Sage Journals: Discover world-class research

Abstract

Objective

Actinomycetes have been known to be the great natural sources to explore antibiotics for the treatment of tuberculosis (TB). The isolation of actinomycetes from the samples in Vietnam followed by the screening of their antimycobacterial activity was performed in this study. The metabolites isolated from the most active strain were further evaluated for their antimycobacterial, antimicrobial and cytotoxic activity.

Methods

Actinomycetes were growth in culture media, isolated and identified by colony, spore chain morphology and 16S rRNA gene sequencing. Agar diffusion assay was used for the screening of the isolated strains against Mycobacterium smegmatis, a safety surrogate for Mycobacterium tuberculosis. The metabolites produced from the most active strain were investigated by actinomycete fermentation, extraction and isolation from biomass and cultures. The structures of the isolated compound were elucidated by spectral data and comparison with the reported literatures.

Results

181 strains were isolated from nine regions along the north to central Vietnam. The five most active strains against Mycobacterium smegmatis were detected. Following the bioassay-guided result, the strain A121 (Streptomyces alboniger) was selected for further isolation of the bioactive metabolites. As a result, obscurolide B_2β (1) and chartreusin (2) were obtained and evaluated for their antimycobacterial activity against M. smegmatis. Compound 2 displayed potential antimycobacterial activity, antimicrobial effect against the Gram positive bacteria Staphylococcus aureus, Bacillus subtilis, Lactobacillus fermentum and cytotoxicity against four cancer cell lines KB, HepG-2, Lu-1 and MCF-7.

Conclusions

Five strains possessing potential antimycobacterial activity were identified from the samples collected in Vietnam. Two compounds including obscurolide B_2β (1) and chartreusin (2) were isolated from the most active strain A121 (Streptomyces alboniger). This is the first time these compounds have been isolated from this strain. Chartreusin (2) exhibited notable antimycobacterial, antimicrobial and cytotoxic activity, making its worthy attention for further drug development, particularly for antituberculosis therapeutic agents.

Keywords

antimycobacterial activity actinomycetes chartreusin cytotoxic activity

Introduction

Tuberculosis (TB), one of the most global health burdens, is an infectious disease caused by Mycobacterium tuberculosis. According to the World Health Organization (WHO), TB has become the thirteen leading cause of death worldwide, with 1.6 million deaths and 10.6 million new infectious cases in 2021.¹ The diagnosis and treatment for TB patients were interrupted during the Covid 19 epidemic, leading to the increasing numbers of TB death and transmission to the community.¹ WHO reported that in 2021 Vietnam ranked 11th out of 30 countries with the highest burden of tuberculosis and multidrug-resistant TB in the world. Each year, there are approximately 170 000 new cases and about 10 400 deaths of TB in Vietnam.² The prevalence of drug-resistant TB has increased considerably, with approximately 8400 new cases of rifampicin-resistant (first-line drug) and multidrug-resistant tuberculosis each year.^3–5 The cost for treatment of multidrug-resistant tuberculosis is ten times more expensive than that of common TB, causing a heavy burden on TB patients.³ Therefore, drug-resistant TB is currently considered as a serious public health problem in Vietnam. Therapeutic treatments for multidrug-resistant tuberculosis (MDR) have been extensively developed by researchers and pharmaceutical companies. However, the spread of drug-resistant TB is still the major challenge for disease control. Hence, finding new antibiotics and more effective drugs are urgently needed to overcome this problem.

Actinomycetes are known to be the largest group of microorganisms for production of antibiotics.⁶ Streptomycin,⁷ kanamycin,⁷ azomycin,⁸ rifamycin,⁹ etc. are the antituberculosis drugs derived from a great number of actinomycetes species. It was proven that the secondary metabolites produced from the actinomycetes that survived in extreme or unique environment may present a wide range of antimicrobial activity.⁶ Vietnam has a great diversity of natural ecosystem including tropical ever-green forest, mangrove forest, coastal island, high mountains, beach, etc. which are the potential resources to discover actinomycetes diversity and natural antibiotics. However, up to now, there are only few reports on the antituberculosis metabolites from actinomycetes in Vietnam.¹⁰

As a part of our ongoing research on antituberculosis agents from actinomycetes, we have collected the samples from various regions along the North to Central Vietnam. The cultivation of actinomycetes from different ecological habitats led to the isolation of 181 strains, which were subsequently screened for their antimycobacterial activity against Mycobacterium smegmatis. This bacteria was used as a surrogate for Mycobacterium tuberculosis in our study, due to its safety, non-pathogenic and eco-friendly properties.^11–13 Unlike other Mycobacterium species, M. smegmatis grows rapidly in cell culture laboratories, and does not live in mammals.^11–13 Thus, this organism does not cause infection in humans.^11,12 It is noteworthy that M. smegmatis contains substantial genome sequences sharing the similarities to M. tuberculosis.^11,12 Therefore, M. smegmatis has been extensively served for antimycobacterial activity assay.^14–21

As the result, the five most active strains were detected including Streptomyces avidinii (A002), Streptomyces spiroverticillatus (A067), Streptomyces wuyanensis (A079), Streptomyces hawaiiensis (A105) and Streptomyces alboniger (A121). We reported herein for the first time the antimycobacterial properties of the above strains against M. smegmatis. From them, the most active one Streptomyces alboniger was fermented in large scale for further investigation of antimicrobial metabolites. Consequently, obscurolide B_2β (1) and chartreusin (2) were isolated and structurally characterized by spectroscopic data. Compound 2 showed strong inhibitory activity against M. smegmatis with an inhibition zone of 15 mm in diameter. This is the first time the isolation of obscurolide B_2β and chartreusin as well as their antimicrobial activity against M. smegmatis were presented. Compound 2 also displayed promising antibacterial activity against gram positive bacteria Staphylococcus aureus, Bacillus subtilis, Lactobacillus fermentum and potential cytotoxicity against four human cancer cell lines KB, HepG-2, Lu-1 and MCF-7.

Results and Discussion

Isolation of Actinomycetes

As shown in Table 1, all the samples were collected in May 2020, in the following regions: Area 1: Mangrove forest in Phu Long commune, Cat Hai district, Hai Phong city; Area 2: Cat Ba National Park, Cat Hai district, Hai Phong; Area 3: On the top of May Bac mountain, Cuc Phuong National Park, Nho Quan district, Ninh Binh province (648 m above sea level); Area 4: On the way to the top of May Bac mountain, Cuc Phuong National Park, Nho Quan district, Ninh Binh province; Area 5: The confluence of Day river and Vac river, Thuong Kiem commune, Kim Son district, Ninh Binh province; Area 6: Mangrove forest in Kim Son district, Ninh Bình province; Area 7: Quynh Luong Mangrove Forest, Quynh Luong commune, Quynh Luu district, Nghe An province; Area 8: Quan Son lake, My Duc district, Ha Noi. Area 9: Yen Stream, Huong Pagoda, My Duc district, Ha Noi. A total of 181 actinomycete strains were recognized based on their differences in colony characteristics such as color, colony size, filamentous structure, mycelium growth, and pigment secretion (Figure 1). Preliminary assessment of actinomycete morphology showed that of 181 total strains there were 135 strains belonging to the genus Streptomyces (74.6%), while the rest one (46 strains) were rare actinomycetes (25.4%). It was observed that the rare actinomycetes were mostly found in the areas 1 and 2, where the mud and decaying leaf samples gave a significant actinomycetes population than those in the soil.

Figure 1.

Colony characteristics of some actinomycetes isolated from different locations.

Table 1.

