Diversity, molecular phylogenetics, and antibiotic biosynthetic potential of endophytic Actinobacteria isolated from medicinal plants in Nigeria

Abstract

Actinobacteria that are found in nature have enormous promise for the growth of the pharmaceutical sector. There is a scarce report on the antimicrobial activities of endophytic Actinobacteria from Nigeria. As a result, this study evaluated the Actinobacteria isolated from Nigerian medicinal plants in terms of their biodiversity, phylogenetics, and ability to produce antimicrobial compounds. Following accepted practices, Actinobacteria were isolated from the surface-sterilized plant parts. They were identified using 16S rRNA sequencing, microscopic, and morphological methods. The cell-free broth of Actinobacteria isolates was subjected to antimicrobial assay by agar well diffusion. Molecular evolutionary and genetic analysis (MEGA) version X was used for phylogenetic analysis, and the interactive tree of life (iTOL) version 6.0 was used to view the neighbour-joining method-drawn tree. A total of 13 Actinobacteria were recovered, belonging to three genera including 10 strains of Streptomyces, 2 strains of Saccharomonospora, and only 1 strain of Saccharopolyspora. They showed inhibitory activity against several bacterial pathogens. The phylogenetic tree generated from the sequences showed that our isolates are divergent and distinct from other closely related strains on the database. Further, optimization of the antibiotic production by selected Saccharomonospora sp. PNSac2 was conducted. It showed that the optimal conditions were the ISP2 medium (1–2% w/v salt) adjusted to pH of 8 at 30–32℃ for 12–14 days. In conclusion, endophytic Actinobacteria dwelling in Nigerian soils could be a promising source of new antibiotics. Future research is warranted because more genomic analysis and characterization of their metabolites could lead to the development of new antibacterial medicines.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-023-01196-8.

Introduction

Recent years have seen an increase in the threat posed by infections that are resistant to known medications especially antibiotics. Now, pathogenic organisms are displaying increased acquired resistance to nearly all commonly used antibiotics [1]. There is a sharp drop in antibiotic discovery and development as multidrug resistance to infectious diseases increases [2]. This raises the possibility of the pre-antibiotic period returning and eliminating all useful antibiotics for treating diseases brought on by pathogens [3]. Therefore, the demand for novel antimicrobials to tackle resistant forms of dangerous bacteria and fungi is ongoing and cyclical. Global attempts to limit drug-resistant diseases are in danger due to a lack of novel antibiotics [4]. Discovering efficient antimicrobial compounds against pathogens is urgently needed [5]. Actinobacteria are majorly found in soil and play a key role in humus production and decomposition [6]. The DNA of the Gram-positive bacteria known as actinomycetes has a high G + C concentration (> 55%). Greek words aktis (a ray) and mykes (fungus) were combined to create the name actinomycetes, also known as Actinobacteria [6]. Gram-positive filamentous bacteria known as endophytic Actinobacteria are found inside the tissues of many plant organs, including the roots, stems, leaves, flowers, fruits, and seeds. They have positive benefits on their host plants as well, such as encouraging plant growth and protecting them from phytopathogens, without harming their plant hosts. Bioactive substances, including antibiotics, enzymes, enzyme inhibitors, and other pharmacologically active substances, are abundant in members of the Streptomyces genus. The isolation of Actinobacteria works best on growth media containing natural substrates. They can be found in a variety of settings, including soil, the ocean, and others [7].

Since the majority of the secondary metabolites of Actinobacteria with antimicrobial properties are too complicated to be generated by combinatorial chemistry, the discovery of novel molecules from these organisms has signalled a new era in antimicrobial research. Additionally, as high-throughput genome sequencing techniques have advanced, numerous genome mining algorithms have been created, which have made it possible to connect newly identified metabolites to their BGCs (biosynthetic gene clusters) [8].

