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Characterization of antibacterial activity of bikaverin from Fusarium sp. HKF15
Journal of Bioscience and Bioengineering VOL. 117 No. 4, 443e448, 2014 www.elsevier.com/locate/jbiosc
Characterization of antibacterial activity of bikaverin from Fusarium sp. HKF15 Radhika Deshmukh,1 Anoop Mathew,2 and Hemant J. Purohit1, * CSIR e National Environmental Engineering Research Institute, Nehru Marg, Nagpur 440020, Maharashtra, India1 and Piramal Enterprises Ltd., 1, Nirlon Complex, Off Western Express Highway, Goregaon (East), Mumbai 400 063, India2 Received 6 August 2013; accepted 28 September 2013 Available online 31 October 2013
Redeemed interest in discovery and characterization of naturally occurring antimicrobials arises from the fact that these compounds possess diverse biological activities useful in the development of new therapeutics. In this study, 35 fungi from extreme environments of effluent treatment plants were screened for antimicrobial activity. An isolate HKF15 produced antimicrobial compound that showed activities against pathogenic and multidrug resistant bacteria; and was identified as Fusarium sp. The active principle was separated through activity-guided HPLC fractionation of 1:1 ethyl acetate crude extract that was identified as bikaverin using HRMS and 1H NMR. Bikaverin is known for antitumor and antifungal activity and in this study; antibacterial activity of bikaverin was assessed, emphasizing the importance of repurposing of antibiotics. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Fusarium; Bikaverin; 18S rRNA gene; Antimicrobial activity; Effluent treatment plant]
Multiple drug resistance (MDR) is an area of major concern today. Due to uncontrolled administration of drugs by humans, MDR strains of pathogenic organisms are rapidly evolving (1). Finding novel natural products or finding novel activities of known natural products can be a key solution to this problem (2). Microbes have a natural survival machinery to compete with other microbes in nature; and this phenomenon can be exploited for the development of antimicrobials (3). Microorganisms including fungi produce a vast range of small molecular weight bioactive secondary metabolites, which show anticancer, antiplasmodial, anti-inflammatory and antiviral potential (4e6). However, the role of these secondary metabolites and survival of microbes in stressed environment is not well understood (7e9). With different types of organic loading, the biological units of effluent treatment plants (ETPs) treating wastewater create an extreme scenario for survival of microbes. The micro flora in these niches is under regular stress due to change in qualitative chemical loading that influences high total dissolved solids (TDS), pH, temperature and salinity (10). In this study, fungi were isolated from different ETPs, and were explored for antimicrobials production. One of the fungal isolates, HKF15 identified as Fusarium produced bikaverin, a polyketide known for antitumor and antifungal property. The study demonstrates the antibacterial property associated with this compound.
* Corresponding author. Tel.: þ91 712 2249883; fax: þ91 712 2243927. E-mail address: [email protected] (H.J. Purohit).
MATERIALS AND METHODS Isolation and identification of fungal isolates Fungal strains were isolated from activated sludge samples from three ETPs located at Ankleshwar, Amlakhadi and Vatwa in Gujarat state of India. Sample (10 g wet weight) was added to 100 ml of 0.1 phosphate buffered saline (PBS), pH 7.0, (Himedia Laboratories, India) in 250 ml conical flasks. To control the undesired bacterial growth the antibiotics were used (11). The antibiotic concentrations used were: Tetracycline, 10 mg ml1; nalidixic acid, 20 mg ml1; ampicillin, 20 mg ml1. Flasks were placed on a shaker at 150 rpm and after overnight shaking, biomass was allowed to settle for 15 min and supernatant liquid was decanted. The decantate was serially diluted in PBS and spread on Potato dextrose agar (PDA) medium (Himedia Laboratories, India). Plates were incubated at 25e28 C for 1 week and fungal colonies were then selected based on differences in colony morphology and streaked on fresh PDA plates. After confirming the purity of the strains, they were maintained on PDA slants/plates at 25e28 C by periodic sub-culturing. Phenotypic identification of the fungi was carried out by microscopic observation on the basis of branching pattern of mycelia and arrangement of conidia and conidiophores after staining with lactophenol cotton blue (12).
Isolation of genomic DNA and identification by 18S rRNA gene sequence analysis Genomic DNA was extracted using ZR Fungal/Bacterial DNA kit (Zymo Research, USA) according to the manufacturer’s instruction and stored at 20 C until further use. The fungal strains were identified by 18S rRNA sequence analysis. PCR was performed using conserved primer sequence (13) encoding 762 bp 18S rRNA product (0817F 50 -TTAGCATGGAATAATRRAATAGGA-30 and 1536R 50 -ATTGCAATGCYCTATCCCCA-30 ) in a Veriti 96-well Thermocycler (Applied Biosystems, USA) at annealing temperature of 56 C. The product was gel extracted using Qiagen gel elution kit (Hilden, Germany) and ligated using a TOPO cloning kit according to the manufacturer instruction (Invitrogen Corporation) followed by sequencing of the amplified product. BLAST searches were made by using the BLAST, version 2.0, software and the nucleotide sequences were deposited in GenBank (http://www.ncbi.nlm.nhi.gov/BLAST). Phylogenetic tree was constructed using BOOTSTRAP tree method from Clustal X software developed by Thompson et al. (14).
