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ORIGINAL ARTICLE/ARTICLE ORIGINAL
Lipopeptide biosurfactant from Bacillus thuringiensis pak2310: A potential antagonist against Fusarium oxysporum Biosurfactant lipopeptidique de Bacillus thuringiensis pak2310 : un antagoniste potentiel contre Fusarium oxysporum R. Deepak, R. Jayapradha * Centre for Research on Infectious Diseases (CRID), School of Chemical and Biotechnology, SASTRA University, Tamilnadu, India Received 15 June 2014; received in revised form 2 October 2014; accepted 6 October 2014
KEYWORDS Fusariosis; Bacillus thuringiensis pak2310; Lipopeptide; Antibiotic; Antifungal; Anti-biofilm
Summary Objectives. — The aims of the study were to evaluate the effects of a biosurfactant obtained from a novel Bacillus thuringiensis on Fusarium oxysporum to determine the morphological changes in the structure of the fungi and its biofilm in the presence of the biosurfactant and to evaluate the toxicity of the biosurfactant on HEp-2 human epithelial cell lines. Materials and methods. — The strain was screened and isolated from petroleum contaminated soil based on the E24 emulsification index. The biosurfactant was produced on glycerol, extracted using chloroform:methanol system and purified using HPLC. The purified fraction showing both surface activity (emulsification and oil-spread activity) and anti-fusarial activity (agar well diffusion method) was studied using FT—IR and MALDI-TOF MS, respectively. The minimum inhibitory concentration (MIC) and the biofilm inhibitory concentration (BIC) were determined using dilution method. The effect of biosurfactant on the morphology of Fusarium oxysporum was monitored using light microscopy and confocal laser scanning microscopy (for biofilm). Results. — The purified surfactant showed the presence of functional groups like that of surfactin in the FT—IR spectra and MALDI-TOF MS estimated the molecular weight as 700 Da. The MIC and BIC were estimated to be 0.05 and 0.5 mg/mL, respectively. The molecule was also non-toxic to HEp-2 cell lines at 10 MIC. Conclusion. — A non-toxic and effective anti-Fusarium biosurfactant, that is both safe for human use and to the environment, has been characterized. The growth and metabolite
* Corresponding author. Centre for Research on Infectious Diseases (CRID), School of Chemical and Biotechnology, SASTRA University, Tirumalaisamudram, Thanjavur, 613 401 Tamilnadu, India. E-mail addresses: [email protected], [email protected] (R. Jayapradha). http://dx.doi.org/10.1016/j.mycmed.2014.10.011 1156-5233/# 2014 Published by Elsevier Masson SAS.
Please cite this article in press as: Deepak R, Jayapradha R. Lipopeptide biosurfactant from Bacillus thuringiensis pak2310: A potential antagonist against Fusarium oxysporum. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.10.011
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R. Deepak, R. Jayapradha production using glycerol (major byproduct of biodiesel and soap industries) also adds up to the efficiency and ecofriendly nature of this biosurfactant. # 2014 Published by Elsevier Masson SAS.
MOTS CLÉS Fusariose ; Bacillus thuringiensis pak2310 ; Lipopeptides ; Antibiotique ; Antifongique ; Anti-biofilm
Re ´sume ´ Objectifs. — Les objectifs de cette étude étaient d’évaluer les effets d’un bio-tensioactif obtenu à partir d’un Bacillus thuringiensis nouveau, sur Fusarium oxysporum, afin de déterminer les changements morphologiques dans la structure du champignon et de son biofilm en présence de l’agent bio-tensioactif et d’évaluer la toxicité du biosurfactant sur des lignées de cellules épithéliales humaines HEp-2. Mate´riels et me ´thodes. — La souche a été criblée et isolée à partir de sols contaminés par du pétrole sur la base de l’indice de mise en émulsion E24. Le bio-tensioactif a été produit sur le glycérol, extrait à l’aide de chloroforme:methanol système et purifié en utilisant une HPLC. La fraction purifiée montrant à la fois une activité de surface (émulsification et l’activité de propagation de l’huile) et de l’activité anti-Fusarium (méthode de diffusion en gélose par puits) a été étudiée à l’aide de FT—IR et MALDI-TOF MS, respectivement. La concentration minimale inhibitrice (CMI) et la concentration inhibitrice biofilm (BIC) ont été déterminées en utilisant la méthode de dilution. L’effet du biosurfactant sur la morphologie de Fusarium oxysporum a été objectivée en utilisant la microscopie optique confocale à balayage laser et la microscopie du biofilm. Re ´sultats. — L’agent tensioactif purifié a montré la présence de groupes fonctionnels, tels que des surfactines dans les spectres FT—IR et MALDI-TOF MS avec un poids moléculaire estimé à 700 Da. La CIM et BIC ont été estimées à 0,05 et 0,5 mg/mL, respectivement. La molécule était également non toxique pour les cellules HEp-2 dans des lignées de cellules 10 MIC. Conclusion. — Un biosurfactant anti-Fusarium non toxique et efficace, à la fois sans danger pour la consommation humaine et l’environnement a été caractérisé. La croissance et la production de métabolites en utilisant du glycérol (sous-produit majeur de biodiesel et de savon d’industrie) ajoute également à l’efficacité écologique et à la protection de la nature de ce biosurfactant. # 2014 Publié par Elsevier Masson SAS.
Introduction Fusarium oxysporum, a plant pathogen, was the etiological agent of the epidemic wilting of bananas in Panama and hence, the name of the disease — Panama Wilt [29] was coined. Several other diseases, such as scabs, crown rot and red blight are caused by this fungus [2]. Although primarily a plant pathogen, recent reports indicate that members of Fusarium, especially F. oxysporum and F. solani are emerging human pathogens as well. Keratitis and onychomycosis are the main superficial infections of F. oxysporum in mankind [5,17,33]. Keratitis has been reported mostly in patients who use soft contact lenses [8,14]. Fusariosis or disseminated Fusarium infection has been reported worldwide in immune compromised patients, especially the ones with prolonged neutropenia [4,34]. The symptoms of the disease are very similar to that of aspergillosis [4]. Intra-venous amphotericin B is used against aspergillosis, while that has been reported to be ineffective against Fusarium infections [4]. Although the infection recedes initially with the administration of azole ring antibiotics, death due to fusariosis seems to be inevitable in immune compromised patients [4,8]. The main route of invasion by the pathogen is through the skin, through cuts, rashes or wounds, like the scratch wounds in cellulitis patients [33]. Once entered, the host body is invaded immensely, releasing toxins that can lead to tissue breakdown and immune suppression [17]. However, the toxins produced by Fusaria can contaminate food and can
be easily ingested. These toxins have been shown to suppress the immune system of the host and in turn make the host more susceptible to the pathogen attack [11,15,17]. Fusarium infections are also accompanied by ecthymagangerosum, a skin infection caused by Pseudomonas spp; osteomyelitis; sinusitis and general fungemia [4,33]. Thus, it is necessary to develop new drugs and therapies that can help to manage the disease effectively. F. oxysporum is capable of producing adherent substances form biofilms and produce tissue damaging enzymes [27,40]. Formation of a biofilm has been reported to be the main reason for keratitis and Fusarium keratitis has gained more prominence in the recent past with 41% of total cases of keratitis [21] as a biofilm is more resistant to drugs in use. Another study reports that people using soft contact lens are 40% susceptible to Fusarium keratitis [1]. Several factors for biofilm formation by the pathogen on contact lens including the material used in fabrication and cleansing solution have also been studied [20]. The same research team has also investigated the formation of biofilms in murine models and its relationship with keratitis [43]. Thus, preventing Fusarium biofilms gains importance in the present scenario. Surfactants are those chemical molecules that have both hydrophobic and hydrophilic groups in them. They are soluble in both polar and non-polar solvents [29]. Surfactants produced by microbes are called biosurfactants. Biosurfactants have many advantages, like biodegradability, lower toxicity, mild production conditions, selectivity and higher
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Lipopeptide biosurfactant from Bacillus thuringiensis pak2310 specific activity in a wider range of pH, temperature and salinity [29]. The applications of biosurfactants can be found in a broad spectrum of industries like food, cosmetic, chemical and pharmaceutical industries [3,6,9,25,29,31,39]. Apart from these industries, the application of biosurfactants also reaches many environmental engineering applications, such as bioremediation, enhanced oil recovery, soil washing, etc. [42]. Biosurfactants also show high anti-microbial, anti-tumour and anti-inflammatory properties [35]. Reports indicate that biosurfactants were discovered as a separate class of compounds during the search for new antibiotics. This study being reported here is hence an investigation into the antifungal property of a lipopeptide biosurfactant against the emerging human pathogen F. oxysporum. In the present study, a lipopeptide biosurfactant from Bacillus thuringiensis pak2310 was produced, characterised and evaluated its efficacy as anti-biofilm and as a probable non-toxic therapeutic agent. This is the first report on a lipopeptide biosurfactant exhibiting significant effect over the emerging human pathogen F. oxysporum biofilm and its efficacy on human epithelial cell lines.
Materials and methods Isolation of biosurfactant producers Petroleum refinery effluent from Chennai Petroleum Corporation and soil sample from a nearby diesel storage yard, contaminated with diesel leakage, were collected for the isolation of biosurfactant producers. The bacteria were isolated using the nutrient agar supplemented with 1% (v/ v) glycerol.
Screening for biosurfactant production Haemolysis test Haemolysis test was performed for all the ten isolated bacteria as stated by Mulligan et al. [32]. The bacteria were streaked over a 5% (v/v) blood agar plate and incubated overnight to visualise the zones of haemolysis. E24 emulsification test An amount of 2 mL of each test culture was inoculated into 250 mL flasks containing 100 mL of test medium [minimal salts (MS) [MgSO4 0.2, CaCl2 0.02, KH2PO4 1.00, K2HPO4 1.00, NH4NO3 1.00, FeCl3 0.05 (g/L)] with 1% w/v yeast extract and 3% v/v glycerol] and left on a shaker incubator at 37 8C at 130 rpm. Culture from each flask was then evaluated for surfactant production as described by Cooper and Goldenberg method [9], post mid stationary phase. To 2 mL of culture supernatant, 2 mL of crude oil was added and vortexed at high speed for 60 s and left undisturbed for 24 h. The percentage ratio of the height of the emulsion formed to the total height of the liquid column is then calculated as E24 indices. Detection of antagonistic activity Antagonistic activity of pak2310 was evaluated using agar well diffusion method on PDA plates with well diameter of 1 cm. Then, 100 mL of cell free supernatant was used after
3 uniformly inoculating the plate with F. oxysporum spores suspension, using sterile cotton swab. The plates were incubated at 25 8C for 120 h.