While the rate of re-isolation of known compounds has grown, the rate of novel compounds discovered from terrestrial actinobacteria has dropped [9]. The discovery of novel compounds during the exploration of previously unexplored ecological niches highlights the requirement to investigate novel actinobacterial groups from these habitats as sources of novel bioactive secondary metabolites [10]. The isolation of bacteria that can produce metabolites using conventional screening techniques has already been well investigated and proven. To prevent the repetitive isolation of the strains that produce known bioactive metabolites and to enhance the quality of the screened natural products, improved approaches for isolating the uncommon and less investigated rare Actinobacteria are needed. Streptomyces kebangsaanenesis, Streptomyces albidoflavus, Actinoallomurus fulvus, Micromonospora lupini, Micromonospora endophytica, Polymorphospora rubra, and Streptosporangium oxazolinicum strains are a few uncommon Actinobacteria that have been reported to produce several significant bioactive agents [11, 12]. It is not understood how the rare actinobacteria Saccharomonospora species can produce antibacterial compounds. Only a small number of authors have described their capacity to contain a wide variety of biosynthetic gene clusters (BCGs), such as polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS), ribosomally synthesized and post-translationally modified peptides (RiPPs), and several hybrid clusters that may be able to synthesize a large number of different putative products [13]. The genus Saccharomonospora had demonstrated a very high degree of innovation and variety in its BCGs when compared to other Actinobacteria genera. Rare Actinobacteria obtained from plant sources have received very little attention in studies on bioactive compounds; instead, the majority have been concentrated on Actinobacteria isolated from soil, marine, or severe environments. Furthermore, under various environmental conditions, various Saccharomonospora strains may create a large variety of secondary metabolites. Thus, altering the fermentation’s incubation period, temperature, salt concentration, and pH could increase the variety of secondary metabolites that are active against different pathogens [14]. Therefore, this work involves the isolation of different genera of Actinobacteria and assessing their potential to produce antibiotics. It also involves improving the antimicrobial production of the strain Saccharomonospora xinjiangensis PNSac2 by optimizing certain growth conditions.

Nigeria’s diversity of medicinal plants is a special advantage that can be a goldmine for the discovery of new isolates. In the current study, Actinobacteria that produce antibiotics have been isolated out of six different ethnomedicinal plants in Nigeria. We speculate that the Actinobacteria that live on these medicinal plants may be responsible for their antibacterial properties and could have the potential to yield novel antimicrobial bio-compounds in the future. Therefore, we describe the isolation of endophytic actinobacterial isolates and performed the phylogenetic analysis as well as their inhibitory or antagonistic activity on diverse bacterial pathogens.

Materials and methods

Sample collection and authentication

On the 25th of May 2022, healthy plant samples were collected from Esebor Medicinal Garden Ojuelegba, Lagos state, western part of Nigeria (6.5111° N, 3.3666° E). The collection was made during the early rainy season. These six medicinal plants are Piper guineense Schumach. (leaf), Euphorbia laterifolia Schum. & Thonn. (stem), Allium ascolonicum L. (root, leaves, and bulb), Crimum glaucum A. Chev. (bulb), Xylopia aethiopica A. Rich (fruit), and Pacrilima nitida Stapf (bark and seed). They were placed in polyethylene bags with appropriate labels. Until they were utilized within 24 h, the bags were kept in the refrigerator at 4 °C. The samples were delivered to the herbarium at the Department of Botany, University of Lagos in Nigeria, where they underwent authentication and were given voucher numbers.

Surface sterilization and pre-treatment of the plant parts

To remove sand and other particles that stuck to the plant components, each plant sample was carefully washed under a running faucet. The plant samples were surface sterilized in accordance with best practices. They were quickly submerged in 4% hypochlorite for 1 min, followed by another minute in 70% ethanol, and then three sets of sterile water were used to rinse them [15, 16]. Serially, surface sterilized plant portions were wet-treated at 50 °C for 10 h to enrich plant samples for more effective isolation of Actinobacteria. On a clean sheet of blotted paper, they were dried. To promote the isolation of rare genera of Actinobacteria, 1 g of the sample was combined with 0.5 g of sterile powdered calcium carbonate in a mortar before being inoculated onto the proper media [17].

