Abstract
Seven endophytes bacteria were isolated from Moringa peregrina and identified using 16S ribosomal RNA sequences, which revealed two bacterial genuses, namely Proteus mirabilis and Bacillus; three species belong to Bacillus: B. licheniformis, B. subtilis subsp. Inaquosorum and B. pumilus. The antimicrobial activity of the isolate was screened against 11 human pathogens, and the potency of the selected isolate was qualitatively and quantitatively analyzed by the cross-streak method and minimum inhibitory concentrations (MICs), respectively. Minimum inhibitory concentration (MIC) of the organic extract of selected isolate B. licheniformis MpKL1 was found to exhibit appreciable growth inhibition between 125 µg/ml and 500 µg/ml against most tested human pathogens and less influence against Acinetobacter baumanii, Enterobacter aerogenes, Streptococcus pyogenes and Candida albicans with MIC > 1000.
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Medicinal plants are the most useful source of lifesaving drugs for humans, animals and plants. Bioactive compounds that are usually extracted from plants are used as medicines, food, insecticides and other industrially important chemicals. One such medicinal plant is Moringa peregrina, belonging to the family Moringaceae, which is mainly found in the tropical and subtropical areas. M. peregrina species is commonly known as the miracle tree, ranging in size from a tiny plant to massive tree [1]. Almost all the parts of M. peregrina are edible and have been consumed as a vegetable and used to treat many ailments such as tumors, hysteria, scurvy, paralysis, sores and skin infections [2, 3]. M. peregrina is an economically important plant of the Arabian and North African region [4, 5]. The occurrence of the associated microbial flora of this important plant is yet to be studied; therefore, looking at the importance and the potential of the plant to withstand extreme environmental conditions, we aimed to assess the endophytes flora of the plant in the present study.
Bacteria and fungi live asymptomatically within the plant tissue without manifesting their presence in the host tissue [6].
A wide variety of endophytes can be observed as colonizing the plant parts, these colonies remain symptomless, and the plant tissue remains unaffected and functional. Several endophytes have been isolated from the tissues of aquatic and terrestrial plants [7, 8]. The wide presence of an endophyte in plant tissue creates an effective barrier preventing an attack of the pathogens to the host plant. It has been observed that metabolites produced by endophytes inhibit the growth of pathogens. Furthermore, the endophytic association of plant enables it to withstand the environmental stress and also from various predations. The plant, in turn, provides shelter and nutrients to the endophytes [9]. The present study aims to isolate endophytes from the selected medicinal plant, M. peregrina, and to explore their antimicrobial activities against a number of human pathogens. This study is driven by the fact that infectious microorganisms are developing resistance to the available antibiotics; hence, there is a need for discovering new antibiotics and therapeutic agents that are highly effective against a wide range of pathogens, less toxic and cost-effective. Endophytes were isolated using the method described by [10]. From the various parts of M. peregrina such as the root, twig and leaves, growing in the eastern region (Dammam and Alkobar) of Saudi Arabia, samples were collected from each region in a sterilized poly-bag during transportation and stored at 4 °C until use. Then, they were thoroughly washed in running tap water and surface sterilized with 75% (v/v) ethanol. The outer tissues of the root and twig were removed and cut into smaller pieces, and the leaf tissues were excised and macerated with sterile distilled water by sterile mortar and pestle. Then, they were plated separately into minimal media (agar 1.5% w/v) supplemented with antibiotic cycloheximide 30 µg/ml and incubated at 37 ± 2 °C. The isolates were characterized and identified both morphologically and physiologically. The identification of isolates was based on the 16S ribosomal RNA sequences by the Genetic Unit in the Institute for Research and Medical Consultations. Test organisms included: E. coli ATCC25922, Pseudomonas aeruginosa ATCC27853, Klebsiella ocytota ATCC700324, K. pneumonia ATCC100324, Acinetobacter baumanii ATCCmra747, Enterobacter aerogenes ATCC13048, Streptococcus dgalacticae ATCC12336, Streptococcus pyogenes ATCC 19615, Staphylococcus epidermis ATCC12228, Staphylococcus aureus ATCC24213 and Candida albicans. The strains were maintained on agar slants at 4 °C and activated at 37 °C for 24 h on nutrient agar prior to any study. All the endophytes isolates were inoculated on nutrient agar by a single streak of inoculum at the center of the Petri dish, while the unstreaked nutrient agar plate was assigned as a control plate. After 2 days of incubation at 37 °C, all the plates including the control plates were seeded with test bacteria by a single streak at a 90° angle (perpendicular) to the endophytes. Both experimental and the control plates were incubated at 37 °C. The assay was carried out in replicates. Based on the results from the