In vitro activity, extraction, separation and structure elucidation of antibiotic produced by Streptomyces anulatus NEAE-94 active against multidrug-resistant Staphylococcus aureus

ABSTRACT During the screening programme for antibiotic produced by actinomycetes, an antibiotic was isolated from the fermentation broth of Streptomyces anulatus NEAE-94. The ethyl acetate extract of S. anulatus NEAE-94 showed high biological activity against Staphylococcus aureus NRRL B-313, multidrug-resistant S. aureus and Bacillus subtilis NRRL B-543. Thin-layer chromatography of the ethyl acetate extract showed one yellowish orange spot with an Rf value of 0.83. The structural elucidation of the isolated compounds was carried out using ultraviolet–visible mass spectrometry (MS), Fourier transform-infrared spectroscopy (FT-IR), proton nuclear magnetic resonance (1H-NMR) and gas chromatography–mass spectrometry (GC–MS) analyses. Characterization of the ethyl acetate extract revealed the presence of 22 compounds, including an unsaturated fatty acid, saturated fatty acids, fatty acid esters, alkanes, alkenes and a triterpene. The antimicrobial efficacy of S. anulatus NEAE-94 might be due to the synergistic effects of the identified compounds.


Introduction
There has been a steady increase in antibiotic resistance during the past several decades, accounting for the long list of currently available antimicrobial agents that are now insufficient for the control of microbial infections [1,2]. Staphylococcus aureus is a bacterial pathogen responsible for serious infections in humans and is becoming increasingly resistant to many commonly used antibiotic treatments [3]. The occurrence of multidrug-resistant S. aureus means high risks especially in surgical intensive care units. Different strains of S. aureus can cause serious infections including boils and pimples, wound infections, pneumonia, osteomyelitis, septicemia, food intoxication and toxic shock syndrome. The rising frequency of antibiotic resistance among pathogenic S. aureus has created an urgent need to discover novel antibiotics with a broad spectrum of activity against resistant organisms [4].
According to Hanson [9], the structure elucidation of natural products can be divided into four stages: (1) preliminary characterization (determination of physical constants, molecular weight and identification of functional groups), (2) structural simplification (identification of carbon skeleton), (3) relative stereochemistry (position of functional groups and their relative configuration in space), (4) absolute configuration. Numerous techniques and analytical systems like mass spectrometry (MS), Fourier transform-infrared spectrum (FT-IR), nuclear magnetic resonance ( 1 H-NMR), elemental analysis and gas chromatography-mass spectrometry (GC-MS) analyses have been developed for the analysis and characterization of active compounds from micro-organisms. The secondary metabolites from micro-organisms have characteristic chromatographic profiles and reactivity towards staining reagents used in thin-layer chromatography (TLC) under specified reaction conditions, which allows the separation and identification of compounds in mixtures [10].
This study aimed at the extraction, isolation of potent antibiotic produced by Streptomyces anulatus NEAE-94 active against multidrug-resistant strains. Furthermore, the antibiotic was characterized by ultraviolet (UV)-visible spectroscopy, MS, 1 H-NMR, FT-IR and GC-MS analyses.