Taxonomical investigation of the potential strain Phenotypic and biochemical characterisation The potential strain (DS-1) was subjected to taxonomic investigation using classical morphological, physiological and biochemical tests according to the Bergey’s manual of determinative bacteriology. Gram staining, motility determination, carbohydrate utilization test (glucose, fructose, sucrose, starch and citrate), caesin hydrolysis, catalase test, indole test, Methyl Red—VogesProskauer test, triple sugar iron agar test were carried out to predict the genus and species. Molecular approach: 16s rRNA sequencing The genomic DNA of the test strain DS-1was isolated following the protocol detailed in Sambrook t al. [30]. A PCR was performed in order to amplify the 16S ribosomal DNA of the test strain. The following universal primers, forward: 50 AAGAGTTTGATCATGGCTCAG-30 and reverse: 50 -GGAGGTGATCCAACCGCA-30 were used and the PCR program was set to denaturation at 94 8C for 1 min annealing at 55 8C for 1 min and extension at 72 8C for 1 min for a total of 35 cycles. The PCR products were excised from agarose gel purified with a GENEI kit and sequenced using DNA sequencer (ABI 3100). Sequence similarity search was made for the 16S rRNA sequence of DS-1 using BLAST. Multiple sequence alignment was done using CLUSTAL W and the phylogenetic tree was constructed using neighbour-joining method.
Extraction and purification of the biosurfactant Five hundred millimeters of mineral salt enriched with 3% (v/ v) of glycerol and 1% beef extract was sterilized and used for the antibiotic production. To this, 0.25% sterile silicone oil was added as anti-foaming agent. The production media was inoculated with 2% seed culture and incubated on shaker incubator at 37 8C at 90 rpm until the onset of deceleration phase (after 145 h). The cells were separated at 10,000 rpm for 10 min at 4 8C. The supernatant was subjected to a triple stage liquid— liquid extraction using 0.3 volume of ice-cold 2:1 chloroform:methanol solution each time and the organic phases was separated and pooled. The solvent was evaporated at room temperature (32 8C 2 8C) in a rotary vacuum evaporator to concentrate the crude extract. The crude extract was further purified using reversed phase HPLC. Then, 0.1% tri fluoro acetic acid in water and 0.1% TFA in methanol were used as solution A and B respectively and the components were eluted out at a flow rate of 0.5 cm3/min with solution B of a linear gradient from 30% to 100%. The elution pattern was monitored at 215 nm and the peaks were eluted out separately and each of them was tested for antiF. oxysporum activity. The antibitoic assay (agar well diffusion and liquid culture inhibition) using F. oxysporum as an indicator organism and emulsification index was performed for each HPLC fractions. Lowry’ total protein estimation was also performed.
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MIC determination Varying final concentrations (0.01 to 0.25 mg/mL) of purified biosurfactant (peak 1) were established along with 2% (v/v) 20 h culture of F. oxysporum spores in potato dextrose broth and the OD as well as colony count on plates were monitored. The liquid culture was incubated on a shaker incubator at 25 8C and at 200 rpm while the plates were incubated at 25 8C and observed after 3 days. The OD values and colony numbers were used to determine the MIC.
Effect of antibiotic on fungal morphology — light microscopy analysis
8 MIC of antibiotics in PBS for 24 h in a CO2 incubator at 37 8C. Twenty microliters of 5 mg mL1 MTT dye was added to the wells and incubated at 37 8C for 4 h. Then, 150 mL of DMSO was then added to each well and agitated gently for 15 min. The absorbance at 590 nm was recorded using an ELISA reader and a graph was plotted between antibiotic concentration and OD590.
Characterisation Test for glycolipids CTAB agar test as described by Seigmund and Wagner [41] and orcinol assay for detection of reducing sugars [7] were performed to screen and identify the surfactant to be a rhamnolipid or a glycolipid.
Following the spectrophotometric studies for a timeline of 8 h, the fungal culture treated with the purified antibiotic and the control were stained using Lacto phenol Cotton Blue and viewed under light compound microscope (Nikon) at an interval of 1 h, the fungal morphology was documented. To check the viability, the treated cultures were inoculated on to PDA plates and incubated at 25 8C for 120 h.
Fourier transform—infra-red spectroscopy The crude extract and the HPLC purified biosurfactant were studied by Fourier transform—infra-red spectroscopy and the characteristic bands between 4000 cm1 and 400 cm1 were recorded.
Biofilm inhibition tests
Mass spectroscopy analysis
Colorimetric assay A loop full of F. oxysporum spores were inoculated in sterile 50 mL PDB and incubated at 25 8C at 130 rpm for 16 h. Then, 1 mL of the suspension was then inoculated in 50 mL sterile PDB to prepare the secondary inoculum. One hundred microliters of secondary inoculum and 100 mL of purified compound in PBS were added to the wells of 96 well polystyrene micro titre plates in triplicates. The concentration of the antibiotic added in the test wells being 0, 0.125, 0.25, 0.5, 1 and 2 mg/mL, the plates were incubated at 25 8C for 16 h. The media was discarded and the wells were washed with PBS to remove non-adherent cells. The cells were dried with 100 mL of absolute ethanol and were stained with 100 mL LCB for 1 min. Excess stain was washed with PBS twice and the stained films were solubilised with 100 mL of 1% SDS solution. OD595 was obtained using ELISA reader and a graph of antibiotic concentration versus OD595 was drawn.
The purified surfactant was subjected to Nano Spray matrix assisted laser desorption/ionisation—time of flight mass spectrometry (MALDI-ToF MS) and the molecular weight was determined from the time of flight.
Results and discussion Isolation and screening of biosurfactant producer
Confocal imaging Secondary inoculum of F. oxysporum was prepared as described in the previous section (Colorimetric assay). Then, 250 mL of secondary inoculum and 250 mL of antibiotic in PBS were added to wells of 8 well cell culture plates containing glass coverslip of 1 cm2 and incubated at 25 8C for 16 h. The concentrations of antibiotic in PBS added to each well were 0, 0.125, 0.25, 0.5, 1 and 2 mg/mL. The medium was aspirated out of the wells and was replaced with FITC dye solution and the adhered cells were incubated in dark for 1 h. The coverslips were then washed with BPBS (PBS with 4% bovine serum albumin), washed thrice with PBS and dried. The samples were imaged using Olympus FluoView FV1000 Confocal microscope at 10 magnification.
Ten different bacterial colonies of bacteria were isolated from the enriched nutrient agar. While performing the haemolysis test, seven out of the total ten bacterial isolates exhibited positive haemolysis activity. However, the strain DS-1 showed the highest haemolytic activity in terms of zone of clearance of 3.53 cm radius (SD = 0.057) on blood agar plate. Emulsion formation is the stable interaction of the hydrophobic phase and the hydrophilic phase. Various E24 indices during mid-log phase and the mid plateau phase were noted. All the cultures showed a relatively higher emulsification index for the idiophase than in the tropophase. DS-1 had the highest emulsification index of 47.22% during the tropophase and hence was selected as the best antibiotic producer. The product was a non-growth associated product as the emulsification shot up significantly during the plateau or stationary phase of the culture. The reduction in interfacial tension is brought about by the surfactant [10,33] with emulsion formation. A higher amount of emulsification indicates higher ability of surfactant production and hence DS-1, exhibiting higher emulsification potential Ep (E24% per mL of live liquid culture) was selected for further studies.
MTT Cell toxicity assay
Screening for biosurfactant production
Fifty microliters of HEp-2 cells at 103 cells per mL were incubated in triplicates with 50 mL of 1 , 2 , 4 , 6 and
Six isolates showed high haemolysis on blood agar plates. Their liquid cultures, 5 out of 6 of them exhibited emulsifica-
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Lipopeptide biosurfactant from Bacillus thuringiensis pak2310 tion activity as well. However, considerable zone of inhibition was observed with the spent broth of DS-1 isolate alone on F. oxysporum. A zone of 1.1 cm radius (standard deviation = 0.11) was observed with the spent broth of DS-1 and hence proved the presence of antifungal molecules in it. Thus, the supernatant produced was subjected to further purification and analysis.
Identification of the DS-1 strain Phenotypic and biochemical characterisation The taxonomical investigation of the strain DS- 1 revealed that the organism is motile, Gram-positive, rod shaped, arranged as chains. The biochemical tests performed for DS-1 showed the organism to be negative for TSI, citrate, MR-VP, tryptophan and caesin hydrolysis tests while positive for starch utilisation and catalase tests. These results were compared with the biosurfactant producing bacterial species and DS-1 was found to be closer to the Bacillus spp. Molecular approach The 16S rRNA studies of the organism showed 99% similarity with B. thuringiensis strains in the Nucleotide database of National Centre for Biotechnological Information (NCBI). The evolutionary relation between the test strain and the existing nearest bacterial species was determined (Fig. 1). It is noteworthy that DS-1 16S rRNAs did not show high identity with any member of the existing database. On this basis, DS-1 isolate described here probably represents a new member of B. thuringiensis and hence was designated as B. thuringiensis pak2310. The nucleotide sequence of this novel bacterium has been deposited with NCBI and GenBank accession number (JF512478) was obtained.