cross-streak method, endophytes isolates from M. peregrina leaves (MpKL1) were selected and further inoculated in five numbers of 250-ml Erlenmeyer flasks, each containing 50 ml nutrient broth at 37 °C for 48 h. The seed culture was transferred to five numbers of the 2-L Erlenmeyer flask, each containing 1000 ml nutrient broth at 37 °C for 7 days to obtain a good quantity of pellet. The culture cell pellet was obtained by centrifugation (15000 rpm), and the supernatant was extracted with two types of organic solvents: chloroform/methanol 5:1 (MpKL1 cm) and ethyl acetate (MpKL1ea) in a separating funnel with vigorous shaking. The organic extract was recovered and then the organic solvent was evaporated by rotary evaporation at 30 °C. The dry weight of organic extract was taken and dissolved in dimethyl sulfoxide (DMSO) for further studies [11]. The Moringa plant species are a valuable source of useful metabolites that has been known for decades. In the present study, seven endophytic isolates were obtained from the samples of M. peregrina, and this result emphasizes that all types of plant species harbor endophytic bacteria. This finding is inconsistent with other researchers where they had stated that the aerial plant parts have a higher endophytic content than underground parts [12]. The result based on the 16S rRNA sequencing revealed two bacterial genus Proteus mirabilis and Bacillus; three species belong to Bacillus: B. licheniformis, B. subtilis subsp. Inaquosorum and B. pumilus. This finding revealed that the Bacillus species is prevalent in this plant (Table 1). Few studies reported endophytic isolate from M. peregrina. Khan et al. [13] isolated two fungi genus Aspergillus caespitosus LK12 and Phoma sp. LK13. Interestingly, there is one study that reported endophytic bacterial isolate from this plant by Khan et al. [14] who isolated five endophytic bacteria: Methylobacterium radiotolerans, two species of Sphingomonas sp. and two strains of Bacillus subtilis. However, isolation of endophytic bacteria from different plants was reported by Amaresan et al. [15] who isolated 37 endophytic bacteria from chili plant. Endophytes are a group of microorganisms that could serve as dependable source of new and highly useful compounds with potential for exploitation in pharmaceutical and agricultural areas [16]. As pathogenic organisms are developing resistance to the majority of antibiotics available, it is important to find new antibiotics to tackle this problem. The result reveals that all endophytic isolates exhibited antagonistic activity against two or more of the pathogenic human microbes (Table 2). Moreover, M. peregrina Alkobar leaf 1 (MpKL1) isolate showed promising antimicrobial activity against all the test organisms with moderate to less activity against only a few organisms such as A. baumanii, S. pyogenes and E. aerogenes. The results of this study in agreement with several researches showed endophytic bacteria exhibit antimicrobial activity against human pathogenic microbes [17]. Also, using low concentrations of the active substance negatively affects their efficacy. Thus, it is important to determine the MIC of the active ingredient and, moreover, to reduce the possible cell toxicity risk of the active substance.
MpKL1 was further studied for its MIC values. The MIC of MpKLea (ethyl acetate extract) showed a high inhibitory effect against gram-negative bacteria E. coli, P. aeruginosa at 125 µg/ml and K. ocytota, K. pneumonia at 250 µg/ml. Also gram-positive bacteria S. epidermis and S. aureus highly inhibited at 250 µg/ml (Fig. 1).
Moreover, MpKLcm (chloroform/methanol extract) also showed high inhibition effect at 250 µg/ml against gram-negative bacteria E.coli, P. aeruginosa, K. ocytota and K. pneumonia, whereas MIC of MpKLcm raised up to 500 µg/ml to inhibit the growth of gram-positive bacteria S. dgalacticae and S. aureus. Also, MIC of MpKLea raised up to 500 µg/ml to inhibit the growth of S. dgalacticae, S. epidermis and Candida albicans. In general, both the extracts were not found very active against A. baumanii, E. aerogenes, S. pyogenes and Candida albicans with MIC > 1000. Ciprofloxacin was used as a standard antibiotic (Table 3).
This assay confirms that there is an appreciable degree of antibacterial activity against human pathogenic organisms. The future prospect of the study is the economic feasibility of the antibacterial drug development from these isolated endophytes. This finding is in agreement with several studies which revealed that endophytic bacteria have been proven as reliable sources of novel bioactive compounds and the richest source of secondary metabolites.
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Acknowledgements
The authors are grateful to the Research Unit—Al-Rayan Camps and to Mr. Badar Alsaqer and Dr. J. Francis Borgio, and they are also grateful to Dr. Suriya Rehman for her research support.
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Aljuraifani, A., Aldosary, S. & Ababutain, I. In Vitro Antimicrobial Activity of Endophytes, Isolated from Moringa peregrina Growing in Eastern Region of Saudi Arabia. Natl. Acad. Sci. Lett. 42, 75–80 (2019). https://doi.org/10.1007/s40009-018-0739-6
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DOI: https://doi.org/10.1007/s40009-018-0739-6