Broad Spectrum Antimicrobial Agent from a Local Isolate of Marine Fungus Strain

12Amany L. Kansoh, Omkalthoum H. Khattab, 2Zainab M. Abd-Elrazek and 3Hemaia M. Motawea

12Microbial Chemistry Dept., Botany and Microbiology Dept., Faculty of science, Helwan University, Cairo, Egypt. 3Pharmacognosy Dept., National Research Centre,Cairo,Egypt.

Abstract: Various samples randomly collected from the sediments and the sea water of Mediterranean Sea in Egypt, were enriched to isolate different fungal isolates. Only, twelve of these isolates showed antimicrobial agent activities against various strains of bacteria, yeast and fungi. These marine fungal isolates were identified and the most active one was designated as Penicillium viridicatum. The optimization of some culture conditions were studied to attain maximum antimicrobial agent production from this strain. Starch 2.0% (w/v) and potassium nitrate 0.24% (w/v) as a carbon and an inorganic nitrogen sources supported the highest level of antimicrobial agent production, respectively. Deletion of organic nitrogen sources from the medium constituent were more preferable. The optimum antimicrobial agent production by Penicillium viridicatum was observed on the 5th day of the shaking culture incubation at 30oC and pH 6.0. The antimicrobial agent produced by Penicillium viridicatum was extracted with ethyl acetate, purified by Sephadex LH-20 column chromatography and it was characterized as fumigaclavine or its isomer (isofumigaclavine).

Key words: Marine fungal species, morphological and physiological characteristics and antimicrobial agent production and characterization.

INTRODUCTION metabolic engineering and post genomic technologies for future supply of these natural products from the Microorganisms adapted to life in an extreme marine environment.[9] environment. Most of them are able to deal with Here we isolated different marine fungal strains moderate concentrations of salts dissolved in water and screened them according to their morphological surrounding them. Obviously, all marine organisms can studies and antimicrobial agent activities against endure, and very often require, the salinity of the various strains of bacteria, yeast and fungi. Studying ocean, which is remarkably constant around the world some culture conditions affecting the biosynthesis of and is close to 3.5% of total salts [1] the effective agent produced by the most active strain These are two major types of biologically to attain the maximum production. Also, Physico- important environments in which the salt factor will chemical properties and structure elucidation of the interact with microbial populations, soil and water produced antimicrobial agent were also, investigated. which account for 70% of the earth's surface. In the oceans, fungi live as saprophytes, parasites, and MATERIALS AND METHODS symbionts on various matrices such as sea, sand, logs, water, soil bubbles as well as algae and other marine Sampling and Isolation of Marine Fungal Isolates: organisms.[2,3] Halophilic fungal isolates were randomly collected from The ocean represents a rich resource for more the sediments and the sea water of Mediterranean Sea novel compounds with great potential as in Egypt, at a depth of 1-3m. The sediments were pharmaceutical, nutritional supplements, cosmetics, homogenized in sterile sea water and shaked for 30 agrichemicals, and enzymes, where each of these minutes. marine bioproducts has a strong potential market value. Isolation was carried out according to the dilution So, the discovery of new biologically active secondary technique of Johnson et al.[10] Plates containing malt- metabolites is a goal for many industries.[4,5,6,7,8] extract and Czapek’s dox agar media [11] having It is clear that the marine environment will play a different sodium chloride concentrations of 5, 10, 15, vital role in the future development and trials of anti – 20, 25 and 30% (w/v) were inoculated with 1.0 ml of infective drugs. Efforts are still needed in terms of the proper dilution. Rose Bengal (1/3000 w/v) and the large scale production and exploitation through antibiotics streptomycin and penicillin (5.0 mg/ml)

