# Purification and Characterization of Bioactive Metabolite fromStreptomyces monomycini RVE129 Derived from the Rift Valley Soil of Hawassa, Ethiopia

Authors: Firew Elias; Sudhamani Muddada; Diriba Muleta; Belachew Tefera
Journal: BioMed Research International
 (2022)

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## Abstract

Streptomyces species have produced a variety of bioactive secondary metabolites with intriguing antimicrobial and anticancer properties. In this study, the bioactive compound obtained from the potent strain RVE129 was purified and characterized. Its bioactivity against various pathogens and its cytotoxicity toward the human cervical cancer (HeLa) cell were also examined. The strain was previously isolated from unexplored areas of the rift valley soil of Hawassa (Ethiopia) and identified by phenotypic characteristics and complete sequencing of the 16S rRNA gene and found to be closely related to Streptomyces monomycini strain NRRL B-24309 (99.65%); accession no. (ON786620). The active fraction undergoes bioassay-guided purification using the TLC method after being extracted by ethyl acetate. Then, it was subjected to physicochemical and structural characteristics using UV–Vis, FTIR, and NMR spectroscopic methods. A minimum inhibitory concentration of the purified antibiotic was achieved by the broth microdilution method. The cytotoxicity of HeLa cells was determined using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The acquired data from spectroscopic studies was compared with that of the reported natural compounds in data bases and found to be the known antibiotic, setamycin. The antibiotic (RVE-02) showed a broad spectrum of bioactivity against both Gram-positive and Gram-negative bacteria, with MIC values that ranged from 1.97 to 125 μg/ml. The bioactivity results also demonstrated antiproliferation and morphological change in HeLa cells with an IC50 value of 24.30μg/ml of antibiotic. The antibiotic, obtained from S. monomycini RVE129, could be a potential candidate to combat pathogens including drug-resistant S. aureus. Further, the effect on HeLa cells suggests that it could be a prominent cancer chemotherapeutic agent.


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## Body

## 1. Introduction

The rapid emergence of resistant pathogens to multiantibiotics along with the limited availability of effective anticancer drugs has led to the urgent need for focused research on effective antimicrobial and anticancer agents with minimal undesired side effects [1]. Microorganisms have been used as a promising source for developing a variety of bioactive metabolites that can be used to fight the spread of infection, deadly diseases such as cancer, and the toxicity associated with currently used cancer drugs [1–3]. Diverse potentially significant species have been researched as a natural source for the production of novel bioactive compounds for the treatment of allergies, autoimmune diseases, infections, cancer, inflammatory diseases, and others [4, 5]. Actinomycetes have been extensively explored and utilized as a group of microorganisms for the development of antibiotics and other important natural bioactive molecules [5]. Among actinomycetes, Streptomyces species are the most potent reservoir of many novel compounds with a broad array of bioactivities [6, 7].Currently, in the route of selection for novel bioactive molecules, many researchers are concentrating on isolating actinomycetes from uncharted and uncommon environments to combat the spread of infectious diseases by pathogens [6] and cancer [7, 8]. However, the number of new microbial natural products has been declining due to the increasing rediscovery of old known molecules. In exploring the poorly studied and unique habitat with varied climatic zones of the Rift Valley of Hawassa, Ethiopia, a promising actinomycete strain, RVE129, belonging to the genus Streptomyces sp., was selected and studied.A few studies have been reported in Ethiopia on the antimicrobial activity of actinomycetes, but no published evidence exists on the antitumor activity. In light of these observations, the current research aims at the purification and characterization of antibacterial compounds as well as evaluating their bioactivity against drug-resistant pathogens. The cytotoxic activity of the bioactive compound RVE-02 was also investigatedin vitro on human cervical carcinoma (HeLa) cells.


## 2. Materials and Methods

### 2.1. Microorganism and Maintenance

The potential antimicrobial metabolite-producing actinomycete isolate,Streptomyces RVE129, was previously recovered from the rhizosphere soils of the Rift Valley areas of Hawassa, Ethiopia [9]. After isolation, the strain was kept in a pure culture over starch casein slants and stored at 4°C. Different bacterial test strains, including Staphylococcus aureus ATCC 259233, Staphylococcus epidermidis ATCC 12228, Klebsiella pneumonia ATCC 700603, Pseudomonas aeruginosa ATCC 27853, Salmonella typhi ATCC 13311, Salmonella typhi ATCC 14028, and Escherichia coli ATCC 25922, were supplied by the Ethiopian Health and Nutrition Research Institute (EHNRI). All bacterial strains were maintained in the refrigerator on Mueller-Hinton’s agar slants.


### 2.2. Morphological and Cultural Characterization

The macromorphology of the strain was visually examined to determine the growth pattern, substrate, and aerial mycelia as well as the colour of diffusible pigment by culturing on starch casein agar [10] and International Streptomyces Project (ISP) media plates as suggested by the ISP and compared with Bergey’s manual [11]. The pure culture of the selected potent isolate was spread on the agar plates of the international Streptomyces project (ISP) medium plates as recommended by the ISP, compared to Bergey’s manual, and incubated for 10 days at 30°C [12]. Micromorphological features such as spore morphology and hyphae were identified by the coverslip method and the Gram staining method using a well-grown sporulated culture plate in yeast extract malt extract agar medium plate at a 45° inclination using a trinocular light microscope at 1000x magnification, and the spore shape and surface were determined by using a scanning electron microscope (Cambridge Stereoscan, model 240) according to the method of Singh et al. [13].


### 2.3. Physiological and Biochemical Characterization

Physiological tests such as different temperatures (15, 20, 25, 30, 37, and 45°C), pH range (4, 5, 6, 7, 8, 9, 10, 11, and 12), and NaCl concentrations (0, 2.5, 5, 7.5, and 10%) for the cultivation of RVE129 isolate were evaluated on ISP2 agar plates after two weeks of incubation. Sugar utilization tests were determined by growth on the ISP-9 medium, added with 1.0% of each of the carbon sources at 30°C as described by Shirling and Gottlieb [11]. The isolate was screened for reduction of nitrate, gelatin degradation, starch hydrolysis, casein, catalase, urease, and H2S production and were determined according to Locci [12], and melanin production was evaluated on tyrosine agar (ISP 7) medium [14].


### 2.4. Molecular-Based Analysis

The amplification and sequencing were carried out by the Microbial Type Culture Collection and Gene Bank in Chandigarh, India. Taxonomic identification of the strain was further verified by performing PCR reactions using universal primers for amplification and subjecting them to the 16S rRNA Genomic DNA gene sequence analysis [15]. The standard basic local alignment search tool (BLAST) program was used to match partial DNA sequences of 16S rRNA from the public database (NCBI) [16, 17]. The 16S RNA gene sequence was compared to other relevant sequences using the Clustal W, and the BLAST search technique was employed to examine for similarities [17]. Individual accession numbers were assigned after the sequences were deposited in the NCBI gene database. Phylogenetic tree construction was conducted using a neighbor-joining approach by MEGA software version 6.0. The isolate and related type strains’ representative nucleotides from the total and partial 16S rRNA sequences in the GenBank database were used to generate the tree from the high similarity sequences.


### 2.5. Production and Extraction of Bioactive Metabolites

Production of active metabolites from the potentialStreptomyces RVE129 sp. isolate followed the shake flask fermentation approach as described by Oskay [18] and Singh et al. [13] with a few modifications. In brief, the seed culture was obtained by inoculating spore suspensions of RVE129 in ISP2 broth medium and incubating for 3 days at 30°C on a shaking incubator (150 rpm). Following incubation, 5% of the culture was inoculated into 200 ml of cultivation medium containing 10 g starch, 2 g NaCl, 2 g (NH4)2SO4, 1 g MgSO4.7H2O, 1 g K2HPO4, and 20 g of agar in 1.0 litre of distilled water. The shake flask was placed in the shaking incubator (150 rpm) for 10 days at 30°C. The fermentation broth was centrifuged at 10,000 rpm for 15 minutes and filtered through Whatman No. 1 to remove the mycelial biomass. The culture filtrates and the ethyl acetate solvent were mixed at a 1 : 1 ratio (w/v) and agitated continuously for 60 min. The organic phase was evaporated and concentrated under a rotary evaporator at 50°C and stored at 4°C. Finally, the dried bioactive metabolite was accumulated and preserved at 4°C for further testing.