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.09.017
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Aspergillus sp HKF16 JF922008
B
Aspergillus flavipes ATCC1030 AY373849 Aspergillus terreus ATCC1012 AY373871 Aspergillus niger TF1 JN974294 Aspergillus clavatus ATCC58869 AY373847 Aspergillus fumigatus SCSGAF0187 JN851056 Fusarium solani RM2 JN887340 Fusarium chlamydosporum SCSGAF0087 JN851018 Fusarium solani NZFS3555 JN595827 Fusarium oxysporum lycopersici ATCCMYA4623 GU327639 Fusarium sp HKF15 JF922007 Fusarium oxysporum ATCC16600 GQ914766 Penicillium sp HKF11 HM773238 Penicillium sp HKF44 HM773240 Penicillium paxilli SCSGAF0173 JN851050 Penicillium citrinum SCSGAF0167 JN851046 Penicillium tricolor ATCC10413 AY373935 Penicillium janthinellum ATCC4845 AY373921 Penicillium oxalicum SCSGAF0143 JN851042
FIG. 1. Diversity of fungal isolates. (A) Phylogenetic tree is inferred from the comparison of 18S rRNA gene sequences of antimicrobial fungal isolates with reference strains. HKF species are lab isolates. Nucleotide sequences of reference strains were retrieved from GenBank data. The values at nodes represent 1000 bootstrap replicates. (B) RAPD analysis with universal RAPD primers 3 and 6. Lanes 1e5: RAPD primer3; lanes 7e11: RAPD primer6. Samples: lane 1: HKF11; lane 2: HKF15; lane 3: HKF16; lane 4: HKF40; lane 5: HKF44; lane 6: 1 kb ladder; lane 7: HKF11; lane 8: HKF15; lane 9: HKF16; lane 10: HKF40; lane 11: HKF44.
Evaluation of genetic diversity of fungal strains by RAPD analysis Genetic diversity of the fungi was evaluated by RAPD by using commercially available RAPD Analysis Primer 3 e (50 -d[GTAGACCCGT]-30 ) and RAPD Analysis Primer 6 - (50 -d [CCCGTCAGCA]-30 ) from the Amersham RAPD kit (Amersham Pharmacia Biotech, Uppsala, Sweden). Medium and culture conditions Potato dextrose medium was suitable for antimicrobial compound production. However, since it is a complex medium, minimal medium was optimized for the production of antimicrobials, the composition of which is described earlier (15). Glusoce (10%) was added as a carbon source. Preliminary screening against pathogens Fungi were grown for seven days in potato dextrose broth at 30 C at 120 rpm on a shaker and the antibacterial activity of culture supernatant was checked preliminarily against five ATCC pathogens namely Escherichia coli (ATCC25922), Bacillus cereus (ATCC10876), Shigella flexneri (ATCC9199), Salmonella paratyphi (ATCC9150) and Staphylococcus epidermidis (ATCC12228) by agar well diffusion method (16) against 107 cells of host pathogen seeded onto an LB agar plate. The measurement of cell OD was carried out at 600 nm on a spectrophotometer (Thermo Scientific). Ceftazidime resistant Pseudomonas aeruginosa strain, vancomycin resistant Enterococcus strain and methicillin resistant Staphylococcus strain (MRSA) and MDR E. coli strains (E. coli 1 and E. coli 2) resistant to vancomycin, oxacillin, ceftazidime and other MDR strains including Serratia sp. GMX1, resistant to sulfametoxazole, ampicillin, azithromycin, tetracycline and netilmycin and Enterobacter resistant to sulfametoxazole, ampicillin, azithromycin and tetracycline were used. Extraction of antimicrobial compound from HKF15 As HKF15 was found to be the most promising potential producer of antibacterial agent, the further studies were focused on it. The fungal strain was cultivated in minimal medium with 10% glucose whose composition is as described earlier. The fungus was grown for 7 days. Incubation was carried out on a rotator shaker at 120 rpm and 30 C. The culture supernatant was extracted with equal volume of ethyl acetate twice, for 3 h each. For this, the supernatant was mixed with equal volume of ethyl acetate and kept on magnetic stirrer for 3 h. It was then allowed to settle. The upper layer of ethyl acetate was separated and extraction process was repeated. The extract was concentrated on rotator evaporator (Buchi type, Sonar) at 40 C to get a solid residue. This residue was suspended in methanol/DMSO and the antimicrobial activity was confirmed. Purification and characterization The 80 mg concentrated crude extract was subjected to HPLC (Perkin Elmer). HPLC separation was carried out on a Gilson 322/215 system equipped with dual wavelength detector using a Water’s Xterra RPC18 50 19 mm column fitted with a 10 mm guard. The HPLC gradient was as in Table S1. Mobile phases were used with 0.1% formic acid added. The crude ethyl acetate extract of HKF15 was dissolved in DMSO and separated by preparative HPLC by injecting 1.5 ml sample; and 70 fractions were collected into custom trays holding Genevac evaporator racks. A flow rate of 24 ml/min was used. Fractions were collected every 0.5 min starting from the solvent front of 0.65 min
into 100 16 mm tubes until 35.5 min. After HPLC fractionation, aliquots from test tubes were added into 39 wells of micro-titer plate according to supplementary Table S2 and Fig. S1. Well 40 was control and contained crude extract. Six active samples were obtained from these wells and only the active D2 sample (17e17.5 min) was characterized by HR-ESI MS (Bruker micro QTOF) and 1 H NMR (Bruker 500 MHz). The QTOF-MS was performed in both positive and negative mode. Thin layer chromatography for partial purification Silica gel (40%) and 8% calcium sulfate was mixed and slurry (50 ml) was poured on thin glass plate (12 cm12 cm). HKF15 crude ethyl acetate extract (5 ml) was spotted thrice. Mobile phase used was chloroform, methanol, acetic acid 94:1:5. Antibacterial activity Bikaverin was analyzed for its antibacterial activity against E. coli ATCC 25922. E. coli was grown overnight in LB and transferred to 1X minimal medium with 10% glucose. The culture was grown overnight at 37 C at 120 rpm. The inoculum of approximately 108 cells was used per 200 ml of fresh medium in micro-titer plate (Corning clear plate with U bottom) in duplicates. The antibacterial activity experiment was done in triplicates and the results show the data points with error bars showing standard deviation from mean of the values. Bikaverin was added in concentrations 20, 40, 60, 80 and 100 ppm each. Suitable control was maintained. The OD was read after every 30 min on Victor X3 multiplate reader set at 37 C for 5 h. The 100% pure bikaverin standard was kindly provided by Dr. Hasumi (The University of Tokyo) (17). The validation of above experiment was done by colony counting. E. coli was grown in LB at 37 C and 120 rpm overnight and inoculated in minimal medium with 10% glucose. This was grown overnight and used as an initial inoculum. The bikaverin concentrations of 10 ppm, 25 ppm and 50 ppm were added to 109 E. coli cells ml1 and the culture was grown at 37 C and 120 rpm. Samples were taken out
TABLE 1. Bioassay results for HKF15 ethyl acetate extract in comparison with standard antibiotics. Organism Pseudomonas aeruginosa Staphylococcus aureus Methicillin resistant Staphylococcus aureus Vancomycin resistant Enterococcus Aspergillus
Diameter of zone in mm for standards
Diameter of zone in mm for HKF15 extract
7 Hazy 8 Clear 8 Clear
7 Hazy 12 Clear 11 Clear
7 Hazy
Nil
9 Hazy
8 Hazy
Standard antibiotics: Ceftazidime for P. aeruginosa, oxacillin for S. aureus and methicillin resistant S. aureus, vancomycin for vancomycin resistant Enterococcus.