Extraction and purification of the biosurfactant After 6 days of fermentation, the exogenous biosurfactant was extracted and purified. Similarly, a Pseudomonas strain has been reported to enter death phase by the
5 end of the 7th day [10], suggesting that a long fermentation time is not uncommon. Bacillus species are known to produce conjugated peptide surfactants, like surfactins, iturins, fengycins, bacillomycin, etc [12,18,24]. The lipid and water soluble, greasy and pale yellow coloured amphiphilic antibiotic was obtained at the end of the triple stage liquid—liquid extraction and the fractions were pooled. The HPLC analysis yielded 3 peaks and the compound corresponding to the first peak eluted from 5 min to 9 min, constituting 42% of the total area under the chromatogram (corresponding to an yield of 100 mg/L) was selected for further study as it had high antifungal property than the other two. There are myriads of studies that claim for the high pH, temperature and salt tolerant nature of biosurfactants [16,23,44]. All these studies fortify the fact that these microbial surfactants have better properties when compared to the chemically synthesised counterparts. Thus, in this study, we have probed into the antibiotic application of the surfactant rather than characterising its chemical stability. A zone of clearance of 2.1 cm radius (standard deviation = 0.66) for HPLC peak 1 was observed on PDA plates spread with actively grown F. oxysporum culture. The plates were observed after 3 days of incubation and after one week of incubation. The effect of the biosurfactant on the growth of F. oxysporum is highly inhibitory. However, the other peaks exhibited no antifungal activity. There have been reports of purified cyclic lipopeptide surfactants from B. thuringiensis strains earlier [23] whose purified forms of about 100 mg/mL alone exhibited comparable activity on Fusarium. There are also reports of surfactin showing high antifungal activity when used synergistically with ketoconazole [28]. Similarly, in liquid broth, both crude and peak 1 biosurfactant exhibited significant inhibition (Fig. 2) of F. oxysporum. Thus, the biosurfactant produced during this study could be an effective antibiotic agent against F. oxysporum. Emulsification of 63% (standard deviation = 0.532) was obtained using the purified antibiotic (HPLC peak 1) on crude
Figure 1 Phylogenetic analysis of DS-1 strain. The distances precede the organisms’ names. ´ ne ´ tique de la souche DS-1. Les distances pre ´ ce ` dent les noms des organismes. L’analyse phyloge Please cite this article in press as: Deepak R, Jayapradha R. Lipopeptide biosurfactant from Bacillus thuringiensis pak2310: A potential antagonist against Fusarium oxysporum. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.10.011
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Figure 2 The effect of (a) crude antibiotic and (b) HPLC purified antibiotic in liquid culture of Fusarium oxysporum. ´ par HPLC sur Fusarium oxysporum en culture liquide. L’effet de (a) l’ antibiotique brut et (b) purifie
oil. There are reports of biosurfactant producer consortium that could emulsify up to 83.4% [24]. However, no significant emulsification or inhibitory effect was exhibited by the other peaks eluted.
MIC determination The purified biosurfactant at a final concentration of 0.05 mg/mL was found to be the MIC. There was no growth
Figure 3 Light microscopy imaging. Effect of HPLC purified antibiotic at MIC on fungal morphology. a: control; b: 1 h incubation; c: 2 h incubation; d: 3 h incubation; e: 4 h incubation; f: 5 h incubation; g: 6 h incubation; h: 7 h incubation; i: 8 h incubation. ´ par HPLC `a la MIC sur la morphologie fongique. a : te ´ moin ; b : 1 h Imagerie : microscopie optique. Effet de l’antibiotique purifie d’incubation ; c : incubation de 2 h ; d : 3 h d’incubation ; e : 4 h d’incubation ; f : 5 h d’incubation ; g : 6 h d’incubation ; h : 7 h d’incubation ; i : 8 h d’incubation. Please cite this article in press as: Deepak R, Jayapradha R. Lipopeptide biosurfactant from Bacillus thuringiensis pak2310: A potential antagonist against Fusarium oxysporum. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.10.011
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Lipopeptide biosurfactant from Bacillus thuringiensis pak2310
7 control (Fig. 3a) showed characteristic microconidia of the fungi imperfecti F. oxysporum while the other samples show deteriorated mass. As there is no sexual mode of reproduction, destruction of the spores ensures high efficacy of antagonistic activity. No fungal growth was seen on the PDA plates swabbed with these cultures subsequent to microscopic analysis. There are three possible modes of action being put forward by various studies on lipopeptide biosurfactants [13,19,26]. Apoptosis in fungi at very low concentration of biosurfactants could also be the contributing factor for the reported activity [37] and this could also be a contributing factor as the MIC and BIC reported here are low.
Figure 4 Colorimetric assay for biofilm inhibition. Increasing concentrations of purified antibiotic VS. OD595. ´ trique pour l’inhibition de biofilm. Des concenDosage colorime ´ VS. OD595. trations croissantes de l’antibiotique purifie
on the plates (triplicates) and the OD600 was comparable with the control (PDB + purified biosurfactant) for final concentrations of 0.05 mg/mL and above.
Effect of antibiotic on fungal morphology—Light Microscopy Analysis Fig. 3 shows the morphology of the spores at different time intervals for the MIC determined in the above section. The
Biofilm inhibition tests Colorimetric assay The increase in optical density is directly proportional to the extent of biofilm formation. Significant reduction of biofilm was observed even at the concentration of 0.063 mg/mL. However, the OD595 almost becomes constant after 0.5 mg/ mL, which is 10 MIC for the antibiotic (Fig. 4). Confocal imaging The number of organisms adhered to the glass coverslip is seen to be decreasing with the increasing concentration of the biosurfactant (Fig. 5). This result is congruent with the results obtained in the colorimetric biofilm assay. At 0.5 mg/ mL concentration and at 1 mg/mL concentration, there are not many hyphae to be seen. Thus, 0.5 mg/mL is being
Figure 5 Confocal images of effect of biosurfactant/antibiotic on biofilm formation at (a) control — 0 mg/mL, (b) 0.063 mg/mL, (c) 0.125 mg/mL, (d) 0.25 mg/mL, (e) 0.5 mg/mL, (f) 1 mg/mL. ´ moin — 0 mg/mL, (b) 0,063 mg/mL, (c) Images confocales de l’effet de biosurfactant/antibiotique sur la formation de biofilm (a) te 0,125 mg/mL, (d) 0,25 mg/mL, (e) 0,5 mg/mL, (f) 1 mg/mL. Please cite this article in press as: Deepak R, Jayapradha R. Lipopeptide biosurfactant from Bacillus thuringiensis pak2310: A potential antagonist against Fusarium oxysporum. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.10.011
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Figure 6 FT—IR spectra of (a) crude extract of antibiotic and (b) HPLC purified antibiotic. ´ par HPLC. Spectres FT—IR de (a) extrait brut d’antibiotique et (b) d’antibiotique purifie
considered as the biofilm inhibitory concentration (BIC) for the surfactant.
MTT cell toxicity assay The cytotoxic effect of the purified biosurfactant on HEp-2 cell lines from 0 to 500 mg/mL (10 MIC) and hence, the IC50 was estimated to be 435 mg/mL (617 mM) using standard MTT assay. The compound was not toxic to human epithelial cells when treated for 24 h. Hence, it can be a good candidate to develop as potential agent for fusariosis.
Characterisation of the antibiotic Test for glycolipids There was no significant coloration of the colonies or of the media even after incubating pak2310 on CTAB agar for 24 h. This proved the absence of glycolipids production. The main classes of microbial surfactants are glycolipids and lipopeptides. Thus, known tests for detection of glycolipid surfactant, the CTAB agar test that works based on coagulation of cationic and anionic surfactants was performed. From the results obtained, it is clear that the absence of a dark blue
Figure 7 a: mass spectroscopy analysis of HPLC purified antibiotic; b: mass reconstruction spectrum. ´ par HPLC ; b : spectre de reconstruction de masse. a : analyse par spectroscopie de masse de l’antibiotique purifie Please cite this article in press as: Deepak R, Jayapradha R. Lipopeptide biosurfactant from Bacillus thuringiensis pak2310: A potential antagonist against Fusarium oxysporum. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.10.011
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Lipopeptide biosurfactant from Bacillus thuringiensis pak2310 halo around the colonies eliminates the possibility of the production of glycolipids [27]. When subjected to orcinol assay, the expected red— orange coloration was not seen. The absence of any red— orange coloration shows that there are no reducing sugars like rhamnose present in the biosurfactant and hence the absence of any glycolipid in the extract [7]. The crude as well as purified antibiotic answered Lowry’s test for proteins and hence, the antibiotic is predicted to be a lipopeptide or cyclic lipopeptide, like fengycin [38] tensin or surfactin. Fourier Transform—infra-red spectroscopy Strong peaks were detected at 3426.37 cm1, 3018.19 cm1, 2923.34 cm1, 1713.25 cm1, 1657.20 cm1, 1432.44 cm1, 1365.03 cm1, 1221.99 cm1, and 765.78 cm1. These peaks were then used to predict the functional groups present in the crude extract of the biosurfactant. The purified biosurfactant showed peaks at 3436.01 cm1, 1627.42 cm1, 1563.95 cm1, 1423.98 cm1, which are characteristics of surfactin like lipopeptide surfactants (Fig. 6). The FT—IR spectrum of the crude biosurfactant with strong bands at 2923.34 cm1 and 1657.2 cm1 indicate the presence of sp3 hybridised carbon and peptide bonds, which show the presence of peptides in the biosurfactant. Further the band at 3426.37 cm1, 1713.25 cm1 and 1221.99 cm1 indicate the presence of hydrophilic groups in the compound, such as —OH and —COOH groups. Furthermore, the bands at 765.78 cm1and at 671.67 cm1 suggest the presence of aromatic side chain groups as they are signatures for aromatic hydrogens. From the above findings, one could conclude that the surfactant extracted could be a lipopeptide [22]. In case of the FT—IR spectrum of purified biosurfactant, the strong signal at 1423.98 cm1 suggests CH2 bending in lipids. Signals at 1627.42 cm1 and 1563.95 cm1 correspond to the peptide (amide) bond vibration, N—H bending and C—N stretching of peptides, respectively. Similarly, the broad peak at 3436.01 cm1 suggests N—H and O—H stretching of peptides [36,45].
Mass spectroscopy analysis The MALDI-TOF MS spectrum and its reconstruction (Fig. 7) confirm the molecular weight of the purified surfactant to be 700.5 Da. An array of surfactants from Bacillus species has been isolated and characterised to have molecular weights from 800 Da to 1500 Da previously [45] which is comparable with the obtained value. The functional groups of the lipopeptide are similar to that of surfactin while the molecule has lesser molecular weight than the commercial surfactin, hence, the isolated lipopeptide from the new strain of Bacillus sp might be a novel liopopeptide which requires the further complete structural elucidation studies.