### 2.6. Purification of Antimicrobial Agent

After organic solvent extraction, the purification of active metabolites was done by thin-layer chromatography (TLC) plates in a mobile solvent technique following the method of Fried and Sherma [19]. Twenty μl of extract (1 mg/mL) was loaded at the bottom of the TLC plate with a capillary tube and placed inside the solvent chamber with a mobile phase of ethyl acetate: hexane (1 : 1). The plates were removed aseptically and allowed to dry at room temperature once the solvent had reached the top of the chromatography board. The spotted TLC plate was kept in a closed iodine chamber to expose iodine vapour and visualized under the ultraviolet light wavelength of 254 nm. The retention factors (RF) values of the different spots were observed and calculated.


### 2.7. Detection of Bioautography

After TLC was performed, the bioautography method was followed as reported by Selvameenal et al. [20] for the identification of the antimicrobial activity of spots separated in TLC. Briefly, TLC strips were placed aseptically on the surface of a sterile Mueller-Hinton’s agar (MHA) plate inoculated with 200 μl of the fresh culture cell count (3×106) of S. aureus ATCC 259233 and S. typhi ATCC 14028. Plates were kept in the refrigerator for 1 hour before being incubated at 37°C for 24 hours. Growth inhibition in the region of the spot suggests that it has an active antimicrobial compound.


### 2.8. Identification of Bioactive Compound

#### 2.8.1. Temperature and pH Stability Study

The solubility of purified antibiotics was evaluated using various solvents such as chloroform, methanol, acetone, ethanol, petroleum ether, and water. The melting point of purified compound was determined by the Fisher-Johns melting point apparatus. The characteristics of purified antibiotics derived fromS. monomycini RVE129 were examined by the standard procedures of Harindran et al.’s [21] slight modification. Briefly, the purified antibiotic sample was placed in test tubes and exposed to various temperatures (0°C, 20°C, 30°C, 40°C, and 50°C) to determine the temperature stability with regard to its antibacterial activity. Then, the solutions were cooled to room temperature and subjected to antibacterial activity tests against Staphylococcus aureus by well diffusion assay [22]. The purified antibiotic sample was placed in separate test tubes, and the pH was adjusted to different ranges (4–12) using 0.1 N NaOH or 0.1 N HCl to evaluate pH stability. The tubes were neutralized and left at room temperature for 60 minutes before being evaluated for bioactivity against S. aureus using an agar well diffusion assay.


#### 2.8.2. Spectroscopic Studies

The active fraction extracted with methanol from TLC was evaluated with several spectrum studies for the identification of the active compound [23]. The UV-vis spectra of the purified fraction was qualitatively observed on a UV-vis spectrophotometer (Shimadzu, Kyoto, Japan) and scanned over the desired wavelength range of 200 and 400 nm using methanol as a reference solvent. The Fourier transformation infrared (FTIR) spectroscopy analysis of the purified antibiotic fraction was done with KBr pellets. The FTIR spectra were observed using (Shimadzu, Kyoto, Japan) spectroscopy at between 400 and 4000 cm−1 wavelength. The results of FTIR spectrum patterns are analyzed and matched to determined compounds in the FTIR library [24]. Nuclear magnetic resonance (NMR) spectroscopy (Bruker DRX500, Germany) was used to record the 1H and 13C NMR spectra of the purified antibiotic in DMSO at 500 MHz for 1H and 150 MHz for 13C. To calibrate chemical shifts in 1H and 13C NMR spectra, an internal standard of 0.1% (v/v) tetramethylsilane (TMS) was used.


### 2.9. Biological Assays

#### 2.9.1. Antimicrobial Assay

The bioactivity of isolated bioactive compounds fromStreptomyces sp. RVE129 against several test pathogens was investigated using the standard reference methods of the Clinical and Laboratory Standards Institute [25]. A stock solution (50 mg/ml) of the active purified fraction was scrapped from TLC, prepared in 50% dimethyl sulfoxide (DMSO), and mixed with Mueller-Hinton’s broth (MHB) to working concentrations, whereas the antibiotics were dissolved in sterile distilled water. A bacteria culture was standardized at 0.5 McFarland and diluted 1 : 100 in saline to have a final cell count of 1×106CFU/mL. The bioactive compound and standard antibiotics (streptomycin) at concentrations (1.97, 3.95, 7.56, 15.12, 31.25, 62.5, 125, and 250 μg/ml) were prepared with sterile water using two-fold serial dilutions. Each well of a microtiter plate was filled with one hundred microliters of test concentration of the compound along with the microbial suspension and 100 μL of sterile MHB. A control was established using MHB media containing cultures of test microorganisms with 50% DMSO as a control. The MICs were determined by incubating microtitre plates for 24 hours at 37°C. The lowest concentration of the test fraction that inhibited observable bacterial growth was determined as the MIC value.


#### 2.9.2. In Vitro Cytotoxicity Assay

The in vitro cytotoxicity of purified antibiotics RVE-02 was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye cell viability assay [26]. HeLa cells were cultured in Dulbecco’s modified eagle’s medium (DMEM), which was supplemented with 0.1 mM sodium pyruvate, 1 mM with 10% fetal bovine serum, and 0.5 mM L-glutamine. The cells were cultured in 96-well microplates at a density of 0.2×106 cells per well with various concentrations of purified fraction (3.78, 7.56, 16.12, 31.25, 62.5, 125, and 250 μg/ml) and incubated for 24 hours in 5% CO2 at 37°C. Then, the medium was treated with 20 μL of MTT solution (5 mg/mL in PBS) in each well and incubated for 4 h at 37°C with 5% CO2. Finally, to dissolve the formazan crystal product, 200 μl of DMSO was added to the solution and well mixed. HeLa cells treated with DMSO under the same conditions were used as a positive control. Optical density (OD) at 570 nm was used to determine absorbance after incubation [27, 28]. The following formula was used to calculate cell viability:
(1)Viabilityofcell%=100−AbssampleAbscontrol×100.The half inhibitory concentration (IC50) value was determined as a 50% reduction in absorbance, compared to the control experiment. Assessment of the cell morphology was performed by using a phase-contrast inverted microscope (Olympus, Japan), and photographs were captured using a digital camera.


## 2.1. Microorganism and Maintenance

The potential antimicrobial metabolite-producing actinomycete isolate,Streptomyces RVE129, was previously recovered from the rhizosphere soils of the Rift Valley areas of Hawassa, Ethiopia [9]. After isolation, the strain was kept in a pure culture over starch casein slants and stored at 4°C. Different bacterial test strains, including Staphylococcus aureus ATCC 259233, Staphylococcus epidermidis ATCC 12228, Klebsiella pneumonia ATCC 700603, Pseudomonas aeruginosa ATCC 27853, Salmonella typhi ATCC 13311, Salmonella typhi ATCC 14028, and Escherichia coli ATCC 25922, were supplied by the Ethiopian Health and Nutrition Research Institute (EHNRI). All bacterial strains were maintained in the refrigerator on Mueller-Hinton’s agar slants.


## 2.2. Morphological and Cultural Characterization

The macromorphology of the strain was visually examined to determine the growth pattern, substrate, and aerial mycelia as well as the colour of diffusible pigment by culturing on starch casein agar [10] and International Streptomyces Project (ISP) media plates as suggested by the ISP and compared with Bergey’s manual [11]. The pure culture of the selected potent isolate was spread on the agar plates of the international Streptomyces project (ISP) medium plates as recommended by the ISP, compared to Bergey’s manual, and incubated for 10 days at 30°C [12]. Micromorphological features such as spore morphology and hyphae were identified by the coverslip method and the Gram staining method using a well-grown sporulated culture plate in yeast extract malt extract agar medium plate at a 45° inclination using a trinocular light microscope at 1000x magnification, and the spore shape and surface were determined by using a scanning electron microscope (Cambridge Stereoscan, model 240) according to the method of Singh et al. [13].


## 2.3. Physiological and Biochemical Characterization

Physiological tests such as different temperatures (15, 20, 25, 30, 37, and 45°C), pH range (4, 5, 6, 7, 8, 9, 10, 11, and 12), and NaCl concentrations (0, 2.5, 5, 7.5, and 10%) for the cultivation of RVE129 isolate were evaluated on ISP2 agar plates after two weeks of incubation. Sugar utilization tests were determined by growth on the ISP-9 medium, added with 1.0% of each of the carbon sources at 30°C as described by Shirling and Gottlieb [11]. The isolate was screened for reduction of nitrate, gelatin degradation, starch hydrolysis, casein, catalase, urease, and H2S production and were determined according to Locci [12], and melanin production was evaluated on tyrosine agar (ISP 7) medium [14].