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FIG. 2. HPLC and bioactivity guided HPLC fraction analysis. (A) Chromatogram for HKF15 crude extract in ethyl acetate. Mobile phase A: water mobile phase B: acetonitrile. Collection started at 0.65 min, stopped at 35.5 min, flow rate: 24 mL min1, run time: 40 min. Dark line represents 195 nm and faint line represents 254 nm. (B) Bioactivity guided HPLC fraction analysis. The HPLC fractions of crude HKF15 extract were checked against drug resistant and pathogenic bacteria. The fractions were first pooled in micro-titer plate and 40 samples were obtained. Graph shows activity of samples from well no. 15 onwards, no activity was detected in previous samples. Open diamond, P. aeruginosa; filled squares, S. aureus; filled triangles, MRSA; open circles, VRE; crosses, E. coli MDR1; filled circles, E. coli MDR2.
periodically and were analyzed for colonies on LB plates. In a parallel experiment, the different cell concentrations treated with the selected bikaverin concentration was studied.
RESULTS AND DISCUSSION Isolation of fungi From ETPs 35 fungal isolates were obtained and it was seen that it is a suitable niche for fungi. The isolated fungi exhibited rapid growth on PDA medium and it was found to be a favorable medium for culturing and storage of fungi from ETPs. Mycological agar, malt yeast extract agar are some other media for the isolation of fungi. Potato dextrose agar is widely used amongst them (18,19). Microscopic observations helped in identification of fungi which was validated by 18S rRNA gene sequencing. Identification by 18S rRNA gene sequence analysis and diversity study by RAPD analysis Amplification and sequencing of the 18S region of rRNA gene for each fungus resulted in 762 bp product, whose sequence has been deposited in NCBI database with accession numbers as, Penicillium sp.: HKF11
(HM773238) and HKF44 (HM773240), Aspergillus sp.: HKF16 (JF922008) and Fusarium sp.: HKF15 (JF922007). Fig. 1A is the phylogenetic tree showing the 18S diversity. RAPD analysis is another important tool to assess genetic diversity (20) which was used. Fig. 1B shows that RAPD primer 3 gave different amplification patterns for all fungi except HKF11 and HKF44. RAPD primer 6 gave different amplification patterns for all fungi except HKF11 and HKF40 where there was similarity in band patterns. So, the RAPD analysis demonstrated the diversity of fungi. Preliminary screening against pathogens Agar diffusion method provides a simple and effective way for rapid screening of antimicrobial activity (21). Activity was observed under nutrient limiting conditions (22). Four fungieHKF11, HKF15, HKF16, and HKF44 showed effective antibacterial activity against ATCC pathogens (Table S3). HKF15 was found to be active against the methicillin resistant Staphylococcus aureus, Vancomycin resistant Enterococcus, Pseudomonas aeruginosa and MDR bacteria (Enterococcus and Serratia sp.) (Fig. S2, Table 1). Though linezolid and vancomycin are used to treat MRSA infections, they have
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FIG. 3. Growth curve showing antibacterial activity of different concentrations of bikaverin against E. coli ATCC 25922. OD was measured at 600 nm. Open circles, culture control; diamonds, 20 ppm; open squares, 40 ppm; filled squares, 60 ppm; open triangles, 80 ppm; filled circles, 100 ppm bikaverin; filled triangles, solvent control. The error bars represent standard deviation from mean values of samples.
