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Anti-Infective Metabolites of a Newly Isolated Bacillus thuringiensis KL1 Associated with Kalmegh (Andrographis paniculata Nees.), a Traditional Medicinal Herb

Anti-Infective Metabolites of a Newly Isolated Bacillus thuringiensis KL1 Associated with Kalmegh... Journal name: Microbiology Insights Journal type: Original Research Year: 2016 Volume: 9 Anti-Infective Metabolites of a Newly Isolated Running head verso: Roy et al Running head recto: Anti-infective metabolites of a newly isolate b d acillus thuringiensis Bacillus thuringiensis KL1 Associated with Kalmegh (Andrographis paniculata Nees.), a Traditional Medicinal Herb 1,2 1 1 1 s udipta r oy , s ahana y asmin , subhadeep g hosh , s omesankar b hattacharya and Debdulal banerjee Department of Botany, Microbiology and Microbial Biotechnology Laboratory, Vidyasagar University, Midnapore, West Bengal, India. PG Department of Biotechnology, Oriental Institute of Science and Technology, Midnapore, West Bengal, India. A BSTR AC T: This study was conducted to isolate endophytic bacteria possessing anti-infective property from Kalmegh ( Andrographis paniculata Nees.), a well-known medicinal plant. A total of 23 strains were isolated from this plant among which the strain KL1, isolated from surface-sterilized leaf of this medicinal herb, showed broad-spectrum antagonism against an array of Gram-positive and -negative bacterial pathogens. Ethyl acetate extract of KL1- fermented media yielded a greenish amorphous substance retaining anti-infective property. Solvent-extracted crude material was separated by thin-layer chromatography, and the active ingredient was located by autobiogram analysis. The purified anti-infective compound was found as anthracene derivative as analyzed by ultraviolet and Fourier transform infrared spectroscopy. The strain was identified as Bacillus thuringiensis KL1 from cultural, physiochemical, and molecular aspects. The above results indicate the pharmaceutical potential of the candidate isolate. K E Y WOR DS: Andrographis paniculata, endophyte, Bacillus thuringiensis, antimicrobial, anthracene, pharmaceutical CITATION: r oy et al. Anti-i nfective Metabolites of a n ewly i solated Bacillus thuringiensis COPYRIGHT: © the authors, publisher and licensee l ibertas Academica l imited. t his is an open-access article distributed under the terms of the c reative c ommons Kl 1 Associated with Kalmegh (Andrographis paniculata n ees.), a t raditional Medicinal cc -by -nc 3.0 l icense. h erb. Microbiology Insights 2016:9 1–7 doi:10.4137/Mbi .s 33394. CORRESPONDENCE: debu33@gmail.com; db@mail.vidyasagar.ac.in TYPE: o riginal r esearch Paper subject to independent expert single-blind peer review. All editorial decisions RECEIVED: n ovember 30, 2015. RESUBMITTED: February 8, 2016. ACCEPTED FOR made by independent academic editor. Upon submission manuscript was subject to PUBLICATION: February 10, 2016. anti-plagiarism scanning. Prior to publication all authors have given signed confirmation ACADEMIC EDITOR: r aúl r ivas, Editor in c hief of agreement to article publication and compliance with all applicable ethical and legal requirements, including the accuracy of author and contributor information, disclosure of PEER REVIEW: t hree peer reviewers contributed to the peer review report. competing interests and funding sources, compliance with ethical requirements relating to Reviewers’ reports totaled 613 words, excluding any confidential comments to the human and animal study participants, and compliance with any copyright requirements of academic editor. third parties. t his journal is a member of the c ommittee on Publication Ethics (co PE). FUNDING: Authors disclose no funding sources. Provenance: the authors were invited to submit this paper. COMPETING INTERESTS: Authors disclose no potential conflicts of interest. Published by l ibertas Academica. l earn more about this journal. microorganisms are also considered as a reservoir of bio active Introduction compounds produced as secondary metabolites. Presently, From the very beginning, pharmaceutical research is pharmaceutical exploration of microbial origin is aimed at signicfi antly dependent on traditional health-care practices endophytic microorganisms. Endophytic microbes are yet with medicinal plants. Application of plants for medicinal less-explored population and presently gaining significant purposes in India has been recognized in ancient literature. However, systemized studies on traditional medicinal plants, field in medicinal research for mankind. They reside symp - especially herbs, were noticed in 1956 and soon achieved recog - tomless within the plant’s internal tissues. Some of them act nition due to loss of traditional knowledge and declining plant synergistically by producing secondary metabolites antagon- is population. At least 7,000 medicinal compounds in the mod - tic to plant pathogens. Both fungi and bacteria are common ern pharmacopoeia are of plant origin. Among the 120 plant- endophytes, and many new antimicrobial compounds have derived bioactive compounds widely used in modern medicine, been already discovered from such class of microorganisms. 80% exhibit a positive correlation between their modern thera- In most cases, the antimicrobial compounds produced by peutic use and the traditional use of source plants. Androgra- such groups of microbes are relatively less toxic as the plant phis paniculata Nees, belonging to Acanthaceae family, is a herb itself serves as a natural selection system M . any bioactive traditionally used as a medicine in ancient oriental, including substances that an endophyte produces were found relatively Indian Ayurveda. Phytochemical exploration revealed diverse new to us. Therefore, there is a huge potential to screen novel, pharmacoactive compounds, including labdane diterpenoid highly active, and low-toxicity antimicrobial compounds from lactones, flavonoids, and others. The leaf extract of this plant endophytic microorganisms. A large number of endophytic is very well known as a therapy for infectious diseases, fever- bacteria have already been isolated possessing antibacte - 5 9 causing diseases, diarrhea colic pain, and loss of appetite. rial or antifungal activity. Endophytic microorganisms also New classes of bioactive compounds are ever demanding, have great contribution in the production of antidiabetic, 7 11 12 and we know that the probability of occurrence of such com- anticancerous, anti-insecticidal, antiviral, and even immu- pounds is higher in any unexplored source. Apart from plants, nosuppressive compounds. Microbiology i nsights 2016:9 1 Roy et al This study was aimed to isolate endophytic bacteria from (Vega© TESCAN) after