Antimicrobial quercetin 3-O-glucoside derivative isolated from Streptomyces antibioticus strain ess_amA8

1 Introduction

Incurable diseases are becoming increasingly commonly, with the rise in non-sensitivity of microbes to antibiotics; the lack of new alternatives poses a threat to world health (Jones et al., 2008). This serious rise in antibiotic resistance of microbes necessitates research on unconventional methodologies for discovering new drugs (Kumar et al., 2017). Biological diversity is a major resource for exploring new products with specific biological activities; exploring natural, biologically active compounds from insect-microbe symbioses is one of the most unconventional methods of drug discovery (Brachmann and Bode, 2013). Oh et al. (2011) isolated Streptomyces sp. from wasp associated symbiotic microbes and those insects are lactam family members. Poulsen et al. (2011) studied the antagonistic activity between Streptomyces sp. associated with solitary wasps, to find whether they produced compounds with antimicrobial activities against fungi and bacteria. Their results support the fact that isolated actinomycetes are associated with insect and they are the completely new source for biologically active novel compounds. A study by Barke et al. (2010) showed the potential role of the symbiotic relationship in the production of antifungal metabolites from Streptomyces sp. associated with leaf-cutting ants; these species could be a valuable source of novel antimicrobial agents. Candicidin macrolides, which are extracted from antagonistic actinomycetes, are effectively kill the fungal pathogen Escovopsis, but not against the ant’s garden fungus Leucoagaricus gongylophorus (Haeder et al., 2009). Previous studies showed that the actinomycetes isolated from termite gut samples were selectively active against some gram-negative and gram-positive pathogenic bacteria, and pathogenic yeast (Arango et al., 2016). Our study aimed to isolate a new strain of Streptomyces species competent to produce antimicrobial compounds combat pathogenic bacteria viz., P. aeruginosa, B. subtilis, S. aureus and E. coli, and pathogenic fungi such as Fusarium moniliforme, A. niger, A. flavus, C. albicans. Further, the biologically active compound was purified and identified.

2 Materials and methods

3 Results

The actinomycete isolate was identified to the species level according to its specific characteristics. Microscopically, it was observed that the morphology of the spore chains of aerial mycelium is retinaculum apertum (Fig. 1). Furthermore, for adequate identification, melanin production is one of the important physiological characters in Streptomyces taxonomy. The finding showed (Table 1) the isolate did not produce melanin pigments on the media used, whereas the aerial mycelium is with different colors accoriding to growth medium. The isolate was inoculated on 9 different media and the growths were observed after 48 h. The results were interpreted as the following values: weak, moderate, good, very good. Good Growth of the isolate was observed on ISP2, ISP4, ISP5, ISP7, Starch nitrate agar, Nutreint agar and Czapek's agar while was weak on ISP3 and ISP6 (Table 1). The Streptomyces sp. utilized protein and starch, but not lipids and no producing H₂S as shown in Table 2. It liquefied gelatin. The actinomycete isolate was selected for an analysis of the enzyme activities of the various species, to identify the isolates exhibiting the highest enzyme potential. The results showed that the amylase, protease, catalase, DNase, and urease tests yielded positive results, while the chitinase and lipase tests yielded negative results; this is shown in Table 2. After 7 days of incubation under different conditions, the growth of isolates was observed and the results were recorded, as shown in Table 2. The results show that the prime temperature for growth of this isolate is 30 °C; it did not grow at 45 °C. Excellent growth of the Streptomyces isolate could be seen within a temperature range of 26–30 °C (Table 2). Good growth was observed within an NaCl range of 1–4 %, and at a pH of 7.0 (Table 2). The results in Table 3 showed that the isolate can utilize rhaffinose, fructose, sucrose, arabinose, lactose, D-galactose, D-xylose, Meso-inositol, cellobiose and maltose, but could not utilize salicine, and cellulose as a carbon source. Good Growth of the Streptomyces sp., as shown in Table 3, was observed when L-valine, lycine, L-methionine, L-arginine, L-aspargine, L-orinthine and L-histidine were used as nitrogen sources; weak growth was seen in case of L-Phenyl alanine, and no growth was observed in case of L-Cysteine. The Streptomyces sp. was sensitive to erythromycin (15 µg), gentamicin (10 µg), kanamycin (1000 µg), colistin sulfate (10 µg), ceftazidime (30 µg), and ciprofloxacin (1 µg), but not to tetracycline (30 µg) and chloramphenicol (30 µg). The isolate was tested using conditions of environmental stress to identify the isolates with a maximum growth range and resistance to stress. Finally, according to biochemical and molecular characterization, the actinomycete isolate was identified as Streptomyces antibioticus strain ess_amA8 with the accession number KF996507.