some side-effects (23) and new molecules are being searched as alternatives for multi-drug resistant organism like MRSA. Extraction and purification of compound The HPLC fractionation gave 6 active samples (Fig. 2A) from the pooled fractions in micro-titer plate. These samples were obtained based on their activity against pathogens. But each sample showed different activity pattern (Fig. 2B). The most active sample was D2 as it showed activity against most of the bacteria used for screening. Sample D2 was subjected to QTOF-MS in both positive and negative mode and 1H NMR analysis. The major compound characterized was bikaverin. The structure and mass was confirmed by HR QTOF-MS and 1H NMR (Fig. S3). It is a red colored pigment. Fig. S4 shows the structure of bikaverin. Bikaverin was first isolated from Fusarium oxysporum sp vasinfectum. It was called lycopersin because of its red color (24). Passiflorine isolated from F. oxysporum sp. passiflorae and mycogonin from Mycogone jaapii are the alternative reports for the same compound (25). A polyketide synthase gene bik1 is responsible for its synthesis (26). The bik gene cluster includes biosynthesis genes bik2 and bik3, regulatory genes bik4 and bik5, and transporter bik6 (27,28). TLC purification and LCMS analysis of purified fraction A purple spot was observed on TLC plate with Rf value: 0.4521 (29). The spot was eluted in ethyl acetate (27) and was compared with standard bikaverin by LCMS as described by Busman et al. (30). Bikaverin was detected in the TLC purified fraction which also showed antibacterial activity. This was indicative of antibacterial potential of bikaverin. Antibacterial activity We observed that bikaverin inhibited E. coli growth significantly in the growth curve study in a microtiter plate reader (Fig. 3). It is the standard method used mostly to determine minimum inhibitory concentration (31). Colony count indicated that 25 ppm concentration was able to inhibit bacterial growth slowly (Table 2). When different cell concentrations were treated with 25 ppm bikaverin, it was observed that after 6 h, the CFU count was reduced to zero (Table 3). While in case of 50 ppm bikaverin, there was a significant inhibition of growth after 1 h. This demonstrated that apart from the other bioactivities (32),
bikaverin also has an antibacterial potential. Repurposing of drugs is a recent area to fulfill the need for new efficient antibiotics where a known compound is exploited for a novel activity. Auranofin therapy against Entamoeba histolytica infection (33), novel activity of 20 FDA approved compounds against drugresistant Acinetobacter baumannii (34) are some examples. The fungal contamination associated with eating of agricultural products contaminated with fungal secondary metabolites like fumonisin has been reported (35); but bikaverin-contaminated products are not reported for any negative impact on human health. It is proven non-genotoxic according to an unscheduled DNA synthesis assay (36). It has some biological effects, but those may be desired ones either as an antibiotic or an antitumor agent rather than deteriorating ones. The antibiotic properties of bikaverin have promising commercial applications in the control of some pathogenic protozoa and fungi or in cancer treatment (32). The screening of such known antibiotics across different targets will surely help understand the biological mechanisms of small antibiotic molecule interactions in a better way, which will be helpful for drug development programs. Most antibiotics have originally come from a small set of molecular scaffolds which are modified by synthetic tailoring for better functionality. The emergence of multidrug resistance suggests that the discovery of new scaffolds should be a priority (37). And, approaches towards scaffold discovery include mining under explored microbial niches for natural products and repurposing synthetic molecule libraries for use as antibiotics (38). With a
TABLE 2. Effect of bikaverin concentrations on E. coli growth with initial inoculum of 109 cells ml1. SN
Colony count in CFU ml1
Time (h) Control
1 2 3 4 5
0 1 2 3 6
3.01 5.04 4.75 2.07 1.55
ND: Not done for zero time.
10 ppm 9
10 109 109 109 109
ND 4.98 4.99 2.14 1.67
109 109 109 109
25 ppm ND 1.03 1.48 0.64 0.36
109 109 109 109
50 ppm ND 0.05 109 0.03 109 0 0
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TABLE 3. Effect of 25 ppm bikaverin on E. coli growth with decreasing inoculum size starting from 106 cells ml1. SN
Colony count in CFU ml1
Time (h) Control
1 2 3
1 3 6
1
6
10 cells ml 6
2.3 10 2.3 106 2.4 106
5
4 10 2 105 0
1
5
10 cells ml 3
1 10 1 103 0
10 cells ml1
103 cells ml1
500 cells ml1
200 cells ml1
100 cells ml1
200 1 0
40 30 0
20 0 0
10 0 0
0 0 0
4
plethora of information on bioactivities of small molecules available through databases like PubChem, it is now a challenging task to mine the actual bioactive potential of a molecule for drug development research. Bioinformatics and chemoinformatics analyses are being used to study the across target activity of bioactive molecules (39,40). Such kinds of surveys might provide insights into a compound’s bioactivity against previously undiscovered target. ETPs provide unique habitat where different microbes survive together and compete for the easily available carbon source under diverse organic loadings. The scenario generates microbial communities which use intermediate generated by other species and provide novel condition for secondary metabolite production. These acquired pathways give molecules that provides defense against other microbes. This study explores such activity associated with a fungal isolate from an ETP, HKF15 towards pathogenic and drug resistant strains. It produces bikaverin that has been reported to be an antifungal molecule. However, this study demonstrates an additional property of antibacterial activity of bikaverin. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2013.09.017. ACKNOWLEDGMENTS We thank Naoko Nishimura and Dr. Keiji Hasumi (Tokyo University of Agriculture and Technology, Tokyo) for providing bikaverin standard; Dr. Saji George, Dr. Arun Balakrishnan (PEL, Mumbai) and Mr. G.S. Kanade (CSIR-NEERI). We acknowledge the financial support from Council of Scientific and Industrial Research (CSIR), Delhi, for one of the authors (Radhika Deshmukh) through CSIR, Senior Research Fellowship. The authors have no conflicts of interest to declare. References 1. Haug, B. E., Stensen, W., Kalaaji, M., Rekdal, O., and Svendsen, J. S.: Synthetic antimicrobial peptidomimetics with therapeutic potential, J. Med. Chem., 51, 4306e4314 (2008). 2. Ekins, S., Williams, A. J., Krasowski, M. D., and Freundlich, J. S.: In silico repositioning of approved drugs for rare and neglected diseases, Drug Discov. Today, 16, 298e310 (2011). 3. Pieters, R. J., Arnuscha, C. J., and Breukinkb, E.: Membrane permeabilization by multivalent anti-microbial peptides, Protein Pept. Lett., 16, 1e7 (2009). 4. Guo, B., Wang, Y., Sun, X., and Tang, K.: Bioactive natural products from endophytes: a review, Appl. Biochem. Microbiol., 44, 136e142 (2008). 5. Onifade, A. K.: Research trends: bioactive metabolites of fungal origin, Res. J. Biol. Sci., 2, 81e84 (2007). 6. Talontsi, F. M., Dittrich, B., Schüffler, A., Sun, H., and Laatsch, H.: Epicoccolides: antimicrobial and antifungal polyketides from an endophytic fungus Epicoccum sp. associated with Theobroma cacao, Eur. J. Org. Chem., 15, 3174e3180 (2013). 7. Mancini, I., Defant, A., and Guella, G.: Recent synthesis of marine natural products with antibacterial activities, Anti-Infect. Agents Med. Chem., 6, 17e48 (2007). 8. Whelan, A. P., Dietrich, L. E. P., and Newman, D. K.: Rethinking ‘secondary’ metabolism: physiological roles for phenazine antibiotics, Nat. Chem. Biol., 2, 71e78 (2006). 9. Pettit, R. K.: Culturability and secondary metabolite diversity of extreme microbes: expanding contribution of deep sea and deep-sea vent microbes to natural product discovery, Mar. Biotechnol., 13, 1e11 (2011).
