Chrome Azurol Research Articles

Fonsecaea pedrosoi produces ferricrocin and can utilize different host iron sources

The survival of living organisms depends on iron, one of the most abundant metals in the Earth's crust. Nevertheless, this micronutrient is poorly available in our aerobic atmosphere as well as inside the mammalian host. This problem is circumvented by the expression of high affinity iron uptake machineries, including the production of siderophores, in pathogenic fungi. Here we demonstrated that F. pedrosoi, the causative agent of the neglected tropical disease chromoblastomycosis, presents gene clusters for siderophore production. In addition, ten putative siderophore transporters were identified. Those genes are upregulated under iron starvation, a condition that induces the secretion of hydroxamates, as revealed by chrome azurol S assays. RP-HPLC and mass spectrometry analysis allowed the identification of ferricrocin as an intra- and extracellular siderophore. F. pedrosoi can grow in different iron sources, including the bacterial ferrioxamine B and the host proteins ferritin, hemoglobin and holotransferrin. Of note, addition of hemoglobin, lactoferrin and holotransferrin to the growth medium of macrophages infected with F. pedrosoi enhanced significantly fungal survival. The ability to produce siderophores in iron limited conditions added to the versatility to utilize different sources of iron are strategies that certainly may contribute to fungal survival inside the host.

Characterization of uropathogenic E. coli from various geographical locations in India

ObjectivesUropathogenic Escherichia coli (UPEC) is the most common causative agent of urinary tract infection, accounting for more than 80% of cases worldwide. This study presents data on prevalent serotypes, resistance profiles, and colonization-aiding virulence characteristics of UPEC from different geographical regions in India. MethodsUPEC were serotyped through microtiter plate agglutination. Standard techniques were used to detect various virulence characteristics, i.e., biofilm formation (tissue culture plate method), siderophore production (screened on Chrome Azurol S agar and categorized with Csaky's and Arnow's methods), colicin release (agar overlay technique), gelatin hydrolysis (on gelatinase agar), and cell surface hydrophobicity (salt aggregation method). Antibiotic resistance profiles (against 20 antimicrobial agents) and extended-spectrum beta-lactamase (ESBL) were evaluated according to Clinical and Laboratory Standards Institute guidelines. ResultsUPEC strains exhibited very high drug resistance rates to most of the commonly used antimicrobial agents; the highest resistance rates were observed for ampicillin (63.4%), nalidixic acid (63.4%), and cefotaxime (62.1%). High rates of multi-drug resistance (63.36%), ESBL-production (34.1%), and carbapenem-resistance (25.0%) were detected in UPEC strains from all geographical regions of India. Hydrophobicity (61.2%), biofilm production (62.5%), and siderophore production (67.7%) were the most common virulence characteristics of UPEC isolates. Co-expression of virulence characteristics was common (69.8%) in UPEC strains. ConclusionUPEC strains with very high antimicrobial-resistance are in circulation in India, and have diverse serotypes and virulence characteristics.

Evaluation of Bio-Friendly Formulations from Siderophore-Producing Fluorescent Pseudomonas as Biocontrol Agents for the Management of Soil-Borne Fungi, Fusarium oxysporum and Rhizoctonia solani

Secretion of siderophores by Pseudomonas aeruginosa F2 and P. fluorescens JY3 was evaluated on chrome azurol S (CAS) agar plates and their inhibitory effect was inspected against Fusarium oxysporum and Rhizoctonia solani. Production of siderophores as biocontrol agents from F2 and JY3 was accomplished in two optimized media. Afterward, cell-free supernatants of the bacterial cultures containing siderophores were used for the preparation of two bio-friendly formulations for the management of F. oxysporum and R. solani under greenhouse conditions. The investigated bacterial isolates, F2 and JY3, showed antagonistic activity in vitro against F. oxysporum and R. solani and produced siderophores in optimized media with high efficiency. Colonies of both bacterial isolates were grown exponentially with a constant specific growth rate of 0.07 h−1 and 0.27 h−1, correspondingly. Siderophores estimated in 10 µL reached their highest value of 16.95% at 47 h and 19.5% at 48 h for isolate F2 and JY3, respectively. Formulations of siderophore-generating F2 and JY3 reduced damping-off caused by F. oxysporum by 40% and 80%, while the reduction percentage of damping-off caused by R. solani reached 87.5% and 62.5%, correspondingly. Moreover, both formulations encouraged the growing of wheat plants where the fresh and dry weight of shoots and roots were increased compared to the treatment with each fungus. In conclusion, bio-friendly formulations resulting from this investigation can play an active role in managing soil-borne diseases.