## 2.4. Molecular-Based Analysis

The amplification and sequencing were carried out by the Microbial Type Culture Collection and Gene Bank in Chandigarh, India. Taxonomic identification of the strain was further verified by performing PCR reactions using universal primers for amplification and subjecting them to the 16S rRNA Genomic DNA gene sequence analysis [15]. The standard basic local alignment search tool (BLAST) program was used to match partial DNA sequences of 16S rRNA from the public database (NCBI) [16, 17]. The 16S RNA gene sequence was compared to other relevant sequences using the Clustal W, and the BLAST search technique was employed to examine for similarities [17]. Individual accession numbers were assigned after the sequences were deposited in the NCBI gene database. Phylogenetic tree construction was conducted using a neighbor-joining approach by MEGA software version 6.0. The isolate and related type strains’ representative nucleotides from the total and partial 16S rRNA sequences in the GenBank database were used to generate the tree from the high similarity sequences.


## 2.5. Production and Extraction of Bioactive Metabolites

Production of active metabolites from the potentialStreptomyces RVE129 sp. isolate followed the shake flask fermentation approach as described by Oskay [18] and Singh et al. [13] with a few modifications. In brief, the seed culture was obtained by inoculating spore suspensions of RVE129 in ISP2 broth medium and incubating for 3 days at 30°C on a shaking incubator (150 rpm). Following incubation, 5% of the culture was inoculated into 200 ml of cultivation medium containing 10 g starch, 2 g NaCl, 2 g (NH4)2SO4, 1 g MgSO4.7H2O, 1 g K2HPO4, and 20 g of agar in 1.0 litre of distilled water. The shake flask was placed in the shaking incubator (150 rpm) for 10 days at 30°C. The fermentation broth was centrifuged at 10,000 rpm for 15 minutes and filtered through Whatman No. 1 to remove the mycelial biomass. The culture filtrates and the ethyl acetate solvent were mixed at a 1 : 1 ratio (w/v) and agitated continuously for 60 min. The organic phase was evaporated and concentrated under a rotary evaporator at 50°C and stored at 4°C. Finally, the dried bioactive metabolite was accumulated and preserved at 4°C for further testing.


## 2.6. Purification of Antimicrobial Agent

After organic solvent extraction, the purification of active metabolites was done by thin-layer chromatography (TLC) plates in a mobile solvent technique following the method of Fried and Sherma [19]. Twenty μl of extract (1 mg/mL) was loaded at the bottom of the TLC plate with a capillary tube and placed inside the solvent chamber with a mobile phase of ethyl acetate: hexane (1 : 1). The plates were removed aseptically and allowed to dry at room temperature once the solvent had reached the top of the chromatography board. The spotted TLC plate was kept in a closed iodine chamber to expose iodine vapour and visualized under the ultraviolet light wavelength of 254 nm. The retention factors (RF) values of the different spots were observed and calculated.


## 2.7. Detection of Bioautography

After TLC was performed, the bioautography method was followed as reported by Selvameenal et al. [20] for the identification of the antimicrobial activity of spots separated in TLC. Briefly, TLC strips were placed aseptically on the surface of a sterile Mueller-Hinton’s agar (MHA) plate inoculated with 200 μl of the fresh culture cell count (3×106) of S. aureus ATCC 259233 and S. typhi ATCC 14028. Plates were kept in the refrigerator for 1 hour before being incubated at 37°C for 24 hours. Growth inhibition in the region of the spot suggests that it has an active antimicrobial compound.


## 2.8. Identification of Bioactive Compound

### 2.8.1. Temperature and pH Stability Study

The solubility of purified antibiotics was evaluated using various solvents such as chloroform, methanol, acetone, ethanol, petroleum ether, and water. The melting point of purified compound was determined by the Fisher-Johns melting point apparatus. The characteristics of purified antibiotics derived fromS. monomycini RVE129 were examined by the standard procedures of Harindran et al.’s [21] slight modification. Briefly, the purified antibiotic sample was placed in test tubes and exposed to various temperatures (0°C, 20°C, 30°C, 40°C, and 50°C) to determine the temperature stability with regard to its antibacterial activity. Then, the solutions were cooled to room temperature and subjected to antibacterial activity tests against Staphylococcus aureus by well diffusion assay [22]. The purified antibiotic sample was placed in separate test tubes, and the pH was adjusted to different ranges (4–12) using 0.1 N NaOH or 0.1 N HCl to evaluate pH stability. The tubes were neutralized and left at room temperature for 60 minutes before being evaluated for bioactivity against S. aureus using an agar well diffusion assay.


### 2.8.2. Spectroscopic Studies

The active fraction extracted with methanol from TLC was evaluated with several spectrum studies for the identification of the active compound [23]. The UV-vis spectra of the purified fraction was qualitatively observed on a UV-vis spectrophotometer (Shimadzu, Kyoto, Japan) and scanned over the desired wavelength range of 200 and 400 nm using methanol as a reference solvent. The Fourier transformation infrared (FTIR) spectroscopy analysis of the purified antibiotic fraction was done with KBr pellets. The FTIR spectra were observed using (Shimadzu, Kyoto, Japan) spectroscopy at between 400 and 4000 cm−1 wavelength. The results of FTIR spectrum patterns are analyzed and matched to determined compounds in the FTIR library [24]. Nuclear magnetic resonance (NMR) spectroscopy (Bruker DRX500, Germany) was used to record the 1H and 13C NMR spectra of the purified antibiotic in DMSO at 500 MHz for 1H and 150 MHz for 13C. To calibrate chemical shifts in 1H and 13C NMR spectra, an internal standard of 0.1% (v/v) tetramethylsilane (TMS) was used.


## 2.8.1. Temperature and pH Stability Study

The solubility of purified antibiotics was evaluated using various solvents such as chloroform, methanol, acetone, ethanol, petroleum ether, and water. The melting point of purified compound was determined by the Fisher-Johns melting point apparatus. The characteristics of purified antibiotics derived fromS. monomycini RVE129 were examined by the standard procedures of Harindran et al.’s [21] slight modification. Briefly, the purified antibiotic sample was placed in test tubes and exposed to various temperatures (0°C, 20°C, 30°C, 40°C, and 50°C) to determine the temperature stability with regard to its antibacterial activity. Then, the solutions were cooled to room temperature and subjected to antibacterial activity tests against Staphylococcus aureus by well diffusion assay [22]. The purified antibiotic sample was placed in separate test tubes, and the pH was adjusted to different ranges (4–12) using 0.1 N NaOH or 0.1 N HCl to evaluate pH stability. The tubes were neutralized and left at room temperature for 60 minutes before being evaluated for bioactivity against S. aureus using an agar well diffusion assay.


## 2.8.2. Spectroscopic Studies

The active fraction extracted with methanol from TLC was evaluated with several spectrum studies for the identification of the active compound [23]. The UV-vis spectra of the purified fraction was qualitatively observed on a UV-vis spectrophotometer (Shimadzu, Kyoto, Japan) and scanned over the desired wavelength range of 200 and 400 nm using methanol as a reference solvent. The Fourier transformation infrared (FTIR) spectroscopy analysis of the purified antibiotic fraction was done with KBr pellets. The FTIR spectra were observed using (Shimadzu, Kyoto, Japan) spectroscopy at between 400 and 4000 cm−1 wavelength. The results of FTIR spectrum patterns are analyzed and matched to determined compounds in the FTIR library [24]. Nuclear magnetic resonance (NMR) spectroscopy (Bruker DRX500, Germany) was used to record the 1H and 13C NMR spectra of the purified antibiotic in DMSO at 500 MHz for 1H and 150 MHz for 13C. To calibrate chemical shifts in 1H and 13C NMR spectra, an internal standard of 0.1% (v/v) tetramethylsilane (TMS) was used.


## 2.9. Biological Assays

### 2.9.1. Antimicrobial Assay

The bioactivity of isolated bioactive compounds fromStreptomyces sp. RVE129 against several test pathogens was investigated using the standard reference methods of the Clinical and Laboratory Standards Institute [25]. A stock solution (50 mg/ml) of the active purified fraction was scrapped from TLC, prepared in 50% dimethyl sulfoxide (DMSO), and mixed with Mueller-Hinton’s broth (MHB) to working concentrations, whereas the antibiotics were dissolved in sterile distilled water. A bacteria culture was standardized at 0.5 McFarland and diluted 1 : 100 in saline to have a final cell count of 1×106CFU/mL. The bioactive compound and standard antibiotics (streptomycin) at concentrations (1.97, 3.95, 7.56, 15.12, 31.25, 62.5, 125, and 250 μg/ml) were prepared with sterile water using two-fold serial dilutions. Each well of a microtiter plate was filled with one hundred microliters of test concentration of the compound along with the microbial suspension and 100 μL of sterile MHB. A control was established using MHB media containing cultures of test microorganisms with 50% DMSO as a control. The MICs were determined by incubating microtitre plates for 24 hours at 37°C. The lowest concentration of the test fraction that inhibited observable bacterial growth was determined as the MIC value.