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Characterization of antibacterial activity of bikaverin from Fusarium sp. HKF15 Radhika Deshmukh,1 Anoop Mathew,2 and Hemant J. Purohit1, * CSIR e National Environmental Engineering Research Institute, Nehru Marg, Nagpur 440020, Maharashtra, India1 and Piramal Enterprises Ltd., 1, Nirlon Complex, Off Western Express Highway, Goregaon (East), Mumbai 400 063, India2 Received 6 August 2013; accepted 28 September 2013 Available online 31 October 2013
Redeemed interest in discovery and characterization of naturally occurring antimicrobials arises from the fact that these compounds possess diverse biological activities useful in the development of new therapeutics. In this study, 35 fungi from extreme environments of effluent treatment plants were screened for antimicrobial activity. An isolate HKF15 produced antimicrobial compound that showed activities against pathogenic and multidrug resistant bacteria; and was identified as Fusarium sp. The active principle was separated through activity-guided HPLC fractionation of 1:1 ethyl acetate crude extract that was identified as bikaverin using HRMS and 1H NMR. Bikaverin is known for antitumor and antifungal activity and in this study; antibacterial activity of bikaverin was assessed, emphasizing the importance of repurposing of antibiotics. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Fusarium; Bikaverin; 18S rRNA gene; Antimicrobial activity; Effluent treatment plant]
Multiple drug resistance (MDR) is an area of major concern today. Due to uncontrolled administration of drugs by humans, MDR strains of pathogenic organisms are rapidly evolving (1). Finding novel natural products or finding novel activities of known natural products can be a key solution to this problem (2). Microbes have a natural survival machinery to compete with other microbes in nature; and this phenomenon can be exploited for the development of antimicrobials (3). Microorganisms including fungi produce a vast range of small molecular weight bioactive secondary metabolites, which show anticancer, antiplasmodial, anti-inflammatory and antiviral potential (4e6). However, the role of these secondary metabolites and survival of microbes in stressed environment is not well understood (7e9). With different types of organic loading, the biological units of effluent treatment plants (ETPs) treating wastewater create an extreme scenario for survival of microbes. The micro flora in these niches is under regular stress due to change in qualitative chemical loading that influences high total dissolved solids (TDS), pH, temperature and salinity (10). In this study, fungi were isolated from different ETPs, and were explored for antimicrobials production. One of the fungal isolates, HKF15 identified as Fusarium produced bikaverin, a polyketide known for antitumor and antifungal property. The study demonstrates the antibacterial property associated with this compound.
* Corresponding author. Tel.: þ91 712 2249883; fax: þ91 712 2243927. E-mail address: [email protected] (H.J. Purohit).
MATERIALS AND METHODS Isolation and identification of fungal isolates Fungal strains were isolated from activated sludge samples from three ETPs located at Ankleshwar, Amlakhadi and Vatwa in Gujarat state of India. Sample (10 g wet weight) was added to 100 ml of 0.1 phosphate buffered saline (PBS), pH 7.0, (Himedia Laboratories, India) in 250 ml conical flasks. To control the undesired bacterial growth the antibiotics were used (11). The antibiotic concentrations used were: Tetracycline, 10 mg ml1; nalidixic acid, 20 mg ml1; ampicillin, 20 mg ml1. Flasks were placed on a shaker at 150 rpm and after overnight shaking, biomass was allowed to settle for 15 min and supernatant liquid was decanted. The decantate was serially diluted in PBS and spread on Potato dextrose agar (PDA) medium (Himedia Laboratories, India). Plates were incubated at 25e28 C for 1 week and fungal colonies were then selected based on differences in colony morphology and streaked on fresh PDA plates. After confirming the purity of the strains, they were maintained on PDA slants/plates at 25e28 C by periodic sub-culturing. Phenotypic identification of the fungi was carried out by microscopic observation on the basis of branching pattern of mycelia and arrangement of conidia and conidiophores after staining with lactophenol cotton blue (12).
Isolation of genomic DNA and identification by 18S rRNA gene sequence analysis Genomic DNA was extracted using ZR Fungal/Bacterial DNA kit (Zymo Research, USA) according to the manufacturer’s instruction and stored at 20 C until further use. The fungal strains were identified by 18S rRNA sequence analysis. PCR was performed using conserved primer sequence (13) encoding 762 bp 18S rRNA product (0817F 50 -TTAGCATGGAATAATRRAATAGGA-30 and 1536R 50 -ATTGCAATGCYCTATCCCCA-30 ) in a Veriti 96-well Thermocycler (Applied Biosystems, USA) at annealing temperature of 56 C. The product was gel extracted using Qiagen gel elution kit (Hilden, Germany) and ligated using a TOPO cloning kit according to the manufacturer instruction (Invitrogen Corporation) followed by sequencing of the amplified product. BLAST searches were made by using the BLAST, version 2.0, software and the nucleotide sequences were deposited in GenBank (http://www.ncbi.nlm.nhi.gov/BLAST). Phylogenetic tree was constructed using BOOTSTRAP tree method from Clustal X software developed by Thompson et al. (14).