The Strain Codes, Natural Source and Numbers of the Strains Isolated from Different Areas Along the Northern to Central Vietnam.

Areas	Locations for actinomycetes collection	Strain Codes	Number of the isolated strains	Natural source
1	Mangrove forest in Phu Long commune, Cat Hai district, Hai Phong city	A001, A002	2	Mud
		A008 to A010	3	Mud
		A012	1	Mud
		A073 to A076	4	Mud
		A110 to A120	11	Mud
		A129 to A131	3	Mud
		A159 to A162	4	Mud
		A003 to A007	5	Soil
		A013 to A018	6	Soil
		A147 to A151	5	Soil
		A163 to A166	4	Soil
		A071, A072	2	Decaying leaf
2	Cat Ba National Park, Cat Hai district, Hai Phong city	A029 to A035	7	Mud
		A019, A020	2	Mud
		A042 to A055	14	Soil
		A167, A168	2	Soil
		A011	1	Decaying leaf
		A021 to A028	8	Decaying leaf
		A036	1	Decaying leaf
		A038 to A041	4	Decaying leaf
3	On the top of May Bac mountain, Cuc Phuong National Park, Nho Quan district, Ninh Binh province (648 m above sea level)	A101 to A109	9	Soil
		A121 to A128	8	Soil
		A132 to A135	4	Soil
4	On the way to the top of May Bac mountain, Cuc Phuong National Park, Nho Quan district, Ninh Binh province	A037	1	Soil
		A056 to A059	4	Soil
		A083 to A100	18	Soil
5	The confluence of Day river and Vac river, Thuong Kiem commune, Kim Son district, Ninh Binh province	A064 to A067	4	Soil
5		A152 to A154	3	Soil
6	Mangrove forest in Kim Son district, Ninh Bình province	A060 to A063	4	Soil
		A068 to A070	3	Soil
		A136 to A146	11	Soil
		A155 to A158	4	Soil
		A177 to A179	3	Soil
7	Quynh Luong Mangrove Forest, Quynh Luong commune, Quynh Luu district, Nghe An province	A077 to A082	6	Soil
7		A180	1	Soil
8	Quan Son lake, My Duc district, Ha Noi	A169 to A176	8	Soil
9	Yen Stream, Huong Pagoda, My Duc district, Ha Noi	A181	1	Soil

Antimycobacterial actinomycetes Against Mycobacterium Smegmatis

Agar plate assay was used for the screening of actinomycete strains against Mycobacterium smegmatis. The evaluation result showed that of 181 actinomycete strains, 14 strains (7.7%) were able to inhibit M. smegmatis. Of which, the most active actinomycete strains against M. smegmatis were A002 (from area 1), A067 (from area 5), A079 (from area 7), A105 (from area 4), and A121 (from area 3) where the ecological environment has been rarely explored by humans. The location, sampling coordination (latitude and longitude), colony color, spore and inhibition zone diameter (mm) of the strains A002, A067, A079, A105 and A121 were illustrated in Table 2. The strains A002, A067 and A079 showed an equivalent activity with an inhibition zone of 9 mm diameter, while the strains A105 was active against M. smegmatis with 8 mm of inhibited zone. The strain A121 was the most active one with an inhibition zone of 11 mm in diameter (Figure 2). Compared with strain A105 whose sample collecting location was also in Cuc Phuong National Park, but in the lower position (on the way to the top of May Bac mountain), the activity of strain A121 was much higher. This may be due to the reason that strain A121 was isolated from a less explored region (on the top of a high mountain) where the ecological environment has not yet been much affected by the activity of humans. Previous literatures reported that many actinomycetes discovered from poorly explored habitats or unusual environments are the sources for production of potential active metabolites.²² Living in such regions may allow the actinomycetes to produce large genomes with many transcription factors that are possible to respond to the particular environments.^23,24 Therefore, the secondary metabolites produced by these organism as well as those biological activity could potentially be increased.²⁵

Figure 2.

Antimicrobial activity illustration of the five most active strains (A002, A067, A079, A105, A121) against M. smegmatis.

Table 2.

Location for Sampling Sites, Colony, Spore Morphology and Inhibition Zone Diameter of the Five Most Active Strains Against M. smegmatis.

Strain code	Strains identification	Location	Latitude	Longitude	Colony color	Spore	Inhibition zone diameter (mm)
A002	Streptomyces avidinii	Mangrove forest in Phu Long commune, Cat Hai district, Hai Phong City	N20°48′35″	E106°55′47.2″	White, powdery	Rectiflexibiles	9
A067	Streptomyces spiroverticillatus	The confluence of Day river and Vac river, Thuong Kiem commune, Kim Son district, Ninh Binh province	N20°3′0.8″	E106°6′52.8″	Light brown, powdery	Spiral	9
A079	Streptomyces wuyuanensis	Quynh Luong Mangrove Forest, Quynh Luong commune, Quynh Luu district, Nghe An province	N19°8′42.1″	E105°41′43.1″	Light brown, powdery	Spiral	9
A105	Streptomyces hawaiiensis	On the way to the top of May Bac mountain, Cuc Phuong National Park, Nho Quan district, Ninh Binh province	N20°21′0.8″	E105°36′15.7″	Brown, powdery	Rectiflexibiles	8
A121	Streptomyces alboniger	On the top of May Bac mountain, Cuc Phuong National Park, Nho Quan district, Ninh Binh province (648 m above sea level)	N21°21′21.9″	E105°36′36.2″	Light brown, powdery	Rectiflexibiles	11

Classification of the Five Most Active Strains

The most active strains (A002, A067, A079, A105, A121) against M. smegmatis were classified based on their cultural characteristic, the color of the substrate mycelia, the colony morphology, the ability to produce pigment, the morphology of the spore chain and 16S rDNA sequences. By analysis of the above data, the actinomycetes A002, A067, A079, A105 and A121 were identified as Streptomyces avidinii, Streptomyces spiroverticillatus, Streptomyces wuyanensis, Streptomyces hawaiiensis and Streptomyces alboniger, respectively (Figure 3, Table 2). All of them belong to the genus Streptomyces, which have been known as promising sources for exploration of antituberculosis drugs. Of the five antimycobacterial strains above, Streptomyces wuyanensis (A079) was first discovered in Wuyan province, China in 2013²⁶ and has not yet been investigated on biological activity and metabolites up to now. This is the first time Streptomyces wuyanensis has been investigated for its antimycobacterial activity against M. smegmatis. Streptomyces avidinii (A002), Streptomyces spiroverticillatus (A067), Streptomyces hawaiiensis (A105) were found to possess antimicrobial, antibiotic and antitumor metabolites.^27–30 However, our study reported for the first time antimycobacterial activity of Streptomyces avidinii and Streptomyces spiroverticillatus against M. smegmatis. Streptomyces hawaiiensis was noted for its production of acyldepsipeptides (ADEPs), the prospective antituberculosis agents against M. tuberculosis.³¹ Streptomyces alboniger (A121) was first isolated in 1954 from the soil samples in Illinois, America³² and was found to exhibit potential cytotoxic effect on many cancer cell lines.³³ In our study, Streptomyces alboniger (A121) was the most active strain against Mycobacterium smegmatis. It is a light brown substrate bacteriophage, a gray white color, and is capable of forming colonies. The investigation of metabolites from these strains led to the isolation of pamamycin, an antibiotic resistance toward many bacterial pathogens, including Mycobacterium smegmatis and Mycobacterium phlei.³⁴

Figure 3.

Colony morphology of the five most active actinomycetes (A002, A067, A079, A105, A121) against M. smegmatis.