Conclusions The lipopeptide obtained from this study has efficient antifungal and anti-biofilm properties against the otherwise recalcitrant F. oxysporum infections, which is evident from the MIC and morphological changes on the fungi observed in the study. At the same time, the lipopeptide is non-toxic to
9 human epithelial cells even at 10-fold of MIC. The promising results might pave way for the development of a promising candidate lipopeptide as drug against fusariosis.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgements The authors sincerely thank SASTRA University for the infrastructure provided and also for funding the project under the TRR Scheme. The authors also express their sincere thanks to Mr. Ankit Rochani of Prof. Utpal S. Tatu’s lab at Indian Institute of Science for extending help with the MALDI-ToF MS analysis, Dr. S. Adline Princy and Mr. C. David Raj of SASTRA University for all the technical support provided promptly.
References [1] Alfanso EC. Genotypic identification of Fusarium species from ocular sources: comparison to morphologic classification and antifungal sensitivity testing (an AOS thesis). Trans Am Opthalmol Soc 2008;105:227—39. [2] Armstrong GM, Armstrong JK. Formaespeciales and races of Fusarium oxysporum causing wilt diseases. In: Nelson PE, editor. Fusarium: Diseases, Biology and Taxonomy. 1981. p. 391—9. [3] Banat IM. Biosurfactants production and possible uses in microbial enhanced oil recovery and oil pollution remediation: a review. Bioresour Technol 1995;51:1. [4] Boutati EI, Anaissie EJ. Fusarium, a significant emerging pathogen in patients with hematological malignancy: ten years’ experience at a cancer centre and implications for management. Blood 1997;90:999—1008. [5] Brilhante RS, Cordeiro RS, Medrano DJ, Rocha MF, Monteiro AJ, Cavalcante CS, et al. Onychomycosis in Ceara (Northeast Brazil): epidemiological and laboratory aspects. Mem Inst Oswaldo Cruz 2005;100:131—5. [6] Cameotra SS, Makkar RS. Recent applications of biosurfactants as biological and immunological molecules. Curr Opin Microbiol 2001;7:1. [7] Chandrasekaran EV, Bemiller JN. Methods in Carbohydrate Chemistry. New York: Academic Press; 1980. [8] Chang DC, Grant GB, O’Donnell K, Wannemuehler KA, NobleWang J, Rao CY, et al. Multistate outbreak of Fusarium keratitis associated with use of a contact lens solution. JAMA 2006;296:953—63. [9] Cooper DG, Goldenberg BG. Surface active agents from two Bacillus spp. Appl Environ Microbiol 1987;53:224. [10] Damasceno FRC, Cammarota MC, Freire DMG. The combined use of a biosurfactant and an enzyme preparation to treat an effluent with a high fat content. Colloids Surfaces B 2012;95:241. [11] Danicke S. Fusarium toxins in animal nutrition. Lohman Info 2002;27:1—9. [12] Darvishi P, Ayatollahi P, Mowla D, Niazi A. Biosurfactant production under extreme environmental conditions by efficient microbial consortium, ERCPPI-2. Colloids Surf B 2011;84:292. [13] Deleu M, Bouffioux O, Razafindralambo H, Paquot M. Interaction of surfactin with membranes: a computational approach. Langmuir 2003;19:3377—85. [14] Donnio A, Van Nuoi DN, Catanese M, Desbois N, Ayebuoa L, Merle H. Outbreak of keratomycosis attributable to Fusarium
Please cite this article in press as: Deepak R, Jayapradha R. Lipopeptide biosurfactant from Bacillus thuringiensis pak2310: A potential antagonist against Fusarium oxysporum. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.10.011
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MYCMED-511; No. of Pages 10
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[15] [16]
[17]
[18]
[19]
[20]
[21] [22] [23]
[24]
[25] [26]
[27]
[28]
[29] [30] [31]
R. Deepak, R. Jayapradha solani in the French West Indies. Am J Ophthalmol 2007;143: 356—8. Doyle ME. Fusarium mycotoxins. UW FRI Briefings; 1997, http:// fri.wisc.edu/docs/pdf/fusarium.pdf. Ghojavand H, Vahabzadeh F, Roayaei E, Shahraki AK. Production and properties of a biosurfactant obtained from a member of the Bacillus subtilis group (PTCC 1696). J Colloid Interface Sci 2008;324:172. Godoy P, Nunes E, Silva V, Tomimori-Yamashita J, Zaror L, Fischman O. Onychomycosis caused by Fusarium solani and Fusarium oxysporum in Sao Paulo, Brazil. Mycopathologia 2004;157:287—90. He H, Silo-Suh LA, Clardy J, Handelsman J. Zwittermicin, an antifungal and plant protection agent from Bacillus cereus. Tetrahedron Lett 1994;34:2499. Heerklotz H, Wieprecht T, Seelig J. Membrane pertubation by the lipopeptide surfactin and detergents as studied by deuterium NMR. J Phys Chem 2004;108:4909—15. Imamura Y, Chandra J, Mukherjee PK, Lattif AA, Szczotka-Flynn LB, Pearlman E, et al. Fusarium and Candida albicans biofilms on soft contact lenses: model development, influence of lens type and susceptibility to lens care solutions. Am Soc Microbiol 2008;52:171—82. Iyer SA, Tuli SS, Wagoner RC. Fungal keratitis: emerging trends and treatment outcomes. Eye Contact Lens 2006;32:267—71. Joseph Hornback M. Organic Chemistry, . 2nd ed., Connecticut: Thomson Brooks/Cole; 2006. Kim PI, Bai H, Bai D, Chae H, Chung S, Kim Y, et al. Purification and characterisation of lipopeptide produced by Bacillus thuringiensis CMB26. J Appl Microbiol 2004;97:942—5. Kluge B, Vater J, Salikow J, Eckhart K. Studies on the biosynthesis of surfactin, a lipopeptide antibiotic from Bacillus subtilis ATCC21332. FEBS Lett 1988;231:107. Kosaric N. Biosurfactants in industry. Pure Appl Chem 1992;64:1731. Kragh-Hansen U, Maire M, Moller J. The mechanism of detergent solubilization of liposomes and protein-containing membranes. Biophys J 1998;75:2932—46. Kratka J, Kovacikova E. The effect of temperature and age of strains of Fusarium oxysporum on its enzymatic activity. Zentbl Bakteriol Naturwiss 1979;134:154—8. Liu X, Ren B, Gao H, Liu M, Dai H, Song F, et al. Optimisation for the production of surfactin with a new synergistic antifungal activity. PLoS One 2002;7:1. Maier RM. Biosurfactant: evolution and diversity in bacteria. Adv Appl Microbiol 2003;52:101. Michael RG, Sambrook J. Molecular Cloning Manual, 2. New York: Cold Spring Harbor Laboratory Press; 2001. Mulligan CN. Environmental applications of biosurfactants. Environ Pollut 2005;133:183.
[32] Mulligan CN, Cooper DG, Neufeld RJ. Selection of microbes producing biosurfactants in media without hydrocarbons. J Ferment Technol 1984;62:311—4. [33] Nussie M, Anaissie E. Fusarium infections in immunocompromised patients. Clin Microbiol Rev 2007;20:695—704. [34] O’ Donell K, Sutton DA, Rinaldi MG, Magnon KC, Cox PA, Revankar SG, et al. Genetic diversity of human pathogenic members of the Fusarium oxysporum complex inferred from multilocus DNA sequencing data and amplified fragment length polymorphism analyses: evidence for the recent dispersion of a geographically widespread clonal lineage and nosocomial origin. J Clin Microbiol 2004;42:5109—20. [35] Perfumo A, Ranchic I, Banat IM. Biosurfactants. In: Sen R, editor. Biosurfactants Book series: Advances in Experimental Medicine and Biology, 627. Texas: Landes Bioscience; 2010. p. 134—45. [36] Prince NPJ, Rooney AP, Swezey JL, Perry E, Cohan FM. Mass spectrometric analysis of lipopeptides from bacillus strains isolated from diverse geographical locations. FEMS Microbiol Lett 2007 [doi:001:1 0.1111-p.1574-6968.2007.00702.x, 2007]. [37] Qi G, Zhu F, Du P, Yang X, Qiu D, Yu Z, et al. Lipopeptide induces apoptosis in fungal cells by a mitochondria dependent pathway. Peptides 2010;31:1978—86. [38] Ramarathnam R, Bo S, Chen Y, Fernando WG, Xuewen G, de Kievit T. Molecular and biochemical detection of fengycin and bacillomycin D-producing Bacillus spp., antagonistic to fungal pathogens of Canola and Wheat. Can J Microbiol 2007;5:901. [39] Ron EZ, Rosenberg E. Biosurfactants oil bioremediation. Curr Opin Biotechnol 2002;13:24. [40] Schneller FR, Gulati SC, Cunningham IB, O’Reilly RJ, Schmitt HJ, Clarkson BD. Fusarium infections in patients with hematologic malignancies. Leuk Res 1990;14:961. [41] Siegmund I, Wagner F. New method for detecting rhamnolipids excreted by Pseudomonas spp during growth on mineral agar. Biotechnol Tech 1991;5:265. [42] Singh P, Cameotra SS. Potential applications of microbial surfactants in biomedical sciences. Trends Biotechnol 2004; 22:142. [43] Sun Y, Chandra J, Mukherjee P, Szczotka-Flynn L, Ghannoum MA, Pearlman E. A murine model of contact lens-associated Fusarium keratitis. Invest Opthalmol Vis Sci 2010;51:1511—6. [44] Vaz DA, Gudina EJ, Alameda EJ, Teixeira JA, Rodrigues LR. Performance of biosurfactant produced by Bacillus subtilis strain isolated from crude oil samples as compared to commercial chemical surfactants. Colloids Surface B 2012;89:167. [45] Yalcin E, Cavusoglu K. Structural analysis and antioxidant activity of a biosurfactant obtained from Bacillus subtilis RW-1, Turk. J Biochem 2010;35:243.
