Phylogenetic insights and antimicrobial biosynthetic potential of Serratia sp. XAFb12 and Pseudomonas sp. XAFb13 from Xylopia aethiopica

Abstract

The extensive distribution of Xylopia aethiopica across the continent of Africa has firmly established its medicinal value in diverse disease management. While its phytochemistry is well established, the diversity, molecular, biochemical, and antimicrobial-biosynthetic characterizations of culturable bacterial endophytes residing in fruits of X. aethiopica have not been studied previously. Additionally, danger continues to loom the global health care and management due to antibiotic resistance; hence, the discovery of microbial natural products especially from endophytes could offer a lasting solution to the quest for novel antimicrobial compounds. In this study, we isolated two bacterial endophytes Serratia sp. XAFb12 and Pseudomonas sp. XAFb13 from fresh X. aethiopica fruit. The 16S rRNA gene sequencing, Vitex biochemical test, Gram staining, and 16S rRNA gene analysis were used to confirm their phenotypic and genotypic profiles. Phylogenetic tree analysis reveals their divergence in a separate branch, indicating their uniqueness. The crude extract of both strains showed inhibition against all tested bacterial and fungal pathogens. The minimum inhibition concentration (MIC) ranged from 2.5 to 10%. Chemical analysis of the crude extracts using gas chromatography-mass spectroscopy (GC–MS) revealed the most abundant compounds to be hydrocinnamic acid, 2-piperidinone, 5-isopropylidene-3,3-dimethyl-dihydrofuran-2-one, and diethyl trisulfide. The bacterial endophytes linked to X. aethiopica were described in this study for the first time in relation to clinically significant pathogens. Our findings imply that crude extracts of the endophytic bacteria from X. aethiopica could be potentially employed as antibiotics. Hence, it is crucial to characterize the active ingredient in further detail for future pharmaceutical applications.

Introduction

The development of antibiotic resistance among microbial pathogens, which is accelerating, has focused research efforts on the synthesis and identification of novel antimicrobials to battle and control the spread of infectious diseases [1]. Studies on the interactions between plants and microbes have inspired researchers all over the world to develop important pharmacologically bioactive compounds, particularly through plant-endophyte research [2].

The microbial symbionts known as endophytes, which include fungi, bacteria, and actinomycetes, occupy the plant tissues, both in aerial and roots without hurting the host plants [3]. The link between plants and endophytes is reciprocal, with plants providing the endophytes with a place to live, food, and protection, while endophytes assist their plant hosts in several ways to promote their growth, well-being, and protection against invading plant pathogens. Additionally, antibiotic resistance is spreading among most disease-causing organisms, making the creation of novel antimicrobial agents from natural sources necessary. Endophytes present themselves as a housekeeper to numerous bioactive metabolites, including phytochemicals like phenolic acids, alkaloids, quinones, steroids, saponins, tannins, and terpenoids, making them a promising candidate for the management of diseases like cancer, malaria, tuberculosis, viral infections, diabetes, and inflammatory diseases. Even though the primary function of the bioactive substances from endophytes is to make the host plants resistant to several unhealthy factors, endophytes are still emerging as an unexpected source for prospective pharmaceuticals [4]. Endophytes, which have been isolated from a wide range of plants, have been demonstrated to be a wealthy source of natural substances for use in a range of industrial, medicinal, agricultural, and pharmaceutical applications [5]. Endophyte-associated enzymes can be employed as substitutes for toxic substances. Recently, potential in the formulation of microbial enzymes and secondary bioactives or metabolites has started to emerge. According to numerous findings now [6], microbes isolated from hitherto undiscovered plants and those from harsh habitats like volcanoes, drought, top mountains, etc. offer enormous pharmaceutical and biotechnological uses.

X. aethiopica (Annonaceae) is described as a perennial, sweet-smelling plant, mostly known as negro pepper, African pepper, Guinea pepper, and spice tree, and is highly regarded in West Africa due to its numerous therapeutic characteristics [7]. According to folklore medicine, the fruit can be used to cure a variety of conditions, including infections, nasopharyngeal infections, diarrhea, dysentery, stomach disorders, menstruation disorders, arthritis, and rheumatism [8].

This plant is progressively becoming an endangered species because of its significant therapeutic use and ongoing deforestation processes [9]. Exploring X. aethiopica-endophyte relationship and the biological functions of their secondary metabolite are urgently needed because of the species’ threatened status. We hypothesize that X. aethiopica fruit could harbor unique endophytic bacteria, capable of affecting their metabolic pattern and producing important bioactive substances, especially antibiotics. So, this study’s objective was to isolate, molecularly describe and identify bacterial endophytes from X. aethiopica as well as the antimicrobial bioassay on pathogenic bacteria and fungi species.