Structural Elucidation of the Compounds Isolated from Streptomyces Alboniger (A121)

Streptomyces alboniger (A121) was chosen for further isolation and characterization of the bioactive metabolites. Compound 1 was obtained from the ethyl acetate extract of the culture solution of strain A121, as a yellow powder. Its ESI- and HR-ESI-MS spectra gave the molecular ion peaks at m/z 316.1 [M + Na + H₂O]⁺, 276.1221 (calculated for C₁₅H₁₈NO₄ 276.1236 [M + H]⁺) and 298.1045 (calculated for C₁₅H₁₇NO₄Na 298.1055 [M + Na]⁺), corresponding to the molecular formula of C₁₅H₁₇NO₄. The NMR data of 1 showed the signals characteristic of an obscurolide, a skeletone possessing 3,4-substituted butyrolactones. In ¹H NMR spectrum, the signals at δ_H 2.44 (1H, dd, J = 18.0, 3.5 Hz, H-2b), 3.18 (1H, dd, J = 18.0, 7.5 Hz, H-2a), 4.35 (1H, m, H-4) and 4.41 (1H, m, H-3) were assigned for a butyrolactone ring. The presence of a para-disubstituted aromatic ring was deduced from the signals at δ_H 6.67 (1H, d, J = 8.5 Hz, H-2′, H-6′), 7.73 (1H, d, J = 8.5 Hz, H-3′, H-5′). Besides, two protons belonging to a double bond with E-configuration were resonated at δ_H 5.88 (1H, dd, J = 15.0, 7.0 Hz, H-6) and 5.64 (1H, dqd, J = 15.0, 7.0, 2.0 Hz, H-7). In addition, a methyl group at δ_H 1,74 (3H, dd, J = 6,5, 1.5 Hz, H-8), an oximethine proton at δ_H 4.32 (1H, m, H-5), two protons belonging to a hydroxyl [δ_H 4.60 (1H, brs, C₅-OH)] and an aldehyde groups [9.77 (1H, s, CHO)] were also observed. In the ¹³C NMR spectrum, an aldehyde carbonyl [δ_C 190.3 (C-7′)], an ester carbonyl [δ_C 174.9 (C-1)], an oximethine carbon [δ_C 73.1], a tertiary oxygenated carbon [δ_C 87.3 (C-4)], one methylene and one methyl carbon [δ_C 36.3 (C-2) and 17.8 (C-8), respectively], together with two olefin carbons [δ_C 131.4 (C-6), 128.2 (C-7)] and the aromatic carbons from δ_C 112.6–150.9 were observed. The structure of 1 was further confirmed by 2D NMR (COSY, HSQC, HMBC and NOESY) spectral data. An aldehyde group was determined to attach at C-4′ of aromatic ring, which was verified by HMBC correlations from H-3′, H-5′ (δ_H 7.73) to C-7′ (δ_C 190.3), C-1′ (δ_C 150.9) (Figure 5). The assignment of the carbons and protons in butyrolactone ring was deduced by the COSY correlations between H-2a (δ_H 3.18) and H-2b (δ_H 2.44)/H-3 (δ_H 4.41) and H-4 (δ_H 4.35), as well as the HMBC correlations from H-2a (δ_H 3.18) and H-2b (δ_H 2.44) to C-1 (δ_C 174.9), C-3 (δ_C 51.1) and C-4 (δ_C 87.4) (Figure 5). The other COSY correlations of H-4 (δ_H 4.35)/H-5 (δ_H 4.32); H-5 (δ_H 4.32)/H-6 (δ_H 5.88); H-6 (δ_H 5.88)/H-7 (δ_H 5.64); H-7 (δ_H 5.64)/H-8 (δ_H 1.74), and the HMBC correlations from H-6 (δ_H 5.88) and H-7 (δ_H 5.64)/C-5 (δ_C 73.1); H-8 (δ_H 1.74)/C-6 (δ_C 131.4) and C-7 (δ_C 128.2); H-5 (δ_H 4.32)/C-3 (δ_C 51.1) also verified the skeleton of obscurolide. The trans configuration at C-6 and C-7 was determined based on the large coupling constant (J = 15.0 MHz) between H-6 and H-7. Proton H-5 was driven as an α-position, deducing from the NOESY correlations of H-5 (δ_H 4.32)/H-7 (δ_H 5.64). Therefore, the hydroxyl group at C-5 was orientated at the β-position. The relative configuration of H-3 and H-4 was defined as trans, based on the NOESY correlations between H-4 (δ_H 4.35)/H-5 (δ_H 4.32). The spectral data of 1 was in agreement with those reported for obscurolide B_2β (Figure 4).³⁵ Therefore, compound 1 was elucidated to be as obscurolide B_2β. Obscurolides are the novel class of phosphodiesterases inhibitors, which were found in many species of the genus Streptomyces. Obscurolide B_2β was first isolated from Streptomyces vitidocbtomogenes in 1993.³⁶ This is the first time this compound has been isolated from Streptomyces alboniger.

Figure 4.

The structures of compound 1 and 2 isolated from Streptomyces alboniger.

Figure 5.

HMBC, COSY and NOESY correlations of compounds 1 and 2.