gold coating. Colony and cellular A. paniculata Nees. and evaluate their anti-infective property details were recorded, and various extracellular enzyme against some bacterial pathogens. One of the isolated strains productions were analyzed for the strain. Sugar utilization identified as Bacillus thuringiensis KL1 from 16s rRNA gene patterns were also determined. The endophytic strain was homology and other characteristics showed potential anti- cultured in TYG medium at pH 7 and 30°C incubation infectivity. Three bioactive metabolites were isolated from this temperature with a shaking speed of 150  rpm to study its endophytic bacterium and one of them was characterized after growth pattern. The growth pattern in batch fashion was spectral analysis. determined after plotting the optical densit y of the cell su - s pension against time. Materials and Methods Genomic DNA isolation and 16s rDNA amplifica - Sampling and endophyte isolation. A. paniculata is tion. The strain was grown in Luria Bertini for 24 hours and native to peninsular India. This herb grows as a bush and centrifuged for 10 minutes at 10,000  rpm. Cell pellets were is a dominant species in the district of Paschim Medinipur, washed with phosphate buffer (0.1  M, pH 7.2) and resus - West Bengal, India. Healthy twigs were collected from d-if pended in 500  µ L TE buffer (25  mM Tris-HCl [pH 8.0], ferent localities of Paschim Medinipur (22.57N–87.11E) 25  mM ethylenediaminetetraacetic acid). After that, it was and stored in refrigerator till isolation of endophytes. Leaves successively treated with lysozyme and sodium dodecyl su -l were separated from twigs and washed under gentle tap fate (10%) for proper cell lysis. DNA was extracted with water. These parts of the plant were aseptically surface steril - phenol–chloroform reagent, and aqueous layers were sep-a ized following standard method. Samples were placed on rated carefully. Genomic DNA was precipitated with isopro - ISP2 agar media supplemented with actidione (50 µ g/mL) panol and washed with ice-cold 70% ethanol. It was dissolved and incubated at 30°C for five days. Endophytic bacteria, in 100  µ L TE buffer (pH 8) and directly used as template which came out from surface-sterilized part of the leaves, for 16s rDNA amplification. For this purpose, the primers were immediately pure cultured and preserved with 30% 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R glycerol at - 20°C. (5′-GGTTACCTTGTTACGACTT-3′) were used. Reac- Evaluation of anti-infectivity. Isolated endophytic tions were performed in a polymerase chain reaction (PCR) strains were individually grown in tryptone yeast extract gl-u machine (Mastercycler® nexus, Eppendorf). Reaction cose liquid media [bacto tryptone, 0.5%; yeast extract, 0.2%; parameters were as follows: initial denaturation at 9°4C for glucose, 1%; K HPO , 0.1%, and MgSO 7H O, 0.05%], pH 5 minutes followed by 30 cycles of denaturation at 9°5C for 2 4 4 2 7.2 for three days at 30°C in a shaker incubator (150 rpm). 60 seconds, annealing at 55°C for 60 seconds, and primer Cultures were taken in a centrifuge tube, and cells were pe -l extension at 72°C for 90 seconds. A final extension was done leted by centrifugation at 6,000 g for eight minutes. Cell-free at 72°C for 10 minutes. PCR products were purified with liquid media were evaluated for pathogen growth inhibition Hi-PurA™ PCR product purification spin kit (Himedia). by agar well diffusion method. Six pathogens available in Forward and reverse DNA-sequencing reactions of purified the laboratory were grown overnight in tryptic soy broth. PCR amplicon was carried out with the same primers using Pathogens included Bacillus subtilis (ATCC 11774), Bacillus BigDye Terminator v3.1 Cycle Sequencing Kit on ABI 3730xl cereus (ATCC 14579), Vibrio parahaemolyticus (ATCC 1782), Genetic Analyzer. Aeromonas caviae (ATCC 15468), Proteus vulgaris (ATCC Phylogeny construction. Consensus sequence of 16s 12453), and Pseudomonas aeruginosa (ATCC 9027). Each pre- rDNA was generated from forward and reverse sequence data grown pathogenic strain was seeded on Müller-Hinton agar using aligner software. The sequence was used to carry out (MHA) media separately, and culture filtrates were applied BLAST with the “nr” database of NCBI GenBank. Based on in the wells on the MHA plates. The MHA plates were the maximum identity, sequences were selected and aligned incubated for another 24 hours, and the zone of inhibition using the multiple alignment software program, ClustalW. produced was recorded. The phylogenetic tree was constructed using MEGA5. The Characterization of potential strain. Endophytic evolutionary history was inferred using the neighbor-joining isolated strain KL1 was characterized from morpholog-i method. The evolutionary distances were computed using cal and biochemical aspects. The strain was obser ved under the Kimura’s two-parameter method. compound and scanning electron microscope. Bacterial Product ion a nd pu r if icat ion of a nt i-infect ive metab- smear (18 hours grown) was prepared on poly-l-lysine- olites. Production of active compound by strain KL1 was coated glass slide and fixed with 0.25% glutaraldehyde carried out in liquid TYG media at 30°C for three days (prepared in Na-phosphate, pH 7.2). Cells were then under shaking conditions (150  rpm). A total of 1.5  L cul - dehydrated for 15 minutes with different grades of etha - ture media was centrifuged (10,000  rpm—15 minutes) to nol (30%–100%) and dried up to the critical point after separate cell mass. Culture filtrate was then extracted t wice which slides were attached to the stub. The materials were with an equal volume of ethyl acetate (Merck—analytical used for imaging through a scanning electron microscope grade). Organic phase was separated and made water free 2 Microbiology i nsights 2016:9 Anti-infective metabolites of a newly isolated bacillus thuringiensis by adding anhydrous Na SO . It was brought to dryness antimicrobial agent and without inocula, respectively. The 2 4 with in vacuo evaporation (rotary evaporator; HS-2005S, IC value was determined by plotting OD values of test Hanshin). The crude metabolites were dissolved in a mini - samples against the concentration of the active compound mum amount of methanol and subjected to activity-guided administered. The activity of purified substance was also thin-layer chromatography (TLC) for purification of an determined against the same pathogen by disk diffusion anti-infective compound. About 15 µ L crude materials was assay, where 25  µ L methanolic solutions were soaked in loaded on silica-coated alumina TLC plate (Silica gel 60 sterile disks and placed on individual plates seeded with F , Merck), and separation was done