Isolation of Actinobacteria from plant segments and their maintenance

Actinobacteria were isolated from plant parts, using the agar plating method in three different culture media including Soya Casein Dextrose Agar (SCDA), Starch Casein Agar (SCA), and International Streptomyces Project 2 (ISP2) agar. All media were supplemented with 2.5 µg/mL of rifampicin, 75 µg/mL of fluconazole, and 50 µg/mL of cycloheximide [18]. The plant segments were inoculated on the media in four different forms: (i) direct inoculation of treated plant segments onto the media; (ii) crushing of the plant segments before inoculating onto agar media; (iii) addition of 5 mL of sterile water to crushed samples, shaking thoroughly, and spreading 0.1 mL aliquots onto the media using sterilized L-shaped glass rod; and (iv) addition of 5 mL of sterilized decoction mixture onto the crushed plant segments, mixed, and spread 0.1 mL aliquot onto the isolation media. All the experiments were performed in triplicate. The plates were incubated at 28 ℃ for 4 weeks and the colonies of actinobacteria isolates were recognized according to their macroscopic and microscopic characteristics [19]. Suspected Actinobacteria were sub-cultured and maintained on International Streptomyces Project 2 (ISP 2) agar slant (Himedia) at 4 ℃ and on 20% glycerol-broth at − 80 ℃ as mycelia suspension [20].

Tested pathogens

The following test organisms were used for the bioassay of the antibiotic during the screening experiment against several clinical pathogens including Staphylococcus aureus LN029, Escherichia coli ML476, Pseudomonas aeruginosa UR978, Klebsiella pneumoniae ML602, Salmonella typhi ATCC 13311, and Bacillus cereus. The bacterial pathogens were provided by the Nigerian Institute of Medical Research (NIMR) and the Department of Microbiology, Lagos University Teaching Hospital (LUTH), Nigeria.

Selection of antibiotic-producing Actinobacteria

The selection of putative Actinobacteria isolates was done by the perpendicular streak method on Soy Casein Dextrose agar (SCDA). Plates containing SCDA were prepared and inoculated with Actinobacterial isolate by a single streak of inoculum at the centre of the petri dish. After 4 days of incubation at 28 °C, the plates were seeded with test organisms after adjusting the turbidity equal to that of 0.5 McFarland with the cell count of 1.5 × 10⁸, by a single streak at a 90° angle to Actinobacterial strains’ growth [18]. The appearance of inhibitory zones was used to examine the microbial interactions. The presence of antibacterial compounds was indicated by the growth inhibition observed close to the centrally streaking isolates. The Actinobacterial isolates found to be active against all or any of the target organisms during the perpendicular streaking assay were further subjected to the fermentation process.

Preliminary screening of Actinobacteria for antimicrobial activity by agar well diffusion method

The possible synthesis of antibiotic compounds was further confirmed by growing all putative Actinobacteria isolates on ISP-2 plates for 7–10 days at 30 °C [21]. Spores from a 1-week-old (SCDA) culture slant were suspended in 5 mL of sterile saline to create the inoculum. In a 250-mL Erlenmeyer flask, 40 mL of sterile soy casein dextrose broth was inoculated with 1 mL of the homogeneous suspension. The culture was then incubated on a rotary shaker at 50 rpm for 7 days at 30 °C. Cell pellets were obtained by centrifuging at 10,000 rpm for 10 min in a 25-mL sterile centrifuge tube [22, 23]. The supernatant was passed through 0.22-µm bacteriological filter and the filtrate was then collected and used for the antibacterial assay.

The antibacterial activity of the cell-free broth of Actinobacteria against reference pathogens was evaluated using the agar well diffusion method. Each reference pathogen colony had two to four loopfuls of material transferred to 5 mL of normal saline in a sterile container with cell density equal to 0.5 McFarland. They were briefly vortexed before being inoculated by swabs on a nutritional agar medium. To carry out a small-scale fermentation, all the isolated Actinobacteria were chosen based on the findings of the perpendicular antimicrobial assay. After a 24-h incubation period at 37 °C, the antibiotic production was assessed by looking at the inhibitory zones.