Micro-organisms and growth conditions
Streptomyces anulatus NEAE-94 was kindly provided by the first author, Dr Noura El-Ahmady El-Naggar (Department of Bioprocess Development, Genetic Engineering and Biotechnology Research Institute, City for Scientific Research and Technological Applications, Alexandria, Egypt). This isolate was maintained on slopes containing starch-nitrate agar medium of the following composition: 20 g/L of starch, 2 g/L of KNO 3 , 1 g/L of K 2 HPO 4 , 0.5 g/L of MgSO 4 .7H 2 O, 0.5 g/L of NaCl, 3 g/L of CaCO 3 , 0.01 g/L of FeSO 4 .7H 2 O, 20 g/L agar and distilled water up to 1 L. Slopes were incubated for a period of 7 days at 30 C. The isolate was stored as spore suspensions in 20% (v/v) glycerol at ¡20 C [11] for subsequent investigation.
Antimicrobial agent activities were tested against a group of multidrug-resistant bacteria isolated from various clinical samples and kindly provided by the Infection Control Unit, Department of Medical Microbiology and Immunology, Faculty of Medicine, Mansoura University, Mansoura, Egypt: Staphylococcus aureus A9897 (this strain is resistant to vancomycin, co-clavulanic acid-amoxicillin, gentamicin, trimethoprim-sulfamethoxazole, oxacillin, amikacin and tobramycin), Pseudomonas aeruginosa T9934 (resistant to ceftriaxone, gentamicin, cefotaxime, trimethoprim-sulfamethoxazole and co-clavulanic acidamoxicillin) and Klebsiella pneumonia A9898 (resistant to trimethoprim-sulfamethoxazole, co-clavulanic acid-amoxicillin, gentamicin, ceftriaxone, amikacin and cefotaxime).
The antimicrobial activities were also tested against a group of bacteria belonging to the Culture Collection of NRRL: Gram-positive (S. aureus NRRL B-313 6538 and Bacillus subtilis NRRL B-543), Gram-negative (Escherichia coli NRRL B-210 and Pseudomonas aeruginosa NRRL B-23) and Candida albicans NRRL Y-477. Stock cultures of the test organisms were maintained on nutrient agar slants. The inoculated agar medium was incubated for 24 h at 30 C, and then maintained at 4 C until further use.

Inoculum preparation
Erlenmeyer (250 mL) flasks containing 50 mL of yeastmalt extract broth (1% malt extract, 0.4% dextrose, 0.4% yeast extract, 2% agar; pH 7.0) were inoculated with three disks of 9 mm diameter taken from the 7-day-old stock culture grown on starch nitrate agar medium [12]. The flasks were incubated for 48-72 h in a rotary incubator shaker at 30 C and 200 r/min and were used as inoculum for subsequent experiments.

Production conditions
Fifty millilitres of fermentation medium were dispensed in 250-mL conical (Erlenmeyer) flasks, inoculated with previously prepared inoculum. The inoculated flasks were incubated on a rotatory incubator shaker at 150-250 rpm and 25-30 C. After the specified incubation time for each set of experimental trials, the mycelium of each isolate was collected by centrifugation at 5000 g (Z326K centrifuge, Hermle Labortechnik GmbH, Germany) for 15 min. The cell-free supernatant was used for antimicrobial activity assays.

Antagonistic action against microbial test strains
The well-diffusion technique was used to test the ability of the isolate to inhibit the growth of several Grampositive, Gram-negative bacteria and C. albicans. Fifty millilitres of nutrient agar medium were poured into Petri plates. After solidifying, plates were inoculated with test strains and wells were punched out using a 9-mm cork borer. One hundred microliters of tested filtrates were transferred into each well. All plates were incubated at 30 C for 24 h. After the incubation period, the plates were observed for zone formation around the wells. The zone of inhibition was calculated by measuring the diameter of the inhibition zone around the well (in mm) including the well diameter.

Extraction and antimicrobial assay of antimicrobial metabolites extracts
The fermentation broth of S. anulatus NEAE-94 was prepared and centrifuged at 5000 g for 20 min at 4 C. The supernatant was filtered; the filtrate was subjected to solvent extraction to recover antibacterial metabolites in pure form. Organic solvents (n-butanol, chloroform, ethyl acetate, n-hexane, benzene, petroleum ether, carbon tetrachloride and diethyl ether) were used in the extraction of the antibacterial metabolites. Different solvents were added to the filtrate in a ratio of 1:1 (v/v) and were shaken vigorously for 1 h for complete extraction. As ethyl acetate was standardized as the best solvent to extract the antibiotic, the organic ethyl acetate phase was collected and concentrated by evaporation to near dryness under reduced pressure by using a rotary evaporator. The antimicrobial activity of the crude extract was tested by using the filter paper disc diffusion method assay on multidrug-resistant S. aureus. The antibiotic was precipitated by addition of petroleum ether to the concentrated ethyl acetate extract with gentle stirring.