### 2.9.2. In Vitro Cytotoxicity Assay

The in vitro cytotoxicity of purified antibiotics RVE-02 was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye cell viability assay [26]. HeLa cells were cultured in Dulbecco’s modified eagle’s medium (DMEM), which was supplemented with 0.1 mM sodium pyruvate, 1 mM with 10% fetal bovine serum, and 0.5 mM L-glutamine. The cells were cultured in 96-well microplates at a density of 0.2×106 cells per well with various concentrations of purified fraction (3.78, 7.56, 16.12, 31.25, 62.5, 125, and 250 μg/ml) and incubated for 24 hours in 5% CO2 at 37°C. Then, the medium was treated with 20 μL of MTT solution (5 mg/mL in PBS) in each well and incubated for 4 h at 37°C with 5% CO2. Finally, to dissolve the formazan crystal product, 200 μl of DMSO was added to the solution and well mixed. HeLa cells treated with DMSO under the same conditions were used as a positive control. Optical density (OD) at 570 nm was used to determine absorbance after incubation [27, 28]. The following formula was used to calculate cell viability:
(1)Viabilityofcell%=100−AbssampleAbscontrol×100.The half inhibitory concentration (IC50) value was determined as a 50% reduction in absorbance, compared to the control experiment. Assessment of the cell morphology was performed by using a phase-contrast inverted microscope (Olympus, Japan), and photographs were captured using a digital camera.


## 2.9.1. Antimicrobial Assay

The bioactivity of isolated bioactive compounds fromStreptomyces sp. RVE129 against several test pathogens was investigated using the standard reference methods of the Clinical and Laboratory Standards Institute [25]. A stock solution (50 mg/ml) of the active purified fraction was scrapped from TLC, prepared in 50% dimethyl sulfoxide (DMSO), and mixed with Mueller-Hinton’s broth (MHB) to working concentrations, whereas the antibiotics were dissolved in sterile distilled water. A bacteria culture was standardized at 0.5 McFarland and diluted 1 : 100 in saline to have a final cell count of 1×106CFU/mL. The bioactive compound and standard antibiotics (streptomycin) at concentrations (1.97, 3.95, 7.56, 15.12, 31.25, 62.5, 125, and 250 μg/ml) were prepared with sterile water using two-fold serial dilutions. Each well of a microtiter plate was filled with one hundred microliters of test concentration of the compound along with the microbial suspension and 100 μL of sterile MHB. A control was established using MHB media containing cultures of test microorganisms with 50% DMSO as a control. The MICs were determined by incubating microtitre plates for 24 hours at 37°C. The lowest concentration of the test fraction that inhibited observable bacterial growth was determined as the MIC value.


## 2.9.2. In Vitro Cytotoxicity Assay

The in vitro cytotoxicity of purified antibiotics RVE-02 was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye cell viability assay [26]. HeLa cells were cultured in Dulbecco’s modified eagle’s medium (DMEM), which was supplemented with 0.1 mM sodium pyruvate, 1 mM with 10% fetal bovine serum, and 0.5 mM L-glutamine. The cells were cultured in 96-well microplates at a density of 0.2×106 cells per well with various concentrations of purified fraction (3.78, 7.56, 16.12, 31.25, 62.5, 125, and 250 μg/ml) and incubated for 24 hours in 5% CO2 at 37°C. Then, the medium was treated with 20 μL of MTT solution (5 mg/mL in PBS) in each well and incubated for 4 h at 37°C with 5% CO2. Finally, to dissolve the formazan crystal product, 200 μl of DMSO was added to the solution and well mixed. HeLa cells treated with DMSO under the same conditions were used as a positive control. Optical density (OD) at 570 nm was used to determine absorbance after incubation [27, 28]. The following formula was used to calculate cell viability:
(1)Viabilityofcell%=100−AbssampleAbscontrol×100.The half inhibitory concentration (IC50) value was determined as a 50% reduction in absorbance, compared to the control experiment. Assessment of the cell morphology was performed by using a phase-contrast inverted microscope (Olympus, Japan), and photographs were captured using a digital camera.


## 3. Results

### 3.1. Cultural, Physiological, and Biochemical Characteristics

The microscopic observation of the isolate, designated asStreptomyces RVE129, showed a Gram positive, long aerial mycelium, filamentous, and rectiflexible (RF) in the arrangement. Scanning electron microscopy analysis also showed long smooth straight spores arising from the aerial mycelia and vegetative hyphae (Figure 1). Hence, the isolate was placed in the group of Streptomyces sp.Figure 1
Morphological characterization ofStreptomyces spp. RVE129 on ISP2 medium. (a) Aerial hyphae bearing spore chains under light microscope (1000x), (b) scanning electron micrograph of strains, (c) substrate mycelium, and (d) aerial mycelium.
(a)(b)(c)(d)The growth pattern ofStreptomyces sp. RVE129 was evaluated on different agar media as illustrated in Table 1. The growth pattern of isolate RVE129 exhibited some variations among the tested media. The results showed good growth on starch casein agar, peptone yeast extract iron agar medium (ISP-6), starch-inorganic salts agar (ISP-4), and yeast extract malt extract dextrose agar (ISP-2) in comparison to the other studied growth media (Table 1). The RVE129 strain displayed brown substrate mycelium and white aerial mycelium without diffusible pigment on ISP 2 plate medium (Figure 1 and Table 1).Table 1
The growth characteristics of theStreptomyces sp RVE129.
Culture mediumGrowth patternAerial myceliumSubstrate myceliumDiffusible pigmentsTryptone yeast extract agar (ISP-1)++GreyPale yellow—Yeast extract malt extract agar (ISP-2)+++WhiteBrown—Oat meal agar (ISP-3)++WhiteGreyBrownInorganic salt starch agar (ISP-4)+++BrownLight ivory—Glycerol asparagines agar (ISP-5)++GreyGreyish white—Peptone yeast extract iron agar (ISP-6)+++GreyDark grey—Tyrosine agar (ISP-7)+CreamBrown—Starch casein agar+++WhiteWhitish brown—+++: good growth; ++: moderate growth; +: poor growth; −: no diffusible pigment.The biochemical and sugar fermentation test results of strain RVE129 are illustrated in Table2. The isolate was grown in a pH range of 4–10, a temperature range of 20–40°C, and a NaCl concentration of up to 5% (w/v). The isolate showed optimal growth at 30°C and at pH 7 as well as in the presence of a 2.5% NaCl (w/v) concentration. The isolate could utilize different sugars as carbon sources, although inositol, arabinose, and rhamnose were not utilized (Table 2). The strain RVE129 exhibited positive results for the production of catalase, urease, H2S, citrate, hydrolysis of starch, casein, gelatin, and esculin degradation, whereas melanin formation, nitrate reduction, production of indole, Voges-Proskauer, and oxidase were shown negative results (Table 2).Table 2
Morphological, biochemical, and physiological characteristics of strain RVE129.
CharacteristicsResultCharacteristicsResultMicroscopic examinationIndole—Gram reaction+Voges-Proskauer—Spore chain morphologyRectiflexibleCarbon utilizationMotilityNonmotileInositol—GrowthAerobicMannose+Physiological characteristicsXylose±Temperature range for growth20-40°CAdonitol±Optimum temperature30°CFructose+pH range for growth4 to 10Sucrose+Optimum pH for growth7Sorbitol+NaCl tolerance range0-5%Trehalose+Optimum NaCl (%) for growth2.5%Arabinose—Biochemical charactersLactose+Melanin production—Galactose+Nitrate reduction—Maltose+Catalase production+Mannitol+Urease production+Glucose+Esculin degradation+Cellulose±H2S production+Starch+Citrate utilization+Fructose+Casein hydrolysis+Rhamnose—Oxidase production—Gelatin hydrolysis++: positive; ±: doubtful/poor; −: negative.


### 3.2. Molecular Characterization

According to the sequencing results, 1435 base pairs were present in the isolate and deposited in NCBI GenBank under the accession number ON786620.https://www.ncbi.nlm.nih.gov/nuccore/ON786620.1. A phylogenetic tree of the RVE129 strain and allied reference strains is shown in Figure 2. Examination of the 16S rDNA gene of the Streptomyces sp. RVE129 sequence showed maximum similarity (99.65%) with the 16S rRNA genes of Streptomyces monomycini NRRL B-24309T as indicated in Figure 2Figure 2
Phylogenetic tree constructed based on 16S rRNA gene sequences of RVE129. The percentage of similarity between the strain and related members of the genusStreptomyces 16S rRNA gene clade is shown next to the strain names.