Genomic DNA extraction, PCR amplification, and sequencing of 16S rRNA gene

Little modification was made to Benhadji et al. 2019’s instructions for extracting genomic DNA [16]. Briefly, the Quick-DNA™ Fungal/Bacterial Miniprep Kit (Zymo Research, Catalogue No. D6005) was used to extract genomic DNA from the cultures. With the aid of a multigene gradient thermocycler (Labnet, with headquarters in Edison, NJ, USA), the 16S rRNA target gene area was amplified using PCR. After the reactions had been incubated at 94 °C for 3 min, they underwent 35 cycles of 94 °C for 45 s, 55 °C for 1 min, and 72 °C for 1 min 30 s. For an additional 10 min, reactions were incubated at 72 °C. The acquired PCR results were examined on a 1% agarose gel stained with ethidium bromide using a UV Gel Documentation System and UV transilluminator (Biorad, Germany) to confirm the existence of a band containing the entire 1.5-kb 16S rRNA gene.

The extracted fragments were purified using Zymo Research’s ZR-96 DNA Sequencing Clean-up Kit™ (Catalogue No. D4050) and then sequenced forward and backward using Nimagen’s Brilliant Dye™ Terminator Cycle Sequencing Kit V3.1, BRD3-100/1000 (Germany). For each reaction and each sample, the purified fragments were examined using the ABI Genetic Analyzer (Applied Biosystems, ThermoFisher Scientific, Germany). The .abl files produced by the ABI 3500XL Genetic Analyzer were examined using BioEdit Sequence Alignment Editor version 7.2 (https://bioedit.software.informer.com/7.2/).

Phylogenetic analysis of 16S rRNA sequences of Actinobacteria isolated in the study

The homology search was performed by comparing the sequences obtained above with those present in the public database (NCBI) using the standard Basic Local Alignment Search Tool (BLAST-N) program (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic analyses were conducted using MEGA software version 11.0 and the 16S rRNA genes of Actinobacteria were aligned against neighbouring nucleotide sequences using Multiple Sequence Alignment by Log Expectation (MUSCLE) (https://www.ebi.ac.uk/Tools/msa/muscle/). The phylogenetic tree was reconstructed by using the neighbour-joining (NJ) method and the topologies were evaluated by bootstrap sampling expressed as a percentage of 1000 replications. The tree was visualized using iTOL version 6.0 (https://itol.embl.de/).

Optimization studies

To maximize Actinobacterial activity, a one-factor-at-a-time strategy was used. The Actinobacteria Saccharomonospora sp. PNSac2 was selected for this study against B. cereus as a reference pathogen. Each experiment was conducted in a 100-ml Erlenmeyer flask with 20 mL of sterile ISP2 broth medium, 2 mL of spore suspension (OD600), and a rotary shaker spinning at 120 rpm. The inhibition zone diameter (IZD) was used to express the activity against B. cereus using the agar well diffusion method.

Four growth media were used to test for antibiotic optimization by Saccharomonospora sp. PNSac2 including International Streptomyces Project 2 (ISP2) broth, nutrient broth (NB), tryptic soy broth (TSB), and soy casein dextrose broth (SCDB) according to the method described in the literature [24]. A 100-mL Erlenmeyer flask was used to inoculate 20 mL of each sterile broth with an aliquot of a 2-mL actinobacteria spore suspension. A sample of 2 mL was taken out and centrifuged for 10 min at 5000 rpm. A 0.22-µm bacteriological filter was employed to filter the filtrate, and 100 µL of cell-free broth was used for agar well diffusion antimicrobial testing against the tested pathogen. Zones of inhibition were measured using a metre rule and recorded appropriately.

ISP-2 broth was inoculated with Saccharomonospora sp. strain PNSac2 for the incubation period. For 14 days, the growth of the mycelium was measured as its dry weight every 48 h. After 10 min of centrifugation at 4000 rpm to separate the biomass, it was transferred to dry, weighted filter paper, and placed in an oven at 60 °C to dry and stabilize in weight. In order to express the growth rate, mg/20 mL culture media were utilized [25]. By using an agar well diffusion experiment, the antibacterial activity was evaluated against B. cereus. To find the ideal pH for Saccharomonospora sp. PNSac2 to produce antimicrobials, ISP-2 broth as a growth medium was changed at various pHs (5–11). About 0.1 N NaOH/0.1 N HCl was used to modify the original pH [26]. The antibacterial activity assay was carried out as previously explained. Furthermore, the antibacterial activity of each salt concentration was examined after adding sodium chloride salt to the fermentation media at varying concentrations ranging from 0 to 7% w/v [26].