Thin-layer chromatography
Readymade pre-coated TLC plates were used for separation of crude compounds. Using the capillary tube, a row of spots of the active fraction of ethyl acetate extract was applied 1.5 cm above the bottom of the TLC plates. The spots were left to dry. The TLC plate was placed vertically in a developing beaker containing the solvent chloroform/ethyl acetate, in a ratio of 1:9, covered with the glass in order to prevent the evaporation of the solvents. The solvent was allowed to run till it moved up to 80% of the TLC plate. After running, the sheet was kept at room temperature for the complete drying of the plate. After drying, the Rf value of the spot separated on the TLC plate was determined. Then the plates were viewed under UV light. The developed yellowish orange spot was scrapped, mixed with ethyl acetate and centrifuged at 3000 g for 15 min. The supernatant was collected and impregnated with sterile filter paper disc and was tested against multidrug-resistant S. aureus spread on the surface of the plate containing nutrient agar medium by the disc diffusion method. The plate seeded was then incubated at 30 C for 24 h. The presence of inhibition zones around the disc was determined.

FT-IR spectroscopy
The infrared (IR) spectrum of the ethyl acetate extract was measured (as KBr discs) in the range of 400-4000 cm ¡1 on FT-IR spectrophotometer (Shimadzu FTIR-8400 S, Kyoto, Japan). The important IR bands, , ʋ (CO) and (CH) symmetric and asymmetric stretching, and stretching frequencies were studied to determine the presence of functional groups in the ethyl acetate crude extracts.

UV spectroscopy
The UV absorption spectrum of the purified active compound (dissolved in chloroform) was scanned in a UVvisible spectrophotometer (Optizen Pop, Daejeon, South Korea). It was used to detect the presence of chromophores like dienes, aromatics, polyenes, and conjugated ketones, etc. [13]. 1

H-NMR spectroscopy
The NMR spectrum of isolated products was obtained on a DRX-400 ( 1 H 400 MHz) instrument from BRUKER (Karlsruhe, Germany). The internal standard was tetramethylsilane.

GC-MS spectroscopy
GC-MS analysis of the isolated products was performed using a Perkin-Elmer GC-Clarus 500 gas chromatograph (Shelton, CT, USA) and gas chromatograph interfaced to a mass spectrometer (GC-MS) equipped with an Elite-I, fused silica capillary column (30 mm £ 0.25 mm 1D £ 1 mm df, composed of 100% dimethylpolysiloxane). For GC-MS detection, an electronionization system with ionizing energy of 70 eV was used. Helium gas (99.999%) was used as the carrier gas at a constant flow rate of 1 mL/min and an injection volume of 2 mL was employed (split ratio of 10:1). The injector temperature was 250 C and the ion-source temperature, 280 C. The oven temperature was programmed from 110 C (isothermal for 2 min) to 200 C, with an increase of 10 C/ min, then 5 C/min to 280 C, ending with a 9 min isothermal at 280 C. Mass spectra were taken at 70 eV; a scan interval of 0.5 s and fragments from 45 to 450 Da. The total GC running time was 36 min. The relative % amount of each component was calculated by comparing its average peak area to the total areas.
The software adopted to handle the mass spectrum and chromatograms was Turbo-Mass ver. 5.2.

Results and discussion
As a part of our search for more potent antibiotic, one promising isolate, S. anulatus NEAE-94, was found to produce a broad spectrum antibiotic with strong antibacterial activity against several pathogenic multidrug-resistant bacteria ( Figure S1 in the Online Supplementary Appendix) and Candida albicans as described in our earlier report [14]. This strain was, therefore, selected for further isolation of its bioactive metabolites and their structure elucidation. Figure 1 shows a schematic representation of the procedures involved in antibiotic isolation, purification, characterization and structure elucidation.
Actinomycetes, and especially Streptomyces species, are commonly recognized as a prolific source of secondary metabolites [15]. Due to their widespread distribution and remarkable capacity to produce a wide range of bioactive compounds [16], Streptomyces species have attracted considerable commercial interest. Soil streptomycetes are major producers of bioactive secondary metabolites [17][18][19][20]. It has been reported that organic solvents always provide a higher efficiency in extracting compounds for antimicrobial activities compared to water-based methods [21,22]. In our study, the antimicrobial products were extracted using ethyl acetate; the extract had different levels of activity against the tested Gram-positive, Gram-negative bacteria and C. albicans. Our previous results on the antimicrobial activity against all tested strains, bacteria and C. albicans [14], showed that the halo diameters obtained with S. aureus NRRL B-313, multidrug-resistant S. aureus and Bacillus subtilis NRRL B-543 were 27, 20 and 20 mm, respectively, whereas those obtained with E. coli NRRL B-210, P. aeruginosa NRRL B-23, multidrug-resistant P. aeruginosa T9934 and C. albicans NRRL Y-477 were 18, 17, 15 and 17 mm, respectively. Different solvents like benzene, diethyl ether, ethyl acetate, hexane, chloroform, petroleum ether, butanol and carbon tetrachloride were used and tested for the extraction of the antibiotic. As seen in Table 1, ethyl acetate was the best solvent for maximum antibiotic extraction. No antimicrobial metabolite activity was seen with petroleum ether and solvent control wells.

