range of 300 to 600 nm, where the peak at OD320 nm confirmed the presence of pyochelin (Figure 4A). Pyochelin quantification was performed by taking spectrophotometric readings at OD320 nm. Calculations were made by dividing OD320/OD600. Isolates MR1, TL7, and J3 showed significantly higher production of pyochelin compared to the reference isolate PAO1 (Figure 4B).
Comparative siderophore production in various bacterial growth media was performed to check the amount of total siderophores produced in various growth media. Four different media were selected, where Luria broth, King&#39;s B media, and Peptone water were extracted with 3% 8-hydroxyquinoline, and unextracted Luria broth was selected for the study. There was no significant difference between siderophore production in Extracted Luria broth, King&#39;s B media, and Peptone water media, while a significant difference was observed for siderophore production in unextracted Luria broth.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65980/65980fig1v2.jpg" /> 
Figure 1: Siderophore production on CAS agar. Growth of P. aeruginosa on CAS agar plates of 24-h-grown cultures were spotted onto CAS agar plates and incubated at 30 &#176;C for 24 h. Siderophore presence is indicated by an orange halo around the bacterial colony (PAO1, MR1, TL7, and J3). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65980/65980fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65980/65980fig2v2.jpg" /> 
Figure 2: Total siderophores production. Total siderophore production was assessed for PAO1 and three clinical isolates (MR1, TL7, and J3). 100 &#181;L of cell-free supernatant (of PAO1, MR1, TL7, and J3 grown for 24 h in peptone water broth) was added to a 96-well microtiter plate, thereafter 100 &#181;L of CAS dye added to it. One-way ANOVA test was performed for statistical significance. Error bars represent &#177; standard deviation (SD) between three biological replicates. *corresponds to p &#60; 0.05; ns corresponds to p &#62; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65980/65980fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65980/65980fig3v2.jpg" /> 
Figure 3: Pyoverdine production. Pyoverdine production of P. aeruginosa strain PAO1 and clinical isolates (MR1, TL7, and J3) assessed after 24 h of growth in peptone water media. The bacterial cultures were centrifuged, and 100 &#181;L of cell-free supernatant was added to a 96-well microtiter plate. Thereafter, 100 &#181;L of 50 mM Tris-HCl (pH 8.0) was added to it. Pyoverdine detection was done by UV spectrophotometry with a range of 350 nm to 600 nm. Spectrometric analysis at 380 nm of pyoverdine production (A). Quantification of pyoverdine production (B). Error bars indicate the standard error over mean of triplicate experiments. Error bars represent &#177; SD between three biological replicates. ****corresponds to p &#60; 0.0001, **corresponds to p &#60; 0.01, and ns corresponds to p &#62; 0.05 based on One-way ANOVA test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65980/65980fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65980/65980fig4v2.jpg" /> 
Figure 4: Pyochelin production. Pyochelin production of PAO1 (used as a reference) and clinical isolates (MR1, TL7, and J3) using extraction with ethyl acetate and magnesium sulfate. Spectrophotometric analysis with a range from 230 nm to 400 nm (A). Quantification of pyochelin (B). Three independent experiments were performed for each isolate. Error bars represent &#177; SD between three biological replicates. ***corresponds to p &#60; 0.001 based on One-way ANOVA test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65980/65980fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Comparative siderophores production in various bacterial growth media. Total siderophores production in four different growth media where King&#39;s B media, Peptone water media, and Luria broth were extracted with 3% 8-hydroxyquinoline to remove any trace iron from it, and Luria broth (unextracted) were selected. Extraction by 3% 8-hydroxyquinoline was performed by adding an equal volume of 3% 8-hydroxyquinoline to the media. Error bars represent &#177; SD between three biological replicates. **corresponds to p &#60; 0.001, and ns corresponds to p &#62; 0.5 based on One-way ANOVA test.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65980/Supplemental Figure 1.pdf" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1: Media recipes used for the present study.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65980/Supplementary File 1.docx" target="_blank">Please click here to view the file.</a>

Watch this Scientific Journal Video about Quantifying Siderophores and Pyochelin for Infection Control at JoVE.com

Author Spotlight: Quantifying Siderophores and Pyochelin for Infection Control 

This protocol provides both qualitative and quantitative analyses of total siderophores, pyoverdine, and pyochelin from Pseudomonas aeruginosa.

