Antibacterial potential and chromatographic profiling of bioactive compounds from endophytic Streptomyces sp. strain MIRK71 isolated from Mirabilis jalapa (L.)

Materials and Method

Collection and preparation of plant samples: The whole plant (root, leaves and stem) of Mirabilis jalapa (L). was collected from Tanhril, Aizawl, Mizoram and was transferred to laboratory using sterile airtight plastic container. The whole plant was washed in running tap water for around 10 min and was dried under room temperature for overnight in the laboratory excluding direct sun light at room temperature and cut into small bits (1.0 × 0.5 cm).

Isolation and morphological identification of endophytic actinobacteria: The small cut bits were surfaced sterilised by immersion in 70% (v/v) ethanol for 60 s, followed by sodium hypochlorite solution (1% w/v, available chlorine) for 120 s and finally in 70% ethanol (v/v) for 30 s and then 4–5 bits were placed on ISP7 (Tyrosine agar) and SCA (Starch Casein Agar) media (Kuster & Williams, 1964). Nalidixic acid and cycloheximide (50 µg/ml) were used to supress the bacterial and fungal growth in the media (Assad et al., 2021). The plates were incubated at 28 ± 2 °C for 3–5 days. Individual colonies with characteristics of actinobacteria morphology were isolated and pure culture of the respective isolates were obtained by repeated streaking on ISP7 plates. The nature of the colony, the colour of aerial and substrate mycelium was studied (Goodfellow & Haynes, 1984). The spore chain morphologies of the isolates were studied using a scanning electron microscope (SEM). The mycelium structures were observed using a phase contrast microscope (Olympus), and the organisms were identified based on their shape and size, Gram reaction, and other pertinent morphological features according to Bergey’s Manual of Determinative Bacteriology 9th edition (Zothanpuia et al., 2018).

Primary screening

Actinobacteria isolates were tested for production of antimicrobial compounds against clinical pathogens (Table 1). The isolates were grown in tryptone yeast extract broth (ISP1) media in shaker incubator at 150 rpm for three days. The broth was centrifuged and the supernatant was collected which was further used for primary antimicrobial screening following agar well diffusion method (Saadoun & Muhana, 2008). Wells with 6 mm diameter were made on the agar plate using sterile cork borer. The test organism was swabbed on the agar surface and 100 µl of the supernatant was added in the wells. The plates were allowed to stand for few minutes and incubated at 37 °C without inverting for 24 h. Clinical pathogens were collected from patients in Synod Hospital, Durtlang, Aizawl, Mizoram. Numbering system was used for marking the pathogens in order to make the patient anonymous. The clinical pathogen was isolated and identified using Vitek^®MS (bioMérieux, Marcy l’Etoile, France) GP colorimetric identification card and the antibiotic susceptibility was determined using Vitek^®MS AST (bioMérieux, Marcy l’Etoile, France).

Extract preparation and antimicrobial screening: The actinobacteria that was selected was inoculated for submerged fermentation. This process was carried out in 700 mL of ISP1 medium with a pH of 7.2, in a 500 mL conical flask under sterile conditions. The flasks were placed on a flask shaker at a speed of 120 rpm at room temperature for one week. After one week of fermentation, the medium was observed to become turbid.. The culture was harvested and centrifuged to remove cells and debris and the resultant broth was mixed with equal volume of solvents (methanol, ethyl acetate and dichloromethane) 1:1 ratio (v/v) to extract secondary metabolites (Selvameenal, Radhakrishnan & Balagurunathan, 2009). After 24 h, the solvents were filtered out using Whatman filter paper. The excess solvent from the filtrated was evaporated under reduced pressure using a rotary evaporator to give crude extract. The extracts were subjected to antimicrobial screening. A concentration of 20 mg/ml of the crude extract was prepared for antimicrobial screening using agar well diffusion method (Saadoun & Muhana, 2008). A total of 4% methanol was used as a negative control. Small wells with a six mm diameter were created on an agar plate using sterile cork borers. The test organism was swabbed onto the agar surface, and 100 µL of a 20 mg/ml crude extract was added to the wells. The plates were then left to stand for a few minutes and were subsequently incubated at 37 °C without being inverted for 24 h.