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.09.017
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Aspergillus sp HKF16 JF922008
B
Aspergillus flavipes ATCC1030 AY373849 Aspergillus terreus ATCC1012 AY373871 Aspergillus niger TF1 JN974294 Aspergillus clavatus ATCC58869 AY373847 Aspergillus fumigatus SCSGAF0187 JN851056 Fusarium solani RM2 JN887340 Fusarium chlamydosporum SCSGAF0087 JN851018 Fusarium solani NZFS3555 JN595827 Fusarium oxysporum lycopersici ATCCMYA4623 GU327639 Fusarium sp HKF15 JF922007 Fusarium oxysporum ATCC16600 GQ914766 Penicillium sp HKF11 HM773238 Penicillium sp HKF44 HM773240 Penicillium paxilli SCSGAF0173 JN851050 Penicillium citrinum SCSGAF0167 JN851046 Penicillium tricolor ATCC10413 AY373935 Penicillium janthinellum ATCC4845 AY373921 Penicillium oxalicum SCSGAF0143 JN851042
FIG. 1. Diversity of fungal isolates. (A) Phylogenetic tree is inferred from the comparison of 18S rRNA gene sequences of antimicrobial fungal isolates with reference strains. HKF species are lab isolates. Nucleotide sequences of reference strains were retrieved from GenBank data. The values at nodes represent 1000 bootstrap replicates. (B) RAPD analysis with universal RAPD primers 3 and 6. Lanes 1e5: RAPD primer3; lanes 7e11: RAPD primer6. Samples: lane 1: HKF11; lane 2: HKF15; lane 3: HKF16; lane 4: HKF40; lane 5: HKF44; lane 6: 1 kb ladder; lane 7: HKF11; lane 8: HKF15; lane 9: HKF16; lane 10: HKF40; lane 11: HKF44.
Evaluation of genetic diversity of fungal strains by RAPD analysis Genetic diversity of the fungi was evaluated by RAPD by using commercially available RAPD Analysis Primer 3 e (50 -d[GTAGACCCGT]-30 ) and RAPD Analysis Primer 6 - (50 -d [CCCGTCAGCA]-30 ) from the Amersham RAPD kit (Amersham Pharmacia Biotech, Uppsala, Sweden). Medium and culture conditions Potato dextrose medium was suitable for antimicrobial compound production. However, since it is a complex medium, minimal medium was optimized for the production of antimicrobials, the composition of which is described earlier (15). Glusoce (10%) was added as a carbon source. Preliminary screening against pathogens Fungi were grown for seven days in potato dextrose broth at 30 C at 120 rpm on a shaker and the antibacterial activity of culture supernatant was checked preliminarily against five ATCC pathogens namely Escherichia coli (ATCC25922), Bacillus cereus (ATCC10876), Shigella flexneri (ATCC9199), Salmonella paratyphi (ATCC9150) and Staphylococcus epidermidis (ATCC12228) by agar well diffusion method (16) against 107 cells of host pathogen seeded onto an LB agar plate. The measurement of cell OD was carried out at 600 nm on a spectrophotometer (Thermo Scientific). Ceftazidime resistant Pseudomonas aeruginosa strain, vancomycin resistant Enterococcus strain and methicillin resistant Staphylococcus strain (MRSA) and MDR E. coli strains (E. coli 1 and E. coli 2) resistant to vancomycin, oxacillin, ceftazidime and other MDR strains including Serratia sp. GMX1, resistant to sulfametoxazole, ampicillin, azithromycin, tetracycline and netilmycin and Enterobacter resistant to sulfametoxazole, ampicillin, azithromycin and tetracycline were used. Extraction of antimicrobial compound from HKF15 As HKF15 was found to be the most promising potential producer of antibacterial agent, the further studies were focused on it. The fungal strain was cultivated in minimal medium with 10% glucose whose composition is as described earlier. The fungus was grown for 7 days. Incubation was carried out on a rotator shaker at 120 rpm and 30 C. The culture supernatant was extracted with equal volume of ethyl acetate twice, for 3 h each. For this, the supernatant was mixed with equal volume of ethyl acetate and kept on magnetic stirrer for 3 h. It was then allowed to settle. The upper layer of ethyl acetate was separated and extraction process was repeated. The extract was concentrated on rotator evaporator (Buchi type, Sonar) at 40 C to get a solid residue. This residue was suspended in methanol/DMSO and the antimicrobial activity was confirmed. Purification and characterization The 80 mg concentrated crude extract was subjected to HPLC (Perkin Elmer). HPLC separation was carried out on a Gilson 322/215 system equipped with dual wavelength detector using a Water’s Xterra RPC18 50 19 mm column fitted with a 10 mm guard. The HPLC gradient was as in Table S1. Mobile phases were used with 0.1% formic acid added. The crude ethyl acetate extract of HKF15 was dissolved in DMSO and separated by preparative HPLC by injecting 1.5 ml sample; and 70 fractions were collected into custom trays holding Genevac evaporator racks. A flow rate of 24 ml/min was used. Fractions were collected every 0.5 min starting from the solvent front of 0.65 min
into 100 16 mm tubes until 35.5 min. After HPLC fractionation, aliquots from test tubes were added into 39 wells of micro-titer plate according to supplementary Table S2 and Fig. S1. Well 40 was control and contained crude extract. Six active samples were obtained from these wells and only the active D2 sample (17e17.5 min) was characterized by HR-ESI MS (Bruker micro QTOF) and 1 H NMR (Bruker 500 MHz). The QTOF-MS was performed in both positive and negative mode. Thin layer chromatography for partial purification Silica gel (40%) and 8% calcium sulfate was mixed and slurry (50 ml) was poured on thin glass plate (12 cm12 cm). HKF15 crude ethyl acetate extract (5 ml) was spotted thrice. Mobile phase used was chloroform, methanol, acetic acid 94:1:5. Antibacterial activity Bikaverin was analyzed for its antibacterial activity against E. coli ATCC 25922. E. coli was grown overnight in LB and transferred to 1X minimal medium with 10% glucose. The culture was grown overnight at 37 C at 120 rpm. The inoculum of approximately 108 cells was used per 200 ml of fresh medium in micro-titer plate (Corning clear plate with U bottom) in duplicates. The antibacterial activity experiment was done in triplicates and the results show the data points with error bars showing standard deviation from mean of the values. Bikaverin was added in concentrations 20, 40, 60, 80 and 100 ppm each. Suitable control was maintained. The OD was read after every 30 min on Victor X3 multiplate reader set at 37 C for 5 h. The 100% pure bikaverin standard was kindly provided by Dr. Hasumi (The University of Tokyo) (17). The validation of above experiment was done by colony counting. E. coli was grown in LB at 37 C and 120 rpm overnight and inoculated in minimal medium with 10% glucose. This was grown overnight and used as an initial inoculum. The bikaverin concentrations of 10 ppm, 25 ppm and 50 ppm were added to 109 E. coli cells ml1 and the culture was grown at 37 C and 120 rpm. Samples were taken out
TABLE 1. Bioassay results for HKF15 ethyl acetate extract in comparison with standard antibiotics. Organism Pseudomonas aeruginosa Staphylococcus aureus Methicillin resistant Staphylococcus aureus Vancomycin resistant Enterococcus Aspergillus
Diameter of zone in mm for standards
Diameter of zone in mm for HKF15 extract
7 Hazy 8 Clear 8 Clear
7 Hazy 12 Clear 11 Clear
7 Hazy
Nil
9 Hazy
8 Hazy
Standard antibiotics: Ceftazidime for P. aeruginosa, oxacillin for S. aureus and methicillin resistant S. aureus, vancomycin for vancomycin resistant Enterococcus.
