Abstract The actinomycete strains SC3-16 and KB-8 isolated from soil samples in Udon Thani and in Krabi Provinces, respectively and were taxonomically studied. The strains SC3-16 and KB-8 showed highest 16S rRNA gene sequence similarity with Streptomyces glycovorans YIM M 10366^T (99.56%) and Micromonospora chalcea DSM 43026^T (99.25%), respectively. During the course of screening for biological activities, the strain SC3-16 exhibited antimicrobial activity against Kocuria rhizophila ATCC 9341 and Staphylococcus aureus ATCC 25923 (inhibition zone 14.2 ± 0.08 – 27.3 ± 0.15 mm). The culture extracts were analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) and the physicochemical properties results of secondary metabolites were searched in database. Chemical analysis of the culture broths suggested that strain SC3-16 contained compounds tentatively assigned as nocardamine, oxotomaymycin- and tomaymycin-related metabolites, whereas strain KB-8 contained compounds tentatively assigned as deferoxamine- and rakicidin-related metabolites. In conclusion, this work contributed to the characterization, antimicrobial activity and their capability for secondary metabolite production of actinomycetes.

Keywords: Antimicrobial activity, Micromonospora, Rakicidins, Deferoxamine, tomaymycin, Streptomyces

Funding: This research was supported by the Grant for International Research Integration: Research Pyramid, Ratchadaphiseksomphot Endowment Fund (CUGRP-61-0133-01), Chulalongkorn University.

Citation:  Kanchanasin, P., Phongsopitanun, W., Tedsree, N., Nakashima, T., and Tanasupawat, S. 2027. Characterization, antimicrobial activity and secondary metabolites of Streptomyces sp. SC3-16 and Micromonospora sp. KB-8 isolated from Thai soils. Natural and Life Sciences Communications. 26(1): e2027003.

Graphical Abstract:

INTRODUCTION

Streptomyces is Gram-positive, aerobic, mesophilic and non-motile actinomycetes. This genus contained cell-wall type I as LL-diaminopimelic acid and no diagnostic sugar in their whole-cell hydrolysates (Tedsree and Tanasupawat, 2021), while Micromonospora strains exhibited a complex life cycle, differentiating into both substrate mycelia and spores. However, no aerial mycelia are produced (Yan et al., 2022). Streptomyces strains were widely distributed in soil, plants, water and other natural environments (Tedsree and Tanasupawat, 2021). They are the most widely group of bacteria that are well known as antibiotic producers and other bioactive compounds (Suksaard et al., 2017; Kanchanasin et al., 2020; Supong and Tanasupawat, 2020; Khan et al., 2023). Micromonospora strains are widely distributed in nature, inhabiting such disparate environments as coastal sediments, marine sediments, peat swamp forests, floodplain meadows and plant rhizospheres. They produced a large number of clinically used antibiotics, such as gentamicin, sisomicin, and erythromycin B, and anti-fungal compounds such as dapiramicins that act to protect plants from pathogens (Hirsch and Valdés, 2010; Yan et al., 2022; Ngamcharungchit et al., 2023). However, the increasing emergence of antimicrobial-resistant microorganisms has created an urgent need to identify additional microbial strains capable of producing structurally and functionally diverse antimicrobial metabolites.

Actinomycetes from underexplored soils could be important sources of bioactive compounds, as their biosynthetic potential is greatly affected by their ecological background (Tedsree et al., 2025; Pooprasert et al., 2026). Thailand encompasses diverse terrestrial ecosystems and soil conditions that may harbor actinomycetes with previously unexplored metabolic capabilities. Therefore, the isolation and characterization of actinomycetes from soils may contribute to the discovery of promising producers of bioactive compounds. Integrating antimicrobial screening with LC-ESI-MS-based metabolite profiling enables the rapid detection and preliminary identification of putative secondary metabolites. In this study, the actinomycete strains designated SC3-16 and KB-8 isolated from soil in Thailand were characterized based on 16S rRNA gene analysis, as well as their antimicrobial activity and secondary metabolite profiles were analyzed using LC-ESI-MS. Compounds with physicochemical properties consistent with oxotomaymycin-related and rakicidin-related metabolites were detected in strains SC3-16 and KB-8, respectively, based on LC-ESI-MS, UV spectra, and database comparison.