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MYCMED-511; No. of Pages 10 Journal de Mycologie Médicale (2014) xxx, xxx—xxx
Available online at
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ORIGINAL ARTICLE/ARTICLE ORIGINAL
Lipopeptide biosurfactant from Bacillus thuringiensis pak2310: A potential antagonist against Fusarium oxysporum Biosurfactant lipopeptidique de Bacillus thuringiensis pak2310 : un antagoniste potentiel contre Fusarium oxysporum R. Deepak, R. Jayapradha * Centre for Research on Infectious Diseases (CRID), School of Chemical and Biotechnology, SASTRA University, Tamilnadu, India Received 15 June 2014; received in revised form 2 October 2014; accepted 6 October 2014
KEYWORDS Fusariosis; Bacillus thuringiensis pak2310; Lipopeptide; Antibiotic; Antifungal; Anti-biofilm
Summary Objectives. — The aims of the study were to evaluate the effects of a biosurfactant obtained from a novel Bacillus thuringiensis on Fusarium oxysporum to determine the morphological changes in the structure of the fungi and its biofilm in the presence of the biosurfactant and to evaluate the toxicity of the biosurfactant on HEp-2 human epithelial cell lines. Materials and methods. — The strain was screened and isolated from petroleum contaminated soil based on the E24 emulsification index. The biosurfactant was produced on glycerol, extracted using chloroform:methanol system and purified using HPLC. The purified fraction showing both surface activity (emulsification and oil-spread activity) and anti-fusarial activity (agar well diffusion method) was studied using FT—IR and MALDI-TOF MS, respectively. The minimum inhibitory concentration (MIC) and the biofilm inhibitory concentration (BIC) were determined using dilution method. The effect of biosurfactant on the morphology of Fusarium oxysporum was monitored using light microscopy and confocal laser scanning microscopy (for biofilm). Results. — The purified surfactant showed the presence of functional groups like that of surfactin in the FT—IR spectra and MALDI-TOF MS estimated the molecular weight as 700 Da. The MIC and BIC were estimated to be 0.05 and 0.5 mg/mL, respectively. The molecule was also non-toxic to HEp-2 cell lines at 10 MIC. Conclusion. — A non-toxic and effective anti-Fusarium biosurfactant, that is both safe for human use and to the environment, has been characterized. The growth and metabolite
* Corresponding author. Centre for Research on Infectious Diseases (CRID), School of Chemical and Biotechnology, SASTRA University, Tirumalaisamudram, Thanjavur, 613 401 Tamilnadu, India. E-mail addresses: [email protected], [email protected] (R. Jayapradha). http://dx.doi.org/10.1016/j.mycmed.2014.10.011 1156-5233/# 2014 Published by Elsevier Masson SAS.
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R. Deepak, R. Jayapradha production using glycerol (major byproduct of biodiesel and soap industries) also adds up to the efficiency and ecofriendly nature of this biosurfactant. # 2014 Published by Elsevier Masson SAS.
MOTS CLÉS Fusariose ; Bacillus thuringiensis pak2310 ; Lipopeptides ; Antibiotique ; Antifongique ; Anti-biofilm
Re ´sume ´ Objectifs. — Les objectifs de cette étude étaient d’évaluer les effets d’un bio-tensioactif obtenu à partir d’un Bacillus thuringiensis nouveau, sur Fusarium oxysporum, afin de déterminer les changements morphologiques dans la structure du champignon et de son biofilm en présence de l’agent bio-tensioactif et d’évaluer la toxicité du biosurfactant sur des lignées de cellules épithéliales humaines HEp-2. Mate´riels et me ´thodes. — La souche a été criblée et isolée à partir de sols contaminés par du pétrole sur la base de l’indice de mise en émulsion E24. Le bio-tensioactif a été produit sur le glycérol, extrait à l’aide de chloroforme:methanol système et purifié en utilisant une HPLC. La fraction purifiée montrant à la fois une activité de surface (émulsification et l’activité de propagation de l’huile) et de l’activité anti-Fusarium (méthode de diffusion en gélose par puits) a été étudiée à l’aide de FT—IR et MALDI-TOF MS, respectivement. La concentration minimale inhibitrice (CMI) et la concentration inhibitrice biofilm (BIC) ont été déterminées en utilisant la méthode de dilution. L’effet du biosurfactant sur la morphologie de Fusarium oxysporum a été objectivée en utilisant la microscopie optique confocale à balayage laser et la microscopie du biofilm. Re ´sultats. — L’agent tensioactif purifié a montré la présence de groupes fonctionnels, tels que des surfactines dans les spectres FT—IR et MALDI-TOF MS avec un poids moléculaire estimé à 700 Da. La CIM et BIC ont été estimées à 0,05 et 0,5 mg/mL, respectivement. La molécule était également non toxique pour les cellules HEp-2 dans des lignées de cellules 10 MIC. Conclusion. — Un biosurfactant anti-Fusarium non toxique et efficace, à la fois sans danger pour la consommation humaine et l’environnement a été caractérisé. La croissance et la production de métabolites en utilisant du glycérol (sous-produit majeur de biodiesel et de savon d’industrie) ajoute également à l’efficacité écologique et à la protection de la nature de ce biosurfactant. # 2014 Publié par Elsevier Masson SAS.
Introduction Fusarium oxysporum, a plant pathogen, was the etiological agent of the epidemic wilting of bananas in Panama and hence, the name of the disease — Panama Wilt [29] was coined. Several other diseases, such as scabs, crown rot and red blight are caused by this fungus [2]. Although primarily a plant pathogen, recent reports indicate that members of Fusarium, especially F. oxysporum and F. solani are emerging human pathogens as well. Keratitis and onychomycosis are the main superficial infections of F. oxysporum in mankind [5,17,33]. Keratitis has been reported mostly in patients who use soft contact lenses [8,14]. Fusariosis or disseminated Fusarium infection has been reported worldwide in immune compromised patients, especially the ones with prolonged neutropenia [4,34]. The symptoms of the disease are very similar to that of aspergillosis [4]. Intra-venous amphotericin B is used against aspergillosis, while that has been reported to be ineffective against Fusarium infections [4]. Although the infection recedes initially with the administration of azole ring antibiotics, death due to fusariosis seems to be inevitable in immune compromised patients [4,8]. The main route of invasion by the pathogen is through the skin, through cuts, rashes or wounds, like the scratch wounds in cellulitis patients [33]. Once entered, the host body is invaded immensely, releasing toxins that can lead to tissue breakdown and immune suppression [17]. However, the toxins produced by Fusaria can contaminate food and can
be easily ingested. These toxins have been shown to suppress the immune system of the host and in turn make the host more susceptible to the pathogen attack [11,15,17]. Fusarium infections are also accompanied by ecthymagangerosum, a skin infection caused by Pseudomonas spp; osteomyelitis; sinusitis and general fungemia [4,33]. Thus, it is necessary to develop new drugs and therapies that can help to manage the disease effectively. F. oxysporum is capable of producing adherent substances form biofilms and produce tissue damaging enzymes [27,40]. Formation of a biofilm has been reported to be the main reason for keratitis and Fusarium keratitis has gained more prominence in the recent past with 41% of total cases of keratitis [21] as a biofilm is more resistant to drugs in use. Another study reports that people using soft contact lens are 40% susceptible to Fusarium keratitis [1]. Several factors for biofilm formation by the pathogen on contact lens including the material used in fabrication and cleansing solution have also been studied [20]. The same research team has also investigated the formation of biofilms in murine models and its relationship with keratitis [43]. Thus, preventing Fusarium biofilms gains importance in the present scenario. Surfactants are those chemical molecules that have both hydrophobic and hydrophilic groups in them. They are soluble in both polar and non-polar solvents [29]. Surfactants produced by microbes are called biosurfactants. Biosurfactants have many advantages, like biodegradability, lower toxicity, mild production conditions, selectivity and higher
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Lipopeptide biosurfactant from Bacillus thuringiensis pak2310 specific activity in a wider range of pH, temperature and salinity [29]. The applications of biosurfactants can be found in a broad spectrum of industries like food, cosmetic, chemical and pharmaceutical industries [3,6,9,25,29,31,39]. Apart from these industries, the application of biosurfactants also reaches many environmental engineering applications, such as bioremediation, enhanced oil recovery, soil washing, etc. [42]. Biosurfactants also show high anti-microbial, anti-tumour and anti-inflammatory properties [35]. Reports indicate that biosurfactants were discovered as a separate class of compounds during the search for new antibiotics. This study being reported here is hence an investigation into the antifungal property of a lipopeptide biosurfactant against the emerging human pathogen F. oxysporum. In the present study, a lipopeptide biosurfactant from Bacillus thuringiensis pak2310 was produced, characterised and evaluated its efficacy as anti-biofilm and as a probable non-toxic therapeutic agent. This is the first report on a lipopeptide biosurfactant exhibiting significant effect over the emerging human pathogen F. oxysporum biofilm and its efficacy on human epithelial cell lines.
Materials and methods Isolation of biosurfactant producers Petroleum refinery effluent from Chennai Petroleum Corporation and soil sample from a nearby diesel storage yard, contaminated with diesel leakage, were collected for the isolation of biosurfactant producers. The bacteria were isolated using the nutrient agar supplemented with 1% (v/ v) glycerol.
Screening for biosurfactant production Haemolysis test Haemolysis test was performed for all the ten isolated bacteria as stated by Mulligan et al. [32]. The bacteria were streaked over a 5% (v/v) blood agar plate and incubated overnight to visualise the zones of haemolysis. E24 emulsification test An amount of 2 mL of each test culture was inoculated into 250 mL flasks containing 100 mL of test medium [minimal salts (MS) [MgSO4 0.2, CaCl2 0.02, KH2PO4 1.00, K2HPO4 1.00, NH4NO3 1.00, FeCl3 0.05 (g/L)] with 1% w/v yeast extract and 3% v/v glycerol] and left on a shaker incubator at 37 8C at 130 rpm. Culture from each flask was then evaluated for surfactant production as described by Cooper and Goldenberg method [9], post mid stationary phase. To 2 mL of culture supernatant, 2 mL of crude oil was added and vortexed at high speed for 60 s and left undisturbed for 24 h. The percentage ratio of the height of the emulsion formed to the total height of the liquid column is then calculated as E24 indices. Detection of antagonistic activity Antagonistic activity of pak2310 was evaluated using agar well diffusion method on PDA plates with well diameter of 1 cm. Then, 100 mL of cell free supernatant was used after
3 uniformly inoculating the plate with F. oxysporum spores suspension, using sterile cotton swab. The plates were incubated at 25 8C for 120 h.