SOIL SALINITY SHIFTS CULTIVABLE MICROBIAL COMMUNITIES OF WHEAT (Triticum turgidum subsp. durum) RHIZOSPHERE IN THE YAQUI VALLEY, MEXICO

Saline soils are a common issue in commercial fields using intensive and irrigated agricultural practices under arid or semi-arid climates, such as the Yaqui Valley in Mexico, one of the most important wheat-producing regions worldwide. This study aimed to assess the cultivable microbial diversity (having plant growth-promoting attributes) associated with wheat rhizosphere in commercial fields, under a saline soil gradient in the Yaqui Valley. Thus, seven wheat commercial zones were studied according to their electrical conductivity (0.9 to 6.4 dS cm-1). The isolation of microorganisms was carried out by dependent culture techniques, sequencing of the 16S (bacteria) and 5.8S (fungi) rRNA genes , and plant growth-promoting attributes were evaluated: phosphate solubilization (Pikovskaya medium), siderophore production (Chrome Azurol S medium), indole production (Salkowsky reagent), and hemolytic activity (Petri dishes 5 % Sheep Blood). Wheat commercial zones with 1.6 dS m-1 showed the highest fungal (2.59 x 106 CFU g-1 dry soil) and bacterial (2.88 x 107 CFU g-1 dry soil) populations, being Rhizopus and Bacillus the most abundant and well-distributed microbial genera, respectively. The impact of saline soils on the microbial metabolic background was not species- or genus-specific; it was at a strain-specific level. In addition, 52 % of fungal and 83 % of bacterial strains showed the ability to produce γ-hemolysis, suggesting it is not harmful to humans and animals. This work provides a microbial culture collection where their members have shown several beneficial traits that potentially increase the yield and quality of wheat growing under saline soil conditions.

Iron Starvation Induces Ferricrocin Production and the Reductive Iron Acquisition System in the Chromoblastomycosis Agent Cladophialophora carrionii.

Iron is a micronutrient required by almost all living organisms. Despite being essential, the availability of this metal is low in aerobic environments. Additionally, mammalian hosts evolved strategies to restrict iron from invading microorganisms. In this scenario, the survival of pathogenic fungi depends on high-affinity iron uptake mechanisms. Here, we show that the production of siderophores and the reductive iron acquisition system (RIA) are employed by Cladophialophora carrionii under iron restriction. This black fungus is one of the causative agents of chromoblastomycosis, a neglected subcutaneous tropical disease. Siderophore biosynthesis genes are arranged in clusters and, interestingly, two RIA systems are present in the genome. Orthologs of putative siderophore transporters were identified as well. Iron starvation regulates the expression of genes related to both siderophore production and RIA systems, as well as of two transcription factors that regulate iron homeostasis in fungi. A chrome azurol S assay demonstrated the secretion of hydroxamate-type siderophores, which were further identified via RP-HPLC and mass spectrometry as ferricrocin. An analysis of cell extracts also revealed ferricrocin as an intracellular siderophore. The presence of active high-affinity iron acquisition systems may surely contribute to fungal survival during infection.

Intermediates and shunt products of massiliachelin biosynthesis in Massilia sp. NR 4-1.

Siderophores are small molecules secreted by microorganisms in order to scavenge iron from the environment. An example is the thiazoline-containing natural product massiliachelin, which is produced by Massilia sp. NR 4-1 under iron-deficient conditions. Based on experimental evidence and genome analysis, it was suspected that this bacterium synthesizes further iron-chelating molecules. After a thorough inspection of its metabolic profile, six previously overlooked compounds were isolated that were active in the chrome azurol S (CAS) assay. Mass spectrometric measurements and nuclear magnetic resonance spectroscopic analyses identified these compounds as possible biosynthetic intermediates or shunt products of massiliachelin. Their bioactivity was tested against one Gram-positive and three Gram-negative bacteria.