Compound 2 was isolated from the methanol extract of the cell residue of strain A121, as a greenish-yellow powder. The molecular formula of compound 2 was determined as C₃₂H₃₂O₁₄, deducing from the molecular ion peaks in ESI- and HR-ESI-MS spectra at m/z 639.1 [M-H]⁻, 641.1876 (calculated for C₃₂H₃₃O₁₄ 641.1870 [M + H]⁺) and 663.1710 (calculated for C₃₂H₃₂O₁₄Na 663.1690, [M + Na]⁺). Detailed analysis of NMR spectral data of compound 2 demonstrated that this compound consisted of a pentacyclic aromatic bilactone ring and a disaccharide unit. The ¹H NMR of 2 indicated the signals of five aromatic protons at δ_H 7.41 (1H, d, J = 8.0 Hz, H-10), 7.44 (1H, d, J = 8.5 Hz, H-17), 7.51 (1H, d, J = 8.5 Hz, H-16), 8.19 (1H, d, J = 8.0 Hz, H-8) and 7.61 (1H, t, J = 8.0 Hz, H-9). Furthermore, one methyl group attached to the aromatic ring at δ_H 2.81 (3H, s, H-19) and one singlet of the hydroxyl group at δ_H 11,63 (C₆-OH) were observed. Besides, two monosaccharides, including ten oxygenated methine protons at δ_H 3.30 (1H, dd, J = 10.0, 3.0 Hz, H-9′), 3.78 (1H, m, H-4′), 3.84 (1H, m, H-8′), 3.87 (1H, m, H-10′), 3.88 (1H, m, H-3′), 4.27 (1H, m, H-2′), 5.33 (1H, d, J = 7.5 Hz, H-1′), 5.70 (1H, d, J = 4.5 Hz, H-7′), δ_H 3.92 (1H, m, H-5′), 4.22 (1H, m, H-11′), together with two methyls at δ_H 1.44 (3H, d, J = 6.5 Hz, H-6′), 1.38 (3H, d, J = 6.5 Hz, H-12′), and one methoxy at δ_H 3,36 (3H, s, H-13′) were also observed. The ¹³C NMR spectrum of compound 2 indicated 32 carbon signals, including an aglycone part and a disaccharide unit. Eleven tertiary aromatic carbons at δ_C 96.6 (C-5), 109.0 (C-4), 117.7 (C-2), 119.0 (C-12), 119.8 (C-3), 126.9 (C-7), 138.5 (C-13), 140.2 (C-18), 146.5 (C-15), 153.1 (C-11), 157.3 (C-6), five aromatic methine carbons at δ_C 115.2 (C-10), 118.4 (C-8), 120.9 (C-16), 127.8 (C-9), 133.0 (C-17), together with two carbonyl lactone at δ_C 159.2 (C-1), 164.7 (C-14) and one methyl carbon at δ_C 22.2 (C-19) were assigned to aglycone part. Additionally, ten oxygenated methine carbons at δ_C 67.1 (C-11′), 67.6 (C-8′), 68.1 (C-10′), 70.9 (C-5′), 71.0 (C-3′), 73.1 (C-4′), 80.1 (C-9′), 82.3 (C-2′), 98.9 (C-1′, C-anomer), 101.4 (C-7′, C-anomer), two methyl carbons at δ_C 16.5 (C-12′), 16.4 (C-6′) and one methoxy group at δ_C 57.0 (C-13′) were determined to belong to the sugar units. Comparison of the ¹H and ¹³C NMR spectral data of the sugar unit with the reported literature suggested that compound 2 was inclusive of D-fucose and D-digitalose in the structure. The assignment of all protons and carbons in compound 2 was further corroborated by 2D NMR (COSY, HSQC, HMBC and NOESY) spectral data. The COSY correlations of H-8 (δ_H 8.19)/H-9 (δ_H 7.61), H-9 (δ_H 7.61)/H-10 (δ_H 7.41), H-16 (δ_H 7.51)/H-17 (δ_H 7.44), as well as the HMBC correlations from H-8 (δ_H 8.19) to C-6 (δ_C 157.3) and C-10 (δ_C 115.2); H-9 (δ_H 7.61) to C-7 (δ_C 126.9), C-10 (δ_C 115.2) and C-11 (δ_C 153.1); H-10 (δ_H 7.41) to C-12 (δ_C 119.0) and C-8 (δ_C 118.4), H-16 (δ_H 7.51) to C-3 (δ_C 119.8); C-15 (δ_C 119.8), and C-18 (δ_C 140.2); H-17 (δ_H 7.44) to C-2 (δ_C 117.7) and C-15 (δ_C 146.5) confirmed the location of the aromatic protons in the aromatic bilactone ring (Figure 5). Furthermore, the HMBC correlation between H-19 (δ_H 2.81)/C-2 (δ_C 117.7), C-18 (δ_C 140.2) and C-17 (δ_C 133.0), along with the correlation of the proton in hydroxyl group C₆-OH (δH 11.63)/C-6 (δ_C 96.6) and C-5 (δ_C 109.0) established the position of the methyl and hydroxyl group in the aglycone unit (Figure 5). The connection of each proton in the sugar unit was affirmed by the COSY spectrum, in which the correlations between H-1′ (δ_H 5.33)/H-2′ (δ_H 4.27), H-2′ (δ_H 4.27)/H-3′ (δ_H 3.88), H-3′ (δ_H 3.88)/H-4′ (δ_H 3.78), H-5′ (δ_H 3.92)/H-6′ (δ_H 1.44) together with H-7′ (δ_H 5.70)/H-8′ (δ_H 3.84), H-8′ (δ_H 3.84)/H-9′ (δ_H 3.30), H-9′ (δ_H 3.30)/ H-10′ (δ_H 3.87), H-10′ (δ_H 3.87)/ H-11′ (δ_H 4.22), H-11′ (δ_H 4.22)/H-12′ (δ_H 1.38) were observed. The D-digitalose unit was located at C-2′ of D-fucose, deducing from the HMBC correlation of proton H-2′ (δ_H 5.33) to C-7′ (δC 101.4). The sugar part was attached to the aglycone at C-1′, approved by the HMBC correlation between H-1′ (δ_H 5.33) to C-11 (δ_C 153.1). The ¹H and ¹³C NMR data of compound 2 were presented in Table 3. The spectral data of this compound was comparable with those reported for chartreusin.³⁷ Therefore, compound 2 was identified as chartreusin (Figure 4), a benzonaphthopyranone glycoside isolated previously from Streptomyces chartreusis. This is the first time chartreusin has been isolated from Streptomyces alboniger.

Table 3.

¹H and ¹³C NMR Spectral Data of Compound 2 and Chartreusin.³⁷

C	2		Chartreusin³⁷
C	δ_H (ppm)^a	δ_C (ppm)^b	δ_H (ppm)^c	δ_C (ppm)^d
1		159.2		159.0
2		117.7		117.5
3		119.8		120.1
4		109.0		108.9
5		96.6		97.4
6		157.3		155.9
7		126.9		126.6
8	8.19 (d, J = 8.0)	118.4	8.09 (d, J = 8.4)	116.8
9	7.61 (t, J = 8.0)	127.8	7.75 (t, J = 8.4)	128.9
10	7.41 (d, J = 8.0)	115.2	7.54 (d, J = 8.4)	114.9
11		153.1		154.6
12		119.0		118.4
13		138.5		139.1
14		164.7		164.3
15		146.5		146.7
16	7.51 (d, J = 8.5)	120.9	7.67 (d, J = 8.3)	121.3
17	7.44 (d, J = 8.5)	133.0	7.63 (d, J = 8.3)	133.5
18		140.2		139.0
19	2.81 (s)	22.2	2.81 (s)	22.2
1′	5.33 (d, J = 7.5)	98.9	5.40 (d, J = 7.7)	100.2
2′	4.27 (m)	82.3	3.98 (m)	78.4
3′	3.88 (m)	71.0	3.67 (m)	71.8
4′	3.78 (m)	73.1	3.65 (m)	72.5
5′	3.92 (m)	70.9	3.96 (m)	70.7
6′	1.44 (d, J = 6.5)	16.4	1.00 (d, J = 6.4)	17.0
7′	5.70 (d, J = 4.5)	101.4	5.44 (d, J = 4.0)	99.9
8′	3.84 (m)	67.6	3.39 (m)	67.4
9′	3.30 (dd, J = 10.0; 3.0)	80.1	3.10 (dd, J = 10.1; 3.1)	80.3
10′	3.87 (m)	68.1	3.63 (m)	67.9
11′	4.22 (m)	67.1	4.26 (q, J = 6.2)	66.3
12′	1.38 (d, J = 6.5)	16.5	1.23 (d, J = 6.2)	16.9
13′	3.36 (s)	57.0	3.15 (s)	56.4
6-OH	11.63 (s)

500 MHz, CDCl₃.

125 MHz, CDCl₃.

400 MHz, DMSO-d₆.

100 MHz, DMSO-d₆

Antimycobacterial Activity

Compounds 1 and 2 were evaluated for their antimycobacterial activity against M. smegmatis. The results showed that compound 2 displayed strong activity against M. smegmatis with 15 mm of inhibition zone diameter, while compound 1 was inactive (Table 4). Chartreusin was reported to have potential antimycobacterial activity against Mycobacterium tuberculosis 607 and Mycobacterium tuberculosis H37Rv.³⁸ Our study reported for the first time the production of chartreusin from Streptomyces alboniger and its antimycobacterial activity against M. smegmatis.

Table 4.

Antimycobacterial Activity of Compound 1 and 2.