MATERIALS AND METHODS

Isolation of actinomycetes

Soil samples were collected from Udon Thani (17° 25′ N 102° 45′ E) and Krabi (7° 44′ N 98° 46′ E) provinces in Thailand, respectively. The soil sample was air-dried and subjected to heat treatment at 100°C for 1 h (Nonomura and Ohara, 1971). Serial dilutions of the samples were prepared using a 10-fold dilution method (10⁻¹, 10⁻², and 10⁻³). Each diluted suspension (100 μL) was spread onto humic acid–vitamin agar (Hayakawa and Nonomura, 1987), supplemented with nalidixic acid (25 μg mL⁻¹) and cycloheximide (50 μg mL⁻¹). The plate was incubated at 30°C for 14-21 days. The colony of actinomycetes was observed using a light microscope and cultivated on ISP 2 agar slants at 30°C for 14 days.

Taxonomic characterization of the isolates

The morphological and cultural characteristics of strain SC3-16 and strain KB-8 were observed on the culture grown on ISP 2 medium at 30°C for 14 days. Cultural characteristics were determined on the colors of aerial mass, upper and reverse colony and soluble pigment using crosshatch streak (Shirling and Gottlieb, 1966) and observed by using the NBS/ISCC color system (Kelly, 1964). Carbon source utilization and several biochemical tests were determined using standard methods as described by Shirling and Gottlieb (1966) and Arai (1975). Utilization of carbon sources was assigned using basal medium supplemented with 1 % (w/v) each of the sugars (Shirling and Gottlieb, 1966). Gelatin liquefaction, starch hydrolysis, skim milk peptonization and coagulation, and nitrate reduction were determined based on standard methods (Arai, 1975). All phenotypic properties were recorded after cultures had been grown at 30°C for 14 days.

The genomic DNA for 16S rRNA gene amplification was extracted from the cultures grown in ISP 2 broth on a rotary shaker as described by Tamaoka and Komagata (1984). The 16S rRNA gene was amplified using two primers 20F (5′-GAGTTTGATCCTGGCTCAG-3′, positions 9-27) and 1500R (5′GTTACCTTGTTACGACTT-3′, positions 1492-1509) as described by Weisburg et al. (1991). The PCR products were sequenced using universal primer, 27F [5’-AGAGTTTGATC (AC) TGGCTCAG-3’] and 1492R [5’-ACGG(CT)TACCTTG TTACGACTT-3’] as described by Lane (1991). An almost complete 16S rRNA gene sequence was analyzed by BLAST using the EzBioCloud database (Yoon et al., 2017). Sequence alignment was performed using BioEdit. Phylogenetic tree was constructed using the Neighbor-Joining method based on the Kimura 2-parameter model in MEGA 11 (Tamura et al., 2021) with bootstrap analysis of 1,000 replicates to assess branch support (Saitou and Nei, 1987).

Screening for antimicrobial activity of isolates

Strains SC3-16 and KB-8 were cultured in ISP 2 broth to prepare seed culture and incubated on a rotary shaker (180 rpm) at 30°C for 4 days. One percent of the seed culture was inoculated into 10 mL of three different production media: A16 medium (0.2% glucose, 1.5% pharmamedia (Iwaki, Tokyo, Japan), 0.3% CaCO₃ (Wako), 1% Diaion HP-20 (Mitsubishi Chemical Ltd, Tokyo, Japan), pH 7.0), 54 medium (2% soluble starch, Wako, 0.5% glycerol, 1% defatted wheat germ, Nisshin Pharma, Tokyo, Japan, 0.3% meat extract, Kyokuto Pharmaceutical Industrial, Tokyo, Japan, 0.3% yeast extract, 0.3% CaCO₃, pH7.0,) and ISP 2 medium, and incubated on a shaker (180 rpm) at 30°C for 6 days. After incubation, 10 mL of 95% ethanol was added to the culture broth, which was then shaken at 200 rpm for 1 h. The culture extract was centrifuged and preserved at -20°C.

The screening of antimicrobial activities was performed using agar disc diffusion assay (Acar and Goldstein, 1991). Four bacteria, Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Kocuria rhizophila ATCC 9341, and the yeast Candida albicans ATCC 10231 were used as the tested microorganisms. The extract solution (20 µL/disc) was dropped on each paper disc (8 mm) and air-dried. The discs were then placed on the surface of agar plates previously inoculated with the test microorganisms. Then, the plates were kept at 4°C for 30 min to allow diffusion before incubation at 37°C for 24 h for bacteria and at 30°C for 48 h for the yeast. The inhibition zones (mm) were measured using a Vernier caliper. The experiments were performed in triplicate and the data are expressed as the mean ± standard deviation. Statistical analysis was conducted using an independent samples t-test to compare the effects of different production media for each tested indicator strain. Differences were considered statistically significant at P < 0.05.