Bacillibactin siderophore induces iron mobilisation responses inside aerobic rice variety through YSL15 transporter

Iron uptake in Oryza sativa primarily occurs with the help of phytosiderophore i.e., deoxymugineic acid in aerobic condition. Internalisation of phytosiderophore-iron complex occurs through YSL15. However, aerobic rice at high pH faces iron deficit condition due to low secretion of plant siderophore and its early diminution. There might be a possibility that microbial siderophore of predominant rhizobacteria present in soil around rice may assist in iron uptake and transport through same transporter providing survival advantage to the plant. Present study aims to explore role of siderophore of Bacillus subtilis in improving root architecture and iron uptake in aerobic rice at high pH and explore possible transporters involved in transporting siderophore-Fe3+ complex inside rice root from rhizosphere. Identification by chrome azurol sulphonate assay and characterisation of purified extract from B. subtilis culture through LC-ESI MS confirmed presence of catecholate siderophore, Bacillibactin. Aerobic rice variety viz. Swarna Shreya grown with 5 ng/ml bacillibactin showed improved morphology and anatomy than untreated sample at alkaline pH 7.5. This is possibly due to enhanced intracellular iron content as observed in atomic absorption spectroscopy. Whole root proteomic analysis of rice root by using LC-ESI MS/MS revealed presence of YSL like transporter and siderophore import proteins in treated sample. RT-qPCR analysis showed enhanced expression of gene YSL15 in siderophore treated sample than control (pH 7.5). The present study possibly suggest role of foreign siderophore (xenosiderophore) in plant growth and development through iron uptake and transport inside rice.

In vitro phenotypic evidence for the utilization of iron from different sources and siderophores production in the fish pathogen Tenacibaculum dicentrarchi.

Iron uptake during infection is an essential pathogenicity factor of several bacteria, including Tenacibaculum dicentrarchi, an emerging pathogen for salmonid and red conger eel (Genypterus chilensis) farms in Chile. Iron-related protein families were recently found in eight T. dicentrarchi genomes, but biological studies have not yet confirmed functions. The investigation reported herein clearly demonstrated for the first time that T. dicentrarchi possesses different systems for iron acquisition-one involving the synthesis of siderophores and another allowing for the utilization of heme groups. Using 38 isolates of T. dicentrarchi and the type strain CECT 7612T , all strains grew in the presence of the chelating agent 2.2'-dipyridyl (from 50 to 150 μM) and produced siderophores on chrome azurol S plates. Furthermore, 37 of the 38 T. dicentrarchi isolates used at least four of the five iron sources (i.e. ammonium iron citrate, ferrous sulfate, iron chloride hexahydrate, haemoglobin and/or hemin) when added to iron-deficient media, although the cell yield was less when using hemin. Twelve isolates grew in the presence of hemin, and 10 of them used only 100 μM. Under iron-supplemented or iron-restricted conditions, whole cells of three isolates and the type strain showed at least one membrane protein induced in iron-limiting conditions (c.a. 37.9 kDa), regardless of the isolation host. All phenotypic results were confirmed by in-silico genomic T. dicentrarchi analysis. Future studies will aim to establish a relationship between iron uptake ability and virulence in T. dicentrarchi through in vivo assays.

Halomonas gemina sp. nov. and Halomonas llamarensis sp. nov., two siderophore-producing organisms isolated from high-altitude salars of the Atacama Desert.