Compound	M. smegmatis *
1	0
2	15 ± 1.0
Rufomycin	12 ± 1.0

*: Inhibition zone diameter (mm)

Additionally, compound 2 was tested for the antimicrobial activity against three gram-positive strains: Staphylococcus aureus, Bacillus subtilis, Lactobacillus fermentum and three gram-negative strains Salmonella enterica, Escherichia coli, Pseudomonas aeruginosa. This compound was active against gram-positive strains Staphylococcus aureus, Bacillus subtilis and Lactobacillus fermentum with IC₅₀ values of 7.09 ± 0.25, 1.68 ± 0.07 và 11.70 ± 0.05 µM, respectively, but distinct inhibited with the gram-negative strains (Table 5). The strain Bacillus subtilis was extremely sensitive to compound 2, (IC₅₀ 1.68 ± 0.07 µM), better than the positive control ampicillin (IC₅₀ 10.36 ± 0.15 µM). Besides, this compound exhibited full inhibition of the growth of Bacillus subtilis strain with MIC value of 12.5 µM. The obtained results corresponded to the literature reported about the antimicrobial effect of chartreusin against Bacillus subtilis³⁸ and Staphylococcus aureus.³⁹ However, our study reported for the first time the MIC value of chartreusin against Bacilus subtilis, and the antibacterial activity of this compound toward Lactobacillus fermentum.

Table 5.

Antibacterial Activity of Compound 2.

	µM	Gram-positive			Gram-negative
	µM	Staphylococcus aureus	Bacillus subtilis	Lactobacillus fermentum	Salmonella enterica	Escherichia coli	Pseudomonas aeruginosa
Compound 2	IC₅₀	7.09 ± 0.25	1.68 ± 0.07	11.70 ± 0.05	>128	>128	>128
Compound 2	MIC	>128	12.5	>128	>128	>128	>128
Ampicillin	IC₅₀	0.06 ± 0.005	10.36 ± 0.15	2.95 ± 0.07
Ampicillin	MIC	0.36	91.58	91.58
Cefotaxime	IC₅₀				0.94 ± 0.05	0.015 ± 0.002	9.53 ± 0.15
Cefotaxime	MIC				70.26	1.10	17.56

Cytotoxic Activity

Compound 2 was evaluated for cytotoxic activity on four human cancer cell lines: KB (Carcinoma cancer), Hep-G2 (Liver cancer), Lu-1 (Lung cancer) and MCF-7 (Breast cancer). The result showed that 2 displayed potential cytotoxicity (IC₅₀ value of 8.23, 0.78, 8.28, and 45.0 µM against KB, Hep-G2, Lu-1 and MCF-7, respectively) (Table 6). Notably, this compound demonstrated a significantly cytotoxic activity against the liver cancer cell line Hep-G2 with IC₅₀ value of 0.78 µM, better than that of the control compound ellipticine (IC₅₀ 1.22 µM). The cytotoxicity of compound 2 against KB cancer cells was in accordance with previous literature.⁴⁰ Chartreusin was reported to have cytotoxic activity against the liver cancer cell Hep3B2.1-7 (IC₅₀ 18.19 µM) and lung cancer cell H1299 (IC₅₀ 19.74 µM).³⁹ This is the first report of cytotoxicity of chartreusin against Hep-G2, Lu-1 and MCF-7 human cancer cells. Chartreusin and its semisynthetic derivatives are the promising antitumor agents acting via breakage of single strands DNA and inhibition of topoisomerase II.³⁷ IST-II was a lead compound derived from chartreusin derivatives, which was advanced to phase II clinical human trials.⁴¹

Table 6.

Cytotoxic Activity of Compound 2.

	IC₅₀ (µM)
	KB	Hep-G2	Lu-1	MCF-7
Compound 2	8.23 ± 0.25	0.78 ± 0.25	8.28 ± 0.31	45.00 ± 0.63
Ellipticine	1.22 ± 0.05	1.22 ± 0.05	1.75 ± 0.05	2.35 ± 0.05

Conclusion

In conclusion, we have discovered and reported for the first time the potential antimycobacterial activity of the actinomycetes collected in the regions along the north to the central of Vietnam, including Streptomyces avidinii (A002), Streptomyces spiroverticillatus (A067), Streptomyces wuyanensis (A079), Streptomyces hawaiiensis (A105), Streptomyces alboniger (A121). Obscurolide B_2β (1) and chartreusin (2) are the metabolites identified from the most active strain Streptomyces alboniger (A121). This is also the first report on the isolation of these compounds from this strain, as well as their antimycobacterial properties against M. smegmatis. Remarkably, chartreusin (2) displayed significant antimycobacterial, antimicrobial and cytotoxic activity, making its attention for further drug development, especially for antituberculosis agents. However, further investigation needs to be conducted to understand the mechanism of its action, efficacy and safety both in vitro and in vivo against multidrug resistant tuberculosis (MDR-TB) strains of Mycobacterium tuberculosis. Additionally, modification of chemical structure of compound 2 may be also performed to enhance the antimycobacterial potency.

Materials and Methods

Chemical and Equipment

NMR spectra were recorded on a Bruker Avance 500 MHz spectrometer, using deuterated solvents and tetramethylsilane as internal standard. ESI-MS spectra were measured on an Agilent LC-MSD-Trap SL instrument. Polar rotation was obtained on a Jasco P-2000 Polarimeter serial A060161232 equipment. Merck 60F254 pre-coated silica gel plates (0.2 mm thickness) were used for thin layer chromatography (TLC). Silica gel 60 (0.040-0.063 mm, 230-400 mesh), Sephadex LH-20 were purchased from Merck. Other chemicals were purchased from Merck and used without purification unless otherwise needed.

Sample Collection

Soils and sediments were collected along the areas from Northern to Central Vietnam (nine areas in Table 1). The coordinates of the collection areas were recorded by GPS navigation device. Moisture, salinity and temperature of the soil samples were determined by soil testing equipment. Each sample was placed in a 50 mL falcon tube and kept in the refrigerator (0-5 °C) during transport to the laboratory.

Isolation and Identification of Actinomycetes

The samples were left drying naturally for 2–3 days and powdered. Each sample (1 g) was diluted to 10⁻³ and 10⁻⁴ concentrations, then spread onto a Petri dish using two different media, NaST21 (solution A: 750 mL sterilized seawater, 1 g K₂HPO₄, 16 g Bactogar; solution B: 250 mL sterilized seawater, 1 g KNO₃, 1 g MgSO₄, 1 g CaCl₂.2H₂O, 0.2 g FeCl₃, 0.1 g MgSO₄.7H₂O) and HV (1 g Humic acid, 0.02 g CaCO₃, 0.01 g FeSO₄.7H₂O, 1.7 g KCl, 0.05 g MgSO₄.7H₂O, 0.5 g Na₂HPO₄, 5 mL B-group vitamins, 16 g Agar, 1 L distilled water, pH 7). Both culture media above were supplemented with nalidixic acid (20 mg/L) and cycloheximide (50 mg/L). The isolation plates were incubated at 28–30 °C for 14 to 21 days. Colonies with different morphology were cultured on YS medium (2 g yeast extract, 10 g agar, 1 L distilled water) and kept at − 80 °C. Identification and classification of actinomycetes were determined based on colony, spore chain morphology and 16S rRNA gene sequencing. Amplifications of the 16S rRNA gene from total DNA were carried out through PCR using the primers pair 27F (5′-AGAGTTTGATCCTGGCTCAG-3) and 1492R (5′-GGTTACCTTGTTACGACTT-3). The PCR product was analyzed by 1% agarose gel electrophoresis, purified by PCR purification Kit (Bioneer, Korea) and finally sequencing. The sequence of the 16S rRNA fragment was used to detect the species by the EZ Taxon tool. Actinomycete strains obtained from the purification step were kept in glycerol (20%) at − 80 °C for further studies.