Material and methods

Plant collection, identification, and hunting for bacterial endophytic isolates

Fresh (healthy) fruits of X. aethiopica were bought on 24th of February 2022, from Olosha medicinal plant market, Mushin Lagos, Nigeria (6.529035 N, 3.35366 E coordinates) in a zip-lock polythene bag and brought to the herbarium in the Department of Botany, the University of Lagos for plant identification by the botanist (Dr. Nodza George). The voucher number LUH 9070 was assigned to our sample for future reference. The plant samples were immediately taken to the Pharmaceutical Microbiology Laboratory in the Idi-araba campus of the University of Lagos for an endophytic bacterial hunt following a modified method described as follows: after thoroughly cleaning the samples with running water, they were aseptically surface-sterilized by serially washing them with 70% ethanol for 30 s, 4% sodium hypochlorite for 2 min, and 70% ethanol for another 30 s [1]. Samples were aseptically dried using sterile dry filter paper after being serially washed three times in sterile distilled water dished in sterile beakers. They were divided into small pieces using a sterile scalpel and placed on tryptic soy agar and nutrition agar plates with fluconazole antifungal powder added at a concentration of 75 µg/mL. For 2–7 days, the plates were incubated at 35–37 °C, and the development of morphologically distinct bacterial colonies surrounding the plant segments was monitored. To verify successful surface sterilization, the water used for the final rinse of the sterile plant samples was likewise streaked on a different agar plate and checked for any microbial growth. The bacterial isolates were further purified using agar plate streaking on the appropriate medium, and the pure cultures were kept on a tryptic soy agar slant at 4 °C with regular sub-culturing done every month.

Morphological identification of endophytic bacterial isolates

It was done to identify endophytic bacteria morphologically. The first features utilized for identification were the colony’s shape, size, smell, and color. According to established techniques, endophytic bacterial isolates were further identified by the conventional Gram stain reaction and examined using a compound bright-field microscope (OLYMPUS CX23 model) at 1000X magnification [10].

Biochemical identification of endophytic bacterial isolates

The biochemical identification of the two endophytic bacterial isolates was done using VITEK 2 compact machine (Biomerieux Automated System) method which uses the fluorogenic principle for organism identification following the manufacturer’s instruction [11]. In brief, bacterial suspension equivalent to McFarland standard was prepared using 0.45% sterile saline. About 3 mL of the suspension was placed in the tube inside a cassette which was gently loaded into the instrument for automatic reading.

Extracting genomic DNA, performing PCR, and sequencing 16S rRNA

This step of our research was carried out in Inqaba Biotec West Africa, Ibadan, Nigeria. Pure colonies of each bacterial endophytic isolate grown on a nutrient agar plate were used for the extraction of the genomic DNA. A bacterial DNA extraction kit (Zymo Research, catalog number R2014) was used to get the DNA. Thermo Fisher Scientific, Waltham, Massachusetts, USA, NanoDrop ND-2000 UV–Vis spectrophotometer was used to quantify the isolated DNA. Each bacterial isolate’s 16S rRNA gene was amplified by PCR [12]. In a nutshell, the 2 × PCR master mix with standard buffer was used to amplify the 16S rRNA gene using the primers (16S-27F: 5′-AGAGTTTGATCMTGGCTCAG-3′ and 16S-1492R: 5′-CGGTTACCTTGTTACGACTT-3′). The extracted fragments were sequenced in the forward and reverse direction (Nimagen, Brilliant Dye™ Terminator Cycle Sequencing kit V3.1, BRD3-100/1000) and purified (Zymo Research, ZR-96 DNA sequencing clean-up kit™, Catalog No. D4050). The purified fragments were analyzed on the ABI 3500XL Genetic Analyzer (Applied Biosystems, Thermo Fisher Scientific) for each reaction for every sample. BioEdit Sequence Alignment Editor version 7.2.5 was used to analyze the.ab1 files generated by the ABI 3500XL Genetic Analyzer [12].

Nucleotide BLAST of the isolates’ sequences

To determine the closest bacterial species, the obtained sequences were analyzed using the basic local alignment search tool (BLASTN) at the National Center for Biotechnology Information (NCBI) website against the prokaryotic rRNA sequence database for bacteria and archaea (http://blast.ncbi.nlm.nih.gov). Bacterial species with 98–100% similarities were aligned with the endophytic bacterial isolates’ sequences for analysis of the phylogeny.

Phylogenetic analysis

MUSCLE (Multiple Sequence Alignment by Log Expectation) was used to align the relevant nucleotide sequences using its default settings. Based on the Tamura-Nei model, the phylogenetic tree was drawn using the neighbor-joining (NJ) method [13]. The interactive tree of life (iTOL) version 6.0 (https://itol.embl.de/) was used to visualize the tree. For the bootstrap test, a total of 1000 replications were used. Every branch that had a bootstrap value above 50% was notable. Gaps and missing nucleotide data were removed from the positions. In MEGA (Molecular Evolutionary and Genetic Analysis) version 11.0, all evolutionary analyses were performed. Bacterial isolates discovered in the study had their 16S rRNA gene sequences deposited in GenBank (www.ncbi.nlm.nih.gov/genbank/) along with their accession numbers; OP363300 and OP363686 for strains XAFb12 and XAFb13, respectively.