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FIG. 2. HPLC and bioactivity guided HPLC fraction analysis. (A) Chromatogram for HKF15 crude extract in ethyl acetate. Mobile phase A: water mobile phase B: acetonitrile. Collection started at 0.65 min, stopped at 35.5 min, flow rate: 24 mL min1, run time: 40 min. Dark line represents 195 nm and faint line represents 254 nm. (B) Bioactivity guided HPLC fraction analysis. The HPLC fractions of crude HKF15 extract were checked against drug resistant and pathogenic bacteria. The fractions were first pooled in micro-titer plate and 40 samples were obtained. Graph shows activity of samples from well no. 15 onwards, no activity was detected in previous samples. Open diamond, P. aeruginosa; filled squares, S. aureus; filled triangles, MRSA; open circles, VRE; crosses, E. coli MDR1; filled circles, E. coli MDR2.
periodically and were analyzed for colonies on LB plates. In a parallel experiment, the different cell concentrations treated with the selected bikaverin concentration was studied.
RESULTS AND DISCUSSION Isolation of fungi From ETPs 35 fungal isolates were obtained and it was seen that it is a suitable niche for fungi. The isolated fungi exhibited rapid growth on PDA medium and it was found to be a favorable medium for culturing and storage of fungi from ETPs. Mycological agar, malt yeast extract agar are some other media for the isolation of fungi. Potato dextrose agar is widely used amongst them (18,19). Microscopic observations helped in identification of fungi which was validated by 18S rRNA gene sequencing. Identification by 18S rRNA gene sequence analysis and diversity study by RAPD analysis Amplification and sequencing of the 18S region of rRNA gene for each fungus resulted in 762 bp product, whose sequence has been deposited in NCBI database with accession numbers as, Penicillium sp.: HKF11
(HM773238) and HKF44 (HM773240), Aspergillus sp.: HKF16 (JF922008) and Fusarium sp.: HKF15 (JF922007). Fig. 1A is the phylogenetic tree showing the 18S diversity. RAPD analysis is another important tool to assess genetic diversity (20) which was used. Fig. 1B shows that RAPD primer 3 gave different amplification patterns for all fungi except HKF11 and HKF44. RAPD primer 6 gave different amplification patterns for all fungi except HKF11 and HKF40 where there was similarity in band patterns. So, the RAPD analysis demonstrated the diversity of fungi. Preliminary screening against pathogens Agar diffusion method provides a simple and effective way for rapid screening of antimicrobial activity (21). Activity was observed under nutrient limiting conditions (22). Four fungieHKF11, HKF15, HKF16, and HKF44 showed effective antibacterial activity against ATCC pathogens (Table S3). HKF15 was found to be active against the methicillin resistant Staphylococcus aureus, Vancomycin resistant Enterococcus, Pseudomonas aeruginosa and MDR bacteria (Enterococcus and Serratia sp.) (Fig. S2, Table 1). Though linezolid and vancomycin are used to treat MRSA infections, they have
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FIG. 3. Growth curve showing antibacterial activity of different concentrations of bikaverin against E. coli ATCC 25922. OD was measured at 600 nm. Open circles, culture control; diamonds, 20 ppm; open squares, 40 ppm; filled squares, 60 ppm; open triangles, 80 ppm; filled circles, 100 ppm bikaverin; filled triangles, solvent control. The error bars represent standard deviation from mean values of samples.
some side-effects (23) and new molecules are being searched as alternatives for multi-drug resistant organism like MRSA. Extraction and purification of compound The HPLC fractionation gave 6 active samples (Fig. 2A) from the pooled fractions in micro-titer plate. These samples were obtained based on their activity against pathogens. But each sample showed different activity pattern (Fig. 2B). The most active sample was D2 as it showed activity against most of the bacteria used for screening. Sample D2 was subjected to QTOF-MS in both positive and negative mode and 1H NMR analysis. The major compound characterized was bikaverin. The structure and mass was confirmed by HR QTOF-MS and 1H NMR (Fig. S3). It is a red colored pigment. Fig. S4 shows the structure of bikaverin. Bikaverin was first isolated from Fusarium oxysporum sp vasinfectum. It was called lycopersin because of its red color (24). Passiflorine isolated from F. oxysporum sp. passiflorae and mycogonin from Mycogone jaapii are the alternative reports for the same compound (25). A polyketide synthase gene bik1 is responsible for its synthesis (26). The bik gene cluster includes biosynthesis genes bik2 and bik3, regulatory genes bik4 and bik5, and transporter bik6 (27,28). TLC purification and LCMS analysis of purified fraction A purple spot was observed on TLC plate with Rf value: 0.4521 (29). The spot was eluted in ethyl acetate (27) and was compared with standard bikaverin by LCMS as described by Busman et al. (30). Bikaverin was detected in the TLC purified fraction which also showed antibacterial activity. This was indicative of antibacterial potential of bikaverin. Antibacterial activity We observed that bikaverin inhibited E. coli growth significantly in the growth curve study in a microtiter plate reader (Fig. 3). It is the standard method used mostly to determine minimum inhibitory concentration (31). Colony count indicated that 25 ppm concentration was able to inhibit bacterial growth slowly (Table 2). When different cell concentrations were treated with 25 ppm bikaverin, it was observed that after 6 h, the CFU count was reduced to zero (Table 3). While in case of 50 ppm bikaverin, there was a significant inhibition of growth after 1 h. This demonstrated that apart from the other bioactivities (32),