Antimycobacterial Activity Assay

Antimycobacterial activity was determined by the agar diffusion assay.⁴² Actinomycete strains were cultured on YS agar medium at 30 °C for 7 days. Mycobacterium smegmatis mc²155 KCTC 9108 strain was obtained from Center for Nutraceutical and Pharmaceutical Materials Myongji University, Korea. This strain was grown on 219 liquid medium (g/L: peptone – 10, yeast extract – 5, malt extract – 5, casamino acid – 5, beef extract – 2, glycerol – 2, tween 80-50 mg, MgSO₄.7H₂O – 1) for 24 h at 30 °C. Bacterial culture was added to the sterilized 219 agar medium with the ratio of 1% and poured into a petri dish. Cultured pieces of agar containing actinomycetes with 5 mm in diameter were placed onto plate. For testing the extract or compounds obtained from fermentation and isolation, holes in the agar plate were made by a plastic tube, following adding a solution of extract or compound (50 µL) to these holes. The agar plates were incubated at 30 °C for 24 h. Antimycobacterial activity was determined by inhibition zone diameter on the agar plate. Agar pieces without bacteria were used as negative control. All experiments were performed in triplicate and the data were obtained as mean ± SD. Rufomycin was used as a reference substance with the test concentration of 10 µg/mL.

Antimicrobial Activity Assay

Antimicrobial activity was determined by Broth microdilution method.⁴³ The test microorganisms included three Gram-positive bacteria Staphylococcus aureus ATCC 13709, Bacillus subtilis ATCC 6633, Lactobacillus fermentum N4 and three Gram-negative bacteria Salmonella enterica, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 15442. MHB (Mueller-Hinton Broth), MHA (Mueller-Hinton Agar); TSB (Tryptic Soy Broth); TSA (Tryptic Soy Agar) were used as culture media. The microbacteria were kept at − 80 °C and activated in culture medium before use, to reach the concentration of 5 × 10⁵ CFU/mL. The test samples were diluted in DMSO and sterilized distilled water to obtain the concentrations of 128.0, 32.0, 8.0, 2.0 and 0.5 µg/mL. 10 µL of sample solution at different concentrations were added into a 96-well plate, following the addition of 200 µL of microbacterial solution. The place was incubated at 37 °C for 24 h. All experiments were tested in triplicate and the data were obtained as mean ± SD. Wells with bacteria suspension in growth medium were used as positive control, while the one containing culture medium without bacteria was considered as negative control. Ampicillin and cefotaxime were used as the reference substance. The MIC (Minimum Bactericidal Concentration) value was determined at the well with the lowest concentration of the test sample that inhibited the growth of bacteria. The IC₅₀ (50% inhibitory concentration) value is calculated by TECAN spectrometer and raw data software.

Cytotoxic Activity Assay

The cytotoxic activity was performed based on MTT asaay.⁴⁴ This method assessed the cell viability through the discoloration of tetrazolium salt (MTT - (3-(4,5-dimethylthiazol-2 - yl)- 2, 5 - diphenyltetrazolium)). The samples were evaluated against carcinoma cancer cells (KB), liver cancer cells (Hep-G2), lung cancer cells (Lu-1) and breast cancer cells (MCF-7). The sample (20 µL) was introduced into the wells of the 96-well plate at the following concentration: 128 µg/mL; 32 µg/mL; 8 µg/mL and 2 µg/mL. The cells were cultured in Dulbecco's modified eagle's medium with 10% fetal bovine serum, 1% penicillin and streptomycin, 1% L-glutamine at 37 °C, 5% CO₂ for 72 h. Cells were separated with trypsin and seed to reach the concentration of 3.10⁴cell/mL. After that, 180 µL of cells was put onto each well of the 96-well plate containing the sample. The plate was incubated at 37 °C, 5% CO₂ for 72 h. After incubation, 10 μL of MTT solution in the phosphate buffer was added and the plate was further incubated for 2–4 h. MTT was removed and 70 μL of DMSO was added. Positive control wells contained culture medium with cells, while the one containing only culture medium served as negative control. Ellipticine was used as the reference substance. All experiments were conducted in triplicate and the data were obtained as mean ± SD. The results were determined by the OD values (optical density), which were measured on a Microplate reader EPOCH 2 spectrophotometer, Biotech, USA at 540 nm. The IC₅₀ value was calculated based on the percentage inhibition value by Table curve software.

Fermentation, Extraction and Isolation of the Metabolites from Strain A121 (Streptomyces alboniger)

Strain A121 was cultured on agar medium for 5–7 days, then it was inoculated into a 1000 mL conical flask containing 350 mL of YS medium, shaking at 160 rpm, 28–30 °C for 72 h. The actinomycete culture broth was inoculated into the 50-liter fermentation equipment with the ratio of 2.5–3% (v/v). The culture strain reached the highest cell density after 144 h, indicating that the growth of actinomycetes was at the equilibrium phase. When the fermentation process ended, the actinomycete culture was collected. Biomass and actinomycete culture were separated by centrifugation, which was used immediately for the extraction and isolation process.

The culture of strain A121 (50 L) was centrifuged at 10 000 rpm (20 min, room temperature). The filtrate (40 L) was extracted with EtOAc (3 × 24 h × 30 L). The EtOAc extracts were grouped and the solvent was evaporated in vacuo to give 6.2 g EtOAc residue (121E). The aqueous fraction remaining after extraction with EtOAc was condensed to yield 10.2 g aqueous residue (121CN). The EtOAc extract (6.2 g) was subjected to a Sephadex LH-20 column (MeOH 100%) to afford five subfractions (121E.1 → 121E.5). Fraction 121E.2 (3.4 g) was purified by a silica gel column (n-hexane/CH₂Cl₂ 98:2→ 1:1) to yield four fractions (121E.2.1 → 121E.2.4). Fraction 121E.2.3 (30 mg) was separated over a silica gel column (CH₂Cl₂/Acetone 9:1→8:2) to give compound 1 (3.7 mg).

The cell residue (biomass) was extracted with MeOH/H₂O (9:1, 3 × 4 h × 1 L), then centrifuged at 10 000 rpm at room temperature to remove the residue. The solvent was evaporated to provide 7 g residue (121X). The fraction 121CN and 121X were examined by thin layer chromatography. As a result, these fractions contained similar compounds, which were therefore grouped together to give fraction 121CX (17 g). Fraction 121 CX was separated on a Sephadex LH-20 column (MeOH/H₂O 9:1) to obtain 8 subfractions (121CX1 → 121CX8). The fraction 121CX5 (0.1 g) was subjected to a Sephadex LH-20 column (MeOH/H₂O 9:1) to obtain 5 fractions (121CX.5.1 →121CX.5.5). Fraction 121CX5.4 (101 mg) was repeatedly purified on a Sephadex LH-20 column (MeOH/H₂O 9:1) to afford compound 2 (5.6 mg).

Obscurolide B_2β (1): Yellow powder; C₁₅H₁₇NO₄; $[α]_{D}^{27} = + 20.4$ (c 0.1, CH₂Cl₂), ESI-MS (m/z): 316.1 [M + Na + H₂O]⁺, HR-ESI-MS (m/z): 276.1221 (calculated for C₁₅H₁₈NO₄ [M + H]⁺), 298.1045 (calculated for C₁₅H₁₇NO₄Na [M + Na]⁺ 298.1055); ¹H-NMR (500 MHz, CDCl₃), δ_H (ppm): 9.77 (1H, s, CHO), 7.73 (1H, d, J = 8.5, H-3′, H-5′), 6.67 (1H, d, J = 8.5, H-2′, H-6′), 5.88 (1H, dd, J = 15.0, 7.0, H-6), 5.64 (1H, dqd, J = 15.0, 7.0, 2.0, H-7), 4.60 (1H, brs, C₅-OH), 4.41 (1H, m, H-3), 4.35 (1H, dd, J = 7.2, 3.0, H-4), 4.32 (1H, m, H-5), 3.18 (1H, dd, J = 18.0, 7.5, H-2a), 2.44 (1H, dd, J = 18.0, 3.5, H-2b), 1.74 (3H, dd, J = 6.5, 1.5 Hz, H-8); ¹³C NMR (125 MHz, CDCl₃), δ_C (ppm): 190.3 (C-7′), 174.9 (C-1), 150.9 (C-1′), 132.3 (C-3′, C-5′), 131.4 (C-6), 128.2 (C-7), 128.1 (C-4′), 112.6 (C-2′, C-6′), 87.3 (C-4), 73.1 (C-5), 51.0 (C-3), 36.3 (C-2), 17.8 (C-8).