Fermentation of the endophytic bacteria isolates

The endophytic bacteria from a one-week-old culture slant (TSA) were suspended in 5 mL of sterile saline to create the inoculum. 40 mL of sterile Soya casein dextrose agar in a 250 mL Erlenmeyer flask was inoculated with 1 mL of the homogeneous suspension, and the culture was incubated at 37 °C for 7 days on a rotational shaker. Centrifugation (12,000 g, 10 min., 4 °C) was used to aseptically separate the biomass, and the cell-free supernatant was then run through a Millipore filter with a 0.22 μm pore size [9]. The cell-free broth was used to perform a preliminary antimicrobial bioassay.

Organisms of interest

The following pathogens of interest were used for the in vitro bioassay of antimicrobial activity of the bacterial endophytes: Staphylococcus aureus ATCC 13311, Streptococcus pneumoniae UR 576, Proteus sp., Bacillus cereus ATCC 12022, Escherichia coli ATCC 25922, Salmonella enterica, Pseudomonas aeruginosa LN056, Klebsiella pneumoniae LN023, Candida tropicalis LUH-5852, Cryptococcus neoformans LUH-5510, and Aspergillus clavatus LUH-5589. The bacteria pathogens were sourced from the Nigerian Institute of Medical Research (NIMR), while the fungi pathogens were obtained from the Department of Mycology, Lagos University Teaching Hospital (LUTH), Nigeria.

Preliminary antimicrobial bioassay

Following perpendicular bioassay and agar well diffusion techniques with the specified pathogenic strains, bacterial endophytes were first tested for their antibacterial efficacy. Using the Mueller–Hinton agar and Sabouraud dextrose agar (HiMedia, India) plates seeded with test bacterial and fungal strains, respectively, approximately 100 µL of filter-sterilized cell-free supernatant was added. The plates were incubated for 48 h at 28 °C for fungal pathogens and overnight at 35 °C for bacteria. The diameter of the inhibition zone that formed around each well was measured to assess the antibacterial activity [14]. The average readings were recorded after experiments were carried out in triplicate.

Secondary antimicrobial screening of the endophytic bacterial extract

The antibacterial and antifungal effects of ethyl acetate extracts were examined using the agar well diffusion method, which was slightly modified from the literature’s descriptions [15]. Succinctly put, a sterile Petri dish had 1 mL of fresh bacterial or fungal culture pipetted into the center of it. Then, a Mueller–Hinton agar (MHA) for bacteria strains or the Potato dextrose agar (PDA) for fungi was molten-cooled and thoroughly mixed after being placed into the Petri plate holding the inoculum. After solidifying, wells were made onto the agar plates-containing inoculums using a sterile cork borer (6 mm in diameter). 10% dimethyl sulfoxide (DMSO) was used to dissolve the crude extract and 100 µL of each extract diluted to 20% w/v concentration was added to the appropriate wells. We chose the extract concentration (20% w/v) based on the results of our preliminary tests. To ensure that the extracts permeated completely into the agar, the plates were kept refrigerated for 30 min. The plates were then incubated for 24 h at 37 ℃ for bacterial pathogens and 48 h at 28 ℃ for fungal pathogens. By measuring the zone of inhibition after the incubation period, antimicrobial activity was predicted. DMSO 10% v/v (100 µL) was employed as negative control while chloramphenicol and amoxicillin (0.1%, 100 µL) were used as a positive control for bacterial pathogens and clotrimazole (0.1%, 100 µL) for fungi. All assays were performed in triplicate and the average readings were noted.

MIC (minimum inhibitory concentration) determination

At a 20% (w/v) concentration, the two tested extracts indicated antimicrobial activity. The agar well diffusion method was used to vary this concentration to ascertain their minimum inhibitory concentrations (MIC) [16]. Through two-fold serial dilution, various concentrations of 10, 5, 2.5, and 1.25% were created. Pipetting exactly 1 mL of each prepared inoculum into sterile Petri dishes was followed by the addition of molten agar and thorough mixing. Four wells were then created on each plate, and 100 µL of each extract’s 10, 5, 2.5, and 1.25% was added to their corresponding wells. After 30 min of refrigeration at 4 ℃, the plates underwent an 18-h incubation period at 37 °C. The MIC was regarded as the lowest concentration at which the tested microorganisms could not grow [17].

The GC–MS analysis of ethyl acetate extracts from Serratia marcescens XAFb12 and Pseudomonas entomophila XAFb13

The GC–MS analysis of bioactive substances present in the ethyl acetate extracts of the endophytic bacteria from the fruit of Xylopia aethiopica was done in the Nigerian Institute of Medical Research (NIMR) Yaba, Lagos using Agilent Technologies GC systems with GC-7890A/MS-5975C model (Agilent Technologies, Santa Clara, CA, USA) equipped with HP-5MS column (30 m in length × 250 μm in diameter × 0.25 μm in thickness of film). A high-energy electron ionization system that made use of electrons with a 70 eV energy level was used for GC–MS’s spectroscopic detection. The carrier gas was used and flowed at a rate of 1 mL/min of pure helium gas (99.995% purity). A holding period of around 10 min was allowed before the temperature increased by 3 °C/min from a starting point of 50 to 150 °C. By adding 10 °C/min, the temperature was eventually raised to 300 °C [18]. In a splitless mode, one microliter of the prepared, 1%-diluted extracts with the appropriate solvents was injected. Based on the peak area produced in the chromatogram, the relative quantity of the chemical components present in each of the extracts was expressed as a percentage.