Taxonomical investigation of the potential strain Phenotypic and biochemical characterisation The potential strain (DS-1) was subjected to taxonomic investigation using classical morphological, physiological and biochemical tests according to the Bergey’s manual of determinative bacteriology. Gram staining, motility determination, carbohydrate utilization test (glucose, fructose, sucrose, starch and citrate), caesin hydrolysis, catalase test, indole test, Methyl Red—VogesProskauer test, triple sugar iron agar test were carried out to predict the genus and species. Molecular approach: 16s rRNA sequencing The genomic DNA of the test strain DS-1was isolated following the protocol detailed in Sambrook t al. [30]. A PCR was performed in order to amplify the 16S ribosomal DNA of the test strain. The following universal primers, forward: 50 AAGAGTTTGATCATGGCTCAG-30 and reverse: 50 -GGAGGTGATCCAACCGCA-30 were used and the PCR program was set to denaturation at 94 8C for 1 min annealing at 55 8C for 1 min and extension at 72 8C for 1 min for a total of 35 cycles. The PCR products were excised from agarose gel purified with a GENEI kit and sequenced using DNA sequencer (ABI 3100). Sequence similarity search was made for the 16S rRNA sequence of DS-1 using BLAST. Multiple sequence alignment was done using CLUSTAL W and the phylogenetic tree was constructed using neighbour-joining method.
Extraction and purification of the biosurfactant Five hundred millimeters of mineral salt enriched with 3% (v/ v) of glycerol and 1% beef extract was sterilized and used for the antibiotic production. To this, 0.25% sterile silicone oil was added as anti-foaming agent. The production media was inoculated with 2% seed culture and incubated on shaker incubator at 37 8C at 90 rpm until the onset of deceleration phase (after 145 h). The cells were separated at 10,000 rpm for 10 min at 4 8C. The supernatant was subjected to a triple stage liquid— liquid extraction using 0.3 volume of ice-cold 2:1 chloroform:methanol solution each time and the organic phases was separated and pooled. The solvent was evaporated at room temperature (32 8C 2 8C) in a rotary vacuum evaporator to concentrate the crude extract. The crude extract was further purified using reversed phase HPLC. Then, 0.1% tri fluoro acetic acid in water and 0.1% TFA in methanol were used as solution A and B respectively and the components were eluted out at a flow rate of 0.5 cm3/min with solution B of a linear gradient from 30% to 100%. The elution pattern was monitored at 215 nm and the peaks were eluted out separately and each of them was tested for antiF. oxysporum activity. The antibitoic assay (agar well diffusion and liquid culture inhibition) using F. oxysporum as an indicator organism and emulsification index was performed for each HPLC fractions. Lowry’ total protein estimation was also performed.
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MIC determination Varying final concentrations (0.01 to 0.25 mg/mL) of purified biosurfactant (peak 1) were established along with 2% (v/v) 20 h culture of F. oxysporum spores in potato dextrose broth and the OD as well as colony count on plates were monitored. The liquid culture was incubated on a shaker incubator at 25 8C and at 200 rpm while the plates were incubated at 25 8C and observed after 3 days. The OD values and colony numbers were used to determine the MIC.
Effect of antibiotic on fungal morphology — light microscopy analysis
8 MIC of antibiotics in PBS for 24 h in a CO2 incubator at 37 8C. Twenty microliters of 5 mg mL1 MTT dye was added to the wells and incubated at 37 8C for 4 h. Then, 150 mL of DMSO was then added to each well and agitated gently for 15 min. The absorbance at 590 nm was recorded using an ELISA reader and a graph was plotted between antibiotic concentration and OD590.
Characterisation Test for glycolipids CTAB agar test as described by Seigmund and Wagner [41] and orcinol assay for detection of reducing sugars [7] were performed to screen and identify the surfactant to be a rhamnolipid or a glycolipid.
Following the spectrophotometric studies for a timeline of 8 h, the fungal culture treated with the purified antibiotic and the control were stained using Lacto phenol Cotton Blue and viewed under light compound microscope (Nikon) at an interval of 1 h, the fungal morphology was documented. To check the viability, the treated cultures were inoculated on to PDA plates and incubated at 25 8C for 120 h.
Fourier transform—infra-red spectroscopy The crude extract and the HPLC purified biosurfactant were studied by Fourier transform—infra-red spectroscopy and the characteristic bands between 4000 cm1 and 400 cm1 were recorded.
Biofilm inhibition tests
Mass spectroscopy analysis
Colorimetric assay A loop full of F. oxysporum spores were inoculated in sterile 50 mL PDB and incubated at 25 8C at 130 rpm for 16 h. Then, 1 mL of the suspension was then inoculated in 50 mL sterile PDB to prepare the secondary inoculum. One hundred microliters of secondary inoculum and 100 mL of purified compound in PBS were added to the wells of 96 well polystyrene micro titre plates in triplicates. The concentration of the antibiotic added in the test wells being 0, 0.125, 0.25, 0.5, 1 and 2 mg/mL, the plates were incubated at 25 8C for 16 h. The media was discarded and the wells were washed with PBS to remove non-adherent cells. The cells were dried with 100 mL of absolute ethanol and were stained with 100 mL LCB for 1 min. Excess stain was washed with PBS twice and the stained films were solubilised with 100 mL of 1% SDS solution. OD595 was obtained using ELISA reader and a graph of antibiotic concentration versus OD595 was drawn.
The purified surfactant was subjected to Nano Spray matrix assisted laser desorption/ionisation—time of flight mass spectrometry (MALDI-ToF MS) and the molecular weight was determined from the time of flight.
Results and discussion Isolation and screening of biosurfactant producer
Confocal imaging Secondary inoculum of F. oxysporum was prepared as described in the previous section (Colorimetric assay). Then, 250 mL of secondary inoculum and 250 mL of antibiotic in PBS were added to wells of 8 well cell culture plates containing glass coverslip of 1 cm2 and incubated at 25 8C for 16 h. The concentrations of antibiotic in PBS added to each well were 0, 0.125, 0.25, 0.5, 1 and 2 mg/mL. The medium was aspirated out of the wells and was replaced with FITC dye solution and the adhered cells were incubated in dark for 1 h. The coverslips were then washed with BPBS (PBS with 4% bovine serum albumin), washed thrice with PBS and dried. The samples were imaged using Olympus FluoView FV1000 Confocal microscope at 10 magnification.
Ten different bacterial colonies of bacteria were isolated from the enriched nutrient agar. While performing the haemolysis test, seven out of the total ten bacterial isolates exhibited positive haemolysis activity. However, the strain DS-1 showed the highest haemolytic activity in terms of zone of clearance of 3.53 cm radius (SD = 0.057) on blood agar plate. Emulsion formation is the stable interaction of the hydrophobic phase and the hydrophilic phase. Various E24 indices during mid-log phase and the mid plateau phase were noted. All the cultures showed a relatively higher emulsification index for the idiophase than in the tropophase. DS-1 had the highest emulsification index of 47.22% during the tropophase and hence was selected as the best antibiotic producer. The product was a non-growth associated product as the emulsification shot up significantly during the plateau or stationary phase of the culture. The reduction in interfacial tension is brought about by the surfactant [10,33] with emulsion formation. A higher amount of emulsification indicates higher ability of surfactant production and hence DS-1, exhibiting higher emulsification potential Ep (E24% per mL of live liquid culture) was selected for further studies.
MTT Cell toxicity assay
Screening for biosurfactant production
Fifty microliters of HEp-2 cells at 103 cells per mL were incubated in triplicates with 50 mL of 1 , 2 , 4 , 6 and
Six isolates showed high haemolysis on blood agar plates. Their liquid cultures, 5 out of 6 of them exhibited emulsifica-
Please cite this article in press as: Deepak R, Jayapradha R. Lipopeptide biosurfactant from Bacillus thuringiensis pak2310: A potential antagonist against Fusarium oxysporum. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.10.011
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Lipopeptide biosurfactant from Bacillus thuringiensis pak2310 tion activity as well. However, considerable zone of inhibition was observed with the spent broth of DS-1 isolate alone on F. oxysporum. A zone of 1.1 cm radius (standard deviation = 0.11) was observed with the spent broth of DS-1 and hence proved the presence of antifungal molecules in it. Thus, the supernatant produced was subjected to further purification and analysis.
Identification of the DS-1 strain Phenotypic and biochemical characterisation The taxonomical investigation of the strain DS- 1 revealed that the organism is motile, Gram-positive, rod shaped, arranged as chains. The biochemical tests performed for DS-1 showed the organism to be negative for TSI, citrate, MR-VP, tryptophan and caesin hydrolysis tests while positive for starch utilisation and catalase tests. These results were compared with the biosurfactant producing bacterial species and DS-1 was found to be closer to the Bacillus spp. Molecular approach The 16S rRNA studies of the organism showed 99% similarity with B. thuringiensis strains in the Nucleotide database of National Centre for Biotechnological Information (NCBI). The evolutionary relation between the test strain and the existing nearest bacterial species was determined (Fig. 1). It is noteworthy that DS-1 16S rRNAs did not show high identity with any member of the existing database. On this basis, DS-1 isolate described here probably represents a new member of B. thuringiensis and hence was designated as B. thuringiensis pak2310. The nucleotide sequence of this novel bacterium has been deposited with NCBI and GenBank accession number (JF512478) was obtained.