Identification of potential endophytic actinobacteria: Genomic DNA was isolated and purified using a DNA extraction kit (Invitrogen) according to the manufacturer’s protocol. Ribosomal RNA (16S rRNA) genes were amplified using universal bacterial primers 16SP1 and 16SP2 as forward and reverse primer (forward 16 s rRNA primer 5′-GTGCCAGCAGCCGCGG-3′ and reverse 16 s rRNA primer 5′-TACGGYTACCTTGTTACDACTT-3′). The reactions and conditions of the PCR were performed in a Veriti thermal cycler (Applied Biosystem, Singapore) in a total volume 25µl and the PCR conditions are as follows: initial denaturation step at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 62 °C for 1 min and extension at 72 °C for 1.2 min with a final extension step at 72 °C for 5 min and sequencing was done commercially at Eurofins Pvt. Ltd. Bangalore, India. Sequences were compared with the reference strains of actinobacteria from the NCBI genomic database using a BLASTn search tool to determine similarity percentages. The strains with the highest similarity percentages were obtained from the NCBI database.

Phylogenetic analysis

The sequences of the 16S rRNA gene were compared with the NCBI database using BlastN and the most similar match sequence was selected. The sequences were aligned with pair wise alignment and multiple alignment using the program Clustal W packaged in the MEGA 11 software. From this data, a phylogenetic tree was constructed using the neighbor-joining tree method (Keklik, 2023). Bootstrap analysis was performed with MEGA 11 using Tamura-Nei (TN93).

Analysis of the chemical components of the extract by Gas chromatography-mass spectrometry (GCMS): Gas chromatography-mass spectrometry (GC-MS) was utilized to identify the volatile organic compounds (VOCs) in the methanolic extracts of Streptomyces sp.For GC–MS, the Clarus 690 Perkin Elmer GC, which was coupled with a mass detector Turbomass gold 5.1 spectrometer, and an Elite 1 (100% Dimethyl poly siloxane) capillary column measuring 123.5 MX 678 M. The instrument was initially set to a temperature of 40 °C and then ramped up to 150 °C at 10 °C/min and held for 5 min. After that the oven temperature was increased to 250 °C at a rate of 40 °C/min and maintained for eight minutes. The injection port temperature was set at 250 °C, while the helium flow rate was maintained at 1.5 mL/min. The ionization voltage used was 70 eV, and the samples were injected in a 10:1 split mode. The mass spectral scanning range was ser to 500–800 (m/z), and the ion source temperature and interface temperature were kept at 230 °C and 240 °C, respectively. The MS start time was three minutes and the end time was 31 min, with a solvent cut time of three minutes. The spectra of volatile compounds detected through GC-MS were compared and matched with NIST17 online library Ver.2.339

Separation of compounds using high-performance thin-layer chromatography (HPTLC): The extract was further analysed using CAMAG HPTLC instrument equipped with winCAT software. A stock solution of 50 mg/ml in distilled water was prepared. TLC plates with 0.2 mm precoated silica gel 60F₂₅₄ (Merck, Germany) of 2 × 10 cm was taken. The sample was spotted using Linomat 5 automated sample spotter (CAMEG) using 100 µl of syringe (Hamilton, Bonaduz, Switzerland). A phenolic, glycosides and mobile phase was used for profiling. Nitrogen gases were supplied on plates for simultaneous drying of bands. The extract loaded plates were kept in TLC twin trough developing chamber with the mobile phase Toluene: Ethyl acetate: Acetic Acid (2:4:0.1). The developed plate was dried and scanned using by TLC scanner with Wincats software. The plates were directly visualized after drying and finger print profile was documented under 254 nm and 366 nm in UV visible light. The peak display, peak densitogram and peak table was recorded

Isolation of endophytic actinobacteria

A total of 18 isolates (MIRK54, MIRK55, MIRK56…MIRK71) were obtained from Mirabilis jalapa Ten isolates were obtained from the roots, five isolates from the leaves and three isolates from the stem. ISP7 and SCA were used for the isolation process, 12 isolates were recovered from ISP7 media and six isolates were recovered from SCA. This clearly indicates that ISP7 is more suitable medium for the isolation of endophytic actinobacteria from Mirabilis jalapa. MIRK71 actinobacterial isolate has white colonies, aerial mass was white while the substrate mycelium was dark red and little pigments were produced on the media (Fig. 1A). A smooth spore surface with branched, filamentous and microspore chain colonies was identified by scanning electron microscope (Fig. 1B).