Compounds’ identification

Based on their retention indices, the volatile bioactive chemicals from the ethyl acetate extracts of the endophytic bacteria XAFb12 and XAFb13 were identified, and the mass spectrum was interpreted using the National Institute of Standards and Technology (NIST) database. The acquired spectra of the ethyl acetate endophytic fraction’s unknown components were compared to the reference mass spectra of those components kept in the NIST library (https://www.nist.gov/nist-research-library).

Statistical analysis

Calculation of means and standard deviations on the data generated during antimicrobial studies of the endophytic bacteria extracts was done using Microsoft Excel Office 2007 version. The values were recorded as average ± standard error of mean (SEM).

Sequencing data

The accession number assigned to Serratia sp. XAFb12 is OP363300 while that of Pseudomonas sp. XAFb13 is OP363686.

Results

Cultural and biochemical identification

The cultural features of our isolates were studied on the nutrient agar (NA) media including the shape, margin, elevation, and pigmentation of each isolate. The biochemical studies showed that both isolates were negative for hydrogen sulfide (H₂S) production, citrate, indole, and urease tests. While only sample XAFb12 tested positive for motility, both isolates were catalase positive and harbored the catalase enzyme. Both isolates were positive for fermentation of certain sugars like glucose, sucrose, maltose, arabinose, and mannitol and negative for fermentation of inositol. Sample XAFb12 showed negative for galactose fermentation and positive for fructose. The reverse was the case in sample XAFb13 (Table 1).

Table 1.

Morphological and biochemical features of bacteria isolated from X. aethiopica

Characteristics/tesr	Isolate XAFb12	Isolate XAFb13
Colony morphology
Shape	Flat
Margin	Fili form
Elevation	Convex
Pigmentation	Light creamy
Cell morphology
Gram’s reaction
Shape	Rod	Tiny rod
Arrangement	Single	Single
Biochemical characteristics
H2S production	-	-
Indole	-	-
Citrate	-	-
Urase	-	-
Motility	+	-
Catalase	+	+
Sugar fermentation
Glucose	+	+
Sucrose	+	+
Maltose	+	+
Arabinose	+	+
Galactose	-	+
Fructose	+	-
Mannitol	+	+
Inositol	-	-
Sorbitol	+	+

(-) stands for negative; ( +) stands for positive test

The colonies of XAFb12 appeared pale white, flat, and mucoid on nutrient agar. While the colonies of isolate XAFb13 appeared raised and creamy. It has an offensive smell and draws upon touching it with a swab stick or inoculating loop. The microscopic identification using Gram’s reaction revealed that both isolates were single Gram-negative rods (fig S1).

Sequence nucleotide blast

The blasting of the 16S rRNA sequence using the BLASTN in the NCBI database revealed the endophytic bacterial isolate XAFb12 to be closely related to Serratia marcescens strain YD25 (NR_169468.1) with a percentage identity of 98.13%. Moreover, the sequence for endophytic bacterial isolate XAFb13 showed 100% similarity with Pseudomonas entomophila strain L48 (NR_102854.1).

Antimicrobial studies

Our endophytic bacterial strains are specifically studied to determine their relevance in antimicrobial production. The antimicrobial studies of our endophytic strains showed their potential application in antimicrobial drug discovery. The preliminary antimicrobial bioassay of the cell-free broth showed inhibitions against several tested pathogens. More so, the two isolates’ ethyl acetate extracts had a broad spectrum of antimicrobial activity that inhibited all the pathogens that were tested.

The results of the antimicrobial assay showed that the two bacterial endophytes XAFb12 and XAFb13 had broad-spectrum antimicrobial activities against both pathogenic fungi and bacteria. Perpendicular antimicrobial bioassay, agar well diffusion of cell-free broth, and ethyl acetate extract of our endophytic bacterial isolates showed observable inhibitory zones against target pathogens (Fig. 1).

Fig. 1.

Representation of the antimicrobial activities of endophytic bacteria strains against clinically relevant pathogens on agar plates. A Plate showing the preliminary antimicrobial activity of endophytic bacterium (XAFb13) against several pathogenic fungi using perpendicular bioassay technique. The bacterial endophyte was first cultured in a straight line on Soya casein dextrose agar (SCDA) at 37 ℃ for 3 days before perpendicular co-culturing of 0.5 McFarland solution of the fungi pathogen and further incubation at 28 ℃ for 48 h. B–D Plates showing the inhibition zones exhibited by cell-free broth from endophytic bacteria (XAFb12 and XAFb13) against Streptococcus pneumoniae, Aspergillus clavatus, and Klebsiella pneumoniae, respectively, using agar well diffusion technique. E, F Plates showing the antimicrobial activity of the ethyl acetate extract of endophytic bacteria against Aspergillus clavatus and Proteus spp., respectively, using agar well diffusion assay. The abbreviations of pathogenic strains are as follows: AC, Aspergillus clavatus; CN, Cryptococcus neoformans; CT, Candida tropicalis; KP, Klebsiella pneumoniae; SP, Streptococcus pneumoniae. The abbreviations of antimicrobials (positive controls) are CHLOR, chloramphenicol; CLOTR, clotrimazole; AMOX, amoxicillin while DMSO, dimethyl sulfoxide is the negative control

The antimicrobial activity assay using cell-free broth of the bacterial isolates showed that Serratia sp. XAFb12 inhibited all fungal pathogens except for bacteria, including Staphylococcus aureus and Bacillus cereus (Table 2).