### 3.3. TLC Profile of the Antibacterial Extract

Thin-layer chromatography (TLC) was used to purify the antibacterial component. The separate spots of the bioactive metabolite in the TLC chromatogram are presented in Figure3. The TLC results show that the extract produced five spots, denoted I, II, III, and IV, with RF values of 0.96, 0.73, 0.63, 0.43, and 0.34, respectively (Figure 3).Figure 3
TLC chromatograph of the extract fromStreptomyces sp. RVE129.The TLC-agar overlay bioautography assay revealed spot II (RF=0.73) as the active spot against S. aureus and S. typhi. The purified antibiotic was denoted as RVE-02.


### 3.4. Characterization of the Active Compound

#### 3.4.1. Physicochemical Characterization

The purified antibiotic RVE-02 was yellowish-brown in colour and had a melting point of 135°C. It was soluble in methanol, ethanol, acetone, chloroform, petroleum ether, ethyl acetate, and DMSO but insoluble in water. The antibacterial activity of purified antibiotic RVE-02 was tested at various temperatures and pH levels, and it was revealed to be stable in the pH range (4–10) and over a wide range of temperatures (20–45°C). The purified antibiotic compound treated at temperatures of 30°C retained maximum antimicrobial activity againstS. aureus ATCC-259233. The antibiotic RVE-02 was most stable between pH 7.0 and 9.0, with activity decreasing slightly at acidic or basic pH.


#### 3.4.2. Spectral Characteristics of Antimicrobial Compound

The active fraction (RF=0.73) obtained from TLC was examined by spectral analysis. The ultraviolet (UV) of antimicrobial compound (dissolved in methanol) showed three characteristic absorption peaks of maximum absorption at 243 nm, 279 nm, and 420 nm and with a shoulder at 327 nm (Figure 4).Figure 4
UV spectra of the purified antibiotic RVE-02.Infrared spectra (IR) showed peaks at 3863.5, indicating the presence of hydroxyl functional group (-OH). FTIR spectra peak absorbance located in the range of 3240 to 3511.01 cm-1, with a maximum peak at 3436.2 cm-1 is indicative of amine function and a broad hydroxyl functional group (Figure 5). Analysis of the FTIR spectrum also showed typical absorption peaks at 1663.12 cm-1 corresponding to conjugated C=O, indicating the presence of an ester group and the absorption peaks 1729.2 cm-1 corresponding to aldehyde. The peak at 1116.58 cm-1 indicates the presence of the alcohol functional group. The presence of alkane groups is indicated by a sharp absorption peak at 2923 cm-1. The spectrum peak at 987.84 cm-1 indicates the presence of methylene compounds with C-C vibrations, and the peak appearing at 797.06–801 cm-1 was assigned to p disubstituted benzene.Figure 5
FTIR spectra of purified antibiotic RVE-02.From the1H NMR spectrum data (Figure 6), the signal at δ 8.84 (N-H) confirmed the existence of the amide functionality, and the chemical shifts observed between 𝛿 H δ 6.5–δ 7.27 ppm indicate the presence of aromatic protons. The signals in the range of 𝛿 H, 4.18–4.35 indicates the presence of the terminal long chain alkane in the compound. The presence of oxygenated methine proton is supported by the chemical shift signals in the region 𝛿 H 3.645–3.79 ppm region for the methoxy group. The chemical shift value in the range of 𝛿 H 0.45–2.69 ppm region corresponds to the occurrence of aliphatic hydrogens i.e., methyl and methylene groups in the side chains (Figure 6).Figure 6
1H NMR (500 MHz, DMSO/CDCl3) spectrum data of antibiotic RVE-02.In addition, the13C NMR spectrum (Figure 7 and Table 3) of antibiotic RVE-02 indicated that the signal in the range of δC 15 to 19 ppm corresponds to methyl (-CH3) groups and peaks in the range δC 21 to 38 ppm represents the presence of different methylene (-CH2-) groups and the peaks in the range δC 39 to 58 ppm a characteristics of methine (CH) group as shown in (Figure 7). The peaks n the range of δC 60-96 ppm represents methoxy groups, and quaternary carbons were detected at δC 100-168 ppm and one α, β-unsaturated lactone carbonyl at δC 144.3 ppm. Finally, the peak at δC 167 to 198 ppm belongs to carbonyl carbon group of either an amide or an ester (Figure 7).Figure 7
13C NMR (500 MHz, DMSO/CDCl3) spectrum data of antibiotic RVE-02.Table 3
NMR spectra data of antibiotic compound RVE-02.
PositionδC of purified compoundδC setamycin [29]PositionδC of purified compoundδC setamycin [29]1167.48167.32176.2876.82141.17141. 22240.040.02-OCH60.0259. 92375.5175.53133.41133.4242827.94133.39133.32512.3512.35143.02143.0267.237.1636.6436.72713.914.0781.1281.22817.4217.3837.1737.12910.009.8941.2241.23020.220.210142.80142.83121.5821.611133.22133.03214.3214.312125.35125.23320.9221.013127.22127.0C-1′164.29164.31470.1670.24C-2′133.59133.614-OCH55.5455.5C-3′133.04133.01577.3677.2C-4′163.6163.61638.2038.2C-5′115.02114.91782.1882.2C-6′175.24175.21842.0542.4C-7′26.0025.81998.4298.2C-8′32.1732.219-OH——C-9′198.00197.72040.0840.09′ OH——


### 3.5. Biological Assays

#### 3.5.1. Antimicrobial Activity of Purified Antibiotic RVE-02

The minimum inhibitory concentration (MIC) of the bioactive compound RVE-02 is presented in Table4. The antibiotic RVE-02 demonstrated broad antibacterial efficacy against Gram-positive and Gram-negative bacteria, with MIC values ranging from 1.97 to 125 μg/ml. When compared to streptomycin (1.97 μg/ml), the lowest MIC was recorded against S. aureus (1.9 μg/ml) and the highest against K. pneumonia and P. aeruginosa (125 μg/ml). Although in most of the cases, control antibiotics had a lower MIC value than the RVE-02 antibiotic; it showed good activity (1.97 μg/ml) against S. aureus when compared to standard antibiotics (1.97 μg/ml).Table 4
Bioactivity potential of antibiotic RVE-02 against different pathogenic bacteria.
Test organismsMIC (μg/ml)Erythromycin (μg/ml)Staphylococcus aureus ATCC-2592331.971.97Staphylococcus epidermidis ATCC-122283.951.97Salmonella typhi ATCC-1331131.257.90Pseudomonas aeruginosa ATCC-2785312531.25Kelbsiella pneumonia ATCC-70060312562.5Escherichia coli ATCC-2592262.57.90Salmonella typhi ATCC-1402862.57.90


#### 3.5.2. In Vitro Cytotoxicity Analysis

The results of the cytotoxicity assay are presented in Figure6. The in vitro cytotoxic experiment showed that the RVE-02 antibiotic obtained from S. monomycini RVE129 inhibited cell growth and viability in the human cervical cancer (HeLa) cell line (Figure 8(a)).Figure 8
(a) Cytoxicity different concentrations of active metabolite on the percentage of HeLa cell viability. The data are represented as themean±SD of three separate experiments, for each experiment. (b) Inverted microscopic (40x) images of HeLa cell lines treated with different concentrations of antibiotic RVE-02.
(a)(b)The cytotoxicity results showed that the purified antibiotic RVE-02 had a considerable antiproliferative influence on HeLa cells, with anIC50 value of 18.30 μg/ml of concentration (Figure 8(a)). The lowest tested concentration (3.78 μg/ml) was able to inhibit cell line proliferation by less than 10%. In contrast, more than 90% of cell line proliferation inhibition was recorded after 24 hours treatment at 250 μg/ml (Figure 8(b)). These results showed that the antibiotic RVE-02 induced cell cytotoxicity against HeLa cell lines in a direct dose-response relationship where the viability of cells was reduced, and a change in the shape of the cell was observed with an increased antibiotic concentration in the treated cells (Figure 8(b)).


