Antibacterial activity of secondary metabolites isolated from Alternaria alternata

The aims of this study were the analysis of the secondary metabolites and evaluation of the antibacterial and antifungal activity of Alternaria alternata. Twenty six bioactive compounds were identified in methanolic extract of Alternaria alternata. The identification of bioactive chemical compounds is based on the peak area, retention time molecular weight and molecular formula. GC-MS analysis of A. alternata revealed the existence of the α-acetyl-L-serine, 2(5H)-furanone, 6-oxabicyclo[3.1.0]hexan-3-one, D-glucose,6-O-α-D-GALACTOPYRANOSYL, DL-arabinose, ƹ-N-fommyl-Llysine, 2-[4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), thrietol, 2-O-heptyl, 2-deoxy-2fluoro-1,6-anhydro-ß-d-glucopyranose, d-ribo-hexos-3-ulose, Α-D-glucopyranoside, O-α-Dglucopyranosyl-(1.fwdarw.3)-ß-D-fru, maltose, 4H-pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl, desulphosinigrin, uric acid, midazole-4-carboxylic acid, 2-fluoro-1-methoxymethyl-ethyl ester, geranyl isovalerate, 1-nitro-ß-d-arabinofuranose, tetraacetate, glycyl-D-asparagine, α-D-xylofuranose, cyclic 1,2:3,5-bis(butylboronate), estra -1,3,5(10)-trien-17ß-ol, glucobrassicin, N-2,4-Dnp-L-arginine, dasycarpidan-1-methanol, acetate(ester) and 5alpha-androstane-3,17-monooxime. The fourier transform infrared (FTIR) analysis of A. alternata proved the presence of aromatic rings, aliphatic fluoro compounds, tetiary amine, C-N stretch, ammonium ions, organic nitrate, methylene-CH. asym, and normal polymeric O-H stretch which shows major peaks at 711.73, 846.57, 873.75, 1026.13, 1149.57, 1205.51, 1238.30, 1409.96, 1631.78, 2517.10, 2854.65, 2924.09, 3059.75 and 3271.27. A. alternate had maximum zone formation (5.04 ± 0.29) mm against Klebsiella pneumonia.


INTRODUCTION
Alternaria spp.are cosmopolitan mould fungi and can be found in soils, plants, food, feed and indoor air (Thomma, 2003).Alternaria species are frequently found on small grains, causing yield losses in production and processing *Corresponding author.E-mail: imad_dna@yahoo.com.Tel: 009647716150716.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License (Ostry, 2008).Many Alternaria species are mycotoxin producers with different toxicological properties.The most important Alternaria toxins are alternariol (AOH), alternariol monomethyl ether (AME), altenuen (ALT), tenuazoic acid (TEA) and altertoxins (ATX-I, II, III) (Logrieco et al., 2009;Hameed et al, 2015a).Alternaria spores are considered to be one of the most prolific fungal allergens, which have been associated with respiratory allergies and skin infections (Corden et al., 2003;Kilic et al., 2010;Pavon et al., 2010).
A. alternata is considered as the most important toxin producing species.A. alternata is a widespread saprophytic species which produces a wide variety of different secondary metabolites, among which are the mutagenic mycotoxins alternariol (AOH) and altertoxin (ATX) (Pfeiffer et al., 2007).The altertoxins ATX-I, -II, and -III are mutagenic in the Ames test and are more potent and acutely toxic to mice than AOH and AME. A. alternata is known as producer of a large spectrum of secondary metabolites.The effect of light on the amount of secondary metabolites by GC-MS and FT-IR was analyzed (Altameme et al., 2015;Hameed et al., 2015b).
The main purpose of this research was the screening of the secondary metabolites products from A. alternata and evaluation of the antibacterial activity.

Collection and growth condition
A. alternata species were isolated from dried fruit and the pure colonies were selected, isolated and maintained on potato dextrose agar slants (Usha and Masilamani, 2013).After the species were identified by the identification key, spores were grown in a liquid culture of potato dextrose broth (PDB) and incubated at 25°C in a shaker for 16 days at 130 rpm.