Thin-layer chromatography
The purification and separation of crude compounds was done using TLC [23]. The R f value was calculated to be 0.83 (Table 2). Then the plates were viewed under UV light. The spot was scrapped out and put in vial, then checked again for antimicrobial activity by the agar well diffusion method. The purified oily, yellowish orange active compound (Table 2) yielded by the filtrate of S. anulatus NEAE-94 was subjected to spectroscopic analyses in an effort to elucidate its structure. The chemical structure of the antibiotic was studied on the basis of UV, FT-IR, MS, 1 H-NMR and GC-MS analysis.

UV spectroscopy
The UV absorption spectrum of the purified active compound (dissolved in chloroform) indicated the presence of R-COOH with a maximum absorption at λ max of 206 nm, C D O group with a maximum absorption at λ max of 218 nm, conjugated polyene with a maximum absorption at λ max of 225 nm, CHCl 3 solvent with a maximum absorption at λ max of 237 nm (CHCl 3 ), aromatic ring with a maximum absorption at a λ max of 256 nm, C D O group with a maximum absorption at λ max of 428 nm, (conjugated double bond system) with a maximum absorption at λ max of 434 nm and polypyrrole with a maximum absorption at λ max of 446 nm.

FT-IR spectroscopy
The FT-IR spectra exhibited bands at 567. 03 [24,25]. The IR spectrum of the compound ( Figure 2) included a diagnostic peak at 3737 cm ¡1 , which is indicative of the -OH group. However, the peak at 3026 cm ¡1 was assigned to aromatic C-H stretching. The peak appearing at 2948 cm ¡1 was assigned to CH 3and -CH 2stretching (alkanes). The peak appearing at 2410 cm ¡1 was assigned to OH (alcohol) symmetric stretching. The peak appearing at 1519 cm ¡1 was assigned to C D C stretching (alkene). The peak appearing at 1421 cm ¡1 was assigned to COO ¡1 stretching (fatty acid ester). The peak appearing at 1215 cm ¡1 was assigned to C-O group. The peak appearing at 1020 cm ¡1 was assigned to ether. The peak appearing at 927 cm ¡1 was assigned to CH 3 . The peak appearing at 757 cm ¡1 was assigned to alkene and the peak appearing at 567 cm ¡1 was assigned to alkyl halide.

H-NMR spectrum
The 1 H-NMR spectrum of the isolated compound (Table 3 and Figure 3) revealed RCH 2 R group in the region of 1.23-1.27 ppm; two solvent residual peaks at 7.2 and 0.86 ppm; a peak at 1.6 ppm, which was assigned to RCH 2 CH 2 R (alkane); a peak at 1.9 ppm, which was assigned to R 2 C D CRCHR 2 (alkene); a peak in the region of 2.1-2.3 ppm, which was assigned to RCOCH 3 ; a peak at 2.5 ppm, which was assigned to COOR (fatty acid ester); a peak at  Benzene  pH 3  20  22  0  23  16  36  27  22  pH 7  21  0  0  22  0  33  22  0  pH 10  23  0  0  27  0  32 23 0  2.8 ppm, which was assigned to ArCH 2 R; a peak at 3.2 ppm, which was assigned to R 2 CHOR; a peak at 3.4 ppm, which was assigned to RCH 2 OH; a peak at 3.6 ppm, which was assigned to OCOR (fatty acid ester); a peak at 3.84 ppm, which was assigned to RCOOCH 3 and ones in the region of 6.8-6.81 ppm and 7.84-7.87 ppm, which were attributed to aromatic H.

