CAS agar diffusion assay for the measurement of siderophores in biological fluids.
ABSTRACT We developed a simple and universal method, by modifying the universal CAS (Chrome azurol S) assay, measuring siderophores in various biological fluids. We named the assay as CAS agar diffusion (CASAD) assay. CAS plate devoid of nutrients was prepared by using Bacto-agar (1.5%, w/v) as a matrix. Holes with 5-mm-diameter were punched on the CAS agar plate. Each hole was added by 35 microl of the test fluids containing Desferal that was twofold serially diluted. After incubating at 37 degrees C or room temperature for 4-8 h, the size of orange haloes formed around the holes was measured. The size of orange haloes correlated well with the concentration of Desferal in all the biological fluids tested in this study. CASAD assay showed consistent results in wide pH range from 5 to 9. Addition of iron to the test fluids containing Desferal decreased the size of orange haloes in a dose-dependent manner, which suggests that the CASAD assay detects only iron non-bound siderophore. These results suggest that CASAD assay would serve as a simple, stable, and highly reproducible test for screening and quantitative siderophore analysis in biological fluids.
SourceAvailable from: Martina Sura-de Jong[Show abstract] [Hide abstract]
ABSTRACT: Selenium (Se)-rich plants may be used to provide dietary Se to humans and livestock, and also to clean up Se-polluted soils or waters. This study focused on endophytic bacteria of plants that hyperaccumulate selenium (Se) to 0.5–1% of dry weight. Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to compare the diversity of endophytic bacteria of hyperaccumulators Stanleya pinnata (Brassicaceae) and Astragalus bisulcatus (Fabaceae) with those from related non-accumulators Physaria bellii (Brassicaceae) and Medicago sativa (Fabaceae) collected on the same, seleniferous site. Hyperaccumulators and non-accumulators showed equal T-RF diversity. Parsimony analysis showed that T-RFs from individuals of the same species were more similar to each other than to those from other species, regardless of plant Se content or spatial proximity. Cultivable endophytes from hyperaccumulators S. pinnata and A. bisulcatus were further identified and characterized. The 66 bacterial morphotypes were shown by MS MALDI-TOF Biotyper analysis and 16S rRNA gene sequencing to include strains of Bacillus, Pseudomonas, Pantoea, Staphylococcus, Paenibacillus, Advenella, Arthrobacter, and Variovorax. Most isolates were highly resistant to selenate and selenite (up to 200 mM) and all could reduce selenite to red elemental Se, reduce nitrite and produce siderophores. Seven isolates were selected for plant inoculation and found to have plant growth promoting properties, both in pure culture and when co-cultivated with crop species Brassica juncea (Brassicaceae) or M. sativa. There were no effects on plant Se accumulation. We conclude that Se hyperaccumulators harbor an endophytic bacterial community in their natural seleniferous habitat that is equally diverse to that of comparable non-accumulators. The hyperaccumulator endophytes are characterized by high Se resistance, capacity to produce elemental Se and plant growth promoting properties.Frontiers in Plant Science 03/2015; 6:1-17. DOI:10.3389/fpls.2015.00113 · 3.64 Impact Factor
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ABSTRACT: Siderophores, a high-affinity chelating agents for ferric iron, is one of the mechanisms by which biocontrol agents' act in inhibiting the growth of phytopathogens. Bacillus subtilis QM3, a siderophore producer, is a potential biocontrol agent. In this study, a simple double-layered chrome azurol S agar (SD-CASA) plate assay was developed. We further studied optimal conditions for detection siderophore production by B. subtilis QM3 using various history cultures, pH value, amino acid, and different carbon-nitrogen ratio as four variables. After incubating different history cultures we found out that the LB medium presented strong significant CAS reaction even for 12 h, and with the help of changing variables on SD-CASA plate assisting paper-disc diffusion, strain QM3 from LB medium with Fe(3+) (10 mu mol/L) showed difference interestingly. In addition, CAS reaction and growth reaction also increased with pH 7.0, tryptophan 10 mu mol/L and carbon-nitrogen ratio 5:1. The findings on solid medium were similar as that in liquid medium. The SD-CASA assay should be a useful tool for screening potential biocontrol microorganisms by siderophore production.African journal of microbiology research 11/2011; 5(25). DOI:10.5897/AJMR11.238 · 0.54 Impact Factor
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ABSTRACT: Context: Escherichia coli is known as causative agent of urinary tract infections (UTIs) tends to form microcolonies in mucosa lining of urinary bladder known as biofilm. These biofilms make the organism to resist the host immune response, more virulent and lead to the evolution of antibacterial drug resistance by enclosing them in an extracellular biochemical matrix. Aims: This study was done to know the association of various virulence factors and biofilm production in uropathogenic E. coli (UPEC) and antibiotic susceptibility pattern. Settings and design: This study was conducted in Pt. B.D. Sharma PGIMS, Rohtak, Haryana during a period of 1 year from January 2011 to December 2011. Methods and material: Biofilm was detected by microtiter plate (MTP) method, and various virulence factors like hemolysin, hemagglutination, gelatinase, siderophore production, serum resistance, and hydrophobicity were detected. The antibiotic susceptibility testing was done by modified Kirby-Bauer disk diffusion and the disk diffusion method was used to confirm the ESBL, AmpC, MBL production by the UPEC statistical analysis used: The data were analyzed by using SPSS version 17.0. A two-sided P-value of less than or equal to 0·05 was considered to be significant. Results: Biofilm production was found in 18 (13·5%) isolates, more commonly in females (two times). These isolates were found to be resistant to antibiotics common in use and were 100% MDR. Conclusions: Biofilm production makes the organism to be more resistant to antibiotics and virulent as compared to non-biofilm producers.