Production, extraction and determination of metabolites
The metabolites were determined and extracted for GC analysis using the method of Hussein et al. (2015) with some modifications.The extraction was performed by adding 25 ml methanol to 100 ml liquid culture in an Erlenmeyer flask after the infiltration of the culture.The mixture was incubated at 4°C for 10 min and then shook for 10 min at 130 rpm.Metabolites were separated from the liquid culture and evaporated to dryness with a rotary evaporator at 45°C.The residue was dissolved in 1 ml methanol, filtered through a 0.2 μm syringe filter, and stored at 4°C for 24 h before being used for GC-MS (Hameed et al., 2015c;Jasim et al., 2015).The identification of the components was based on comparison of their mass spectra with those of NIST mass spectral library as well as on comparison of their retention indices either with those of authentic compounds or with literature values.

Gas chromatography-mass spectrometry (GC-MS) analysis
Bioactive compound were examined for the chemical composition using GC-MS (Agilent 7890 A) equipped with a DB-5MS column (30 m × 0.25 mm i.d., 0.25 um film thickness, J&W Scientific, Folsom, CA).Helium was used as the carrier gas at the rate of 1.0 mL/min (Imad et al., 2014a;Kareem et al., 2015).Effluent of the GC column was introduced directly into the source of the MS via a transfer line (250°C) (Tabaraie et al., 2012).Ionization voltage was 70 eV and ion source temperature was 230°C.Scan range was 41 to 450 amu.The constituents were identified after comparison with available data in the GC-MS library in the literatures (Mohammed and Imad, 2013).

Fourier transform infrared spectrophotometer (FTIR)
The powdered sample of the A. alternata specimen was treated for fourier transform infrared spectroscopy (Shimadzu, IR Affinity 1, Japan).The sample was run at infrared region between 400 and 4000 nm (Imad et al., 2014b).

Determination of antibacterial activity of crude fraction of A. alternata compounds
The test pathogens (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus) were swabbed in Muller Hinton agar plates.90 μl of fungal extracts was loaded on the bored wells.The wells were bored in 0.5 cm in diameter (Suja et al., 2013;Huda et al., 2015b;Imad et al., 2015).The plates were incubated at 37°C for 24 h and examined.After the incubation the diameter of inhibition zones around the discs were measured.

Statistical analysis
Data were analyzed using analysis of variance (ANOVA) and differences among the means were determined for significance at P < 0.05 using Duncan's multiple range test (by SPSS software) Version 9.1.

Isolation of fungi from dried fruit
The fungi were isolated from dried fruit by serial dilution method (Perfect et al., 2001;Mogensen et al., 2003).Based on morphological characteristics, fungi was isolated in selective media of potato dextrose agar media.Morphological and microscopical characteristics of fungal strains were determined using specific media light and compound microscope (Figure 1).

Production of secondary metabolites
The 400 ml of fermentation broth (PDA broth) which contained 200 μl of the standardized fugal suspensions were used to inoculate the flasks and incubated at 37°C on a shaker at 90 rpm for 7 days.After fermentation, the secondary metabolites were produced by isolated microorganisms.

Identification of secondary metabolites from the methanolic crude extract of A. alternata by gas chromatography and mass spectrometry
Gas chromatography and mass spectroscopy analysis of   2. The first set up peak was determined to be α-acetyl-L-serine (Figure 3).The second peak indicated to be 2(5H)-furanone (Figure 4).Sharma et al., 2011;Chacko et al., 2012).

Antibacterial activity
Four clinical pathogens were selected for antibacterial activity namely, K. pneumoniae, P. aeroginosa, E. coli and S. aeureus.Maximum zone formation against K. pneumonia was found (5.04 ± 0.29) as shown in Table 3 and Figure 30.

Conclusion
The results of this study showed that A. alternata have high biological activities and produce many important secondary metabolites.

Conflict of interests
The author(s) did not declare any conflict of interest.

Table 1 .
Major bioactive chemical compounds identified in methanolic extract of Alternaria alternata.
compounds was carried out in methanolic extract of A. alternate as shown in Table1.The GC-MS chromatogram of the 26 peaks of the compounds detected are shown in Figure

Table 3 .
Zone of inhibition (mm) of test bacterial strains to fungal products and standard antibiotics.