Corresponding Author: Amany L. Kansoh, Microbial Chemistry Dept., E-mail: [email protected] 580 J. Appl. Sci. Res., 6(6): 580-588, 2010 were added to each agar plate to inhibit the growth of agent production of the selected strain. Bacillus subtilis, bacteria. The plates were incubated for 2 to 7 days at Candida albicans and Aspergillus terreus were takes as 28°C (±2°C) and examined daily for the presence of representative test organisms. developing fungal hyphae. Distinct fungal colonies on Sucrose as a carbon source in Czapek’s dox the agar plates were then transferred to new plates for medium (containing 5% NaCl concentration) was further isolation and purification. The single purified replaced by various carbon sources at a concentration fungal colonies were picked up and subcultured on of 2% (w/v) to study the effects of different carbon agar slants of the dox medium. sources on antimicrobial agent production by Penicillium viridicatum. Detection of the Antimicrobial Agent Activities: The The effect of addition of equimolar amounts of purified genera and types isolated were screened for different inorganic and organic nitrogen sources to their antimicrobial activities by the agar diffusion enhance the antimicrobial agent production by method [12] in dox culture media supplemented with 5% Penicillium viridicatum were also, studied. Since, Yeast v/v NaCl (B.S.M). The most active isolates that give extract might be considered as a growth factor and/or broad spectrum antimicrobial agents were completely nitrogen source. So, different concentrations of yeast identified and chosen to prepare spore suspensions as extract were added to test their effect on the inoculum for the fermentation media. antimicrobial agent production. The modified B.S.M was adjusted to appropriate Identification of the Most Active Fungal Isolates: All PH value of 4.0 to 9.0 to study the effect of the initial fungal isolates that showed antimicrobial agent PH values on antimicrobial agent production. activities were identified according to Domsch et al.[13] at the Regional Centre of Micology and Biotechnology Time Course of Antimicrobial Agent Production by (RCMB), Al-Azhar University. Penicillium Viridicatum: Time course from one to eight days was followed in shaking incubated flasks Fermentation Studies: In all experiments, each containing the modified B.S.M having the optimized Erlenmeyer flask (250ml) containing 50ml of the liquid culture conditions. The optimum period of incubation culture (B.S.M) contained gm/l :Sucrose,20; KNO3, 2.0; for maximum antimicrobial agent production was

KCl,0.5; MgSO42.7H O,0.5; FeSO42.7H O, 0.01; determined. NaCl,50.0; trace elements solution, 1.0ml and agar in solidified medium. It was inoculated with the spore Extraction and Purification of the Antimicrobial suspension (2×106 spore/ml) of the selected fungal Agent Produced by Penicillium Viridicatum: About strains and incubated at 30°C with initial PH of 6.0 on 50 liters of the modified B.S.M broth were centrifuged a rotary shaker (180 rpm) for five days. The culture and the supernatant was concentrated under vacuum was freed from mycelium debris by centrifugation at and extracted with ethyl acetate trice at neutral PH 4000 (rpm) under cooling. The supernatant was used as value. The crude extracted residue was fractionated on a crude antimicrobial agent preparation. a Sephadex LH-20 column chromatography The most active strain was chosen to determine the (18×400mm) preequiliberated with methanol, and eluted physiological and physical conditions that would affect using a mixture of methanol - chloroform (9:1) at a antimicrobial agent production. rate of 40-50ml/h.[15] The active fractions were collected, concentrated under vacuum and bioassayed Effect of Sea Water and Various Sodium Chloride against the test organism. [16] Concentrations on the Mycelial Growth of the Selected Strain: The colonial growth diameter of the Structural Characterization of the Purified selected strain was determined on Czapek’s dox agar Antimicrobial Agent Produced by Penicillium media supplemented with different sodium chloride Viridicatum: Antimicrobial agent was investigated by concentrations of 5, 10, 15, and 20% (w/v) and sea different spectral method via ultraviolet UV and water at different depths for 2-8 days at 28°C.[14] infrared IR spectra using T80 + UV/VIS spectrometer, Antimicrobial agent production were tested in the PG Instruments Ltd (190-1000 MHZ) and Fourier same NaCl concentrations and seawater as previous one Transform infrared spectrometer, using JASCO (Japan), by agar diffusion method [12] and inhibition zone FT / IR-6100 (50-4000 MHZ) at N.R.C, Cairo-Egypt. diameters were measured. The electron impact spectrometric spectrum MS was estimated using Finnigan Mat-SSQ 7000 with full Factors Affecting the Antimicrobial Agent scan, (5 sec) scan time and 70 ev, at N.R.C, Cairo- Production by Penicillium Viridicatum: All Egypt. experiments in this study were carried out in duplicates The proton 11H NMR and 3C NMR spectra were shaking cultures to determine the physiological and estimated by Joel-Eca 500 MHZ NMR spectrometer, at physical conditions that would affect the antimicrobial the Micro-analysis Center, N.R.C, Cairo-Egypt.