Extraction and purification of the biosurfactant After 6 days of fermentation, the exogenous biosurfactant was extracted and purified. Similarly, a Pseudomonas strain has been reported to enter death phase by the
5 end of the 7th day [10], suggesting that a long fermentation time is not uncommon. Bacillus species are known to produce conjugated peptide surfactants, like surfactins, iturins, fengycins, bacillomycin, etc [12,18,24]. The lipid and water soluble, greasy and pale yellow coloured amphiphilic antibiotic was obtained at the end of the triple stage liquid—liquid extraction and the fractions were pooled. The HPLC analysis yielded 3 peaks and the compound corresponding to the first peak eluted from 5 min to 9 min, constituting 42% of the total area under the chromatogram (corresponding to an yield of 100 mg/L) was selected for further study as it had high antifungal property than the other two. There are myriads of studies that claim for the high pH, temperature and salt tolerant nature of biosurfactants [16,23,44]. All these studies fortify the fact that these microbial surfactants have better properties when compared to the chemically synthesised counterparts. Thus, in this study, we have probed into the antibiotic application of the surfactant rather than characterising its chemical stability. A zone of clearance of 2.1 cm radius (standard deviation = 0.66) for HPLC peak 1 was observed on PDA plates spread with actively grown F. oxysporum culture. The plates were observed after 3 days of incubation and after one week of incubation. The effect of the biosurfactant on the growth of F. oxysporum is highly inhibitory. However, the other peaks exhibited no antifungal activity. There have been reports of purified cyclic lipopeptide surfactants from B. thuringiensis strains earlier [23] whose purified forms of about 100 mg/mL alone exhibited comparable activity on Fusarium. There are also reports of surfactin showing high antifungal activity when used synergistically with ketoconazole [28]. Similarly, in liquid broth, both crude and peak 1 biosurfactant exhibited significant inhibition (Fig. 2) of F. oxysporum. Thus, the biosurfactant produced during this study could be an effective antibiotic agent against F. oxysporum. Emulsification of 63% (standard deviation = 0.532) was obtained using the purified antibiotic (HPLC peak 1) on crude
Figure 1 Phylogenetic analysis of DS-1 strain. The distances precede the organisms’ names. ´ ne ´ tique de la souche DS-1. Les distances pre ´ ce ` dent les noms des organismes. L’analyse phyloge Please cite this article in press as: Deepak R, Jayapradha R. Lipopeptide biosurfactant from Bacillus thuringiensis pak2310: A potential antagonist against Fusarium oxysporum. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.10.011
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Figure 2 The effect of (a) crude antibiotic and (b) HPLC purified antibiotic in liquid culture of Fusarium oxysporum. ´ par HPLC sur Fusarium oxysporum en culture liquide. L’effet de (a) l’ antibiotique brut et (b) purifie
oil. There are reports of biosurfactant producer consortium that could emulsify up to 83.4% [24]. However, no significant emulsification or inhibitory effect was exhibited by the other peaks eluted.
MIC determination The purified biosurfactant at a final concentration of 0.05 mg/mL was found to be the MIC. There was no growth
Figure 3 Light microscopy imaging. Effect of HPLC purified antibiotic at MIC on fungal morphology. a: control; b: 1 h incubation; c: 2 h incubation; d: 3 h incubation; e: 4 h incubation; f: 5 h incubation; g: 6 h incubation; h: 7 h incubation; i: 8 h incubation. ´ par HPLC `a la MIC sur la morphologie fongique. a : te ´ moin ; b : 1 h Imagerie : microscopie optique. Effet de l’antibiotique purifie d’incubation ; c : incubation de 2 h ; d : 3 h d’incubation ; e : 4 h d’incubation ; f : 5 h d’incubation ; g : 6 h d’incubation ; h : 7 h d’incubation ; i : 8 h d’incubation. Please cite this article in press as: Deepak R, Jayapradha R. Lipopeptide biosurfactant from Bacillus thuringiensis pak2310: A potential antagonist against Fusarium oxysporum. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.10.011
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Lipopeptide biosurfactant from Bacillus thuringiensis pak2310
7 control (Fig. 3a) showed characteristic microconidia of the fungi imperfecti F. oxysporum while the other samples show deteriorated mass. As there is no sexual mode of reproduction, destruction of the spores ensures high efficacy of antagonistic activity. No fungal growth was seen on the PDA plates swabbed with these cultures subsequent to microscopic analysis. There are three possible modes of action being put forward by various studies on lipopeptide biosurfactants [13,19,26]. Apoptosis in fungi at very low concentration of biosurfactants could also be the contributing factor for the reported activity [37] and this could also be a contributing factor as the MIC and BIC reported here are low.
Figure 4 Colorimetric assay for biofilm inhibition. Increasing concentrations of purified antibiotic VS. OD595. ´ trique pour l’inhibition de biofilm. Des concenDosage colorime ´ VS. OD595. trations croissantes de l’antibiotique purifie
on the plates (triplicates) and the OD600 was comparable with the control (PDB + purified biosurfactant) for final concentrations of 0.05 mg/mL and above.
Effect of antibiotic on fungal morphology—Light Microscopy Analysis Fig. 3 shows the morphology of the spores at different time intervals for the MIC determined in the above section. The
Biofilm inhibition tests Colorimetric assay The increase in optical density is directly proportional to the extent of biofilm formation. Significant reduction of biofilm was observed even at the concentration of 0.063 mg/mL. However, the OD595 almost becomes constant after 0.5 mg/ mL, which is 10 MIC for the antibiotic (Fig. 4). Confocal imaging The number of organisms adhered to the glass coverslip is seen to be decreasing with the increasing concentration of the biosurfactant (Fig. 5). This result is congruent with the results obtained in the colorimetric biofilm assay. At 0.5 mg/ mL concentration and at 1 mg/mL concentration, there are not many hyphae to be seen. Thus, 0.5 mg/mL is being
Figure 5 Confocal images of effect of biosurfactant/antibiotic on biofilm formation at (a) control — 0 mg/mL, (b) 0.063 mg/mL, (c) 0.125 mg/mL, (d) 0.25 mg/mL, (e) 0.5 mg/mL, (f) 1 mg/mL. ´ moin — 0 mg/mL, (b) 0,063 mg/mL, (c) Images confocales de l’effet de biosurfactant/antibiotique sur la formation de biofilm (a) te 0,125 mg/mL, (d) 0,25 mg/mL, (e) 0,5 mg/mL, (f) 1 mg/mL. Please cite this article in press as: Deepak R, Jayapradha R. Lipopeptide biosurfactant from Bacillus thuringiensis pak2310: A potential antagonist against Fusarium oxysporum. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.10.011
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Figure 6 FT—IR spectra of (a) crude extract of antibiotic and (b) HPLC purified antibiotic. ´ par HPLC. Spectres FT—IR de (a) extrait brut d’antibiotique et (b) d’antibiotique purifie
considered as the biofilm inhibitory concentration (BIC) for the surfactant.
MTT cell toxicity assay The cytotoxic effect of the purified biosurfactant on HEp-2 cell lines from 0 to 500 mg/mL (10 MIC) and hence, the IC50 was estimated to be 435 mg/mL (617 mM) using standard MTT assay. The compound was not toxic to human epithelial cells when treated for 24 h. Hence, it can be a good candidate to develop as potential agent for fusariosis.
Characterisation of the antibiotic Test for glycolipids There was no significant coloration of the colonies or of the media even after incubating pak2310 on CTAB agar for 24 h. This proved the absence of glycolipids production. The main classes of microbial surfactants are glycolipids and lipopeptides. Thus, known tests for detection of glycolipid surfactant, the CTAB agar test that works based on coagulation of cationic and anionic surfactants was performed. From the results obtained, it is clear that the absence of a dark blue
Figure 7 a: mass spectroscopy analysis of HPLC purified antibiotic; b: mass reconstruction spectrum. ´ par HPLC ; b : spectre de reconstruction de masse. a : analyse par spectroscopie de masse de l’antibiotique purifie Please cite this article in press as: Deepak R, Jayapradha R. Lipopeptide biosurfactant from Bacillus thuringiensis pak2310: A potential antagonist against Fusarium oxysporum. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.10.011
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Lipopeptide biosurfactant from Bacillus thuringiensis pak2310 halo around the colonies eliminates the possibility of the production of glycolipids [27]. When subjected to orcinol assay, the expected red— orange coloration was not seen. The absence of any red— orange coloration shows that there are no reducing sugars like rhamnose present in the biosurfactant and hence the absence of any glycolipid in the extract [7]. The crude as well as purified antibiotic answered Lowry’s test for proteins and hence, the antibiotic is predicted to be a lipopeptide or cyclic lipopeptide, like fengycin [38] tensin or surfactin. Fourier Transform—infra-red spectroscopy Strong peaks were detected at 3426.37 cm1, 3018.19 cm1, 2923.34 cm1, 1713.25 cm1, 1657.20 cm1, 1432.44 cm1, 1365.03 cm1, 1221.99 cm1, and 765.78 cm1. These peaks were then used to predict the functional groups present in the crude extract of the biosurfactant. The purified biosurfactant showed peaks at 3436.01 cm1, 1627.42 cm1, 1563.95 cm1, 1423.98 cm1, which are characteristics of surfactin like lipopeptide surfactants (Fig. 6). The FT—IR spectrum of the crude biosurfactant with strong bands at 2923.34 cm1 and 1657.2 cm1 indicate the presence of sp3 hybridised carbon and peptide bonds, which show the presence of peptides in the biosurfactant. Further the band at 3426.37 cm1, 1713.25 cm1 and 1221.99 cm1 indicate the presence of hydrophilic groups in the compound, such as —OH and —COOH groups. Furthermore, the bands at 765.78 cm1and at 671.67 cm1 suggest the presence of aromatic side chain groups as they are signatures for aromatic hydrogens. From the above findings, one could conclude that the surfactant extracted could be a lipopeptide [22]. In case of the FT—IR spectrum of purified biosurfactant, the strong signal at 1423.98 cm1 suggests CH2 bending in lipids. Signals at 1627.42 cm1 and 1563.95 cm1 correspond to the peptide (amide) bond vibration, N—H bending and C—N stretching of peptides, respectively. Similarly, the broad peak at 3436.01 cm1 suggests N—H and O—H stretching of peptides [36,45].
Mass spectroscopy analysis The MALDI-TOF MS spectrum and its reconstruction (Fig. 7) confirm the molecular weight of the purified surfactant to be 700.5 Da. An array of surfactants from Bacillus species has been isolated and characterised to have molecular weights from 800 Da to 1500 Da previously [45] which is comparable with the obtained value. The functional groups of the lipopeptide are similar to that of surfactin while the molecule has lesser molecular weight than the commercial surfactin, hence, the isolated lipopeptide from the new strain of Bacillus sp might be a novel liopopeptide which requires the further complete structural elucidation studies.