with solvent mix- pathogens separately. ture (chloroform–methanol; 10:1). Separated compounds Spectroscopic characterization of purified active were detected upon ultraviolet (UV) rays (longer wave compound. The purified active compound produced by length), and R values were determined. Bioautogram was endophytic bacterium KL1 was dissolved in chloroform, and studied by overlaying the TLC plates with molten MHA absorption spectrum was recorded with ultraviolet-visible 5 - 1 media (45°C) containing B. cereus (50 µ L of 10  cfu mL ). (UV-Vis) spectrophotometer (UV-1800; Shimadzu) from The TLC plates were kept inside a sterile Petri plate under 200 to 600  nm with respect to chloroform as control. The moist conditions and incubated at 37°C for 24 hours. Cell active compound was also analyzed for its spectrum in infr- a viabilit y was checked by spraying methylthiazoltetrazolium red (IR) region. Dried samples were mixed well with KBr and (5  mg/mL). The position of active compound was located sample—KBr pellet was analyzed from 600 to 4,000 nm in by comparing pathogen inhibition zone and band position Fourier transform infrared spectrophotometer (Spectrum T; on TLC plates. The most active compound was scrapped PerkinElmer). from TLC plates, dissolved in methanol, and  ∼5  µ L was subjected for further separation with a different solvent Results mixture (chloroform–methanol; 10:0.5). Separation was Endophytic bacteria appeared within a 48-hour incubation of detected by exposition of TLC plates on UV, and active surface-sterilized leaves of Andrographis on the isolation media. fraction was located by the same bioautogram study. R A total of 23 endophytic bacteria were isolated in this study, value was determined for the active compound and consid- and most of the strains were found to inhibit at least one of the ered spectral characterization. test pathogens. However, the rational of selecting strain KL1 Determination of IC of active compound. It is an was due to its broad-spectrum anti-infective property. It inhib- essential parameter to determine the effective concentra- ited all of the test pathogenic bacteria except Pseudomonas tion of any purif ied antimicrobial agent. Slightly modif ied (Table 1). This strain was our choice of interest due to its anti- microbroth dilution assays were employed to determine infectivity against both Gram-positive and -negative bacteria. the IC value of the most active purified metabolite pro - The strain was found to be Gram-positive, rod-shaped duced by endophytic strain KL1. For this purpose, one (Fig.  1), and with endospore. It produced characteristics of Gram-positive strain B. cereus and one Gram-negative various extracellular enzymes. Sugar utilization patterns and strain P. vugaris were selected. Stock solution was pre - other detailed characterization parameters are summarized in pared by dissolving 17  mg (final TLC purified) of active Table 2. The strain utilized most of test sugars though could metabolite in 5  mL of methanol (high-performance li-q not utilize fructose, xylose, and raffinose at all. Genomic DNA uid chromatography grade). It was then serially diluted was isolated from 24-hour grown culture of KL1 from which (200:1–10:1) in Müller-Hinton broth (1  mL) and inocu - 16s rDNA (∼1.5 kb) was amplified. Approximately 900 bases lated separately with test pathogens (25 µ L of OD  0.6). of the amplified product were sequenced, and homology was Positive and negative controls were prepared without an determined in NCBI database. The constructed phylogenetic Table 1. Antimicrobial activities of strain Kl 1 against several bacterial pathogens. PATHOGENS ZONE OF INHIBITION* (mm) ZONE OF INHIBITION* (mm) IC (µ g/mL) BY KL1 CULTURE FILTRATE OF PURIFIED SUBSTANCE Bacillus subtilis (Atcc 11774) 119.00 12.5 ± 0.311 27.3 ± 0.033 Bacillus cereus (Atcc 14579) 12 ± 0.613 nt nt Vibrio parahaemolyticus Atcc 1782 nt nt 10.5 ± 0.133 Aeromonas caviae (Atcc 15468) 10 ± 0.235 nt nt Proteus vulgaris (Atcc 12453) 11.5 ± 0.073 24.6 ± 0.133 153.00 Pseudomonas aeruginosa (Atcc 9027) – nt nt Notes: *Values are represented as mean ± standard deviation of triplicate experiments. Abbreviations: nt , not tested; –, no inhibition. Microbiology i nsights 2016:9 3 Roy et al Figure 1. Endophytic strain B. thuringiensis Kl1; ( A) scanning electron microscopy, (B) optical microscopy (100×), and (C) position of the strain in phylogenetic tree showing similarities with related strains. - 1 tree revealed that the strain showed close sequence similarity was found with (KBr) cm : 2,924, 2,854, 1,634–1,632, 1,592, (99% similarity with 100% query coverage and E value 0) with 1,508–1,494, 1,372, and 758 (Fig. 5). These spectroscopic data different strains of B. thuringiensis (Fig. 1). The isolated endo - are characteristic features of different structural properties phtyic strain KL1 was proposed to be identified as B. thuringi- and indicate the presence of a few functional groups within ensis KL1. It showed a regular growth fashion in batch culture the active compound. Antimicrobial activity determination (Fig. 2) with ∼5 hours as lag phase in TYG medium and other revealed that the purified substance was active against both mentioned growth conditions though it reached the maximum types of microorganisms with varying IC values. It was cell density at 22 hours. found that the IC values are 119.0 and 153.0 µ g/mL, respec- The solvent-extracted crude metabolites appeared as tively, against B. cereus and P. vulgaris (Table 1). greenish amorphous substances with a sharp smell. This mixture was separated through TLC, and several fractions Discussion were obtained among which three compounds were detected The large number of secondary metabolites so far isolated (R —0.813, 0.480, and 0.053 in chlororform–methanol; 10:1) from microbial cultures and plant extracts have no doubt to inhibit the growth of B. subtilis (Fig. 3A). The compound significant industrial values. Like many other microbial with an R value of 0.813 was found as the major anti-infective species, Bacillus also produces many antibacterial second - compound with the highest activity. However, a further puri- ary metabolites. Bacillus, traditionally isolated from the fication of this compound indicated that the compound with soil, is renowned for the production of various antibiotics an R value of 0.784 (chlororform–methanol; 10:0.5) was the such as bacitracin, polymyxin, zwittermicin A, and several most active anti-infective compound produced by endophytic others. Unfortunately, reisolation of the same microorgan - B. thuringiensis KL1 (Fig. 3B). The pure active material was ism from the same source and the throttled success of com- found to absorb UV at 259 and 371 nm (Fig. 4), while IR