Chartreusin (2): Greenish-yellow powder; C₃₂H₃₂O₁₄; ESI-MS (m/z): 639.1 [M-H]⁻, HR-ESI-MS (m/z): 641.1876 (calculated for C₃₂H₃₃O₁₄ 641.1870 [M + H]⁺), 663.1710 (calculated for C₃₂H₃₂O₁₄Na 663.1690 [M + Na]⁺). ¹H and ¹³C NMR data: see Table 3.

Footnotes

Acknowledgments

The authors are grateful to the Ministry of Science and Technology of Vietnam (MOST) for financial support for this study (Vietnam-Korea International bilateral cooperative project, Project Nr: NĐT.47.KR/18). PhD Huynh Thi Ngoc Ni and Master Ngo Van Hieu acknowledge the financial support by the Domestic Master/Ph.D. Scholarship Programme of Vingroup Innovation Foundation.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Approval

Our institution does not require ethical approval for reporting individual cases or case series.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Vietnamese Ministry of Science and Technology, (grant number NDT.47.KR/18)

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

ORCID iDs

Chien Van Tran

Tran Thi Phuong Thao

Supplemental Material

Supplemental material for this article is available online.

References

Bagcchi

. WHO’s global tuberculosis report 2022. Lancet Microbe. 2023;4(1):e20. https://doi.org/10.1016/S2666-5247(22)00359-7

Wortberg

Perscheid

Moormann

LNQ

. Towards the design of a gamified application to increase tuberculosis treatment adherence in Vietnam. In: Proceedings of the 56th Hawaii International Conference on System Sciences; 2023:3146–3155. https://hdl.handle.net/10125/103018

World Health Organization. Global Tuberculosis report 2022. 2022. https://www.who.int/publications/i/item/9789240061729.

Ngoc

Dinh

Thuy

, et al. Active surveillance for adverse events in patients on longer treatment regimens for multidrug-resistant tuberculosis in Viet Nam. Plus one. 2021;16(9):e0255357. https://doi.org/10.1371/journal.pone.0255357

Redwood

Fox

Nguyen

, et al. Good citizens, perfect patients, and family reputation: stigma and prolonged isolation in people with drug-resistant tuberculosis in Vietnam. PLOS Glob Public Health. 2022;2(6):e0000681. https://doi.org/10.1371/journal.pgph.0000681

De Simeis

Serra

. Actinomycetes: a never-ending source of bioactive compounds—an overview on antibiotics production. Antibiotics. 2021;10(5):483. https://doi.org/10.3390/antibiotics10050483

Arbex

Varella

MdCL

Siqueira

HRd

Mello

FAFd

. Antituberculosis drugs: drug interactions, adverse effects, and use in special situations-part 1: first-line drugs. J Bras Pneumol. 2010;36(5):626-640. https://doi.org/10.1590/S1806-37132010000500016

Stover

Warrener

VanDevanter

, et al. A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis. Nature. 2000;405(6789):962-966. https://doi.org/10.1038/35016103

Falzari

Zhu

Pan

Liu

Hongmanee

Franzblau

. In vitro and in vivo activities of macrolide derivatives against Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2005;49(4):1447-1454. https://doi.org/10.1128/aac.49.4.1447-1454.2005

10.

Tuan

Hieu

Huong

DTM

, et al. Secondary compounds from the marine actinomycete strain Micromonospora sp.(G043). Vietnam J Chem. 2016;54(5):581-585. https://doi.org/10.15625/0866-7144.2016-00368

11.

Ranjitha

Rajan

Shankar

. Features of the biochemistry of Mycobacterium smegmatis, as a possible model for Mycobacterium tuberculosis. J Infect Public Health. 2020;13(9):1255-1264. https://doi.org/10.1016/j.jiph.2020.06.023

12.

Zhou

Wei

Fleming

, et al. Mycobacterium smegmatis MSMEG_0129 is a nutrition-associated regulator that interacts with CarD and ClpP2. Int J Biochem Cell Biol. 2020;124(7):105763. https://doi.org/10.1016/j.biocel.2020.105763

13.

Golichenari

Yari

Tasbiti

Behravan

Vaziri

Ghazvini

. First conjugation directed traverse of gene cassettes harboring α1, 3GT from fast-growing Mycobacterium smegmatis mc2 155 to slow-growing pathogen Mycobacterium tuberculosis H37Rv, presumably opening up new scopes in tuberculosis treatment. Enzyme Microb Technol. 2022;156(5):110003. https://doi.org/10.1016/j.enzmictec.2022.110003

14.

Tapfuma

Nyambo

Adu-Amankwaah

, et al. Antimycobacterial activity and molecular docking of methanolic extracts and compounds of marine fungi from Saldanha and False Bays, South Africa. Heliyon. 2022;8(12):e12406. https://doi.org/10.1016/j.heliyon.2022.e12406

15.

Tuyiringire

Mugisha

Tusubira

Munyampundu

J-P

Muvunyi

Vander Heyden

. Invitro antimycobacterial activity of medicinal plants Lantana camara, Cryptolepis sanguinolenta, and Zanthoxylum leprieurii. J Clin Tuberc Other Mycobact Dis. 2022;27(3):100307. https://doi.org/10.1016/j.jctube.2022.100307

16.

Wang

Feng

Dong

Luo

Shang

. Membrane-active and DNA binding related double-action antimycobacterial mechanism of antimicrobial peptide W3R6 and its synthetic analogs. Biochim Biophys Acta -General Subjects. 2023; 1876(9):130415. https://doi.org/10.1016/j.bbagen.2023.130415

17.

Ramadhani

Dyah Kurniati

Dian Rakhmawatie

. Antimicrobial activity of Pecut Kuda leaf extract (Stachytarpheta jamaicensis (L. Vahl) against Mycobacterium smegmatis. Mutiara Medika: Jurnal Kedokteran dan Kesehatan. 2023;23(1):21-27. https://doi.org/10.18196/mmjkk.v22i2.15599

18.

Alelaiwi

Heindl

Sivaganesh

Peethambaran

McKee

. Structure–activity relationship of 2-aminodibenzothiophene pharmacophore and the discovery of aminobenzothiophenes as potent inhibitors of Mycobacterium smegmatis. Bioorganic Med Chem Lett. 2022;63(5):128650. https://doi.org/10.1016/j.bmcl.2022.128650

19.

Rakhmawatie

Mustofa

Pratiwi

Wibawa

. Identification of antimycobacterial from actinobacteria (INACC A758) secondary metabolites using metabolomics data. Sains Malays. 2022;51(5):1465-1473. https://doi.org/10.17576/jsm-2022-5105-16

20.

Chaturvedi

Dwivedi

Tripathi

Sinha

. Evaluation of Mycobacterium smegmatis as a possible surrogate screen for selecting molecules active against multi-drug resistant Mycobacterium tuberculosis. J Gen Appl Microbiol. 2007;53(6):333-337. https://doi.org/10.2323/jgam.53.333

21.