Conclusions The lipopeptide obtained from this study has efficient antifungal and anti-biofilm properties against the otherwise recalcitrant F. oxysporum infections, which is evident from the MIC and morphological changes on the fungi observed in the study. At the same time, the lipopeptide is non-toxic to
9 human epithelial cells even at 10-fold of MIC. The promising results might pave way for the development of a promising candidate lipopeptide as drug against fusariosis.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgements The authors sincerely thank SASTRA University for the infrastructure provided and also for funding the project under the TRR Scheme. The authors also express their sincere thanks to Mr. Ankit Rochani of Prof. Utpal S. Tatu’s lab at Indian Institute of Science for extending help with the MALDI-ToF MS analysis, Dr. S. Adline Princy and Mr. C. David Raj of SASTRA University for all the technical support provided promptly.
References [1] Alfanso EC. Genotypic identification of Fusarium species from ocular sources: comparison to morphologic classification and antifungal sensitivity testing (an AOS thesis). Trans Am Opthalmol Soc 2008;105:227—39. [2] Armstrong GM, Armstrong JK. Formaespeciales and races of Fusarium oxysporum causing wilt diseases. In: Nelson PE, editor. Fusarium: Diseases, Biology and Taxonomy. 1981. p. 391—9. [3] Banat IM. Biosurfactants production and possible uses in microbial enhanced oil recovery and oil pollution remediation: a review. Bioresour Technol 1995;51:1. [4] Boutati EI, Anaissie EJ. Fusarium, a significant emerging pathogen in patients with hematological malignancy: ten years’ experience at a cancer centre and implications for management. Blood 1997;90:999—1008. [5] Brilhante RS, Cordeiro RS, Medrano DJ, Rocha MF, Monteiro AJ, Cavalcante CS, et al. Onychomycosis in Ceara (Northeast Brazil): epidemiological and laboratory aspects. Mem Inst Oswaldo Cruz 2005;100:131—5. [6] Cameotra SS, Makkar RS. Recent applications of biosurfactants as biological and immunological molecules. Curr Opin Microbiol 2001;7:1. [7] Chandrasekaran EV, Bemiller JN. Methods in Carbohydrate Chemistry. New York: Academic Press; 1980. [8] Chang DC, Grant GB, O’Donnell K, Wannemuehler KA, NobleWang J, Rao CY, et al. Multistate outbreak of Fusarium keratitis associated with use of a contact lens solution. JAMA 2006;296:953—63. [9] Cooper DG, Goldenberg BG. Surface active agents from two Bacillus spp. Appl Environ Microbiol 1987;53:224. [10] Damasceno FRC, Cammarota MC, Freire DMG. The combined use of a biosurfactant and an enzyme preparation to treat an effluent with a high fat content. Colloids Surfaces B 2012;95:241. [11] Danicke S. Fusarium toxins in animal nutrition. Lohman Info 2002;27:1—9. [12] Darvishi P, Ayatollahi P, Mowla D, Niazi A. Biosurfactant production under extreme environmental conditions by efficient microbial consortium, ERCPPI-2. Colloids Surf B 2011;84:292. [13] Deleu M, Bouffioux O, Razafindralambo H, Paquot M. Interaction of surfactin with membranes: a computational approach. Langmuir 2003;19:3377—85. [14] Donnio A, Van Nuoi DN, Catanese M, Desbois N, Ayebuoa L, Merle H. Outbreak of keratomycosis attributable to Fusarium
Please cite this article in press as: Deepak R, Jayapradha R. Lipopeptide biosurfactant from Bacillus thuringiensis pak2310: A potential antagonist against Fusarium oxysporum. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.10.011
+ Models
MYCMED-511; No. of Pages 10
10
[15] [16]
[17]
[18]
[19]
[20]
[21] [22] [23]
[24]
[25] [26]
[27]
[28]
[29] [30] [31]
R. Deepak, R. Jayapradha solani in the French West Indies. Am J Ophthalmol 2007;143: 356—8. Doyle ME. Fusarium mycotoxins. UW FRI Briefings; 1997, http:// fri.wisc.edu/docs/pdf/fusarium.pdf. Ghojavand H, Vahabzadeh F, Roayaei E, Shahraki AK. Production and properties of a biosurfactant obtained from a member of the Bacillus subtilis group (PTCC 1696). J Colloid Interface Sci 2008;324:172. Godoy P, Nunes E, Silva V, Tomimori-Yamashita J, Zaror L, Fischman O. Onychomycosis caused by Fusarium solani and Fusarium oxysporum in Sao Paulo, Brazil. Mycopathologia 2004;157:287—90. He H, Silo-Suh LA, Clardy J, Handelsman J. Zwittermicin, an antifungal and plant protection agent from Bacillus cereus. Tetrahedron Lett 1994;34:2499. Heerklotz H, Wieprecht T, Seelig J. Membrane pertubation by the lipopeptide surfactin and detergents as studied by deuterium NMR. J Phys Chem 2004;108:4909—15. Imamura Y, Chandra J, Mukherjee PK, Lattif AA, Szczotka-Flynn LB, Pearlman E, et al. Fusarium and Candida albicans biofilms on soft contact lenses: model development, influence of lens type and susceptibility to lens care solutions. Am Soc Microbiol 2008;52:171—82. Iyer SA, Tuli SS, Wagoner RC. Fungal keratitis: emerging trends and treatment outcomes. Eye Contact Lens 2006;32:267—71. Joseph Hornback M. Organic Chemistry, . 2nd ed., Connecticut: Thomson Brooks/Cole; 2006. Kim PI, Bai H, Bai D, Chae H, Chung S, Kim Y, et al. Purification and characterisation of lipopeptide produced by Bacillus thuringiensis CMB26. J Appl Microbiol 2004;97:942—5. Kluge B, Vater J, Salikow J, Eckhart K. Studies on the biosynthesis of surfactin, a lipopeptide antibiotic from Bacillus subtilis ATCC21332. FEBS Lett 1988;231:107. Kosaric N. Biosurfactants in industry. Pure Appl Chem 1992;64:1731. Kragh-Hansen U, Maire M, Moller J. The mechanism of detergent solubilization of liposomes and protein-containing membranes. Biophys J 1998;75:2932—46. Kratka J, Kovacikova E. The effect of temperature and age of strains of Fusarium oxysporum on its enzymatic activity. Zentbl Bakteriol Naturwiss 1979;134:154—8. Liu X, Ren B, Gao H, Liu M, Dai H, Song F, et al. Optimisation for the production of surfactin with a new synergistic antifungal activity. PLoS One 2002;7:1. Maier RM. Biosurfactant: evolution and diversity in bacteria. Adv Appl Microbiol 2003;52:101. Michael RG, Sambrook J. Molecular Cloning Manual, 2. New York: Cold Spring Harbor Laboratory Press; 2001. Mulligan CN. Environmental applications of biosurfactants. Environ Pollut 2005;133:183.
[32] Mulligan CN, Cooper DG, Neufeld RJ. Selection of microbes producing biosurfactants in media without hydrocarbons. J Ferment Technol 1984;62:311—4. [33] Nussie M, Anaissie E. Fusarium infections in immunocompromised patients. Clin Microbiol Rev 2007;20:695—704. [34] O’ Donell K, Sutton DA, Rinaldi MG, Magnon KC, Cox PA, Revankar SG, et al. Genetic diversity of human pathogenic members of the Fusarium oxysporum complex inferred from multilocus DNA sequencing data and amplified fragment length polymorphism analyses: evidence for the recent dispersion of a geographically widespread clonal lineage and nosocomial origin. J Clin Microbiol 2004;42:5109—20. [35] Perfumo A, Ranchic I, Banat IM. Biosurfactants. In: Sen R, editor. Biosurfactants Book series: Advances in Experimental Medicine and Biology, 627. Texas: Landes Bioscience; 2010. p. 134—45. [36] Prince NPJ, Rooney AP, Swezey JL, Perry E, Cohan FM. Mass spectrometric analysis of lipopeptides from bacillus strains isolated from diverse geographical locations. FEMS Microbiol Lett 2007 [doi:001:1 0.1111-p.1574-6968.2007.00702.x, 2007]. [37] Qi G, Zhu F, Du P, Yang X, Qiu D, Yu Z, et al. Lipopeptide induces apoptosis in fungal cells by a mitochondria dependent pathway. Peptides 2010;31:1978—86. [38] Ramarathnam R, Bo S, Chen Y, Fernando WG, Xuewen G, de Kievit T. Molecular and biochemical detection of fengycin and bacillomycin D-producing Bacillus spp., antagonistic to fungal pathogens of Canola and Wheat. Can J Microbiol 2007;5:901. [39] Ron EZ, Rosenberg E. Biosurfactants oil bioremediation. Curr Opin Biotechnol 2002;13:24. [40] Schneller FR, Gulati SC, Cunningham IB, O’Reilly RJ, Schmitt HJ, Clarkson BD. Fusarium infections in patients with hematologic malignancies. Leuk Res 1990;14:961. [41] Siegmund I, Wagner F. New method for detecting rhamnolipids excreted by Pseudomonas spp during growth on mineral agar. Biotechnol Tech 1991;5:265. [42] Singh P, Cameotra SS. Potential applications of microbial surfactants in biomedical sciences. Trends Biotechnol 2004; 22:142. [43] Sun Y, Chandra J, Mukherjee P, Szczotka-Flynn L, Ghannoum MA, Pearlman E. A murine model of contact lens-associated Fusarium keratitis. Invest Opthalmol Vis Sci 2010;51:1511—6. [44] Vaz DA, Gudina EJ, Alameda EJ, Teixeira JA, Rodrigues LR. Performance of biosurfactant produced by Bacillus subtilis strain isolated from crude oil samples as compared to commercial chemical surfactants. Colloids Surface B 2012;89:167. [45] Yalcin E, Cavusoglu K. Structural analysis and antioxidant activity of a biosurfactant obtained from Bacillus subtilis RW-1, Turk. J Biochem 2010;35:243.
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