Primary screening

The primary screening of antimicrobial was initially done for all the 18 isolates using supernatant of three days old broth culture grown in ISP1 media against 33 MDR clinical pathogens namely MRSA, VRE, Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli. Out of the 18 isolates, MIRK55, MIRK68, MIRK69 and MIRK71 showed inhibition of pathogens growth to at least five or more of the test organisms with an inhibition zone ranging from 5–18 mm in diameter with the best inhibition shown by MIRK71.

Extract preparation and secondary screening

Based on the primary screening, the best four isolates i.e MIRK55, MIRK68, MIRK69 and MIRK71 were subjected for extract preparation and secondary screening. Methanol, ethyl acetate and dichloromethane were used as a solvent for each isolate and a total of 12 extract were prepared and subjected to secondary screening using Agar well diffusion method. The extracts were tested only against 23 MDR clinical pathogens that showed positive results from the primary screening, including MRSA, VRE and Pseudomonas aeruginosa. Of the 12 extracts, methanolic crude extract of MIRK71 showed the best antimicrobial activity by inhibiting 18 pathogens with zone of inhibition ranging from 3–10 mm in diameter. Results of secondary antimicrobial screening suggest that the most commonly inhibited bacterial growth for methanolic extract of actinobacteria includes MRSA and VRE. Consequently, methanolic crude extract of MIRK71 was selected for further detailed characterization.

Molecular identification

The best isolate MIRK71 was subjected to molecular identification using 16S rRNA gene sequencing, the isolate MIRK71 was identified as Streptomyces sp. that share 99.3% similarity with Streptomyces sp. NSC30 (MG833381). The sequence was deposited in NCBI GenBank database with an accession number OR896092.

Phylogenetic analysis

16S rRNA gene sequences of MIRK71 and the most similar match sequences were used for the construction of phylogenetic tree. The phylogenetic tree was constructed using neighbor-joining method and the bootstrap analysis was performed using Tamura nei model as given in Fig. 2.

Determination of volatile compounds using GC-MS

The volatile organic compounds (VOCs) was determined using Gas chromatography–mass spectroscopy (GC-MS). The methanolic crude extract of Streptomyces sp. MIRK71 revealed 20 VOCs within the retention time of 15–29 min (Table 2). The area percentage ranges from 10.111 (1H-indole-3-acetic acid, 5-chloro-2-methyl-1-(trimethylsilyl) silane) to 0.835% (Perylene). Of the 20 VOCs, most of the compounds were found to have antimicrobial activity.

HPTLC profiling of the potential extract

Three optimized solvent system for mobile phase namely ethyl acetate: methanol: water (20:3:2), cyclohexane: ethyl acetate: formic acid (4:6:1) and toluene: ethyl acetate: methanol: acetic acid (3:5:1:0.5) for glycosides (SS1), phenols (SS2) and anthracene (SS3) respectively were used at a volume of eight µl of methanolic extract performed at UV 254 nm and 366 nm in visualizer and visible light and single brown spots were detected (Fig. 3). The images were captured and scanned in CAMAG TLC Scanner using VisionCATS 3.2 SP2 software.

The peak display, peak densitogram, and peak table were recorded and analysed and Rf values were measured then the presence of positive result was recorded on 254 nm and 366 nm. five peaks were recorded in SS1 (Fig. 4) whereas, seven peaks were recorded in SS2 (Fig. 5) and five peaks were recorded in SS3 (Fig. 6) at 254 nm. The peaks with Rf value and area (%) of SS1, SS2 and SS3 are shown in Tables 3, 4 and 5 respectively.