Analyses of antimicrobial compounds

Strains SC3-16 and KB-8 were inoculated into seed medium, consisting of 2.4% starch (Wako), 0.1% glucose, 0.3% peptone (Kyokuto Pharmaceutical Industrial, Tokyo, Japan), 0.3% meat extract, 0.5% yeast extract and 0.4% CaCO₃ (adjusted to pH 7.0 before sterilization). The flask was incubated on a rotary shaker (210 rpm) at 27°C for 3 days. The seed culture was transferred into 10 mL of production medium 54 (composition described above), and the cultivation was carried out on a rotary shaker (210 rpm) at 27°C for 7 days. After cultivation, an equal volume of 95% ethanol was added directly to the whole culture broth, including both the supernatant and mycelial biomass. The mixture was shaken vigorously and centrifuged, and the resulting supernatant was subjected to LC-ESI-MS analysis.

The LC-ESI-MS spectra were measured using an AB Sciex TripleTOF 5600⁺ System coupled to an ExionLC^TM AC (Framingham, MA, USA).  The culture extract was applied to LC-ESI-MS on a CAPCELL CORE C18 (3.0 × 100 mm, Osaka Soda Co. Ltd., Osaka, Japan) at 40°C. Gradient elution was performed using methanol with 2 mM ammonium acetate (AA), 5% methanol from 0 to 2 min, increased from 5-100% methanol from 2 to 12 min, and 100% methanol from 12-14 min. The flow rate was 0.5 mL/min, the injection volume was 2 μL, and a photodiode array detector monitored UV detection. ESI-MS was recorded for 14 min in the m/z region from 100 to 2,000 Da. LC-MS analysis was undertaken by high-resolution ESI-MS (R ≥ 30,000; tolerance for mass accuracy = 5 ppm). The physicochemical properties (retention time, UV absorbance, and pseudomolecular ion) were compared with the reported chemical profile using the Dictionary of Natural Product database (DNP; http://dnp.chemnetbase.com).

RESULTS

Isolation and characterization of the isolates

Strain SC3-16 was isolated from a soil sample in Udon Thani Province, Thailand. The upper and reverse colony color of this strain were yellowish white and brilliant yellow, respectively. It produced spiral spore chains.  The aerial mass was white. Yellow-tone pigment was observed. The strain SC3-16 utilized D-cellobiose, D-fructose, galactose, glycerol, myo-inositol, D-mannitol, D-mannose, raffinose and sucrose, and weakly utilized L-rhamnose as sole carbon source, but not L-arabinose, D-cellulose and D-xylose. Starch hydrolysis, skim milk peptonization and coagulation were positive. Gelatin liquefaction was negative. Nitrate was reduced to nitrite (Table 1). Strain KB-8 was isolated from a soil sample in Krabi Province, Thailand. The upper and reverse colony color of strain KB-8 were brownish black and brownish gray, respectively. It produced single spore. No aerial mass and pigment were observed. The strain KB-8 utilized L-arabinose, D-cellobiose, D-fructose, D-galactose, glycerol, myo-inositol, D-mannitol, D-mannose, D-raffinose, L-rhamnose, sucrose and D-xylose as sole carbon sources. Gelatin liquefaction, starch hydrolysis, skim milk peptonization and coagulation were positive. Nitrate was reduced to nitrite. Both strains characteristics were compared with S. glycovorans YIM M 10366^T and M. chalcea DSM 43026^T (Table 1).

Table 1. Phenotypic characteristics of strains SC3-16, KB-8, YIM M 10366^T (Xu et al., 2012) and DSM 43026^T (Songsumanus et al., 2013; Carro et al., 2018; Camacho Pozo et al., 2020).

Note: ⁺, positive; w, weakly positive; -, negative. ND, no data.

The BLAST analysis of the 16S rRNA gene sequences showed that strain SC3-16 (1,368 nt, accession LC554833) indicated that the strain was closely related to Streptomyces glycovorans YIM M 10366^T (99.56%), while strain KB-8 (1,339 nt, accession LC554834) showed the highest similarity to Micromonospora chalcea DSM 43026^T (99.25%) (Figure 1).

The GenBank/EMBL/DDBJ accession numbers of strains SC3-16 and KB-8 for the 16S rRNA gene sequences of the identified isolates are LC554833 and LC554834, respectively.

Antimicrobial activities of the isolate

The antimicrobial activity of strain SC3-16 was significantly affected by the production medium. For S. aureus ATCC 25923, the inhibition zone in medium 54 (20.0 ± 0.10 mm) was significantly higher than that in ISP 2 (14.2 ± 0.08 mm). Similarly, for K. rhizophila ATCC 9341, medium 54 (27.3 ± 0.15 mm) showed significantly greater inhibition compared to ISP 2 (20.7 ± 0.06 mm) but no antimicrobial activity against B. subtilis ATCC 6633, E. coli ATCC 25922 and C. albicans ATCC 10231. The strain KB-8 exhibited no antimicrobial activity (Table S1 and Figure S1).