Statistical analysis

All antibacterial assay results were collected in triplicates, and using the Microsoft Excel office 2007 edition, the values were expressed as mean ± standard error of mean (SEM).

Results

Isolation of Actinobacteria

Among the six plant samples, a total of thirteen strains of Actinobacteria were isolated (Fig. 1).

Fig. 1.

Distribution of Actinobacteria isolated from medicinal plants in Nigeria

They were isolated using different media like the Soy casein dextrose agar (SCDA), starch casein agar (SCA) medium, and ISP2 agar. The crushing of the plant parts, then mixing with its decoction mixture before inoculation of 0.1 mL aliquots on the ISP2 media yielded the highest number of Actinobacteria isolates. Direct inoculation of the segmented plant parts and crushing the segments before inoculation gave two actinobacteria isolates each (Table S1). The Actinobacteria colonies that appeared macroscopically different from other isolates were selected and further purified by streak plate technique. Colonies of Actinobacteria were recognized by their characteristic chalky to leathery appearance (Fig. 2).

Fig. 2.

Macroscopic features of Actinobacteria isolated from medicinal plants. They represent a 7-day-old culture on soya casein dextrose agar incubated at 30 ℃. A, D, E, F, and H are strains that belonged to the genus Streptomyces sp. showing different colours of aerial mycelium; B and G belonged to the genus Saccharomonospora showing white aerial mycelium, while C belonged to genus Saccharopolyspora, with limited white aerial growth

The Actinobacteria isolates showed different morphological features in different media. Most isolates could grow on starch casein agar, International Streptomyces Project 2 media, and soy casein dextrose agar while some isolates seem to be selective to growth media showing distinct colours. Streptomyces sp. XAFac8 showed white aerial mycelium with distinct faint yellow pigmentation in SCDA and SCA while Streptomyces sp. AARac1 showed golden yellow colonies with deep wine agar pigmentation in the same media. Streptomyces sp. CGBac4y showed greyish ash colour colonies with dark-brown pigment in SDA and SCDA media (Table S2).

The outcome of the sequence analysis between the isolates in this study and those on the Genbank database revealed similarities in the range of 96.59–99.65%. The sequences of our isolates have been deposited in the NCBI GenBank with their submission and accession numbers well documented. Thirteen Actinobacteria strains were identified to belong to Streptomyces (10), Saccharomonospora (2), and Saccharopolyspora (1). The numbers in parenthesis represent the accession numbers of most similar hits obtained from the NCBI database (Table 1).

Table 1.

Identification of endophytic Actinobacteria based on similarity searches on the NCBI Database

E-value, expect value; % identity, percentage identity; Submission No, submission number assigned to our strains; Accession no, accession number assigned to our strains

Antimicrobial activity of endophytic Actinobacteria

The inhibitory activity of the Actinobacteria isolates was indicated by the absence of growth of the tested pathogen near the central strips of Actinobacterial growth. The results showed that 8 Actinobacteria isolates including Streptomyces sp. XAFac8, Saccharomonspora sp. ELSac7, Streptomyces sp. AARac1, Saccharopolyspora sp. PGLac3, Saccharomonospora sp. PNSac2, Streptomyces sp. CGBac4x, Streptomyces sp. CGBac4y, and Streptomyces sp. PGLac3x) had an antagonistic interaction against the tested pathogens (Figure S1).