Grzelak

Choules

Gao

, et al. Strategies in anti-Mycobacterium tuberculosis drug discovery based on phenotypic screening. J Antibiot (Tokyo). 2019;72(10):719-728. https://doi.org/10.1038/s41429-019-0205-9

22.

Jose

Jebakumar

SRD

. Unexplored hypersaline habitats are sources of novel actinomycetes. Front Microbiol: Front Media SA. 2014;5(5):242. https://doi.org/10.3389/fmicb.2014.00242

23.

Singh

Sharma

Talukdar

. Production of potent antimicrobial agent by actinomycete, Streptomyces sannanensis strain SU118 isolated from phoomdi in Loktak Lake of Manipur, India. BMC Microbiol. 2014;14(1):1-13. https://doi.org/10.1186/s12866-014-0278-3

24.

Sarkar

Suthindhiran

. Diversity and biotechnological potential of marine actinomycetes from India. Indian J Microbiol. 2022;62(4):475-493. https://doi.org/10.1007/s12088-022-01024-x

25.

Manikkam

Venugopal

Subramaniam

Ramasamy

Kumar

. Bioactive potential of actinomycetes from less explored ecosystems against Mycobacterium tuberculosis and other nonmycobacterial pathogens. Int Sch Res Notices. 2014;2014(5):1-9. https://doi.org/10.1155/2014/812974

26.

Zhang

Zheng

Xin

Pang

. Streptomyces wuyuanensis sp. nov., an actinomycete from soil. Int J Syst Evol Microbiol. 2013;63(Pt_8):2945-2950. https://doi.org/10.1099/ijs.0.047050-0

27.

Ivanova

Lyutskanova

Kolarova

Aleksieva

Raykovska

Stoilova-Disheva

. Structural elucidation of a bioactive metabolites produced by Streptomyces Avidinii SB9 strain, isolated from permafrost soil in Spitsbergen, Arctic. Biotechnol Biotechnol Equip. 2010;24(4):2092-2095. https://doi.org/10.2478/v10133-010-0080-9

28.

Adler

Cook

Luo

, et al. Tautomycetin and tautomycin suppress the growth of medullary thyroid cancer cells via inhibition of glycogen synthase kinase-3β. Mol Cancer Ther. 2009;8(4):914-920. https://doi.org/10.1158/1535-7163.MCT-08-0712

29.

Nogawa

Terai

Amagai

, et al. Heterologous expression of the biosynthetic gene cluster for verticilactam and identification of analogues. J Nat Prod. 2020;83(12):3598-3605. https://doi.org/10.1021/acs.jnatprod.0c00755

30.

Goodreid

Wong

Leung

, et al. Total synthesis and antibacterial testing of the A54556 cyclic acyldepsipeptides isolated from Streptomyces hawaiiensis. J Nat Prod. 2014;77(10):2170-2181. https://doi.org/10.1021/np500158q

31.

Michel

Kastner

. A54556 antibiotics and process for production thereof. U.S. Patent No. 4,492,650; 1985.

32.

Hesseltine

Porter

Deduck

Hauck

Bohonos

Williams

. A new species of Streptomyces. Mycologia. 1954;46(1):16-23. https://doi.org/10.1080/00275514.1954.12024337

33.

Yan

Zhang

Tian

, et al. Puromycin A, B and C, cryptic nucleosides identified from Streptomyces alboniger NRRL B-1832 by PPtase-based activation. Synth Syst Biotechnol. 2018;3(1):76-80. https://doi.org/10.1016/j.synbio.2018.02.001

34.

McCann

Pogell

. Pamamycin: a new antibiotic and stimulator of aerial mycelia formation. J Antibiot (Tokyo). 1979;32(7):673-678. https://doi.org/10.7164/antibiotics.32.673

35.

Luo

Yang

Y-B

Yang

X-Q

, et al. The streptazolin-and obscurolide-type metabolites from soil-derived Streptomyces alboniger YIM20533 and the mechanism of influence of γ-butyrolactone on the growth of Streptomyces by their non-enzymatic reaction biosynthesis. RSC Adv. 2018;8(61):35042-35049. https://doi.org/10.1039/C8RA06690F

36.

Ritzau

Philips

Zeeck

Hoff

Zähner

. Obscurolides, a novel class of phosphodiesterase inhibitors from Streptomyces. II. Minor components belonging to the obscurolide B to D series. J Antibiot. 1993;46(10):1625-1628. https://doi.org/10.7164/antibiotics.46.1625

37.

Wang

Zhang

Zhu

, et al. Molecular basis for the final oxidative rearrangement steps in chartreusin biosynthesis. J Am Chem Soc. 2018;140(34):10909-10914. https://doi.org/10.1021/jacs.8b06623

38.

Leach

Calhoun

Johnson

Teeters

Jackson

. Chartreusin, a new antibiotic produced by Streptomyces chartreusis, a new species. J Am Chem Soc. 1953;75(16):4011-4012. https://doi.org/10.1021/ja01112a040

39.

Zhao

Chen

, et al. New peptidendrocins and anticancer chartreusin from an endophytic bacterium of Dendrobium officinale. Ann Transl Med. 2020;8(7):455-464. https://doi.org/10.21037/atm.2020.03.227

40.

Smith

Lummis

Grady

. An improved tissue culture assay II. Cytotoxicity studies with antibiotics, chemicals, and solvents. Cancer Res. 1959;19(8):847-852.

41.

Mohan

Rangappa

Nayak

, et al. Bacteria as a treasure house of secondary metabolites with anticancer potential. Semin Cancer Biol. 2022;86(2):998-1013. https://doi.org/10.1016/j.semcancer.2021.05.006

42.

Balouiri

Sadiki

Ibnsouda

. Methods for in vitro evaluating antimicrobial activity: a review. J Pharm Anal. 2016;6(2):71-79. https://doi.org/10.1016/j.jpha.2015.11.005

43.

Lone

Rather

Bhat

Tantry

Prakash

. Synthesis and in vitro evaluation of 2-(((2-ether) amino) methylene)-dimedone derivatives as potential antimicrobial agents. Microb Pathog. 2018;114(1):431-435. https://doi.org/10.1016/j.micpath.2017.12.022

44.

NTT

Van Cuong

Tra

Van Tuyen

Cham

Tung

. Cytotoxic naphthoquinones from Diospyros fleuryana leaves. Discov Phytomed. 2020;7(1):42-46. https://doi.org/10.15562/phytomedicine.2020.117

Screening for Antimycobacterial Activity of Actinomycetes Collected in Vietnam – Isolation and Activity of Metabolites from Streptomyces alboniger (A121)

Abstract

Objective

Methods

Results

Conclusions

Keywords

Introduction

Results and Discussion

Isolation of Actinomycetes

Antimycobacterial actinomycetes Against Mycobacterium Smegmatis

Classification of the Five Most Active Strains

Structural Elucidation of the Compounds Isolated from Streptomyces Alboniger (A121)

Antimycobacterial Activity

Cytotoxic Activity

Conclusion

Materials and Methods

Chemical and Equipment

Sample Collection

Isolation and Identification of Actinomycetes

Antimycobacterial Activity Assay

Antimicrobial Activity Assay

Cytotoxic Activity Assay

Fermentation, Extraction and Isolation of the Metabolites from Strain A121 (Streptomyces alboniger)

Footnotes

Acknowledgments

Declaration of Conflicting Interests

Ethical Approval

Funding

Statement of Human and Animal Rights

Statement of Informed Consent

ORCID iDs

Supplemental Material

References