This study aimed to identify and characterize novel siderophore-producing organisms capable of secreting high quantities of the iron-binding compounds. In the course of this, two not yet reported halophilic strains designated ATCHAT and ATCH28T were isolated from hypersaline, alkaline surface waters of Salar de Llamará and Laguna Lejía, respectively. The alkaline environment limits iron bioavailability, suggesting that native organisms produce abundant siderophores to sequester iron. Both strains were characterized by polyphasic approach. Comparative analysis of the 16S rRNA gene sequences revealed their affiliation with the genus Halomonas. ATCHAT showed close similarity to Halomonas salicampi and Halomonas vilamensis, while ATCH28T was related closest to Halomonas ventosae and Halomonas salina. The ability of both strains to secrete siderophores was initially assessed using the chromeazurol S (CAS) liquid assay and subsequently further investigated through genomic analysis and NMR. Furthermore, the effect of various media components on the siderophore secretion by strain ATCH28T was explored. The CAS assay confirmed the ability of both strains to produce iron-binding compounds. Genomic analysis of strain ATCHAT revealed the presence of a not yet reported NRPS-dependant gene cluster responsible for the secretion of siderophore. However, as only small amounts of siderophore were secreted, further investigations did not lie within the scope of this study. Via NMR and genomic analysis, strain ATCH28T has been determined to produce desferrioxamine E (DFOE). Although this siderophore is common in various terrestrial microorganisms, it has not yet been reported to occur within Halomonas, making strain ATCH28T the first member of the genus to produce a non-amphiphilic siderophore. By means of media optimization, the produced quantity of DFOE could be increased to more than 1000 µM. Phenotypic and genotypic characteristics clearly differentiated both strains from other members of the genus Halomonas. Average nucleotide identity (ANI) values and DNA-DNA relatedness indicated that the strains represented two novel species. Therefore, both species should be added as new representatives of the genus Halomonas, for which the designations Halomonas llamarensis sp. nov. (type strain ATCHAT = DSM 114476 = LMG 32709) and Halomonas gemina sp. nov. (type strain ATCH28T = DSM 114418 = LMG 32708) are proposed.

Determination of trace aluminum using resonance Rayleigh scattering method with chrome azurol S and hexadecyl trimethyl ammonium bromide system

Determination of trace aluminum using resonance Rayleigh scattering method with chrome azurol S and hexadecyl trimethyl ammonium bromide system

Biogeochemical networks in the abandoned historical gold mines affecting mobilization and transport of arsenic in Kolar

Enormous water-logging in ancient abandoned mining shafts of Kolar Gold Fields (KGFs), has largely induced the leaching of sulfide-rich gold minerals contaminating the aquifer system with hazardous elements. Transport of these contaminant has posed threat to the health of the urban population of Kolar township. A detailed survey of borewells, covering radius of 10 km of the KGF was carried out during pre and post-monsoon seasons and various parameters were assessed. Almost 80% of the water samples exceeded the regulatory limits of potable water criteria with excess arsenic (As; 12–127 μg/L), fluoride (F; <0.005 μg/L), dissolved salts (>500 mg/L). Water Quality Index (WQI) was used to understand the overall urban groundwater quality. At the centre of sampling circle core, mineral dissolution was found to be the function of pH, induced by acidophilic sulfur oxidizing bacteria . Modelling of predicted microbial metabolic pathways in metagenomics libraries using PICRUSt, indicated complex functional networks. High expression of siderophore proteins (> 2 cm halo in the chrome azurol test) caused Fe-sequestration, secondary Fe-mineral formation and subsequent release of As. Sulfide bearing Au-rich minerals (Arsenopyrite, Scorodite, Jarosite) were bio-weathered leading to release of H3AsO3+ at low pH, resulted in groundwater composition of Ca–HCO3 type and Ca–Na–HCO3 or Ca–Mg–Cl type.

Plant Growth-promoting Ability and Pathogen Inhibitory Effect of Actinomycetes Isolated from Fecal Pellets of the Giant Millipede Thyropygus resimus (Diplopoda)

