Diversity and characterization of bioactive compounds of endophytic bacteria from medicinal plants

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Endophytes are believed to produce a number of beneficial bioactive compounds of pharmacological importance. The aims of this study are to evaluate the diversity and characterize the bioactive compounds of bacterial endophytes from selected local medicinal plants. Eighty-seven species of endophytic bacteria were isolated from surface sterilized tissues of six local medicinal plants of which 29 were isolated from Aloe vera, 15 from Mentha spicata, eight from Ocimum basilicum, 16 from Cymbopogon citratus, 10 from Morinda citrifolia and eight from Triticum aestivum. A. vera has the highest diversity of endophytic bacteria based on its richness and evenness of the genera and species of endophytic bacteria obtained from its tissues. Pseudomonas, Bacilli and Enterobacter were the most common genera from the six medicinal plants. The roots gave the highest population of 41%, stems 34%, and leaves 25%. Five isolates: - Bacillus tequilensis ALR-2, Chryseobacterium indologenes ALR-13, Pseudomonas entomophila ALR-12, Pseudomonas putida MSS-3 and Sphingomonas yabuuchiae TAR-1 were selected for further evaluations of their antimicrobial and antioxidant activities. The free radical scavenging effect of the five isolates’ extracts (10 mg/mL) on the DPPH radical decreased in the order of ALR-2>ALR-13>ALR-12>TAR-1>MSS-3 (77.4, 69.5, 65.7, 62.6 and 60.8% respectively). In addition, extracts from ALR-2 exhibited the highest phenolic, flavonoid contents, ferric reducing antioxidant power and relatively high antioxidant activity with a relatively low IC₅₀ (1.95 ±0.1 mg/mL) which was significantly different (p < 0.05) when compared with other four. In addition, the ethylacetate extract of ALR-2 isolate showed the strongest antimicrobial effects (9.2 to 13.7 ±0.8 mm zone of inhibition) on B. cereus, S. pyogenes, S. aureus, E. faecalis and C. albicans pathogens. The MIC and MBC values of ALR-12 and ALR-2 extracts gave the lowest MIC (0.625 mg/mL, 1.25 mg/mL) and MBC (1.25 mg/mL, 2 mg/mL) respectively. Consequently, Bacillus tequilensis ALR-2 extract was partially purified to obtain its bioactive compounds. The partially purified compound SF5 (5 mg mLˉ¹) indicated strong inhibition against B. cereus, S. pyogenes, S. aureus, E. faecalis and K. pneumoniae with zone of inhibition from 8.6 to 30.3 ±0.5 mm. These were significantly more effective than the standard antibiotics, Ciprofloxacin 5 µg (18.6 ±0.5 to 23.6 ±0.5 mm) and Kanamycin 30 µg (13.6 ±0.5 to 20.3 ±0.5 mm). The UPLC-MS analysis of SF5 showed three series of ion peaks. A further electrospray ionization/collision-induced dissociation spectrum revealed the three ion peaks series represented different fengycin homologues (lipopeptides) and surfactin homologues. Predominant peaks at [M+H] m/z = 1356.0151; 1358.0664; 1105.8010; 1119.8870; 1120.8992 and 1121.9119 revealed ions which were compatible with lipopeptides and surfactins. One of the ion [M+H] m/z = 1120.8992 was compatible with antibiotic 61-26 (accurate mass =1119.6750 and molecular weight = 1120.348) which was isolated from a Bacillus sp. as reported in the literature. Conclusively, in this study Bacillus tequilensis ALR-2 was found to be co-producer of fengycins and surfactins as suggested by the UPLC-MS analysis and antimicrobial activities. These natural lipopeptide compounds can be of use in the food preservation, control of plant diseases and pharmacological applications