binatorial chemistry in providing new compounds indicate max max 4 Microbiology i nsights 2016:9 Anti-infective metabolites of a newly isolated bacillus thuringiensis Table 2. c ultural and physiological and biochemical characteristics reported for endophytic origin such as Azorhizobium, Bacillus, of endophytic B. thuringiensis Kl1. Bradyrhizobium, Gluconacetobacter, Klebsiella, Burkholderia, Enterobacter, Pseudomonas, and Streptomyces. B. thuringiensis CHARACTERIZATION APPEARANCE is an aerobic, Gram-positive endospore-forming bacterium PARAMETERS and well known for crystalline inclusions o δr -endotoxins. c olony morphology White to off white color, slightly raised elevation and regular margin These proteins are selectively insecticidal, but the strain also c ell morphology r od shaped, slightly swollen cen- secretes zwittermicin A, a linear aminopolyol antibiotic effec- trally, 1.5–1.8 µ m length, 0.25 µ m tive against Gram-negative bacteria mostly. A number of diameter lipopeptides with anti-infectivity were also purified from g ram character Positive different species of Bacillus. A fengycin-like substance was Endospore Positive purified from a soil-dwelling B. thuringiensis CMB26, which Extracellular enzymes exhibited fungicidal, bactericidal, and insecticidal activities. c atalase + Present findings report the isolation of the endophytic isolate Amylase + KL1, which is identified as B. thuringiensis KL1. The strain c ellulase + was isolated on ISP2 agar media from surface-sterilized leaves l ipase + of A. paniculata Nees. ISP2 agar media is an effective isola - c aseinase - tion media for endophytic bacteria as it contains minimum g elatinase + nutritional requirements for growth. Endophytes are able to emerge from plant parts and grow using plant extracts and Urease - nutrients provided by media, whereas other microorganisms Phenyl alanine hydrolase + are unable to access plant extracts and their growth is not sup - sugar utilization ported so much. Sugar utilization pattern of this strain indi- Dextrose + cates that it can use some polysaccharides such as cellulose Fructose - and starch as carbon source, which are very usual in plants. g alactose + Besides, its utilization of disaccharides and few monosac- r hamnose + charides explores its wide carbon utilization property that Xylose - might help it to grow in the specialized microenvironment. Raffinose - Presence of different extracellular enzymes also helps such Maltose + group of microbes to exist in such an association as was found s ucrose in this endophytic strain. A report states that its leaves contain 3 4 - 1 lactose + about 10 –10  cfu mL when 1 g of surface-sterilized leaves s tarch were crushed in 10 mL distilled water and subjected for iso - lation of endophytic bacteria, though this study reports of c ellulose + 23 bacteria l isolates from the same. Interestingly B , . thuringiensis var israelensis (Bti), a nonendophytic isolate, and ethano - lic extracts of A. paniculata were found for their larvicidal as uncertainty in future antimicrobial therapy. Thus, finding well as pupicidal activity againsA t nopheles stephensi Liston in new groups of microbes from unexplored or less-explored a synergistic fashion. Here, we report that the endophytic habitats became mandatory by pursuing them as sources of strain B. thuringiensis KL1 was able to inhibit both Gram- novel bioactive compounds. Different bacteria are already positive and Gram-negative pathogens employed though it could not inhibit Pseudomonas at all. TLC along with bioautogram analysis revealed that the compound is slightly nonpolar in nature and the strain produced at least three different compounds that were inhibitory to test pathogen. Spectroscopic characterization of most active compounds showed strong UV absorption at 259 and 361  nm. UV at max 259  nm suggests the presence of conjugated diene in ben- zene ring, while at 361  nm sharply, it indicates the anthr- a cene nature of the active compound. IR absorbance values at - 1 2,854 and 2,924 cm specify some alkane stretch, whereas the presence of monosubstituted alkene was indicated by 1,634– - 1 1,632  cm . –COOH attached with the aromatic ring was - 1 indicated at 1,592 cm , and the presence of aromatic ring was - 1 Figure 2. g rowth pattern of strain Kl 1 in batch culture. confirmed by IR absorption at 1,508–1,494  cm . The active Microbiology i nsights 2016:9 5 Roy et al Figure 3. Activity-guided TLC-based purification of the most active anti-infective metabolite produced by KL1; ( B, C) represent thin layer chromatogram in which (B) indicates three active metabolites and (C) represents the most active band indicated inside the oval shape. (A, D) Demonstrate bioautogram analysis indicating activity among the separated compounds. compound consisted –NO as the functional group that was effectivity to both Gram-positive and -negative pathogens - 1 suggested by absorption at 1,372  cm . Therefore, the spec - indicating the probability of similar inhibition mechanisms. troscopic characterization recommends the active compound However, this compound does not bear any similarity with as anthracene derivative consisting poly alkane or alkene, either compound isolated from B. thuringiensis previously 30,31 where phenyl group may be substituted with COOH and NO reported. Internal plant tissue is a specialized ecosystem groups. Anthracenes are reported to inhibit at different steps in that may be responsible of such an alteration in gene sequence 34 35 DNA/RNA biosynthesis, which is considered a potent tar- during coevolution. Here is the opportunity of getting vari- get site for antibiotics. The active principal here shows marked ations in metabolites from similar microorganisms isolated Figure 5. ir spectrum of the most active anti-infective metabolite of Figure 4. 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Anti-Infective Metabolites of a Newly Isolated Bacillus thuringiensis KL1 Associated with Kalmegh (Andrographis paniculata Nees.), a Traditional Medicinal Herb

Microbiology Insights , Volume 9 – Mar 13, 2016

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Abstract