The microbial properties of millipede fecal pellets have been studied mainly in Glomerida (pill millipedes), and much less in the significant majority of other millipede groups. Therefore, the present study examined actinomycetes isolated from the fecal pellets of the non-glomerid giant millipede Thyropygus resimus Attems, 1938 (Spirostreptida) to (1) test their plant growth-promoting ability, and (2) evaluate their potential to control and inhibit plant pathogenic microorganisms. Millipedes were collected from Phu Kum Khao, Kalasin Province, Thailand. A total of 59 actinomycete isolates were obtained and identified as belonging to the genus Streptomyces using 16S rRNA sequencing. The plant growth-promoting properties of the isolates were tested by screening four characteristics: nitrogen fixation, phosphate solubility, siderophore production, and indole-3-acetic acid (IAA) production. A nitrogen-fixation test on nitrogen-free solid malate media (NFM) showed that 54 isolates were capable of fixing nitrogen. Phosphate solubility was tested on double-layered glucose yeast extract agar (GYA) medium containing tricalcium phosphate. This showed that 42 isolates formed a clear zone around the colonies due to phosphate dissolution. Siderophore production was tested on chrome azurol sulfate (CAS) agar. This showed that 55 isolates could grow on this medium and form clear yellow to orange zones around their colonies. IAA production tests revealed that 41 isolates could produce IAA. Based on the combined results of these four tests, eight of the 59 isolates were the most effective in promoting plant growth: KLS-AC04, KLD-AC01, KLD-AC02-1, KLD-AC08, KLD-AC09, KLD-AC16, KLD-AC29-1, and KLD-AC30. Seventeen isolates inhibited the growth of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight disease in rice, more effectively than rifampicin (100 ppm), with isolate KLS-AC02-1-1 being the most effective (inhibition zone, 58.25 mm in diameter). Therefore, these isolates can be used for growth promotion and rice disease control in the future.

Siderophore production in fungi from asbestos biofilms: The first step towards bioremediation of a carcinogenic mineral

Asbestos refers to six types of fibrous, silicate minerals, historically used for a wide range of household, commercial and industrial applications. Asbestos exposure is known to cause diseases such as asbestosis, pleural mesothelioma and lung cancer, and is responsible for around 220 deaths per year in Aotearoa / New Zealand. Asbestos is disposed of using a designated hazardous landfill facility, an expensive and unsustainable practice. As an alternative, research has focused on bioremediation to manage asbestos contamination. Bioremediation research has shown that asbestos fibres can be partially degraded, and surface reactivity decreased, by the activity of iron-chelating siderophores, produced by some fungi and bacteria. This paper presents initial results of siderophore-detecting chrome azurol S (CAS)-agar plate assays on mycobiota collected from biofilms from asbestos-containing building products in Tāmaki Makaurau / Auckland and natural asbestos mineral deposits in Kahurangi National Park, northwest of Whakatū / Nelson.

Colorimetric Determination of Adenylation Domain Activity in Nonribosomal Peptide Synthetases by Using Chrome Azurol S.

Adenylation domains are the main contributor to structural complexity among nonribosomal peptides due to their varied but stringent substrate selection. Several in vitro assays to determine the substrate specificity of these dedicated biocatalysts have been implemented, but high sensitivity is often accompanied by the cost of laborious procedures, expensive reagents or the requirement for auxiliary enzymes. Here, we describe a simple protocol that is based on the removal of ferric iron from a preformed chromogenic complex between ferric iron and Chrome Azurol S. Adenylation activity can be rapidly followed by a decrease in absorbance at 630 nm, visualized by a prominent color change from blue to orange.

Siderophore Production and its Role as Therapeutic Agent

Siderophores are iron chelators, which are produced by bacteria under iron-deficient conditions required for their growth. Therefore, siderophores can be used as a carrier to direct drugs into the bacteria and kill them. The present study was designed to screen siderophore production using different bacteria using an iron-deficient medium and its synergistic capability to kill drug-resistant bacteria. Siderophore under iron-deprived condition was evaluated by chrome azurol S (CAS) assay. Whereas, broth micro-dilution method and checkerboard assay were used to determine the antimicrobial properties of selected drugs or epigallocatechin gallate (EGCG) individually or in combination with synthetic siderophore. Results demonstrated that the entire tested microorganisms produced siderophore under the iron-deprived condition as evidenced by orange halo zones in CAS agar plates. Gram-negative bacteria produced more siderophores as reflected by orange color with bacterial zone inhibition of 17-22mm as compared to Gram-positive bacteria (13-15mm). As compared to antibiotics and EGCG, acetohydroxamic acid (aHa; synthetic siderophore) showed no antibacterial properties (1500 - 6500 µg/ml). The synergism of aHa with tetracycline, ceftriaxone, and EGCG (FIC index &lt;0.5) against S. typhi, methicillin-resistant and sensitive Staphylococcus aureus, and E. coli were evident. In conclusion, siderophore may be considered a potential candidate to design different combination therapy against emerging antimicrobial-resistant pathogens.