In vitro antibacterial activity of fermentation filtrates of selected Actinobacteria

After screening by cross streak method, 8 strains were selected for the anti-bacterial activity against the selected pathogens. All the isolates showed antibacterial activity against two or more tested pathogens. The formation of an inhibition zone around the pathogenic strain was due to the production of antimicrobial secondary metabolites by actinobacterial strains isolated in this study. In the antibacterial results of selected actinobacterial culture filtrates, Streptomyces sp. CGBac5, Saccharopolyspora sp. PGLac3, Saccharomonospora sp. ELSac7, Saccharomonospora sp. PNSac2, and Streptomyces sp. XAFac8 showed inhibitory activity against all tested pathogenic organisms. Streptomyces sp. PGLac1 strain showed inhibitory activity against only two pathogens. It was found that Streptomyces sp. AARac1, Streptomyces sp. PGLac1, and Streptomyces sp. AABac1 could not show any inhibitory activity against Pseudomonas aeruginosa (Table 2).

Table 2.

Primary screening of antibacterial activity using the cell-free broth of selected antibacterial strains

K, Klebsiella pneumoniae; E, Escherichia coli; P, Pseudomonas aeruginosa; B, Bacillus cereus; PR, Proteus sp.; ST, Salmonella typhi; SA, Staphylococcus aureus; CEF, ceftriaxone

Phylogenetics studies

The phylogenetic analysis showed the ancestral relationship between our isolates and those from Genbank. The phylogenetic trees are made of different species of Streptomyces (Fig. 3), Saccharomonospora, and Saccharopolyspora (Fig. 4). They were branched into several clades which were made up of several sister clades. They were divergent with some isolates forming a monophyletic clade signaling potential novel isolates. Saccharomonospora sp. ELSac7 and Saccharomonospora sp. PNSac2 seemed to be genetically similar isolates from different plants. They were gathered away from other isolates and shared similar roots.

Fig. 3.

Phylogenetic tree based on 16S rRNA gene sequences, showing the position of the strains (labelled in red) in this study among other strains of Streptomyces genus from Genbank. The phylogenetic analysis was done using MEGA X bioinformatic programme and the sequences were aligned by MUSCLE while the phylogenetic tree was drawn by neighbour-joining method using default options. Numbers at the nodes indicate the level of bootstrap support (%); Genbank accession numbers are given in parentheses while scale 0.1 represents substitutions per base

Fig. 4.

Phylogenetic tree based on 16S rRNA gene sequences, showing the position of rare endophytic actinobacteria strains from this study (labelled in red) among other strains from the NCBI database. The phylogenetic analysis was done using MEGA X bioinformatic program and the sequences were aligned by MUSCLE while the phylogenetic tree was drawn by neighbour-joining method using default options. Numbers at the nodes indicate the level of bootstrap support (%); Genbank accession numbers are given in parentheses. The scale 0.1 represents substitutions per base

The ISP2 medium exhibited an optimal growth medium for the putative strain (PNSac2) at an optimal incubation temperature between 30 and 32 ℃ and an incubation period of 12–14 days (Fig. 5).

Fig. 5.

Graphic representation of the optimal medium and temperature for growth and antibiotic production by Saccharomonospora sp. PNSac2. a The relationship or effect of the duration of incubation period and cultivation medium on the antibiotic production. b The relationship between antibiotic production by Saccharomonospora sp. PNSac2 and temperature of incubation

The optimal medium for antibiotic production by Saccharomonospora sp. PNSac2 was ISP2 medium. The growth came to an optimum point on the 12th day. The optimal antibiotic production was maintained from the 12th day up till the 14th day after which a sharp decline was observed. The media support for antibacterial compound production for the rare Actinobacteria Saccharomonospora sp. PNSac2 showed the highest inhibition in ISP2, followed by SCDA, TSA, and NB. Its optimal temperature for antibiotic production was at 30 ℃ up to 32 ℃ after which a sharp decline was noticed in the antibiotic production.

The pH 8 was the optimum for the growth of Saccharomonospora sp. PNSac2 while 1–2% w/v was the optimal salt concentration required (Fig. 6).

Fig. 6.