Table 2.

Antimicrobial activity of cell-free broth from bacterial endophytes by agar well diffusion against pathogenic strains*

Endophytic bacteria		Inhibition zone diameter (mm)
Codes	Name	SA	BC	SP	PS	KP	SE	EC	PA_	CT	CN	AC
XAFb12	Serratia marcescens	0	0	9 ± 1.4	6 ± 0.8	12 ± 0.5	0	15 ± 1.4	9 ± 0.3	12 ± 1.2	15 ± 0.5	9 ± 0.2
	Pseudomonas entomophila	25 ± 0.8	22 ± 0.3	25 ± 1.2	26 ± 0.2	15 ± 1.3	22 ± 0.4	20 ± 0.6	17 ± 1.2	10 ± 0.8	8 ± 1.3	13 ± 0.4
AMOX**		8 ± 2.0	6 ± 1.3	0	6 ± 0.5	8 ± 0.7	6 ± 1.4	8 ± 1.8	8 ± 2.2	-	-	-
CLOTR**		-	-	-	-	-	-	-	-	11 ± 1.3	9 ± 0.4	0

^*SA, Staphylococcus aureus; BC, Bacillus cereus; SP, Streptococcus pneumoniae; PS, Proteus spp.; KP, Klebsiella pneumoniae; SE, Salmonella enterica; EC, Escherichia coli; PA, Pseudomonas aeruginosa; CT, Candida tropicalis; CN, Cryptococcus neoformans; AC, Aspergillus clavatus. **Control antimicrobials: AMOX, amoxicillin; CLOTR, clotrimazole

Cell-free broth of Pseudomonas sp. XAFb13 showed inhibitory activities against all tested pathogens. The ethyl acetate extracts of both samples inhibited all the tested pathogens. Larger inhibition zones were recorded against the fungal pathogens (Candida tropicalis 12 mm, Cryptococcus neoformans 25 mm, and Aspergillus clavatus 20 mm) for both endophytic bacterial strains than in bacterial pathogens such as Bacillus cereus, Proteus sp., Salmonella enterica, and Escherichia coli with (10, 10, 7, and 9 mm), respectively, for strain XAFb12 and (18, 12, 10, and 10 mm) for the endophytic strain XAFb13 (Fig. 2).

Fig. 2.

Antimicrobial activity of ethyl acetate extract from bacterial endophytes by agar well diffusion against pathogenic strains. SA, Staphylococcus aureus; BC, Bacillus cereus; SP, Streptococcus pneumoniae; PS, Proteus spp.; KP, Klebsiella pneumoniae; SE, Salmonella enterica; EC, Escherichia coli; PA, Pseudomonas aeruginosa; CT, Candida tropicalis; CN, Cryptococcus neoformans; AC, Aspergillus clavatus. Control antimicrobials: AMOX, amoxicillin; CLOTR, clotrimazole

The minimum inhibition concentrations (MICs) of the ethyl acetate extract of our endophytes against all tested pathogens were determined to be within the range of 2.5 to 10% w/v (Table 3).

Table 3.

MIC of endophytic bacteria’s ethyl acetate extract against pathogenic strains*

Endophytic bacteria extract	Minimum inihibitory concentration (MIC)%_W/V
Extract codes	SA	BC	SP	PS	KP	SE	EC	PA	CT	CN	AC
XAFb12	10	5	10	10	10	5	5	10	5	5	2.5
XAFb3	5	5	5	5	10	5	5	10	2.5	2.5	2.5

^*SA, Staphylococcus aureus; BC, Bacillus cereus; SP, Streptococcus pneumoniae; PS, Proteus spp.; KP, Klebsiella pneumoniae; SE, Salmonella enterica; EC, Escherichia coli; PA, Pseudomonas aeruginosa; CT, Candida tropicalis; CN, Cryptococcus neoformans; AC, Aspergillus clavatus

Phylogenetic analysis

The phylogenetic analysis tree was done using up to eighteen organism strains available in the NCBI GenBank. The strain Serratia sp. XAFb12 was found to be located within sisters’ strains including Serratia entomophila strain DSM 12358 (NR_025338.1), Serratia ficaria strain DSM 4569 (NR_041979.1), Serratia odorifera strain PADG 1073 (NR_037110.1), and Serratia rubidaea strain DSM 4480 (NR_114716.1). The phylogenetic analysis of Serratia sp. XAFb12 showed a more recent ancestral relationship with Serratia rubidaea strain DSM 4480 (NR_114716.1) than other strains. Pseudomonas sp. XAFb13 showed a more recent evolutionary relationship with Pseudomonas entomophila strain L48 (NR_102854.1). These two strains of ours formed a sub-clade different from other sisters’ clades. The phylogenetic tree for Serratia sp. XAFb12 revealed three major clades (a, b, and c) (Fig. 3).