## 3.1. Cultural, Physiological, and Biochemical Characteristics

The microscopic observation of the isolate, designated asStreptomyces RVE129, showed a Gram positive, long aerial mycelium, filamentous, and rectiflexible (RF) in the arrangement. Scanning electron microscopy analysis also showed long smooth straight spores arising from the aerial mycelia and vegetative hyphae (Figure 1). Hence, the isolate was placed in the group of Streptomyces sp.Figure 1
Morphological characterization ofStreptomyces spp. RVE129 on ISP2 medium. (a) Aerial hyphae bearing spore chains under light microscope (1000x), (b) scanning electron micrograph of strains, (c) substrate mycelium, and (d) aerial mycelium.
(a)(b)(c)(d)The growth pattern ofStreptomyces sp. RVE129 was evaluated on different agar media as illustrated in Table 1. The growth pattern of isolate RVE129 exhibited some variations among the tested media. The results showed good growth on starch casein agar, peptone yeast extract iron agar medium (ISP-6), starch-inorganic salts agar (ISP-4), and yeast extract malt extract dextrose agar (ISP-2) in comparison to the other studied growth media (Table 1). The RVE129 strain displayed brown substrate mycelium and white aerial mycelium without diffusible pigment on ISP 2 plate medium (Figure 1 and Table 1).Table 1
The growth characteristics of theStreptomyces sp RVE129.
Culture mediumGrowth patternAerial myceliumSubstrate myceliumDiffusible pigmentsTryptone yeast extract agar (ISP-1)++GreyPale yellow—Yeast extract malt extract agar (ISP-2)+++WhiteBrown—Oat meal agar (ISP-3)++WhiteGreyBrownInorganic salt starch agar (ISP-4)+++BrownLight ivory—Glycerol asparagines agar (ISP-5)++GreyGreyish white—Peptone yeast extract iron agar (ISP-6)+++GreyDark grey—Tyrosine agar (ISP-7)+CreamBrown—Starch casein agar+++WhiteWhitish brown—+++: good growth; ++: moderate growth; +: poor growth; −: no diffusible pigment.The biochemical and sugar fermentation test results of strain RVE129 are illustrated in Table2. The isolate was grown in a pH range of 4–10, a temperature range of 20–40°C, and a NaCl concentration of up to 5% (w/v). The isolate showed optimal growth at 30°C and at pH 7 as well as in the presence of a 2.5% NaCl (w/v) concentration. The isolate could utilize different sugars as carbon sources, although inositol, arabinose, and rhamnose were not utilized (Table 2). The strain RVE129 exhibited positive results for the production of catalase, urease, H2S, citrate, hydrolysis of starch, casein, gelatin, and esculin degradation, whereas melanin formation, nitrate reduction, production of indole, Voges-Proskauer, and oxidase were shown negative results (Table 2).Table 2
Morphological, biochemical, and physiological characteristics of strain RVE129.
CharacteristicsResultCharacteristicsResultMicroscopic examinationIndole—Gram reaction+Voges-Proskauer—Spore chain morphologyRectiflexibleCarbon utilizationMotilityNonmotileInositol—GrowthAerobicMannose+Physiological characteristicsXylose±Temperature range for growth20-40°CAdonitol±Optimum temperature30°CFructose+pH range for growth4 to 10Sucrose+Optimum pH for growth7Sorbitol+NaCl tolerance range0-5%Trehalose+Optimum NaCl (%) for growth2.5%Arabinose—Biochemical charactersLactose+Melanin production—Galactose+Nitrate reduction—Maltose+Catalase production+Mannitol+Urease production+Glucose+Esculin degradation+Cellulose±H2S production+Starch+Citrate utilization+Fructose+Casein hydrolysis+Rhamnose—Oxidase production—Gelatin hydrolysis++: positive; ±: doubtful/poor; −: negative.


## 3.2. Molecular Characterization

According to the sequencing results, 1435 base pairs were present in the isolate and deposited in NCBI GenBank under the accession number ON786620.https://www.ncbi.nlm.nih.gov/nuccore/ON786620.1. A phylogenetic tree of the RVE129 strain and allied reference strains is shown in Figure 2. Examination of the 16S rDNA gene of the Streptomyces sp. RVE129 sequence showed maximum similarity (99.65%) with the 16S rRNA genes of Streptomyces monomycini NRRL B-24309T as indicated in Figure 2Figure 2
Phylogenetic tree constructed based on 16S rRNA gene sequences of RVE129. The percentage of similarity between the strain and related members of the genusStreptomyces 16S rRNA gene clade is shown next to the strain names.


## 3.3. TLC Profile of the Antibacterial Extract

Thin-layer chromatography (TLC) was used to purify the antibacterial component. The separate spots of the bioactive metabolite in the TLC chromatogram are presented in Figure3. The TLC results show that the extract produced five spots, denoted I, II, III, and IV, with RF values of 0.96, 0.73, 0.63, 0.43, and 0.34, respectively (Figure 3).Figure 3
TLC chromatograph of the extract fromStreptomyces sp. RVE129.The TLC-agar overlay bioautography assay revealed spot II (RF=0.73) as the active spot against S. aureus and S. typhi. The purified antibiotic was denoted as RVE-02.


## 3.4. Characterization of the Active Compound

### 3.4.1. Physicochemical Characterization

The purified antibiotic RVE-02 was yellowish-brown in colour and had a melting point of 135°C. It was soluble in methanol, ethanol, acetone, chloroform, petroleum ether, ethyl acetate, and DMSO but insoluble in water. The antibacterial activity of purified antibiotic RVE-02 was tested at various temperatures and pH levels, and it was revealed to be stable in the pH range (4–10) and over a wide range of temperatures (20–45°C). The purified antibiotic compound treated at temperatures of 30°C retained maximum antimicrobial activity againstS. aureus ATCC-259233. The antibiotic RVE-02 was most stable between pH 7.0 and 9.0, with activity decreasing slightly at acidic or basic pH.


### 3.4.2. Spectral Characteristics of Antimicrobial Compound

The active fraction (RF=0.73) obtained from TLC was examined by spectral analysis. The ultraviolet (UV) of antimicrobial compound (dissolved in methanol) showed three characteristic absorption peaks of maximum absorption at 243 nm, 279 nm, and 420 nm and with a shoulder at 327 nm (Figure 4).Figure 4
UV spectra of the purified antibiotic RVE-02.Infrared spectra (IR) showed peaks at 3863.5, indicating the presence of hydroxyl functional group (-OH). FTIR spectra peak absorbance located in the range of 3240 to 3511.01 cm-1, with a maximum peak at 3436.2 cm-1 is indicative of amine function and a broad hydroxyl functional group (Figure 5). Analysis of the FTIR spectrum also showed typical absorption peaks at 1663.12 cm-1 corresponding to conjugated C=O, indicating the presence of an ester group and the absorption peaks 1729.2 cm-1 corresponding to aldehyde. The peak at 1116.58 cm-1 indicates the presence of the alcohol functional group. The presence of alkane groups is indicated by a sharp absorption peak at 2923 cm-1. The spectrum peak at 987.84 cm-1 indicates the presence of methylene compounds with C-C vibrations, and the peak appearing at 797.06–801 cm-1 was assigned to p disubstituted benzene.Figure 5
FTIR spectra of purified antibiotic RVE-02.From the1H NMR spectrum data (Figure 6), the signal at δ 8.84 (N-H) confirmed the existence of the amide functionality, and the chemical shifts observed between 𝛿 H δ 6.5–δ 7.27 ppm indicate the presence of aromatic protons. The signals in the range of 𝛿 H, 4.18–4.35 indicates the presence of the terminal long chain alkane in the compound. The presence of oxygenated methine proton is supported by the chemical shift signals in the region 𝛿 H 3.645–3.79 ppm region for the methoxy group. The chemical shift value in the range of 𝛿 H 0.45–2.69 ppm region corresponds to the occurrence of aliphatic hydrogens i.e., methyl and methylene groups in the side chains (Figure 6).Figure 6
1H NMR (500 MHz, DMSO/CDCl3) spectrum data of antibiotic RVE-02.In addition, the13C NMR spectrum (Figure 7 and Table 3) of antibiotic RVE-02 indicated that the signal in the range of δC 15 to 19 ppm corresponds to methyl (-CH3) groups and peaks in the range δC 21 to 38 ppm represents the presence of different methylene (-CH2-) groups and the peaks in the range δC 39 to 58 ppm a characteristics of methine (CH) group as shown in (Figure 7). The peaks n the range of δC 60-96 ppm represents methoxy groups, and quaternary carbons were detected at δC 100-168 ppm and one α, β-unsaturated lactone carbonyl at δC 144.3 ppm. Finally, the peak at δC 167 to 198 ppm belongs to carbonyl carbon group of either an amide or an ester (Figure 7).Figure 7
13C NMR (500 MHz, DMSO/CDCl3) spectrum data of antibiotic RVE-02.Table 3
NMR spectra data of antibiotic compound RVE-02.
PositionδC of purified compoundδC setamycin [29]PositionδC of purified compoundδC setamycin [29]1167.48167.32176.2876.82141.17141. 22240.040.02-OCH60.0259. 92375.5175.53133.41133.4242827.94133.39133.32512.3512.35143.02143.0267.237.1636.6436.72713.914.0781.1281.22817.4217.3837.1737.12910.009.8941.2241.23020.220.210142.80142.83121.5821.611133.22133.03214.3214.312125.35125.23320.9221.013127.22127.0C-1′164.29164.31470.1670.24C-2′133.59133.614-OCH55.5455.5C-3′133.04133.01577.3677.2C-4′163.6163.61638.2038.2C-5′115.02114.91782.1882.2C-6′175.24175.21842.0542.4C-7′26.0025.81998.4298.2C-8′32.1732.219-OH——C-9′198.00197.72040.0840.09′ OH——


## 3.4.1. Physicochemical Characterization

The purified antibiotic RVE-02 was yellowish-brown in colour and had a melting point of 135°C. It was soluble in methanol, ethanol, acetone, chloroform, petroleum ether, ethyl acetate, and DMSO but insoluble in water. The antibacterial activity of purified antibiotic RVE-02 was tested at various temperatures and pH levels, and it was revealed to be stable in the pH range (4–10) and over a wide range of temperatures (20–45°C). The purified antibiotic compound treated at temperatures of 30°C retained maximum antimicrobial activity againstS. aureus ATCC-259233. The antibiotic RVE-02 was most stable between pH 7.0 and 9.0, with activity decreasing slightly at acidic or basic pH.