A graphical representation of the effects of pH and salt concentration on antibiotic production by Saccharomonospora sp. PNSac2. a The media’s pH effect on the antimicrobial biosynthetic potential of Saccharomonospora sp. PNSac2. b The effect of salt concentration of the medium on the antimicrobial biosynthetic ability of the Actinobacterium. The Actinobacterium was grown on ISP2 medium at different pHs of 5 to 11, with a salt concentration of 0 to 7% w/v, and then incubated at 30 ℃ for 12 days

The acidity of the medium was not favourable to the growth of Saccharomonospora sp. PNSac2, hence affected negatively its antimicrobial bioactive production. The antibiotic production by this strain increased significantly towards neutral pH 7 and became highest at pH 8 before it started to decline at extreme alkalinity. The inhibitory activity obtained at neutral pH 7 was equal to the same obtained at alkaline pH 9. The pH favourability for this strain was highest at 8 followed by 7 and 9, 10, 11, 6, and 5 (Fig. 6a). A high salt concentration of 6% or more did not allow the growth of this strain at all. At 0% salt concentration, growth was prominent with antibacterial activity against the target strains, but the antibacterial activity was higher when the salt concentration increased from 1 up to 2%. Above 2% salt concentration, its antibiotic production began to decline. The salt tolerance for antibiotic production in this strain was best at 1–2% w/v and least at 6% w/v upwards (Fig. 6b).

Discussion

The study was carried out to discover the antibiotic-producing Actinobacteria from various parts of Nigerian ethnomedicinal plants. A total of 6 medicinal plants were used in this study. At least one endophytic Actinobacterium was isolated from each sampled plant material. Piper guineense (leaves) and Crinum glaucum (bulb) yielded the highest number of isolates (3). The root of A. ascolonicum yielded 2 strains of Actinobacteria while each of E. laterifolia (stem), A. ascolonicum (bulb), X. aethiopica (fruit), and P. nitida (seed) yielded only one strain of Actinobacterium. A total of thirteen Actinobacteria strains were isolated from the six medicinal plants used and were identified to belong to three different genera: Streptomyces (76.92%), Saccharomonospora (15.38%), and Saccharopolyspora (7.69%). This report concurs with that of Sapkota et al. [27] that Streptomyces species dominate natural samples by up to 70.7%.

The direct inoculation method of the treated plant segments and crushing the plant segments before inoculation yielded the least number (2) of Actinobacteria isolates. It was noted that the number of Actinobacteria isolates doubled to 4 when the plant samples were crushed with 5 mL of sterile water, then thoroughly shaken and 0.1 mL aliquot spread on the isolation media. The highest number of isolates (5) was recorded when 5 mL of decoction mixture of the plant part was mixed and shaken thoroughly with the crushed segment before spreading 0.1 mL aliquot onto the isolation media. More so, we recorded that the majority of the Actinobacteria were isolated using ISP2 agar followed by starch casein agar while the soy casein dextrose agar isolated only one Actinobacterium. This may be attributed to the nutrient variation among these media. However, our findings revealed that a reduction in the surface area of the plant segment and shaking vigorously with sterile water or decoction mixture could promote the release of Actinobacteria from the plant tissues, hence increasing the isolation rate of endophytic Actinobacteria.

Several Streptomyces strains were retrieved from our study including Streptomyces sp. XAFac8, Streptomyces sp. AARac1, Streptomyces sp. AABac1, Streptomyces sp. PGLac3x, and Streptomyces sp. AALac1. Their growth was supported by several known media including the nutrient agar, starch casein agar, tryptic soy agar, ISP2 agar, and soy casein dextrose agar. Similar studies showed that casein dextrose agar and ISP2 agar are very good supporting media for Actinobacteria’s isolation [28, 29]. These isolates have a percentage identity that varies between 96.59 and 99.65% with other strains blasted from the NCBI GenBank. Streptomyces sp. XAFac8 is 99.57% identical with Streptomyces ardesiacus NR_112454.1. Saccharomonospora sp. PNSac2 is 96.59% identical with Saccharomonospora xinjiangensis NR_042059.1. Similarly, several studies on endophytes in Nigeria had focused on endophytic fungi while very few studies on endophytic Actinobacteria succeeded in isolating only Streptomyces species. Our study targeted diversified species of Actinobacteria which resulted in distinct strains that were not previously documented to have been isolated from Nigerian medicinal plants [30–33].