Journal name: Microbiology Insights Journal type: Original Research Year: 2016 Volume: 9 Anti-Infective Metabolites of a Newly Isolated Running head verso: Roy et al Running head recto: Anti-infective metabolites of a newly isolate b d acillus thuringiensis Bacillus thuringiensis KL1 Associated with Kalmegh (Andrographis paniculata Nees.), a Traditional Medicinal Herb 1,2 1 1 1 s udipta r oy , s ahana y asmin , subhadeep g hosh , s omesankar b hattacharya and Debdulal banerjee Department of Botany, Microbiology and Microbial Biotechnology Laboratory, Vidyasagar University, Midnapore, West Bengal, India. PG Department of Biotechnology, Oriental Institute of Science and Technology, Midnapore, West Bengal, India. A BSTR AC T: This study was conducted to isolate endophytic bacteria possessing anti-infective property from Kalmegh ( Andrographis paniculata Nees.), a well-known medicinal plant. A total of 23 strains were isolated from this plant among which the strain KL1, isolated from surface-sterilized leaf of this medicinal herb, showed broad-spectrum antagonism against an array of Gram-positive and -negative bacterial pathogens. Ethyl acetate extract of KL1- fermented media yielded a greenish amorphous substance retaining anti-infective property. Solvent-extracted crude material was separated by thin-layer chromatography, and the active ingredient was located by autobiogram analysis. The purified anti-infective compound was found as anthracene derivative as analyzed by ultraviolet and Fourier transform infrared spectroscopy. The strain was identified as Bacillus thuringiensis KL1 from cultural, physiochemical, and molecular aspects. The above results indicate the pharmaceutical potential of the candidate isolate. K E Y WOR DS: Andrographis paniculata, endophyte, Bacillus thuringiensis, antimicrobial, anthracene, pharmaceutical CITATION: r oy et al. Anti-i nfective Metabolites of a n ewly i solated Bacillus thuringiensis COPYRIGHT: © the authors, publisher and licensee l ibertas Academica l imited. t his is an open-access article distributed under the terms of the c reative c ommons Kl 1 Associated with Kalmegh (Andrographis paniculata n ees.), a t raditional Medicinal cc -by -nc 3.0 l icense. h erb. Microbiology Insights 2016:9 1–7 doi:10.4137/Mbi .s 33394. CORRESPONDENCE: debu33@gmail.com; db@mail.vidyasagar.ac.in TYPE: o riginal r esearch Paper subject to independent expert single-blind peer review. All editorial decisions RECEIVED: n ovember 30, 2015. RESUBMITTED: February 8, 2016. ACCEPTED FOR made by independent academic editor. Upon submission manuscript was subject to PUBLICATION: February 10, 2016. anti-plagiarism scanning. Prior to publication all authors have given signed confirmation ACADEMIC EDITOR: r aúl r ivas, Editor in c hief of agreement to article publication and compliance with all applicable ethical and legal requirements, including the accuracy of author and contributor information, disclosure of PEER REVIEW: t hree peer reviewers contributed to the peer review report. competing interests and funding sources, compliance with ethical requirements relating to Reviewers’ reports totaled 613 words, excluding any confidential comments to the human and animal study participants, and compliance with any copyright requirements of academic editor. third parties. t his journal is a member of the c ommittee on Publication Ethics (co PE). FUNDING: Authors disclose no funding sources. Provenance: the authors were invited to submit this paper. COMPETING INTERESTS: Authors disclose no potential conflicts of interest. Published by l ibertas Academica. l earn more about this journal. microorganisms are also considered as a reservoir of bio active Introduction compounds produced as secondary metabolites. Presently, From the very beginning, pharmaceutical research is pharmaceutical exploration of microbial origin is aimed at signicfi antly dependent on traditional health-care practices endophytic microorganisms. Endophytic microbes are yet with medicinal plants. Application of plants for medicinal less-explored population and presently gaining significant purposes in India has been recognized in ancient literature. However, systemized studies on traditional medicinal plants, field in medicinal research for mankind. They reside symp - especially herbs, were noticed in 1956 and soon achieved recog - tomless within the plant’s internal tissues. Some of them act nition due to loss of traditional knowledge and declining plant synergistically by producing secondary metabolites antagon- is population. At least 7,000 medicinal compounds in the mod - tic to plant pathogens. Both fungi and bacteria are common ern pharmacopoeia are of plant origin. Among the 120 plant- endophytes, and many new antimicrobial compounds have derived bioactive compounds widely used in modern medicine, been already discovered from such class of microorganisms. 80% exhibit a positive correlation between their modern thera- In most cases, the antimicrobial compounds produced by peutic use and the traditional use of source plants. Androgra- such groups of microbes are relatively less toxic as the plant phis paniculata Nees, belonging to Acanthaceae family, is a herb itself serves as a natural selection system M . any bioactive traditionally used as a medicine in ancient oriental, including substances that an endophyte produces were found relatively Indian Ayurveda. Phytochemical exploration revealed diverse new to us. Therefore, there is a huge potential to screen novel, pharmacoactive compounds, including labdane diterpenoid highly active, and low-toxicity antimicrobial compounds from lactones, flavonoids, and others. The leaf extract of this plant endophytic microorganisms. A large number of endophytic is very well known as a therapy for infectious diseases, fever- bacteria have already been isolated possessing antibacte - 5 9 causing diseases, diarrhea colic pain, and loss of appetite. rial or antifungal activity. Endophytic microorganisms also New classes of bioactive compounds are ever demanding, have great contribution in the production of antidiabetic, 7 11 12 and we know that the probability of occurrence of such com- anticancerous, anti-insecticidal, antiviral, and even immu- pounds is higher in any unexplored source. Apart from plants, nosuppressive compounds. Microbiology i nsights 2016:9 1 Roy et al This study was aimed to isolate endophytic bacteria from (Vega© TESCAN) after gold coating. Colony and cellular A. paniculata Nees. and evaluate their anti-infective property details were recorded, and various extracellular enzyme against some bacterial pathogens. One of the isolated strains productions were analyzed for the strain. Sugar utilization identified as Bacillus thuringiensis KL1 from 16s rRNA gene patterns were also determined. The endophytic strain was homology and other characteristics showed potential anti- cultured in TYG medium at pH 7 and 30°C incubation infectivity. Three bioactive metabolites were isolated from this temperature with a shaking speed of 150  rpm to study its endophytic bacterium and one of them was characterized after growth pattern. The growth pattern in batch fashion was spectral analysis. determined after plotting the optical densit y of the cell su - s pension against time. Materials and Methods Genomic DNA isolation and 16s rDNA