Fig. 3.

Phylogenetic tree constructed by the neighbor-joining method showing the relationships of Serratia sp. XAFb12 based on 16S rRNA gene sequence compared with the reference sequences from the GenBank. Alignment was conducted with MUSCLE and MEGA 11.0 software for drawing the tree using the neighbor-joining method while the annotation was done using iTOL version 6.0. The GenBank taxa and our strain are designated by the strain codes followed by the accession numbers, with our strain highlighted in red. Numbers at nodes are bootstrap percentages based on 1000 replications. The bar indicates 0.01 nucleotide substitutions per site. The labeling a, b, and c refer to three clades formed by different strains while the solid red lines represent the clade bearing the endophytic strain

The phylogenetic analysis of Pseudomonas sp. XAFb13 showed three major clades (a, b, and c) (Fig. 4). These several clades revealed the diversity of our isolates.

Fig. 4.

Phylogenetic tree constructed by the neighbor-joining method showing the relationships of Pseudomonas sp. XAFb13 based on 16S rRNA gene sequence compared with the reference sequences from the GenBank. Alignment was conducted with MUSCLE and MEGA 11.0 software for drawing the tree using the neighbor-joining method while the annotation was done using iTOL version 6.0. The GenBank taxa and our strain are designated by the strain codes followed by the accession number, with our strain highlighted in red. Numbers at nodes are bootstrap percentages based on 1000 replications. The bar indicates 0.01 nucleotide substitutions per site. The labeling a, b, and c refer to the three clades formed by the strains while the solid red line represents the endophytic strain of interest

Gas chromatography-mass spectroscopy profiling of ethyl acetate extracts

The GC–MS analysis revealed several chemical compositions of bacterial endophytes’ crude extracts. The chromatogram revealed the three most abundant bioactive compounds in each of the isolate’s extracts (Fig. 5).

Fig. 5.

GC–MS chromatogram of ethyl acetate extract of endophytic bacteria from X. aethiopica fruit. A GC–MS chromatogram of ethyl acetate extract of endophytic bacterium (XAFb12). Twenty-nine bioactive compounds were identified, and hydrocinnamic acid (48.02%), 2-piperidinone (8.36%), and 5-Isopropylidene-3,3-dimethyl-dihydrofuran-2-one (6.42%) were the most abundant. B GC–MS chromatogram of ethyl acetate extract of endophytic bacterium (XAFb13). Thirty-three bioactive compounds were identified, and hydrocinnamic acid (32.28%), 2-piperidinone (23.11%), and diethyl trisulfide (5.29%) are the most abundant

The chromatograms showed different peaks for each identified compound at different time intervals. The data showing their biochemical constituents with their retention time (RT), molecular formula, molecular weight (MW), and percentage concentration (%) in the endophytic extract are presented (Table 4).

Table 4.

The constituents of ethyl acetate extracts of bacterial endophytes- XAFb12 and XAFb13