MS spectroscopy
The electron impact (EI) mass spectrum confirmed that the molecular weight of the antibiotic was 1255.40 g/mole (M C peak) ( Figure 4 and Table 2).        (6), 207 (7), 191 (7), 183 (7), 169 (7), 155 (8) Some of these fatty acids have been detected from actinobacteria. Fatty acids are promising as antimicrobial agents in medicine and the food industry due to their safety, non-specific mode of action and the lack of resistance mechanisms against the actions of these fatty acids [26,27]. When S. aureus colonization occurs in the skin, human skin summons a battery of host-defence mechanisms responsible for natural protective immunity, which include fatty acids and antimicrobial peptides [28]. The most potent antibacterial fatty acid in mice is 9-hexadecenoic acid, and in humans, its isomer 6-hexadecenoic acid [29]. In humans and mice, these two 16-carbon monounsaturated fatty acids block the growth of S. aureus in the skin [29][30][31]. It has been hypothesized that fatty acids act by destabilizing the lipid bilayers of the bacterial membrane and disrupting the membrane functions [32] due to a range of effects on cellular metabolism [33][34][35][36][37][38].
Cis-2-decenoic acid (C2DA) is an unsaturated fatty acid. It disperses biofilm in many strains of micro-organisms [39]. C2DA has been reported to inhibit the growth of multidrug-resistant S. aureus and biofilm in vitro [39]. In 2006, Karanja et al. [40] identified non-adecane from Chy 4-10, a novel Streptomyces isolate from Chyulu National Park, by GC-MS. Nonadecane is a component of the bacteriocin produced by a mixed culture of Aeromonas sp. and Enterobacter sp. and is responsible for its high antibiofilm activity [41]: it has cytotoxic and antimicrobial effect [42]. Selvin et al. [43] detected hexadecanoic acid and hexadecanoic acid methyl ester from Nocardiopsis dassonvillei MAD08 by GC-MS analysis. They act as antioxidant, hypocholesterolemic nematicide, pesticide, lubricant, antiandrogenic, flavour, hemolytic and 5-alpha reductase inhibitor. Other constituents of the Streptomyces Chy 4-10 extract detected by GC-MS are n-hexadecanoic acid, squalene and tetracosane [40]. Tetracosane acts as antioxidant [44] and squalene has antibacterial, antioxidant, antitumour, cancer preventive and immunostimulant activity [45].
Overall, the ethyl acetate extract of S. anulatus NEAE-94 with its 22 bioactive compounds identified by GC-MS analysis in this study showed antimicrobial activity comparable to conventional drugs. According to Lancini et al. [60], the activity of an antibiotic is defined and measured in terms of its ability to inhibit microbial growth (bacteria, fungi) and protozoa. The wide range of bioactive constituents in the ethyl acetate extract of S. anulatus NEAE-94 is in good agreement with other recent studies. For example, Nandhini et al. [48] reported the presence of 19 bioactive compounds in the ethyl acetate extract of Streptomyces cavouresis KUV39, using GC-MS analysis. These results make S. anulatus NEAE-94 an attractive strain for further investigation and industrial exploitation.

Conclusions
In the present study, S. anulatus NEAE-94 showed a broad antimicrobial spectrum, as it inhibited Gram-positive, Gram-negative bacteria, as well as Candida albicans. Based on the data analysis, the S. anulatus NEAE-94 extract could be considered to have biological activity as good as other conventional drugs against such microorganisms. Further studies are suggested in the future to evaluate the pharmacological potentials of the extracted bioactive metabolites. On the basis of the results of this work, we can recommend the wild isolate S. anulatus NEAE-94 as a good source of bioactive metabolites that inhibited Gram-positive, Gram-negative bacteria as well as fungi. It shows promising potential to be employed as antibiotic for treatment of human pathogenic bacterial and fungal infections.