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RESULTS AND DISCUSSION antimicrobial agent production, while no obvious effect was detected with organic nitrogen sources (Fig.4). Detection of Antimicrobial Agent Activities of 0.24% (w/v) was the best concentration of potassium Marine Fungal Isolates: From various fungal isolates nitrate for optimum antimicrobial agent production by collected from the sediments and the sea water of Penicillium viridicatum (Fig.5). Mediterranean Sea in Egypt, twelve fungal isolates The requirements for specific nitrogen supplement showed antimicrobial agent activities against various differ from one microorganism to another. In most strains of bacteria, yeast and fungi (Table.1). The most microorganisms both inorganic and organic forms of active strain no.12 that showed a broad spectrum nitrogen are metabolized to produce amino acids, activities was chosen to study the physiological and nucleic acids, proteins and cell wall components. physical conditions that would affect the antimicrobial However, it was found that some carbon and nitrogen agent production. sources had an inhibitory effect on the antimicrobial agent production and this may be due to organic acid Identification: All fungal isolates that showed accumulation, oxygen depletion or sugar catabolic antimicrobial agent activities were identified. The most repression. These conclusions are in agreement with active fungal isolate was identified as Penicillium those reported by Bernan, et al.[17] and Burgess, et al.[2] viridicatum (Fig.1) which based on current universal No noticeable increase in antimicrobial agent keys Penicillium with its teleomorphic states and using production was detected at various yeast extract an image analysis system. Other isolates are belonging concentrations (Fig.6). Since, yeast extract was used as to various Penicillium spp, Aspergillus flavus, an organic nitrogen source and/or as growth factor in Aspergillus clavutus, Cladosporium sp. and the medium. So, deletion of yeast extract and organic Scopulariopsis brevicaulis. nitrogen sources from the medium constituent were more preferable. Effect of Sea Water and Various Sodium Chloride The antimicrobial agent production was affected by Concentrations on the Growth and Antimicrobial the initial pH values of the medium, ranging from pH Agent Production by Penicillium Viridicatum: Table 5.0 to 9.0. Results indicated that the suitable pH for 2, showed the growth of Penicillium viridicatum on antimicrobial agent production by Penicillium different concentrations of NaCl and on sea water at viridicatum was at pH range from 6.0 to 7.0 , while it different incubation period from 2 to 10 days. 5% was optimum at pH 6.0 (Fig.7). During fermentation, NaCl concentration was more suitable for growth and the culture pH strongly affected many microbiological the antimicrobial agent production was optimum at 5% processes and the transport of various components (w/v) NaCl concentration on the 5th day of incubation across the cell membrane.[18,19] period. Time Course of the Antimicrobial Agent Production Factors Affecting Antimicrobial Agent Production on the Optimized Medium by Penicillium by Penicillium Viridicatum: This part of work aims at Viridicatum: Time course from one to eight days was the optimization of some culture conditions to attain followed in shaking incubated flasks containing the maximum antimicrobial agent production. optimized culture conditions inoculated with Penicillium Of all the tested carbon sources (1% w/v) in the viridicatum (Fig.8). The antimicrobial agent production basal salt medium (B.S.M) inoculated and incubated seems to be stable on the 5th and 6th days of the shaken for 5 days, starch supported the highest level incubation period, having maximum on the 5th day. The of antimicrobial agent production by Penicillium time course for antimicrobial agent production differs viridicatum (Fig.2). Other sugars though they supported according to the strain and cultivation conditions. For the growth of the fungal strain, they repressed the instance, the maximum antimicrobial agent production antimicrobial agent production than the starch level. was achieved after 4 days of incubation of Fig.3, shows that the concentration of 2.0% (w/v) Cladosporium sp.[20], 5 days of incubation of starch supported the highest antimicrobial agent Penicillium corylophilum [21] and 14 days of incubation production by Penicillium viridicatum. Further increase of Penicillium waksmanii.[6] of starch concentration in the medium cause gradual decrease in activity. Isolation and Structural Characterization of the Addition of equimolar amounts of various Purified Antimicrobial Agent Produced by inorganic and organic nitrogen sources in the B.S.M Penicillium Viridicatum: It is known that clavine ergot supplemented with 2.0% (w/v) show that Potassium alkaloids are among metabolites produced by genus nitrate was a good inorganic nitrogen source on the Penicillium. The antimicrobial metabolite (A) produced

582 J. Appl. Sci. Res., 6(6): 580-588, 2010 by Penicillium viridicatum in this work. was closely UV showed absorption at 238, 276 and round similar to fumigaclavine B and/or its isomer produced 295nm within the absorption rang of fumigaclavines. by species related to the genus Penicillium [22]. IR showed absorption bands at cm-1 3370 (OH & NH),

Antimicrobial (A) was extracted with ethyl acetate at 3741 (aromatic ring), 2927 (N-CH3) and 1032 (OH on neutral pH value, fractionated on a Sephadex LH-20 six membered ring). column chromatography (18õ400mm) and eluted with The base peak at m/z 122 is given by cleavage at a mixture of methanol-chloroform (9:1). The active ring C to give ring D in ionic form as shown in Fig. fractions having the highest antimicrobial agent (10). activities were collected and concentrated under The MS (Fig.9) showed in addition to fragments of vacuum to give the purified antimicrobial agent. fumigaclavine, some fragments of its isomer such as Antimicrobial (A) was investigated by different spectral m/z 237, 183, 168. Also the mass showed fragment at 13 method via MS, IR, UV, HNMR and C NMR (Fig.9). m/z 104 due to loss of H2O from fragment 122 (base MS of (A) showed M+ at m/z 256.23 for molecular peak, 122-104=18) and this supports the deduced formula C16H20ON2. It has a double bond equivalent structure and deduced base peak.The HNMR and number 8 which means a total number of 8 rings C13NMR (Table 3) are very closed to fumigaclavines. and/or unsaturations. All spectral data of antimicrobial In conclusion from the available data the (A) were closely coincided with fumigaclavin B or its antimicrobial (A) is fumigaclavine (B) or its isomer isomer. (isofumigaclavine).