S/N	RT (min)		Compound name	Molecular formula	Molecular weight (g/mol)	Peak area (%)
	XAFb12	XAFb13				XAFb12	XAFb13
1	17.570	12.180	Pyrrolo[1,2-a]pyrazine-1,4-dione***	C10H16N2O2	196.25	6.13	1.36
2	13.667	12.700	Diethyl trisulfide***	C4H10S3	154.3	5.04	5.29
3	13.507	-	l-Norvaline*	C5H11NO2	117.148	4.43	-
4	12.689	-	5-Isopropylidene-3,3-dimethyl-dihydrofuran-2-one*	C9H14O2	154.21	6.42	-
5	8.111	8.128	Hydrocinnamic acid***	C9H10O2	150.1745	48.02	32.28
6	6.040	6.154	2-Piperidinone***	C5H9NO	99.13	8.36	23.11
7	13.507	12.958	L-Proline***	C5H9NO2	115.13	4.43	3.27
8	6.503	6.509	Methenamine***	C6H12N4	140.186	1.73	0.75
9	-	14.852	11-Octadecenoic acid**	C19H36O2	296.5	-	2.20
10	10.829	10.829	8-Azahypoxanthine***	C4H3N5O	137.10	2.48	1.27
11	-	4.134	1H-Pyrazole-3,4-diamine**	C3H6N4	98.11	-	1.37
12	7.076	7.076	Benzene propanoic acid***	C9H12O2	152.19	1.93	1.07
13	15.041	15.041	Methyl stearate***	C19H38O2	298.5	1.04	1.56
14	10.829	-	p-Benzoquinone*	C6H4O2	108.095	2.48	-
15	4.792	4.798	Benzoic acid***	C7H6O2	122.12	1.65	1.29
16	3.659	3.665	Triethanolamine***	C6H15NO3	149.188	0.47	0.27
17	4.604	-	1-Pentanol*	C5H12O	88.15	0.27	-
18	4.729	-	Undecane*	C11H24	156.31	0.33	-
19	5.422	-	Heptane*	C7H16	100.21	0.35	-
20	6.269	-	2-Imidazolidinone*	C3H6N2O	86.09	0.44	-
21	7.138	-	2-Methylpiperidine*	C6H13N	99.17	0.77	-
22	7.447	7.448	Indole***	C8H7N	117.15	0.50	0.75
23	8.380	-	1,2-Diphenyl-3-pyrrolidin-1-yl-propan-1-ol*	C19H23NO	281.4	0.49	-
24	10.091	-	(E)-2-Fluoro-3-(4-N,N-dimethylaminephenyl)-propenoic acid*	C11H16FNO2	213.4	0.32	-
25	11.642	11.642	Carbonic acid***	H2CO3	62.03	0.68	0.45
26	11.756	-	Cyclodecanome*	C10H18O	154.25	0.84	-
27	12.929	-	Phenol*	C6H6O	94.11	1.62	-
28	13.433	13.433	Hexadecanoic acid***	C16H32O2	256.4	0.99	0.97
29	14.354	-	9-Octadecenamide*	C18H35NO	281.477	0.76	-
30	14.852	-	Oleic acid*	C18H34O2	282.47	0.60	-
31	4.792	14.806	Methyl ester***	C2H3O2R	59.044	1.65	0.28
32	-	4.014	1-Butanamine**	C4H11N	73.14	-	0.67
33	-	4.587	2-Pyrrolidinone**	C4H7NO	85.11	-	0.97
34	-	5.096	2-Hexene**	C6H12	84.16	-	0.60
35	-	5.325	Pyridine**	C5H5N	79.1	-	0.22
36	-	5.439	Acetic acid**	CH3COOH	60.052	-	0.27
37	-	6.629	Benzofuran**	C8H6O	118.1	-	0.44
38	-	7.150	L-Glutamic acid**	C5H9NO4	147.13	-	0.52
39	-	8.352	Benzylmalonic acid**	C10H10O4	194.18	-	0.38
40	-	8.397	Pipecolic acid**	C6H11NO2	129.157	-	0.50
41	-	10.892	Maleimide**	C4H3NO2	97.07	-	1.28
42	-	11.762	3-Pyrrolidin-2-yl-propionic acid**	C7H13NO2	143.18	-	1.20
43	-	11.997	Ethanol**	C2H5OH	46.07	-	0.99
44	-	13.433	Pentadecanoic acid**	C15H30O2	242.40	-	0.97
45	-	13.524	3-Mercapto-N-norvaline**	C5H11NO2S	149.21	-	3.33
46	-	13.667	l-Leucine**	C6H13NO2	131.17	-	3.46
47	-	14.806	9,12-Octadecadienoic acid**	C18H32O2	280.447	-	0.28

^*Peculiar compounds to Serratia sp. XAFb12. **Peculiar compounds to Pseudomonas sp. XAFb13. ***Compounds shared by the two endophytic bacteria strains. RT, retention time

The ethyl acetate extract of XAFb12 yielded twenty-nine (29) bioactive compounds. Among these, hydrocinnamic acid (48.02%), 2-piperidinone (8.36%), and 5-isopropylidene-3,3-dimethyl-dihydrofuran-2-one (6.42%) are the most abundant compounds (Fig. 5A). More so, the GC–MS analysis carried out on ethyl acetate extract of XAFb13 yielded 33 bioactive compounds. It is worth noting that 2-piperidinone (23.11%), hydrocinnamic acid (32.28%), and diethyl trisulfide (5.29%) were the major identified bioactive compounds (Fig. 5B).

Based on the peak area percentage, it can be deduced that the two extracts contain greater percentage of hydrocinnamic acid followed by 2-piperidinone. There is greater abundance of hydrocinnamic acid (48.02%) than 2-piperidinone (8.36%) produced by Serratia sp. XAFb12 when compared with Pseudomonas sp. XAFb13 which has 32.28% of hydrocinnamic acid and 23.11% abundance for 2-piperidinone. Both endophytic isolates shared 15 bioactive compounds in common. Fourteen (14) bioactive compounds were peculiar to Serratia sp. XAFb12, while eighteen (18) compounds were peculiar to Pseudomonas sp. XAFb13. Hence, a total of forty-seven unique compounds were identified to be produced by our endophytic bacterial isolates.