Figure 1. Neighbor-joining tree based on the 16S rRNA gene sequences of strains SC3-16 and strain KB-8, showing the relationships among them and related species. The numbers on the branches represent the percentage of bootstrap values from 1,000 replicates. Staphylococcus aureus ATCC 12600ᵀ was used as the outgroup. The bar represents 0.02 substitutions per nucleotide position.

Identification of secondary metabolites by LC/MS

The secondary metabolites in the culture broths cultivated in medium 54 of strains SC3-16 and KB-8 were analyzed by UV and MS spectrometry (Figures 2 and 3).

In strain SC3-16, five secondary metabolites were detected and classified into three groups (Figure 2A). Around a retention time of 6.5 min, the HR-ESIMS spectra of compounds A1 and A2 both exhibited a [M+H]⁺ ion at m/z 565.1569. This ion corresponded to the molecular formula C₂₆H₂₈O₁₄ (calcd for C₂₆H₂₉O₁₄, 565.1552) (Figure 2B). The physicochemical properties of compounds A1 and A2 were consistent with those of isoschaftoside and schaftoside based on comparison with data from the DNP (Figure S2). However, these two compounds are known structural isomers, and discrimination between isoschaftoside and schaftoside was not possible based solely on the MS data.

Around a retention time of 7.5 min, the HR-ESIMS spectra of compound B exhibited a [M+H]⁺ ion at m/z 601.3569. This ion corresponded to the molecular formula C₂₇H₄₈N₆O₉ (calcd for C₂₇H₄₉N₆O₉, 601.3556) (Figure 2B). Compound B was tentatively assigned as nocardamine based on its physicochemical properties and MS/MS fragmentation pattern.  Because nocardamine shares a partial structure with deferoxamine, the MS/MS fragmentation patterns of the two compounds were compared. The MS/MS fragmentation pattern of compound B was in good agreement with that of compound D2, deferoxamine (Figure S3).

Around a retention time of 8.5 min, the HR-ESIMS spectra of compounds C1 and C2 exhibited a [M+H]⁺ ion at m/z 289.1182 and 305.1502, respectively. Because the UV spectra of the two compounds (C1 and C2) were generally similar, these compounds are structural analogs (Figure 2C). These ions corresponded to the molecular formula C₁₅H₁₆N₂O₄ (calcd for C₁₅H₁₇N₂O₄, 289.1183) and C₁₆H₂₀N₂O₄ (calcd for C₁₆H₂₁N₂O₄, 305.1496) (Figure 2B). Compounds C1 and C2 were tentatively assigned as oxotomaymycin- and tomaymycin- related compounds, respectively, by comparison with DNP data. In addition, the MS/MS fragmentation patterns of compounds C1 and C2 were in agreement with those of oxotomaymycin and tomaymycin (Figure S4).

Figure 2. LC-MS analysis of secondary metabolites produced by strain SC3-16.

In strain KB-8, six secondary metabolites were detected and classified into two groups (Figures 3A and 3B). Around a retention time of 6 min, the HR-ESIMS spectra of compounds D1, D2, and D3 exhibited [M+H]⁺ ions at m/z 547.3463, 561.3641, and 575.3757, respectively. These ions corresponded to the molecular formulas C₂₄H₄₆N₆O₈ (calcd for C₂₄H₄₇N₆O₈, 547.3450), C₂₅H₄₈N₆O₈ (calcd for C₂₅H₄₉N₆O₈, 561.3606), and C₂₆H₅₀N₆O₈ (calcd for C₂₆H₅₁N₆O₈, 575.3763), respectively (Figure 3B). Compound D2 was tentatively assigned as deferoxamine by comparison with data from the DNP. In addition, the MS/MS fragmentation pattern of compound D2 was in good agreement with that of deferoxamine (Figure S5). The MS/MS fragmentation patterns of compounds D1 and D3 were similar to those of D2. Therefore, compound D1 was estimated to be a homolog with one fewer methylene unit, whereas compound D3 was estimated to possess one additional methylene unit, based on the MS data.