## 3.4.2. Spectral Characteristics of Antimicrobial Compound

The active fraction (RF=0.73) obtained from TLC was examined by spectral analysis. The ultraviolet (UV) of antimicrobial compound (dissolved in methanol) showed three characteristic absorption peaks of maximum absorption at 243 nm, 279 nm, and 420 nm and with a shoulder at 327 nm (Figure 4).Figure 4
UV spectra of the purified antibiotic RVE-02.Infrared spectra (IR) showed peaks at 3863.5, indicating the presence of hydroxyl functional group (-OH). FTIR spectra peak absorbance located in the range of 3240 to 3511.01 cm-1, with a maximum peak at 3436.2 cm-1 is indicative of amine function and a broad hydroxyl functional group (Figure 5). Analysis of the FTIR spectrum also showed typical absorption peaks at 1663.12 cm-1 corresponding to conjugated C=O, indicating the presence of an ester group and the absorption peaks 1729.2 cm-1 corresponding to aldehyde. The peak at 1116.58 cm-1 indicates the presence of the alcohol functional group. The presence of alkane groups is indicated by a sharp absorption peak at 2923 cm-1. The spectrum peak at 987.84 cm-1 indicates the presence of methylene compounds with C-C vibrations, and the peak appearing at 797.06–801 cm-1 was assigned to p disubstituted benzene.Figure 5
FTIR spectra of purified antibiotic RVE-02.From the1H NMR spectrum data (Figure 6), the signal at δ 8.84 (N-H) confirmed the existence of the amide functionality, and the chemical shifts observed between 𝛿 H δ 6.5–δ 7.27 ppm indicate the presence of aromatic protons. The signals in the range of 𝛿 H, 4.18–4.35 indicates the presence of the terminal long chain alkane in the compound. The presence of oxygenated methine proton is supported by the chemical shift signals in the region 𝛿 H 3.645–3.79 ppm region for the methoxy group. The chemical shift value in the range of 𝛿 H 0.45–2.69 ppm region corresponds to the occurrence of aliphatic hydrogens i.e., methyl and methylene groups in the side chains (Figure 6).Figure 6
1H NMR (500 MHz, DMSO/CDCl3) spectrum data of antibiotic RVE-02.In addition, the13C NMR spectrum (Figure 7 and Table 3) of antibiotic RVE-02 indicated that the signal in the range of δC 15 to 19 ppm corresponds to methyl (-CH3) groups and peaks in the range δC 21 to 38 ppm represents the presence of different methylene (-CH2-) groups and the peaks in the range δC 39 to 58 ppm a characteristics of methine (CH) group as shown in (Figure 7). The peaks n the range of δC 60-96 ppm represents methoxy groups, and quaternary carbons were detected at δC 100-168 ppm and one α, β-unsaturated lactone carbonyl at δC 144.3 ppm. Finally, the peak at δC 167 to 198 ppm belongs to carbonyl carbon group of either an amide or an ester (Figure 7).Figure 7
13C NMR (500 MHz, DMSO/CDCl3) spectrum data of antibiotic RVE-02.Table 3
NMR spectra data of antibiotic compound RVE-02.
PositionδC of purified compoundδC setamycin [29]PositionδC of purified compoundδC setamycin [29]1167.48167.32176.2876.82141.17141. 22240.040.02-OCH60.0259. 92375.5175.53133.41133.4242827.94133.39133.32512.3512.35143.02143.0267.237.1636.6436.72713.914.0781.1281.22817.4217.3837.1737.12910.009.8941.2241.23020.220.210142.80142.83121.5821.611133.22133.03214.3214.312125.35125.23320.9221.013127.22127.0C-1′164.29164.31470.1670.24C-2′133.59133.614-OCH55.5455.5C-3′133.04133.01577.3677.2C-4′163.6163.61638.2038.2C-5′115.02114.91782.1882.2C-6′175.24175.21842.0542.4C-7′26.0025.81998.4298.2C-8′32.1732.219-OH——C-9′198.00197.72040.0840.09′ OH——


## 3.5. Biological Assays

### 3.5.1. Antimicrobial Activity of Purified Antibiotic RVE-02

The minimum inhibitory concentration (MIC) of the bioactive compound RVE-02 is presented in Table4. The antibiotic RVE-02 demonstrated broad antibacterial efficacy against Gram-positive and Gram-negative bacteria, with MIC values ranging from 1.97 to 125 μg/ml. When compared to streptomycin (1.97 μg/ml), the lowest MIC was recorded against S. aureus (1.9 μg/ml) and the highest against K. pneumonia and P. aeruginosa (125 μg/ml). Although in most of the cases, control antibiotics had a lower MIC value than the RVE-02 antibiotic; it showed good activity (1.97 μg/ml) against S. aureus when compared to standard antibiotics (1.97 μg/ml).Table 4
Bioactivity potential of antibiotic RVE-02 against different pathogenic bacteria.
Test organismsMIC (μg/ml)Erythromycin (μg/ml)Staphylococcus aureus ATCC-2592331.971.97Staphylococcus epidermidis ATCC-122283.951.97Salmonella typhi ATCC-1331131.257.90Pseudomonas aeruginosa ATCC-2785312531.25Kelbsiella pneumonia ATCC-70060312562.5Escherichia coli ATCC-2592262.57.90Salmonella typhi ATCC-1402862.57.90


### 3.5.2. In Vitro Cytotoxicity Analysis

The results of the cytotoxicity assay are presented in Figure6. The in vitro cytotoxic experiment showed that the RVE-02 antibiotic obtained from S. monomycini RVE129 inhibited cell growth and viability in the human cervical cancer (HeLa) cell line (Figure 8(a)).Figure 8
(a) Cytoxicity different concentrations of active metabolite on the percentage of HeLa cell viability. The data are represented as themean±SD of three separate experiments, for each experiment. (b) Inverted microscopic (40x) images of HeLa cell lines treated with different concentrations of antibiotic RVE-02.
(a)(b)The cytotoxicity results showed that the purified antibiotic RVE-02 had a considerable antiproliferative influence on HeLa cells, with anIC50 value of 18.30 μg/ml of concentration (Figure 8(a)). The lowest tested concentration (3.78 μg/ml) was able to inhibit cell line proliferation by less than 10%. In contrast, more than 90% of cell line proliferation inhibition was recorded after 24 hours treatment at 250 μg/ml (Figure 8(b)). These results showed that the antibiotic RVE-02 induced cell cytotoxicity against HeLa cell lines in a direct dose-response relationship where the viability of cells was reduced, and a change in the shape of the cell was observed with an increased antibiotic concentration in the treated cells (Figure 8(b)).