amplifica - Sampling and endophyte isolation. A. paniculata is tion. The strain was grown in Luria Bertini for 24 hours and native to peninsular India. This herb grows as a bush and centrifuged for 10 minutes at 10,000  rpm. Cell pellets were is a dominant species in the district of Paschim Medinipur, washed with phosphate buffer (0.1  M, pH 7.2) and resus - West Bengal, India. Healthy twigs were collected from d-if pended in 500  µ L TE buffer (25  mM Tris-HCl [pH 8.0], ferent localities of Paschim Medinipur (22.57N–87.11E) 25  mM ethylenediaminetetraacetic acid). After that, it was and stored in refrigerator till isolation of endophytes. Leaves successively treated with lysozyme and sodium dodecyl su -l were separated from twigs and washed under gentle tap fate (10%) for proper cell lysis. DNA was extracted with water. These parts of the plant were aseptically surface steril - phenol–chloroform reagent, and aqueous layers were sep-a ized following standard method. Samples were placed on rated carefully. Genomic DNA was precipitated with isopro - ISP2 agar media supplemented with actidione (50 µ g/mL) panol and washed with ice-cold 70% ethanol. It was dissolved and incubated at 30°C for five days. Endophytic bacteria, in 100  µ L TE buffer (pH 8) and directly used as template which came out from surface-sterilized part of the leaves, for 16s rDNA amplification. For this purpose, the primers were immediately pure cultured and preserved with 30% 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R glycerol at - 20°C. (5′-GGTTACCTTGTTACGACTT-3′) were used. Reac- Evaluation of anti-infectivity. Isolated endophytic tions were performed in a polymerase chain reaction (PCR) strains were individually grown in tryptone yeast extract gl-u machine (Mastercycler® nexus, Eppendorf). Reaction cose liquid media [bacto tryptone, 0.5%; yeast extract, 0.2%; parameters were as follows: initial denaturation at 9°4C for glucose, 1%; K HPO , 0.1%, and MgSO 7H O, 0.05%], pH 5 minutes followed by 30 cycles of denaturation at 9°5C for 2 4 4 2 7.2 for three days at 30°C in a shaker incubator (150 rpm). 60 seconds, annealing at 55°C for 60 seconds, and primer Cultures were taken in a centrifuge tube, and cells were pe -l extension at 72°C for 90 seconds. A final extension was done leted by centrifugation at 6,000 g for eight minutes. Cell-free at 72°C for 10 minutes. PCR products were purified with liquid media were evaluated for pathogen growth inhibition Hi-PurA™ PCR product purification spin kit (Himedia). by agar well diffusion method. Six pathogens available in Forward and reverse DNA-sequencing reactions of purified the laboratory were grown overnight in tryptic soy broth. PCR amplicon was carried out with the same primers using Pathogens included Bacillus subtilis (ATCC 11774), Bacillus BigDye Terminator v3.1 Cycle Sequencing Kit on ABI 3730xl cereus (ATCC 14579), Vibrio parahaemolyticus (ATCC 1782), Genetic Analyzer. Aeromonas caviae (ATCC 15468), Proteus vulgaris (ATCC Phylogeny construction. Consensus sequence of 16s 12453), and Pseudomonas aeruginosa (ATCC 9027). Each pre- rDNA was generated from forward and reverse sequence data grown pathogenic strain was seeded on Müller-Hinton agar using aligner software. The sequence was used to carry out (MHA) media separately, and culture filtrates were applied BLAST with the “nr” database of NCBI GenBank. Based on in the wells on the MHA plates. The MHA plates were the maximum identity, sequences were selected and aligned incubated for another 24 hours, and the zone of inhibition using the multiple alignment software program, ClustalW. produced was recorded. The phylogenetic tree was constructed using MEGA5. The Characterization of potential strain. Endophytic evolutionary history was inferred using the neighbor-joining isolated strain KL1 was characterized from morpholog-i method. The evolutionary distances were computed using cal and biochemical aspects. The strain was obser ved under the Kimura’s two-parameter method. compound and scanning electron microscope. Bacterial Product ion a nd pu r if icat ion of a nt i-infect ive metab- smear (18 hours grown) was prepared on poly-l-lysine- olites. Production of active compound by strain KL1 was coated glass slide and fixed with 0.25% glutaraldehyde carried out in liquid TYG media at 30°C for three days (prepared in Na-phosphate, pH 7.2). Cells were then under shaking conditions (150  rpm). A total of 1.5  L cul - dehydrated for 15 minutes with different grades of etha - ture media was centrifuged (10,000  rpm—15 minutes) to nol (30%–100%) and dried up to the critical point after separate cell mass. Culture filtrate was then extracted t wice which slides were attached to the stub. The materials were with an equal volume of ethyl acetate (Merck—analytical used for imaging through a scanning electron microscope grade). Organic phase was separated and made water free 2 Microbiology i nsights 2016:9 Anti-infective metabolites of a newly isolated bacillus thuringiensis by adding anhydrous Na SO . It was brought to dryness antimicrobial agent and without inocula, respectively. The 2 4 with in vacuo evaporation (rotary evaporator; HS-2005S, IC value was determined by plotting OD values of test Hanshin). The crude metabolites were dissolved in a mini - samples against the concentration of the active compound mum amount of methanol and subjected to activity-guided administered. The activity of purified substance was also thin-layer chromatography (TLC) for purification of an determined against the same pathogen by disk diffusion anti-infective compound. About 15 µ L crude materials was assay, where 25  µ L methanolic solutions were soaked in loaded on silica-coated alumina TLC plate (Silica gel 60 sterile disks and placed on individual plates seeded with F , Merck), and separation was done with solvent mix- pathogens separately. ture (chloroform–methanol; 10:1). Separated compounds Spectroscopic characterization of purified active were detected upon ultraviolet (UV) rays (longer wave compound. The purified active compound produced by length), and R values were determined. Bioautogram was endophytic bacterium KL1 was dissolved in chloroform, and studied by overlaying the TLC plates with molten MHA absorption spectrum was recorded with ultraviolet-visible 5 - 1 media (45°C) containing B. cereus (50 µ L of 10  cfu mL ). (UV-Vis) spectrophotometer (UV-1800; Shimadzu) from The TLC plates were kept inside a sterile Petri plate under 200 to 600  nm with respect to chloroform as control. The moist conditions and incubated at 37°C for 24 hours. Cell active compound was also analyzed for its spectrum in infr- a viabilit y was checked by spraying methylthiazoltetrazolium red (IR) region. Dried samples