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Fig. 1

Morphological characterization of strain ess_amA8. a) Culture of S. antibioticus strain ess_amA8 on ISP 4 agar. b) Shape of the spore chain under light microscope. c) Shape of the spore chain under scanning electron microscope.

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In this study, the antagonistic activity of strain ess_amA8 was tested against F. moniliforme, A. niger, A. flavus, C. albicans, P. aeruginosa, B. subtilis, S. aureus, and E. coli. The results showed that the strain has the ability to inhibit the growth of all the tested pathogenic microbes except A. flavus, as shown in Table 4. The most potent species from the Actinomycetes isolate was isolated on the basis of the wide spectrum of its antimicrobial activity. The results recorded in Table 5 indicated that the maximum extraction of the bioactive substance occurred at a pH range of 2–7 (acidic range) using ethyl acetate, chloroform, and petroleum ether, while the extraction decreased or failed when pH values rose to the alkaline range (>7.0). Additionally, n-butanol failed to extract the active substance at different pH values. Hence, ethyl acetate was chosen as the most suitable organic solvent to extract the active substance, especially when the active filtrate was adjusted to a pH of 7.0. The best solvent system was ethanol: Chloroform: acetic acid (6: 2: 2 v/v/v), and it was utilized at room temperature. This solvent system separated the crude extract into six different spots, and each spot was bioassayed with S. aureus. The active spot was at R_f 0.85. The pure, bioactive compound was also tested with other pathogenic organisms. Finally, the pure active compound had a melting point of 232° C. The purified compound was isolated as an amorphous pale yellow powder with no characteristic odor; it appeared yellow in acidic and alkaline solutions. The isolated compound was soluble in chloroform, ethyl alcohol, ethyl acetate and methanol, and it was insoluble in petroleum ether, hexane, benzene and water. The absorption peaks of compound showed at 252 and 494 nm that is features of highly conjugated systems. Structural elucidation of the compound could be achieved by analysis of its UV, NMR, and MS spectral data (Fig. 2 and Fig. 3). According to physicochemical characterizations, the purified compound produced by strain ess_amA8 was identified as a quercetin 3-O-glucoside derivative (Fig. 2 and Fig. 3).

Table 5 Bioactivity of strain ess_amA8 extract using different organic solvents and different pH values.

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Fig. 2

a) UV spectrum; b) FTIR spectrum of active compound.

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Fig. 3

NMR Spectrum of the bioactive compound produced by S. antibioticus strain ess_amA8.

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4 Discussion

The isolated actinomycete was identified to the species level according to its specific characteristics. Spore masses were matched against the seven color wheels of Tresner and Backus (1963) as used in the ISP (Shirling and Gottlieb, 1966). Microscopically, it was observed that spore chains of its aerial mycelium showed Retinaculum-Apertum morphology (Pridham et al., 1958). The strain wasidentified as a member of S. antibioticus by 16S rRNA gene sequencing and the sequence has been deposited in GenBank (Accession Number: KF996507). The sequenced 16S rRNA gene has revealed that the strain ess_am8 showed 99.0% similarity with S. antibioticus. Classification of the isolate was initially established, based on its morphological and physiological characterizations. Nowadays, molecular approaches by 16S rRNA gene analysis were commonly used for identification of actinomycetes (Nimaichand et al., 2013). The foremost intention of this research was to discover novel, biologically active secondary metabolites. Insect bodies are the most common habitats of Streptomyces; symbiotic associations occur between the insects and the actinomycetes. Our research was centered on actinomycetes and more particularly on genus Streptomyces, the species of which have documented with their remarkable antimicrobial activities (Thakur et al., 2003). As on day the genus Streptomyces has been assessed at least 1,00,000 novel biocompounds with medically interest (Watve et al., 2001). In an earlier report, the strain PE7 of Streptomyces was found to produce antimicrobial compounds (Gopikrishnan et al., 2013). In our current study, the active antimicrobial compound was identified as the quercetin and this chemical name was 3-O-glucoside derivative, 2- (3,4 - dihydroxyphenyl) − 3,5,7 –trihydroxy-4H-chromen-4-one. Quercetin is a kind of flavonoid commonly present in herbal medicines and plant foods (Materska, 2008). In addition, it also possessed antiviral, anticarcinogenic, and anti-inflammatory effects (Lee et al., 2013).