Functional Divergence of the N-Lobe and C-Lobe of Transferrin Gene in Pungitius sinensis (Amur Stickleback).

Transferrin is an important iron-binding glycosylated protein and plays key roles in iron-binding and immune response. Here, a 2037-bp open reading frame was obtained from our previous transcriptome sequencing data of Amur stickleback, which encoded a 679 amino acid putative transferrin protein harbored obvious N-lobe and C-lobe domains. The tissue-specific expression pattern showed that the transcript was detected in a variety of tissues, with the highest signal in liver. Moreover, Streptococcus iniae pathogen stimulation can increase the expression level of this transcript, implying important immune properties for organisms. Next, N-lobes and C-lobes were obtained from 45 fish species. The phylogenetic tree showed that N-lobes and C-lobes were in two different evolutionary branches, and they had different motif composition. Functional divergence indicated a higher evolutionary rate or site-specific alteration among the N-lobe and C-lobe groups. Ka/Ks value of C-lobe group was relatively higher than that of N-lobe group, indicating a faster change rate of C-lobe sequences in evolution. Moreover, some sites experiencing positive selection were also found, which may be involved in the iron- or anion-binding, pathogen resistance and diversification of transferrin protein. Differential iron-binding activity was also detected between N-lobe and C-lobe of Amur stickleback transferrin protein with Chrome Azurol S assay. Compared with the C-lobe, the N-lobe showed stronger growth inhibitory activity of Escherichia coli, implying their potential antibacterial properties. This study will give a reference for subsequent research of transferrin proteins.

Whole-genome analysis revealed the growth-promoting mechanism of endophytic bacterial strain Q2H1 in potato plants.

Endophytes are non-pathogenic inhabitants of healthy plant tissues and have been found to promote plant growth and health. The endophytic bacterial strain Q2H1 was isolated from the roots of the potato and was identified to exhibit growth-promoting effects in potato plants. Whole-genome sequencing was performed to reveal the mechanism underlying its growth-promoting effect. The obtained sequencing data of approximately 5.65 MB encompassed 5,533 coding sequences. Of note, nine secondary metabolite gene clusters, including siderophore gene clusters, closely associated with plant growth promotion (PGP) were predicted by antiSMASH software. Comparative genomic analysis revealed that Q2H1 belongs to the genus Peribacillus. By gene function annotation, those genes related to plant growth-promoting activities, including indole-3-acetic acid (IAA) synthesis in tryptophan metabolism, siderophore biosynthetic activity, phosphate solubilization, nitrogen fixation, and related genes, were summarized. IAA (14.4 μg/ml) was presumptively produced by Q2H1 using the Salkowski colorimetric method. A total of five genes, namely, phoU, pstB, pstA1, pstC, and pstS, were annotated for phosphate solubilization, which is associated with the ability of the Q2H1 strain to solubilize phosphate under in vitro conditions. It is revealed that genes in the Q2H1 genome associated with nitrogen fixation belonged to three groups, namely, nitrogen fixation (nifU, sufU, salA, and nifS), nitrogen metabolism (nirA, nrtB, and nasA), and glutamate synthesis (glnA, gltB, gltD, and gudB), supported by evidence that Q2H1 grew on medium without nitrogen. We have also identified a siderophore gene cluster located on the chromosome of Q2H1, including seven genes (viz., rbsR, rhbf, rhbE, rhbD, rhbC, rhbA, ddc, and an unknown gene). In the in vitro assay, a prominent brown circle around the colony was produced on the chrome azurol S medium at 48 and 72 h post-inoculation, indicating that the siderophore gene cluster in Q2H1 harbored the ability to produce siderophores. In summary, these findings implied that identifying strain-specific genes for their metabolic pathways in bacterial endophytes may reveal a variety of significant functions of plant growth-promoting mechanisms.