Actinobacterial strains from this study showed remarkable antibacterial activity against several tested pathogens. Similarly, studies have shown that Actinobacteria isolated from natural samples could show inhibitory activity against several pathogens including S. aureus, E. coli, and Candida albican [34, 35]. Streptomyces sp. XAFac8, Saccharomonospora sp. ELSac7, Saccharopolyspora sp. PGLac3, Streptomyces sp. PGLac3x, and Streptomyces sp. CGBac5 inhibited all the bacteria pathogens tested. The recorded inhibition zone diameters (IZD) ranged from 10 to 24 mm. Streptomyces sp. AARac1 and Streptomyces sp. AABac1 did not have anti-Pseudomonas activity while Streptomyces sp. PGLac1 had activity only against E. coli and Salmonella typhi with IZD of 10 mm which could serve as an indication that they may be specific in their antimicrobial activity.

The phylogenetic tree formed by the endophytic Actinobacterial strains showed that they are divergent and have evolutionary relationships with other strains from the GenBank. The phylogenetic analysis of putative Streptomyces species (Streptomyces sp. PGLac1, Streptomyces sp. CGBac5, and Streptomyces sp. AARac1) showed a monophyletic clade formed by three strains and showed a distant relationship with those from the Genbank. Saccharomonospora sp. PNSac2 and Saccharomonospora sp. ELSac7 formed a tight taxonomic position at a bootstrap value of 80% and were separated away from other strains. Saccharopolyspora sp. PGLac3 had a close ancestral relationship with Saccharopolyspora cebuensis SPE 10–1 (NR_044047.1) at a 100% bootstrap value. There was no documentation on the phylogenetic study of endophytic Actinobacteria from Nigeria at the time of this report.

Optimization of antibiotic production by Actinobacteria is an important step for their biotechnological applications [36, 37]. The optimal conditions for antibiotic production by distinct Actinobacteria Saccharomonospora sp. PNSac2 studied against Bacillus cereus showed that it required ISP 2 medium for optimal growth at an incubation temperature between 30 and 32 ℃ for 12–14 days. The assessment of optimal pH revealed that the Actinobacterium performed optimally at a pH of 8 after which any increase in pH leads to a decline in antibacterial activity by reduced size of IZD. This agrees with the report by Pansomsuay et al. [38] whose work determined the optimal pH for Actinobacteria to be 32℃. It could tolerate salt up to 1–2% w/v concentration above which there was a sharp decline in the antibacterial activity of the Actinobacteria. This is also in line with the report by an author whose report indicated 0–2% w/v as the optimal salt concentration for antibiotics production among Actinobacteria [39]. There is scarce information on the optimization of antimicrobial production among endophytic Actinobacteria from Nigeria [40]. However, some groups of researchers reported that their endophytic Actinobacteria showed optimal antimicrobial activity within salt tolerance of 0–1% and pH between 7 and 9 [41–43], which agrees with our findings. These findings provide valuable insights for future studies focusing on enhancing antibiotic production among endophytic Actinobacteria.

Conclusion

In summary, the findings of this study suggest that endophytic Actinobacteria from Nigerian ethnomedicinal plants hold promise as potential sources of novel antimicrobial compounds. Further genomic analysis and characterization of their metabolites are warranted to uncover the biosynthetic gene clusters that link new antimicrobial agents. These future investigations would contribute to advancing the field of antibiotic discovery and have significant implications in the pharmaceutical industry.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contribution

CEE: conceptualization, methodology, validation, formal analysis, investigation, resources, data collection, writing—original draft, writing—review and editing, visualization. NHI: writing—review and editing, project administration. CFO: visualization, editing, methodology. SCM: data collection, project administration, writing and editing of manuscript. UEM: supervision, writing—review and editing, project administration. DHA: writing—review and editing, supervision, project administration, funding acquisition, formal analysis. All authors read and approved the final manuscript.

Funding

Partial financial support was received from Ladipo Mobolaji Abisogun-Afodu Annual Lecture in Pharmacy Grant 2021. Grant No: VC/OA/L.12/Vol.5.

Data availability

Sequence data are available at NCBI Genbank (https://www.ncbi.nlm.nih.gov/). Accession numbers are available in this report (Table 1).

Declarations

Competing interests

The authors declare no competing interests.