Table 1: Detection of antimicrobial agent activities of marine fungal isolates. Test organisms Activities of marine fungal isolates numbers against the test organisms * ------5 8 9 12 14 15 18 19 24 26 27 28 Bacteria: ------Bacillus cereus 15 14 - 19 12 15 - 12 13 16 12 14 ------Bacillus subtilis 14 14 - 21 13 - 15 15 18 - 19 21 ------Escherichia coli ---15------Staphylococus aureus 11 15 16 18 20 - 16 15 13 14 13 14 Yeast: ------Candida albicans - 16 - 20 12 - 17 17 ------Candida paseudotropicans 15 13 13 19 12 12 12 14 19 15 14 18 ------Saccharomyces cervesia ---13------Saccharomyces charles 15 15 11 14 14 12 15 15 12 15 15 14 ------Saccharomyces cheva -15------Rhodetula - 15 12 14 12 12 - 13 16 - 14 16 Fungi: ------Aspergillus flavus 12 14 12 - 13 - 11 11 11 ------Aspergillus niger - - - 14 14 - 16 13 - - 13 11 ------Aspergillus terrus 14 15 - 16 11 14 - - - 14 - 13 ------Botyrites alli - - - 12 - 11 - 11 - 11 12 12 ------Deplodia - 11 - 12 16 13 11 12 11 12 11 ------Fusarium oxysporium 11--11- ----121211 ------Helmthosporium turcicum - 12 - 15 14 - 14 11 15 ------Macrophylumn -12------13- *: Activities of the fungal isolates no: 5, 8, 9, 12, 14, 15, 18, 19, 24, 26, 27, and 28 were measured as the inhibition zone diameters (mm), at the 5th day incubation period.

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Table 2: Effect of sea water and various NaCl concentrations on the growth and the antimicrobial agent production by Penicillium viridicatum Sea Water and Mycelial growth width (mm) at different incubation period (days) Inhibition zone diameter (mm)* at NaCl concentrations different incubation periods. ------2 3 45 6 7810 4th 5th 6th 5% 9 15 20 25 32 37 39 45 18 20 15 ------10% 5 10 16 21 27 30 34 40 15 19 11 ------15% - 5 7 10 15 18 20 25 14 17 ------20% - - - - - 4 4 8 ------Surface sea water 9 10 20 25 32 35 39 49 18 21 15 ------Deep sea water 7 11 16 22 28 33 35 42 15 18 13 *Activity was tested as inhibition zone diameters (mm) on the 4th , 5th and 6th days of incubation against Bacillus Subtilis as a representative test organism.

Table 3: HNMR and C13NMR of antimicrobial (A) No HNMR ä C13NMR ä 1 1.5 34.6 ------2 1.9 , 2.06 61.8 ------3 3.1 65.1 ------4 2.9 , 2.4 25.6 ------5 - 127 ------6 6.699 125 ------7 2.2 ------8 _ 128.7 ------9 7.03 108 ------10 7.2 119.9 ------11 7.1 119.7 ------12 - 145 ------13 2.76 37.09 ------14 2.04 ------15 0.9 , 1.09 16.2 ------16 2.27 41 ------NH 2.2 -

Fig. 1: Penicillium viridicatum

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Fig. 2: Effect of various Carbon sources on antimicrobial agent production by Penicillium viridicatum

Fig. 3: Effect of different concentrations of starch on antimicrobial agent production by Penicillium viridicatum

Fig. 4: Effect of different inorganic and organic nitrogen sources for antimicrobial agent production by Penicillium viridicatum

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Fig. 5: Effect of different concentrations of potassium nitrate on antimicrobial agent production by Penicillium viridicatum

Fig. 6: Effect of addition of the different yeast extract concentrations for antimicrobial agent production by Penicillium viridicatum

Fig. 7: Effect of initial pH values on antimicrobial agent production by Penicillium viridicatum

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Fig. 8: Time course of the antimicrobial agent production by Penicillium viridicatum. *Activity was determined against Bacillus subtilis as a representative test organism.

Fig. 9: Structural characterization of the purified antimicrobial agent produced by Penicillium viridicatum.

Fig. 10: Ring D in an ionic form Ring (D) C7OH8N (m/z 122)

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