were mixed well with KBr and (5  mg/mL). The position of active compound was located sample—KBr pellet was analyzed from 600 to 4,000 nm in by comparing pathogen inhibition zone and band position Fourier transform infrared spectrophotometer (Spectrum T; on TLC plates. The most active compound was scrapped PerkinElmer). from TLC plates, dissolved in methanol, and  ∼5  µ L was subjected for further separation with a different solvent Results mixture (chloroform–methanol; 10:0.5). Separation was Endophytic bacteria appeared within a 48-hour incubation of detected by exposition of TLC plates on UV, and active surface-sterilized leaves of Andrographis on the isolation media. fraction was located by the same bioautogram study. R A total of 23 endophytic bacteria were isolated in this study, value was determined for the active compound and consid- and most of the strains were found to inhibit at least one of the ered spectral characterization. test pathogens. However, the rational of selecting strain KL1 Determination of IC of active compound. It is an was due to its broad-spectrum anti-infective property. It inhib- essential parameter to determine the effective concentra- ited all of the test pathogenic bacteria except Pseudomonas tion of any purif ied antimicrobial agent. Slightly modif ied (Table 1). This strain was our choice of interest due to its anti- microbroth dilution assays were employed to determine infectivity against both Gram-positive and -negative bacteria. the IC value of the most active purified metabolite pro - The strain was found to be Gram-positive, rod-shaped duced by endophytic strain KL1. For this purpose, one (Fig.  1), and with endospore. It produced characteristics of Gram-positive strain B. cereus and one Gram-negative various extracellular enzymes. Sugar utilization patterns and strain P. vugaris were selected. Stock solution was pre - other detailed characterization parameters are summarized in pared by dissolving 17  mg (final TLC purified) of active Table 2. The strain utilized most of test sugars though could metabolite in 5  mL of methanol (high-performance li-q not utilize fructose, xylose, and raffinose at all. Genomic DNA uid chromatography grade). It was then serially diluted was isolated from 24-hour grown culture of KL1 from which (200:1–10:1) in Müller-Hinton broth (1  mL) and inocu - 16s rDNA (∼1.5 kb) was amplified. Approximately 900 bases lated separately with test pathogens (25 µ L of OD  0.6). of the amplified product were sequenced, and homology was Positive and negative controls were prepared without an determined in NCBI database. The constructed phylogenetic Table 1. Antimicrobial activities of strain Kl 1 against several bacterial pathogens. PATHOGENS ZONE OF INHIBITION* (mm) ZONE OF INHIBITION* (mm) IC (µ g/mL) BY KL1 CULTURE FILTRATE OF PURIFIED SUBSTANCE Bacillus subtilis (Atcc 11774) 119.00 12.5 ± 0.311 27.3 ± 0.033 Bacillus cereus (Atcc 14579) 12 ± 0.613 nt nt Vibrio parahaemolyticus Atcc 1782 nt nt 10.5 ± 0.133 Aeromonas caviae (Atcc 15468) 10 ± 0.235 nt nt Proteus vulgaris (Atcc 12453) 11.5 ± 0.073 24.6 ± 0.133 153.00 Pseudomonas aeruginosa (Atcc 9027) – nt nt Notes: *Values are represented as mean ± standard deviation of triplicate experiments. Abbreviations: nt , not tested; –, no inhibition. Microbiology i nsights 2016:9 3 Roy et al Figure 1. Endophytic strain B. thuringiensis Kl1; ( A) scanning electron microscopy, (B) optical microscopy (100×), and (C) position of the strain in phylogenetic tree showing similarities with related strains. - 1 tree revealed that the strain showed close sequence similarity was found with (KBr) cm : 2,924, 2,854, 1,634–1,632, 1,592, (99% similarity with 100% query coverage and E value 0) with 1,508–1,494, 1,372, and 758 (Fig. 5). These spectroscopic data different strains of B. thuringiensis (Fig. 1). The isolated endo - are characteristic features of different structural properties phtyic strain KL1 was proposed to be identified as B. thuringi- and indicate the presence of a few functional groups within ensis KL1. It showed a regular growth fashion in batch culture the active compound. Antimicrobial activity determination (Fig. 2) with ∼5 hours as lag phase in TYG medium and other revealed that the purified substance was active against both mentioned growth conditions though it reached the maximum types of microorganisms with varying IC values. It was cell density at 22 hours. found that the IC values are 119.0 and 153.0 µ g/mL, respec- The solvent-extracted crude metabolites appeared as tively, against B. cereus and P. vulgaris (Table 1). greenish amorphous substances with a sharp smell. This mixture was separated through TLC, and several fractions Discussion were obtained among which three compounds were detected The large number of secondary metabolites so far isolated (R —0.813, 0.480, and 0.053 in chlororform–methanol; 10:1) from microbial cultures and plant extracts have no doubt to inhibit the growth of B. subtilis (Fig. 3A). The compound significant industrial values. Like many other microbial with an R value of 0.813 was found as the major anti-infective species, Bacillus also produces many antibacterial second - compound with the highest activity. However, a further puri- ary metabolites. Bacillus, traditionally isolated from the fication of this compound indicated that the compound with soil, is renowned for the production of various antibiotics an R value of 0.784 (chlororform–methanol; 10:0.5) was the such as bacitracin, polymyxin, zwittermicin A, and several most active anti-infective compound produced by endophytic others. Unfortunately, reisolation of the same microorgan - B. thuringiensis KL1 (Fig. 3B). The pure active material was ism from the same source and the throttled success of com- found to absorb UV at 259 and 371 nm (Fig. 4), while IR binatorial chemistry in providing new compounds indicate max max 4 Microbiology i nsights 2016:9 Anti-infective metabolites of a newly isolated bacillus thuringiensis Table 2. c ultural and physiological and biochemical characteristics reported for endophytic origin such as Azorhizobium, Bacillus, of endophytic B. thuringiensis Kl1. Bradyrhizobium, Gluconacetobacter, Klebsiella, Burkholderia, Enterobacter, Pseudomonas, and Streptomyces. B. thuringiensis CHARACTERIZATION APPEARANCE is an aerobic, Gram-positive endospore-forming bacterium PARAMETERS and well known for crystalline inclusions o δr -endotoxins. c olony morphology White to off white color, slightly raised elevation and regular margin These proteins are selectively insecticidal, but the strain also c ell morphology r od shaped, slightly swollen cen- secretes zwittermicin A, a linear aminopolyol antibiotic effec- trally, 1.5–1.8 µ m length, 0.25 µ m tive against Gram-negative bacteria mostly. A number of diameter lipopeptides with anti-infectivity were also purified from g ram character Positive different species of Bacillus. A fengycin-like substance was Endospore Positive purified from a soil-dwelling B. thuringiensis CMB26, which Extracellular enzymes exhibited fungicidal, bactericidal, and insecticidal activities. c atalase + Present findings report the isolation of the endophytic isolate Amylase + KL1, which is identified as B. thuringiensis KL1. The strain c ellulase + was isolated on ISP2 agar media from surface-sterilized leaves l ipase + of A. paniculata Nees. ISP2 agar media is an effective isola - c aseinase - tion media for endophytic bacteria as it contains minimum g elatinase + nutritional requirements for growth. Endophytes are able to emerge from plant parts and grow using plant extracts and Urease - nutrients provided by media, whereas other microorganisms Phenyl alanine hydrolase + are unable to access plant extracts and their growth is not sup - sugar utilization ported so much. Sugar utilization pattern of this strain indi- Dextrose + cates that it can use some polysaccharides such as cellulose Fructose - and starch as carbon source, which are very usual in plants. g alactose + Besides, its utilization of disaccharides and few monosac- r hamnose + charides explores its wide carbon utilization property that Xylose - might help it to grow in the specialized microenvironment. Raffinose - Presence of different extracellular enzymes also helps such Maltose + group of microbes to exist in such an association as was found s ucrose in this endophytic strain. A report states that its leaves contain 3 4 - 1 lactose + about 10 –10  cfu mL when 1 g of surface-sterilized leaves s tarch were crushed in 10 mL distilled water and subjected for iso - lation of endophytic bacteria, though this study reports of c ellulose + 23 bacteria l isolates from the same. Interestingly B , . thuringiensis var israelensis (Bti), a nonendophytic isolate, and ethano - lic extracts of A. paniculata were found for their larvicidal as uncertainty in future antimicrobial therapy. Thus, finding well as pupicidal activity againsA t nopheles stephensi Liston in new groups of microbes from unexplored or less-explored a synergistic fashion. Here, we report that the endophytic habitats became mandatory by pursuing them as sources of strain B. thuringiensis KL1 was able to inhibit both Gram- novel bioactive compounds. Different bacteria are already positive and Gram-negative pathogens employed though it could not inhibit Pseudomonas at all. TLC along with bioautogram analysis revealed that the compound is slightly nonpolar in nature and the strain produced at least three different compounds that were inhibitory to test pathogen. Spectroscopic characterization of most active compounds showed strong UV absorption at 259 and 361  nm. UV at max 259  nm suggests the presence of conjugated diene in ben- zene ring, while at 361  nm sharply, it indicates the anthr- a cene nature of the active compound. IR absorbance values at - 1 2,854 and 2,924 cm specify some alkane stretch, whereas the presence of monosubstituted alkene was indicated by 1,634– - 1 1,632  cm . –COOH attached with the aromatic ring was - 1 indicated at 1,592 cm , and the presence of aromatic ring was - 1 Figure 2. g rowth pattern of strain Kl 1 in batch culture. confirmed by IR absorption at 1,508–1,494  cm . The active Microbiology i nsights 2016:9 5 Roy et al Figure 3. Activity-guided TLC-based purification of the most active anti-infective metabolite produced by KL1; ( B, C) represent thin layer chromatogram in which (B) indicates three active metabolites and (C) represents the most active band indicated inside the oval shape. (A, D) Demonstrate bioautogram analysis indicating activity among the separated compounds. compound consisted –NO as the functional group that was effectivity to both Gram-positive and -negative pathogens - 1 suggested by absorption at 1,372  cm . Therefore, the spec - indicating the probability of similar inhibition mechanisms. troscopic characterization recommends the active compound However, this compound does not bear any similarity with as anthracene derivative consisting poly alkane or alkene, either compound isolated from B. thuringiensis previously 30,31 where phenyl group may be substituted with COOH and NO reported. Internal plant tissue is a specialized ecosystem groups. Anthracenes are reported to inhibit at different steps in that may be responsible of such an alteration in gene sequence 34 35 DNA/RNA biosynthesis, which is considered a potent tar- during coevolution. Here is the opportunity of getting vari- get site for antibiotics. The active principal here shows marked ations in metabolites from similar microorganisms isolated Figure 5. ir spectrum of the most active anti-infective metabolite of Figure 4. UV-Vis spectrum of most active metabolites by strain Kl 1. strain Kl1. 6 Microbiology i nsights 2016:9 Anti-infective metabolites of a newly isolated bacillus thuringiensis 12. Guo B, Dai J, Huang NY, Leong C, Ong W, Carte BK. Cytonic acids A, and B: from other common habitats. The isolated endophytic strain novel tridepside inhibitors of hCMV protease from the endophytic fungus may be a good source for the antimicrobial compound due to Cytonema sp. J Nat Prod. 2000;63:602–604. 13. Lee J, Lobkovsky E, Pliam NB, Strobel GA, Clardy J. Subglutinols A and B: its broad-spectrum antibacterial property. immunosuppressive compounds from the endophytic fungus Fusarium subgluti- nans. J Org Chem. 1995;61:7076–7077. Conclusion 14. Zin NM, Loi CS, Sarmin MN, Rosli AN. Cultivation-dependent characteriza - tion of endophytic actinomycetes. Res J Microbiol. 2010;5:717–724. doi:10.3923/ Endophytic bacteria from medicinal plants are good sources jm.2010.717.724. of new class anti-infective compounds. This study showed 15. Roy S, Banerjee D. Bioactive endophytic actinomycetes of Cinnamomum sp.; iso- lation, identification, activity guided purification and process optimization of potential existence of endophytic bacteria in the studied plant. active metabolite. Am J Microbiol. 2015;6:4–13. Moreover, the strain, identified as B. thuringiensis KL1, is 16. Perez C, Paul M, Bazerque P. Antibiotic assay by agar-well diffusion method. Act Biol Med Exp. 1990;15:113–115. found to possess broad-spectrum anti-infective property. The 17. Franson TR, Sheth NK, Rose HD, Sohnle PG. Scanning electron microscopy of ethyl acetate extract of this isolate yielded three different anti- bacteria adherent to intravascular catheters. J Clin Microbiol. 1984;20:500 –505. 18. Lee O, Choi GJ, Choi YH, et al. 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