Qualitative and Quantitative Analysis of Siderophore Production from Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is known for its production of a diverse range of virulence factors to establish infections in the host. One such ...

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Nanyang Technological University

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3.8K Views.  The Maharaja Sayajirao University of Baroda. This protocol provides both qualitative and quantitative analyses of total siderophores, pyoverdine, and pyochelin from Pseudomonas aeruginosa.

Video: Author Spotlight: Quantifying Siderophores and Pyochelin for Infection Control 

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the abundances of key metabolites to which regulatory factors respond, adjusting cellular metabolism. In recent years, a number of proteins and RNA have emerged as important regulators of virulence gene expression. For example, the CodY protein responds to levels of branched-chain amino acids and GTP and is widely conserved in low G+C Gram-positive bacteria. As a global regulator in Staphylococcus aureus, CodY controls the expression of dozens of virulence and metabolic genes. We hypothesize that S. aureus uses CodY, in part, to alter its metabolic state in an effort to adapt to nutrient-limiting conditions potentially encountered in the host environment. This manuscript describes a method for extracting and analyzing metabolites from S. aureus using liquid chromatography coupled with mass spectrometry, a protocol that was developed to test this hypothesis. The method also highlights best practices that will ensure rigor and reproducibility, such as maintaining biological steady state and constant aeration without the use of continuous chemostat cultures. Relative to the USA200 methicillin-susceptible S. aureus isolate UAMS-1 parental strain, the isogenic codY mutant exhibited significant increases in amino acids derived from aspartate (e.g., threonine and isoleucine) and decreases in their precursors (e.g., aspartate and O-acetylhomoserine). These findings correlate well with transcriptional data obtained with RNA-seq analysis: genes in these pathways were up-regulated between 10- and 800-fold in the codY null mutant. Coupling global analyses of the transcriptome and the metabolome can reveal how bacteria alter their metabolism when faced with environmental or nutritional stress, providing potential insight into the physiological changes associated with nutrient depletion experienced during infection. Such discoveries may pave the way for the development of novel anti-infectives and therapeutics.

Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry.

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Biochemistry

High-throughput Siderophore Screening from Environmental Samples: Plant Tissues, Bulk Soils, and Rhizosphere Soils

Siderophores (low-molecular weight metal chelating compounds) are important in various ecological phenomenon ranging from iron (Fe) biogeochemical cycling in soils, to pathogen competition, plant growth promotion, and cross-kingdom signaling. Furthermore, siderophores are also of commercial interest in bioleaching and bioweathering of metal-bearing minerals and ores. A rapid, cost effective, and robust means of quantitatively assessing siderophore production in complex samples is key to identifying important aspects of the ecological ramifications of siderophore activity, including, novel siderophore producing microbes. The method presented here was developed to assess siderophore activity of in-tact microbiome communities, in environmental samples, such as soil or plant tissues. The samples were homogenized and diluted in a modified M9 medium (without Fe), and enrichment cultures were incubated for 3 days. Siderophore production was assessed in samples at 24, 48, and 72 hours (h) using a novel 96-well microplate CAS (Chrome azurol sulphonate)-Fe agar assay, an adaptation of the traditionally tedious and time-consuming colorimetric method of assessing siderophore activity, performed on individual cultivated microbial isolates. We applied our method to 4 different genotypes/Lines of wheat (Triticum aestivum L.), including Lewjain, Madsen, and PI561725, and PI561727 commonly grown in the inland Pacific Northwest. Siderophore production was clearly impacted by the genotype of wheat, and in the specific types of plant tissues observed. We successfully used our method to rapidly screen for the influence of plant genotype on siderophore production, a key function in terrestrial and aquatic ecosystems. We produced many technical replicates, yielding very reliable statistical differences in soils and within plant tissues. Importantly, the results show the proposed method can be used to rapidly examine siderophore production in complex samples with a high degree of reliability, in a manner that allows communities to be preserved for later work to identify taxa and functional genes.

We present a protocol for rapid screening of environmental samples for siderophore potential contributing to micronutrient bioavailability and turnover in terrestrial systems.

Siderophores (low-molecular weight metal chelating compounds) are important in various ecological phenomenon ranging from iron (Fe) biogeochemical cycling ...

Environment

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

Immunology and Infection

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Bioengineering

Comparative Analysis of Rhamnolipid Production in Pseudomonas aeruginosa Strains

Detection and Quantification of Mono-Rhamnolipids and Di-Rhamnolipids Produced by Pseudomonas aeruginosa

The environmental bacterium Pseudomonas aeruginosa is an opportunistic pathogen with high antibiotic resistance that represents a health hazard. This bacterium produces high levels of biosurfactants known as rhamnolipids (RL), which are molecules with significant biotechnological value but are also associated with virulence traits. In this respect, the detection and quantification of RL may be useful for both biotechnology applications and biomedical research projects. In this article, we demonstrate step-by-step the technique to detect the production of the two forms of RL produced by P. aeruginosa using thin-layer chromatography (TLC): mono-rhamnolipids (mRL), molecules constituted by a dimer of fatty acids (mainly C10-C10) linked to one rhamnose moiety, and di-rhamnolipids (dRL), molecules constituted by a similar fatty acid dimer linked to two rhamnose moieties. Additionally, we present a method to measure the total amount of RL based on the acid hydrolysis of these biosurfactants extracted from a P. aeruginosa culture supernatant and the subsequent detection of the concentration of rhamnose that reacts with orcinol. The combination of both techniques can be used to estimate the approximate concentration of mRL and dRL produced by a specific strain, as exemplified here with the type strains PAO1 (phylogroup 1), PA14 (phylogroup 2), and PA7 (phylogroup 3).

Author Spotlight: Understanding Rhamnolipid Regulation in Pseudomonas aeruginosa

Pseudomonas aeruginosa&#160;produces the rhamnolipid biosurfactants. Thin-layer chromatography detects and determines the proportion of mono- and di-rhamnolipids produced by each strain. Quantification of total rhamnolipids involves assessing rhamnose equivalents present in these biosurfactants extracted from the culture supernatants using the orcinol method.

The environmental bacterium Pseudomonas aeruginosa is an opportunistic pathogen with high antibiotic resistance that represents a health hazard. This ...

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based ELISA.

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype ...

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a large proportion of the infecting bacteria form biofilms within airway sputum. Pa biofilms isolated from CF sputum have been shown to grow in small, dense aggregates of ~10-1,000 cells that are spatially organized and exhibit clinically relevant phenotypes such as antimicrobial tolerance. One of the biggest challenges to studying how Pa aggregates respond to the changing sputum environment is the lack of nutritionally relevant and robust systems that promote aggregate formation. Using a synthetic CF sputum medium (SCFM2), the life history of Pa aggregates can be observed using confocal laser scanning microscopy (CLSM) and image analysis at the resolution of a single cell. This in vitro system allows the observation of thousands of aggregates of varying size in real time, three dimensions, and at the micron scale. At the individual and population levels, having the ability to group aggregates by phenotype and position facilitates the observation of aggregates at different developmental stages and their response to changes in the microenvironment, such as antibiotic treatment, to be differentiated with precision.

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Synthetic cystic fibrosis sputum medium (SCFM2) can be utilized in combination with both confocal laser scanning microscopy and fluorescence-activated cell sorting to observe bacterial aggregates at high resolution. This paper details methods to assess aggregate populations during antimicrobial treatment as a platform for future studies.

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a ...

Isolation of Total RNA from Pseudomonas aeruginosa within Biofilms for Measuring Gene Expression

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes infections in the airways of cystic fibrosis (CF) patients. P. aeruginosa is known for its ability to form biofilms that are protected by a matrix of exopolysaccharides. This matrix allows the microorganisms to be more resilient to external factors, including antibiotic treatment. One of the most common methods of biofilm growth for research is in microtiter plates or chambered slides. The advantage of these systems is that they allow for the testing of multiple growth conditions, but their disadvantage is that they produce limited amounts of biofilm for RNA extraction. The purpose of this article is to provide a detailed, step by step protocol on how to extract total RNA from small amounts of biofilm of sufficient quality and quantity for high throughput sequencing. This protocol allows for the study of gene expression within these biofilm systems.