bikaverin also has an antibacterial potential. Repurposing of drugs is a recent area to fulfill the need for new efficient antibiotics where a known compound is exploited for a novel activity. Auranofin therapy against Entamoeba histolytica infection (33), novel activity of 20 FDA approved compounds against drugresistant Acinetobacter baumannii (34) are some examples. The fungal contamination associated with eating of agricultural products contaminated with fungal secondary metabolites like fumonisin has been reported (35); but bikaverin-contaminated products are not reported for any negative impact on human health. It is proven non-genotoxic according to an unscheduled DNA synthesis assay (36). It has some biological effects, but those may be desired ones either as an antibiotic or an antitumor agent rather than deteriorating ones. The antibiotic properties of bikaverin have promising commercial applications in the control of some pathogenic protozoa and fungi or in cancer treatment (32). The screening of such known antibiotics across different targets will surely help understand the biological mechanisms of small antibiotic molecule interactions in a better way, which will be helpful for drug development programs. Most antibiotics have originally come from a small set of molecular scaffolds which are modified by synthetic tailoring for better functionality. The emergence of multidrug resistance suggests that the discovery of new scaffolds should be a priority (37). And, approaches towards scaffold discovery include mining under explored microbial niches for natural products and repurposing synthetic molecule libraries for use as antibiotics (38). With a
TABLE 2. Effect of bikaverin concentrations on E. coli growth with initial inoculum of 109 cells ml1. SN
Colony count in CFU ml1
Time (h) Control
1 2 3 4 5
0 1 2 3 6
3.01 5.04 4.75 2.07 1.55
ND: Not done for zero time.
10 ppm 9
10 109 109 109 109
ND 4.98 4.99 2.14 1.67
109 109 109 109
25 ppm ND 1.03 1.48 0.64 0.36
109 109 109 109
50 ppm ND 0.05 109 0.03 109 0 0
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TABLE 3. Effect of 25 ppm bikaverin on E. coli growth with decreasing inoculum size starting from 106 cells ml1. SN
Colony count in CFU ml1
Time (h) Control
1 2 3
1 3 6
1
6
10 cells ml 6
2.3 10 2.3 106 2.4 106
5
4 10 2 105 0
1
5
10 cells ml 3
1 10 1 103 0
10 cells ml1
103 cells ml1
500 cells ml1
200 cells ml1
100 cells ml1
200 1 0
40 30 0
20 0 0
10 0 0
0 0 0
4
plethora of information on bioactivities of small molecules available through databases like PubChem, it is now a challenging task to mine the actual bioactive potential of a molecule for drug development research. Bioinformatics and chemoinformatics analyses are being used to study the across target activity of bioactive molecules (39,40). Such kinds of surveys might provide insights into a compound’s bioactivity against previously undiscovered target. ETPs provide unique habitat where different microbes survive together and compete for the easily available carbon source under diverse organic loadings. The scenario generates microbial communities which use intermediate generated by other species and provide novel condition for secondary metabolite production. These acquired pathways give molecules that provides defense against other microbes. This study explores such activity associated with a fungal isolate from an ETP, HKF15 towards pathogenic and drug resistant strains. It produces bikaverin that has been reported to be an antifungal molecule. However, this study demonstrates an additional property of antibacterial activity of bikaverin. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2013.09.017. ACKNOWLEDGMENTS We thank Naoko Nishimura and Dr. Keiji Hasumi (Tokyo University of Agriculture and Technology, Tokyo) for providing bikaverin standard; Dr. Saji George, Dr. Arun Balakrishnan (PEL, Mumbai) and Mr. G.S. Kanade (CSIR-NEERI). We acknowledge the financial support from Council of Scientific and Industrial Research (CSIR), Delhi, for one of the authors (Radhika Deshmukh) through CSIR, Senior Research Fellowship. The authors have no conflicts of interest to declare. References 1. Haug, B. E., Stensen, W., Kalaaji, M., Rekdal, O., and Svendsen, J. S.: Synthetic antimicrobial peptidomimetics with therapeutic potential, J. Med. Chem., 51, 4306e4314 (2008). 2. Ekins, S., Williams, A. J., Krasowski, M. D., and Freundlich, J. S.: In silico repositioning of approved drugs for rare and neglected diseases, Drug Discov. Today, 16, 298e310 (2011). 3. Pieters, R. J., Arnuscha, C. J., and Breukinkb, E.: Membrane permeabilization by multivalent anti-microbial peptides, Protein Pept. Lett., 16, 1e7 (2009). 4. Guo, B., Wang, Y., Sun, X., and Tang, K.: Bioactive natural products from endophytes: a review, Appl. Biochem. Microbiol., 44, 136e142 (2008). 5. Onifade, A. K.: Research trends: bioactive metabolites of fungal origin, Res. J. Biol. Sci., 2, 81e84 (2007). 6. Talontsi, F. M., Dittrich, B., Schüffler, A., Sun, H., and Laatsch, H.: Epicoccolides: antimicrobial and antifungal polyketides from an endophytic fungus Epicoccum sp. associated with Theobroma cacao, Eur. J. Org. Chem., 15, 3174e3180 (2013). 7. Mancini, I., Defant, A., and Guella, G.: Recent synthesis of marine natural products with antibacterial activities, Anti-Infect. Agents Med. Chem., 6, 17e48 (2007). 8. Whelan, A. P., Dietrich, L. E. P., and Newman, D. K.: Rethinking ‘secondary’ metabolism: physiological roles for phenazine antibiotics, Nat. Chem. Biol., 2, 71e78 (2006). 9. Pettit, R. K.: Culturability and secondary metabolite diversity of extreme microbes: expanding contribution of deep sea and deep-sea vent microbes to natural product discovery, Mar. Biotechnol., 13, 1e11 (2011).
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