## 3.5.1. Antimicrobial Activity of Purified Antibiotic RVE-02

The minimum inhibitory concentration (MIC) of the bioactive compound RVE-02 is presented in Table4. The antibiotic RVE-02 demonstrated broad antibacterial efficacy against Gram-positive and Gram-negative bacteria, with MIC values ranging from 1.97 to 125 μg/ml. When compared to streptomycin (1.97 μg/ml), the lowest MIC was recorded against S. aureus (1.9 μg/ml) and the highest against K. pneumonia and P. aeruginosa (125 μg/ml). Although in most of the cases, control antibiotics had a lower MIC value than the RVE-02 antibiotic; it showed good activity (1.97 μg/ml) against S. aureus when compared to standard antibiotics (1.97 μg/ml).Table 4
Bioactivity potential of antibiotic RVE-02 against different pathogenic bacteria.
Test organismsMIC (μg/ml)Erythromycin (μg/ml)Staphylococcus aureus ATCC-2592331.971.97Staphylococcus epidermidis ATCC-122283.951.97Salmonella typhi ATCC-1331131.257.90Pseudomonas aeruginosa ATCC-2785312531.25Kelbsiella pneumonia ATCC-70060312562.5Escherichia coli ATCC-2592262.57.90Salmonella typhi ATCC-1402862.57.90


## 3.5.2. In Vitro Cytotoxicity Analysis

The results of the cytotoxicity assay are presented in Figure6. The in vitro cytotoxic experiment showed that the RVE-02 antibiotic obtained from S. monomycini RVE129 inhibited cell growth and viability in the human cervical cancer (HeLa) cell line (Figure 8(a)).Figure 8
(a) Cytoxicity different concentrations of active metabolite on the percentage of HeLa cell viability. The data are represented as themean±SD of three separate experiments, for each experiment. (b) Inverted microscopic (40x) images of HeLa cell lines treated with different concentrations of antibiotic RVE-02.
(a)(b)The cytotoxicity results showed that the purified antibiotic RVE-02 had a considerable antiproliferative influence on HeLa cells, with anIC50 value of 18.30 μg/ml of concentration (Figure 8(a)). The lowest tested concentration (3.78 μg/ml) was able to inhibit cell line proliferation by less than 10%. In contrast, more than 90% of cell line proliferation inhibition was recorded after 24 hours treatment at 250 μg/ml (Figure 8(b)). These results showed that the antibiotic RVE-02 induced cell cytotoxicity against HeLa cell lines in a direct dose-response relationship where the viability of cells was reduced, and a change in the shape of the cell was observed with an increased antibiotic concentration in the treated cells (Figure 8(b)).


## 4. Discussion

The development and spread of multidrug-resistant bacteria that are resistant to currently available antibiotics have become a global challenge [1, 2]. This will threaten our potential to treat common infectious diseases and cancer [3]. As a result, screening for highly efficient, innovative, and novel broad-spectrum antimicrobial metabolites is still urgently needed in the development of antimicrobial metabolites to combat these pathogens. The focus of this research is to purify and characterize a bioactive metabolite produced by Streptomyces sp. RVE129, which was previously isolated from the rift valley areas of Hawassa, Ethiopia. The bioactive metabolite was then examined for its bioactivity against various pathogens, including drug-resistant opportunistic pathogens and for its cytotoxicity against the human cervical cancer (HeLa) cell.Furthermore, in accordance with Barka et al. [5], the genus Streptomyces can be distinguished from all other bacterial groups by a broad range of stable exhibited phenotypic features such as morphology, physiology, and biochemical traits. The RVE129 strain was aerobic, Gram positive, exhibited good growth on different growth medium, aerial, and substrate mycelia of different colours which are typical characteristics of actinomycete. The features of strain RVE129 were compared with those of actinomycete species noted in Bergey’s manual of determinative bacteriology [12], and obtained characteristics strongly suggested that strain RVE129 belonged to the genus Streptomyces. The strain assimilated the majority of the tested sugars, demonstrating a diverse pattern of carbon utilization. These results were highly congruent with the findings of Holt [14]. One of the key criteria for identifying Streptomyces is its spore morphology, which differs substantially between species. It has been discovered that the spores were arranged in chains, were rectiflexible, and had smooth spore surfaces, which were typical features of the genus Streptomycetes [15].Molecular examination of the 16S rRNA gene of a selected isolate further supports this preliminary identification. According to the findings of a 16S rRNA phylogenetic analysis, the strain RVE129 is 99.65% related toStreptomyces monomycini NRRLB 24309. Similarly, various studies have noted the importance of 16S rRNA gene sequence matching as a technique for confirming the characterization of actinomycetes [5, 13, 16, 17, 23].The characterization of major bioactive compounds has been published in prior studies using various approaches [23, 30–32]. The use of a range of techniques could aid in determining the nature of the bioactive antibiotic chemical isolation in the future. The purified antibiotic compound RVE-02 was soluble in ethanol, methanol, acetone, chloroform, petroleum ether, and DSMO but minimally soluble in water. Similar findings have previously been reported [23, 31]. The results showed that when the antibiotic compound was exposed to different temperatures and pH levels, it demonstrated relatively high temperature (45°C) and pH (pH=9) stability. Thirumurugan and Vijayakumar [30] found that the Streptomyces sp. ECR77 polyketide antibiotic could be subjected to 45°C for 30 minutes with no loss of its antibacterial activity. The findings are also in agreement with those of Vijayakumar et al. [31], who observed that the antimicrobial compound of Streptomyces sp. VPTSA18 maintains its stability at pH 9 and 50°C. The result suggests that the antibiotic RVE-02, produced by S. monomycini RVE129, has potential applications in the pharmaceutical and food industry.A bioactive metabolite fraction with anRF value of (RF=0.73) was recovered via bioassay-guided fractionation and demonstrated significant bioactivity against Gram-positive and Gram-negative bacteria. Sanghvi et al. [23] have determined the antimicrobial potential of two bioactive fractions obtained from ethyl acetate extract of Streptomyces werraensis by thin-layer chromatography. The bioactive fraction-subjected UV spectroscopy has shown strong absorption maxima in the UV-visible spectrum at 277 nm and 327 nm, indicating the existence of conjugated systems in the chemical structure of the substance, such as unsaturated aldehydes, ester group, and aromatic ring compounds [32, 33] and the absence of polyenic structures [34]. These distinctive absorption bands at the wavelengths suggested that the purified compound could be a polyketide group of macrolide antibiotics. Similar observations were also made by other authors [20–23, 32–38].The IR spectra data of purified fraction confirms the presence of carbonyls (ketone or ester group), hydroxyls, aromatic rings, and lactone moiety and the presence of prominent saturated carbons in various places, which are typical features of macrolide polyketide antimicrobial molecules [36]. Further, the IR spectra of purified fraction RVE-02 are almost identical to functional groups of the polyketide antimicrobial metabolite as reported for setamycin by Omura et al. [29], exhibiting peaks in the range of 3400–3500 cm−1, around 1660 cm−1, and 1717–1730 cm−1 which indicated hydroxyl, a hydrogen bonded carbonyl group and ketonic group or ester carbonyl, respectively. The chemical shift values of NMR 1H and 13C shown by the purified antibiotic RVE-02 were very similar to that of setamycin [39].In general, the purified antibiotic compound produced byS. monomycini was identified using various analyses, and 1H and 13C NMR spectral data as well as a comparative evaluation of present data with Pubchem data was proposed that the antibiotic RVE-02 belongs to the polyketide macrolide antibiotic family to be setamycin with a molecular formula of C44H65NO13. The literature survey indicated that there were no reports on the production of polyketide antibiotics from S. monomycini. However, more research investigation is needed to elucidate the chemical structure of the antibiotic RVE-02.The MIC values of the purified polyketide antibiotic RVE-02 against test bacteria ranged between 1.97 and 125μg/ml. The lowest MIC was recorded against S. aureus (1.97 μg/ml). The findings of this study were comparable to those of broad-spectrum antibacterial bioactivity of polyketide antibiotics produced by Streptomyces puniceus strain AS13 [16], and Streptomyces sp. AP-123 [34].The cytotoxicity result of the polyketide antibiotic RVE-02 demonstrated a strong antiproliferating effect on the HeLa cells with anIC50 value of 24.3 μg/ml of the concentration. This is in line with the findings of Anuradha et al. [35], who reported that the cytotoxicity on HeLa cells had an IC50 value 26.20 μg/ml as determined by the MTT assay. Earlier studies have also documented that the antimicrobial metabolite extracted from Streptomyces sp. causes antiproliferation activity and morphological changes in HeLa cells [16, 27, 28, 34]. These results showed that the antibiotic RVE-02 induced cell cytotoxicity against HeLa cell lines in a direct dose-response relationship where viability of cells was reduced, and a change in the shape of the cell was observed with increased antibiotic concentration in the treated cells.


## 5. Conclusion

This study demonstrated that the isolate RVE129 showed high similarity withS. monomycini based on phenotypic and genotypic characteristics. The antibiotic compound RVE-02 produced by strain can be identified and has close similarity with setamycin. This is the first report on the production of the polyketide antibiotic with antimicrobial and antitumour activity by the S. monomycini strain. The antibacterial bioactivity test indicated that the identified antibiotic RVE-02 is effective against a wide range of pathogens, including drug-resistant bacteria such as S. aureus. The antibiotic RVE-02 has antitumor activity against HeLa cells as tested in vitro. Thus, it is a promising candidate as an anticancer antibiotic. Current findings suggest the need for further investigations regarding the complete structure of the active compound and pharmacology of RVE-02.



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