Isolation of Total RNA from Pseudomonas aeruginosa within Biofilms for Measuring Gene Expression

This protocol presents a method to isolate RNA from Pseudomonas aeruginosa biofilms grown in chamber slides for high throughput sequencing.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes infections in the airways of cystic fibrosis (CF) patients. P. aeruginosa is ...

T3SS Activity in Yersinia pseudotuberculosis Under Iron Depletion & Anaerobic Stress

Quantifying Yersinia pseudotuberculosis Type III Secretion System Activity Following Iron Starvation and Anaerobic Growth

A key virulence mechanism for many Gram-negative pathogens is the type III secretion system (T3SS), a needle-like appendage that translocates cytotoxic or immunomodulatory effector proteins into host cells. The T3SS is a target for antimicrobial discovery campaigns since it is accessible extracellularly and largely absent from non-pathogenic bacteria. Recent studies demonstrated that the T3SS of Yersinia and Salmonella are regulated by factors responsive to iron and oxygen, which are important niche-specific signals encountered during mammalian infection. Described here is a method for iron starvation of Yersinia pseudotuberculosis, with subsequent optional supplementation of inorganic iron. To assess the impact of oxygen availability, this iron starvation process is demonstrated under both aerobic and anaerobic conditions. Finally, incubating the cultures at the mammalian host temperature of 37 &#176;C induces T3SS expression and allows quantification of Yersinia T3SS activity by visualizing effector proteins released into the supernatant. The steps detailed here offer an advantage over the use of iron chelators in the absence of iron starvation, which is insufficient for inducing robust iron starvation, presumably due to efficient Yersinia iron uptake and scavenging systems. Likewise, acid-washing laboratory glassware is detailed to ensure the removal of residual iron, which is essential for inducing robust iron starvation. Additionally, using a chelating agent is described to remove residual iron from media, and culturing the bacteria for several generations in the absence of iron to deplete bacterial iron stores. By incorporating standard protocols of trichloroacetic acid-induced protein precipitation, SDS-PAGE, and silver staining, this procedure demonstrates accessible ways to measure T3SS activity. While this procedure is optimized for Y. pseudotuberculosis, it offers a framework for studies in pathogens with similar robust iron uptake systems. In the age of antibiotic resistance, these methods can be expanded to assess the efficacy of antimicrobial compounds targeting the T3SS under host-relevant conditions.

Bacteria colonize host tissues that vary in oxygen and iron bioavailability, yet most approaches to studying bacteria use aerated, rich media. This protocol describes culturing the human pathogen Yersinia pseudotuberculosis under varying iron concentrations and oxygen tension, and quantifying activity of the Yersinia type III secretion system, which is an important virulence factor.

A key virulence mechanism for many Gram-negative pathogens is the type III secretion system (T3SS), a needle-like appendage that translocates cytotoxic or ...

FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with F&#246;rster resonance energy transfer (FRET) provide accurate information about PPIs in live cells. FLIM-FRET relies on measuring the fluorescence lifetime decay of a FRET donor at each pixel of the FLIM image, providing quantitative and accurate information about PPIs and their spatial cellular organizations. We propose here a detailed protocol for FLIM-FRET measurements that we applied to monitor PPIs in live Pseudomonas aeruginosa in the particular case of two interacting proteins expressed with highly different copy numbers to demonstrate the quality and robustness of the technique at revealing critical features of PPIs. This protocol describes in detail all the necessary steps for PPI characterization - starting from bacterial mutant constructions up to the final analysis using recently developed tools providing advanced visualization possibilities for a straightforward interpretation of complex FLIM-FRET data.

We describe here a protocol to characterize protein-protein interactions between two highly-differently expressed proteins in live Pseudomonas aeruginosa using FLIM-FRET measurements. The protocol includes bacteria strain constructions, bacteria immobilization, imaging and post-imaging data analysis routines.

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with F&#246;rster ...

Qualitative and Quantitative Analysis of Siderophore Production from Pseudomonas aeruginosa

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A Tandem Liquid Chromatography–Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

High-throughput Siderophore Screening from Environmental Samples: Plant Tissues, Bulk Soils, and Rhizosphere Soils

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Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

Detection and Quantification of Mono-Rhamnolipids and Di-Rhamnolipids Produced by Pseudomonas aeruginosa

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FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.