Isolation, screening, characterization, and optimization of bacteria isolated from calcareous soils for siderophore production

The most effective agricultural practice to prevent iron deficiency in calcareous soils is fertilizing with synthetic chelates. These compounds are non-biodegradable, and persistent in the environment; hence, there is a risk of leaching metals into the soil horizon. To tackle iron deficiency-induced chlorosis (IDC) in crops grown on calcareous soils, environmentally friendly solutions are needed rather than chemical application as it affects the soil health further. Hence, the present work focused on isolating and screening calcareous soil-specific bacteria capable of producing iron-chelating siderophores. Siderophore-producing bacteria (SPB) was isolated from the groundnut (Arachis hypogeaL.) rhizosphere region, collected from Coimbatore district, Tamil Nadu, of which 17 bacterial isolates were positive for siderophore production assayed by chrome azurol sulphonate. The performance of SPB isolates was compared for siderophore kinetics, level of siderophore production, type of siderophore produced and iron-chelating capacity under 15mM KHCO3. Four best performing isolates were screened, with average siderophores yield ranging ∼60-80% under pH 8, with sucrose as carbon source and NH2SO4 as nitrogen source at 37°C. The four efficient SPB were molecularly identified asB. licheniformis,B. subtilis,B. licheniformis,andO. grignonensebased on 16S rDNA sequencing. The simultaneous inhibition methodshowedT.viridehas the highest antagonistic effect againstS.rolfsii,andM.phaseolinawith a reduction of mycelial growth by 69.3 and 65.1%, respectively,compared to control.Our results indicate that the optimized conditions enhanced siderophores chelation by suppressing the stem and root rot fungi, which could help in a cost-effective and environmentally friendly manner.

Aspergillus oryzae attenuates quorum sensing -associated virulence factors and biofilm formation in Klebsiella pneumoniae extended-spectrum beta-lactamases

Background: Klebsiella pneumoniae communicate between and among species using quorum sensing (QS). Biofilm formation and virulence factors are regulated by QS. This QS is indirectly responsible for K. pneumoniae pathogenicity. Inhibiting QS is a novel and highly effective method for controlling K. pneumoniae extended-spectrum beta-lactamases (KP-ESBL) infections. This study aimed to investigate how Aspergillus oryzae extracellular protein (AOEP) affected QS and KP-ESBL virulence factors. Methods: Methods used included minimal inhibitory concentration (MIC) through the microdilution method, biofilms with crystal violet staining, extracellular polysaccharides using the Congo Red assay, quantifying the expression of genes coding for capsular polysaccharide (wzI gene) and adhesion (mrkA gene) through quantitative reverse-transcription polymerase chain reaction (RT-qPCR), siderophore level measurement using Chrome Azurol sulphonate assay (CAS assay), biofilm morphology using a scanning electron microscope (SEM), and confirmation using the life span killing assay method on Caenorhabditis elegans (C. elegans). Results: In vitro studies revealed that AOEP inhibited biofilms and exopolysaccharides (EPS) in KP-ESBL at the sub-MIC level. In addition, AOEP inhibited the expression of the mrkA gene, which is involved in the adhesion process. Furthermore, an in vivo study revealed that AOEP levels of 75 and 150 µg/mL respectively increased C. elegans survival rates by 72.67% and 80.76% against K. pneumoniae infection. Conclusions: Our findings suggest that the extracellular protein of A. oryzae may be an effective QS inhibitor and a novel anti-virulence agent to control bacterial pathogens.

Isolation and Characterization of Siderophores Producing Chemolithotrophic Bacteria from the Coal Samples of the Aluminum Industry

Siderophores are iron grabbers and are classified according to the ligands used to chelate the ferric iron. The role of siderophores is to scavenge iron and make it accessible to the cell. It has crucial applications in industries, medical, research, and environmental clean-up. In the current research work, the authors attempted to isolate and characterize a few siderophores producing bacteria from the coal samples, collected from the Bharat Aluminum Company Ltd. Chhattisgarh. The quantitative and qualitative assay for siderophores producing bacteria (SPB) was carried by Chrome Azurol Sulfonate agar test for all the ten samples and positive results were observed only in two strains, i.e., B6 and B7 when grown in iron-deficient succinate medium in an incubator shaker for 48 h at 120 rpm. The biochemical tests revealed that the B6 and B7 strains have similarities to Bacillus sp. and Vibrio sp., respectively. The study reflects the potential of these strains as effective siderophores producers and holds a noteworthy perspective in further industrial and research applications.