Discussion

There are numerous known uses for the fruit of X. aethiopica in folk medicine in some African nations, particularly Nigeria [19]. There is yet no documentation on its association with endophytic bacteria. Our study isolated two endophytic bacteria from the fruit of X. aethiopica, namely, Serratia sp. XAFb12 and Pseudomonas sp. XAFb13. Most studies documented endophytic association with roots and leaves of different medicinal plants with greater diversity in the isolated endophytes. At the time of our report, there was no documentation on the association of endophytes with X. aethiopica. However, several endophytic bacterial genera have been reported in medicinal plants’ leaves and roots, including Bacillus, Staphylococcus, Enterobacter, Pantoea, and Stenotrophomonas [20]. These genera are not consistence with our result which identified only two genera: Serratia and Pseudomonas. Endophytic Serratia marcescens have been reported in the leaves of Achyranthes aspera L. It was claimed that Serratia marcescens experimentally exhibits a plant-promoting property [21]. It has also been isolated from the inner bark of a Cameroonian Maytenus serrata plant, and it was confirmed that it promotes the growth of this plant [22]. Additionally, Endophytic Serratia marcescens S-JSI isolated by some researchers was demonstrated to promote plant growth and improve plants’ resistance to fungi pathogens and pests [23]. This corresponds to our finding on the good antifungal activity of our strain on human pathogenic fungi. Similarly, several endophytic Pseudomonas spp. have been demonstrated to have several bioactivities. Pseudomonas putida isolated from the root of black pepper was demonstrated to have antimicrobial activities against fungi pathogens affecting the growth of black pepper [24]. More so, endophytic Pseudomonas putida has been documented to control the shoot growth in apples, cellular-redox balance, and expression of proteins under laboratory conditions [25]. Pseudomonas aeruginosa from leaves of Achyranthes aspera L. demonstrated strong antioxidant and growth-stimulating properties. It also demonstrated good in vitro antifungal activity against Rhizoctonia solani, Fusarium oxysporum, and Pyricularia oryzae [26].

The endophytic bacterial strains from this research were further characterized through molecular phylogenetic analysis. The phylogenetic analysis of their phylograms revealed their divergence. Serratia sp. XAFb12 showed a close ancestral relationship with Serratia spp. forming a monophyletic clade. The strain, Pseudomonas sp. XAFb13, was revealed to have a more recent common ancestral relationship with entomophila strain L48 (NR_102854.1). It also revealed a long branch length of this strain which points to the fact that it has more nucleotide substitutions per site in the gene sequence when compared to Serratia sp. XAFb12. In contrast, an endophytic Pseudomonas stutzeri isolated from Taxus chinensis revealed a similar topology to our strain but had a very short branch length and a close ancestral relationship with Pseudomonas psychrotolerans [27]. These confirm our hypothesis that medicinal plants like X. aethiopica could be a good source of distinct bacteria endophytes. More so, the broad antimicrobial activity exhibited by the isolated strains agrees with the therapeutic activities recorded by some authors on the host plant (X. aethiopica) [7].

Twenty-nine volatile chemical components were found via GC–MS of ethyl acetate extract of Serratia sp. XAFb12, while thirty-three chemical constituents were identified from Pseudomonas sp. XAFb13 crude extract. The identified compounds belong to different chemical groups of acid (benzoic acid and carbonic acid), alcohol (phenol and 1-pentanol), amino acids (leucine and proline), amine (triethanolamine), esters (methyl stearate), organic trisulfide (diethyl trisulfide), lactam or cyclic amide (2-piperidinone), and heterocyclic organic compounds (methenamine and benzofuran). Two bioactive compounds with the greatest abundance (hydrocinnamic acid and 2-Piperidinone) showed the highest percentage peak area in both isolates. These two abundant compounds are of great significance in antimicrobial activities exhibited by our endophytic bacterial strains. In contrast to our report, extract from endophytic Pseudomonas putida isolated from black pepper root was revealed to yield dimethyl trisulfide and pyrazine as major constituents with plant growth-promoting activity [23].

Hydrocinnamic acid and its derivatives have been documented to possess antimicrobial, antioxidant, anti-inflammatory, and ultra-violet (UV) protective effects [28]. 2-Piperidinone belongs to a group of a chemical compound known as delta lactam comparable to the penicillin group of antibiotics. Al-Bahadily et al. (2019) demonstrated the significant antimicrobial activity of 2-piperidinone extracted from pomegranate peels against diverse pathogenic organisms including Pseudomonas aeruginosa, Proteus mirabilis, and Candida albicans [29]. Their result concurs with the broad antimicrobial spectrum of our extracts, which contained this compound. The inhibition zones produced by Pseudomonas sp. XAFb13 free broth and crude extract were found to be higher than that of Serratia sp. XAFb12. This could be attributed to a higher abundance of 2-piperidinone in the former than in the latter. Other identified bioactive compounds from our extracts that have been documented for their antimicrobial activities include benzene propanoic acid, acetic acid, benzoic acid, imidazolidinone, phenol, and benzyl malonic acid which confirmed our hypothesis [30]. However, to the best of our knowledge, there was no documentation of endophytic bacteria from X. aethiopica at this report’s time. Hence, the endophytic relationship of Serratia sp. and Pseudomonas sp. in X. aethiopica is described in this work for the first time.

Conclusion

To our knowledge, this study is the first to document the presence of distinctive Serratia sp. and Pseudomonas sp. bacterial endophytes in the fruit of X. aethiopica, showing their antimicrobial potential under in vitro conditions. The extracts of bacterial strains Serratia sp. XAFb12 and Pseudomonas sp. XAFb13 showed a significant, broad-spectrum antimicrobial activity against clinically important bacterial and fungal pathogens. They are promising strains, capable of producing hydrocinnamic acid, 2-piperidinone, 5-isopropylidene-3,3-dimethyl-dihydrofuran-2-one, and diethyl trisulfide for further exploitation.

Author contribution

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

Funding

Partial financial support was received from the 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/). The accession number assigned to Serratia sp. XAFb12 is OP363300, while that of Pseudomonas sp. XAFb13 is OP363686.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.