Around a retention time of 11.5 min, four signals were observed in the UV chromatogram monitored at 254 nm. The UV spectra of three compounds (E1, E3, and E4) were generally similar, whereas that of compound E2 was distinct (Figure 3C). This observation suggests that compounds E1, E3, and E4 are structural analogs. Compound E2 was excluded from further analysis because its MS signal intensity was low and the obtained data were insufficient for reliable molecular formula determination and structural characterization. The HR-ESIMS spectra of compounds E1, E3, and E4 exhibited [M+H]⁺ ions at m/z 593.3932, 607.4080, and 621.4234, respectively. These ions corresponded to the molecular formulas C₃₁H₅₂N₄O₇ (calcd for C₃₁H₅₃N₄O₇, 593.3909), C₃₂H₅₄N₄O₇ (calcd for C₃₂H₅₅N₄O₇, 607.4065), and C₃₃H₅₆N₄O₇ (calcd for C₃₃H₅₇N₄O₇, 621.4222), respectively (Figure 3B). Compounds E3 and E4 were tentatively assigned as rakicidin A- and rakicidin B-related compounds, respectively, by comparison with data from the DNP. In addition, the MS/MS fragmentation patterns of compounds E3 and E4 were in agreement with those reported for rakicidin A and B (Figure S6). The MS/MS fragmentation pattern of compound E1 was similar to that of the rakicidins. A fragment ion at m/z 225 was observed in the MS/MS spectrum of rakicidin A but was absent in that of compound E1. Instead, a fragment ion at m/z 211 was detected in compound E1. This fragment ion corresponds to the molecular formula C₁₅H₃₁. Therefore, compound E1 was estimated to be a homolog of rakicidin A with one fewer methylene unit.

Figure 3. LC-MS analysis of secondary metabolites produced by strain KB-8.

DISCUSSION

Actinomycetes distributed in terrestrial and marine ecosystems are highly proficient in producing a wide range of natural products with diverse antitumor, immunosuppressive, antimicrobial, and antiviral activities (Ngamcharungchit et al., 2023). This study, 2 actinomycetes isolated from soils, strain SC3-16 produced spiral spore chains and the upper and reverse colony color were yellowish white and brilliant yellow and was closely related to S. glycovorans YIM M 10366^T (99.56% similarity).  Strain SC3-16 exhibited cultural characteristics the same as the strain S. glycovorans YIM M 10366^T. However, it showed different characteristics to S. glycovorans YIM M 10366^T (Table 1, Xu et al., 2012). One of the secondary metabolites of strain SC3-16, was tentatively assigned as nocardamine, which shared a partial structure with deferoxamine, a known siderophore (Hossain et al., 1983). It was reported to exhibit a mild antimalarial activity (Lopez et al., 2019) and inhibited the enzyme sortase B.  This enzyme was covalently linked to the peptidoglycan of the bacterial cell wall. The pathogens become resistant to antibiotics, inhibition of sortases may offer a novel strategy against Gram-positive bacterial infections (Lee et al., 2005). In addition, the other detected metabolites showed physicochemical properties consistent with oxotomaymycin- and tomaymycin-related compounds, the antibiotic originally isolated from Streptomyces achromogenes and has been reported to exhibit antimicrobial activity against Gram-positive bacteria (Arima et al., 1972; Hurley et al., 1976). Tomaymycin showed antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Corynebacterium xerosis, Sarcina lutea, and Penicillium chrysogenum and was weakly active against yeasts and fungi.  Additionally, this compound has been reported to have an inhibitory effect on leukemia L1210 cells (Hurley, 1977).  In this study, the strain SC3-16 could exhibit antimicrobial activity against Gram-positive bacteria, K. rhizophila ATCC 9341 and S. aureus ATCC 25923. The detection of metabolites tentatively assigned as tomaymycin- and oxotomaymycin- related compounds in strain SC3-16 suggests that this strain has the potential to produce bioactive secondary metabolites with antibacterial activity.

Strain KB-8 produced a single spore and the upper and reverse colony color were brownish black and brownish gray and was closely related to M. chalcea DSM 43026^T (99.25% similarity) based on 16S rRNA gene similarity (Stackebrandt and Goebel, 1994; Chun et al., 2018). It showed some characteristics different to M. chalcea DSM 43026^T (Songsumanus et al., 2013; Carro et al., 2018; Camacho Pozo et al., 2020) as in Table 1.

Strain KB-8 contained metabolites tentatively assigned as rakicidin-related compounds. Rakicidins isolated from Micromonospora sp. No. R385-2 has been reported and showed cytotoxicity against the M109 cell line (McBrien et al., 1995), while those from Micromonospora sp. Guam1582 exhibited antibacterial activity against Staphylococcus aureus and Enterococcus faecium (Wiebke et al., 2016). Rakicidin F and rakacidin C showed inhibitory activity against bacteria produced from a marine sponge-derived Streptomyces sp. GKU 220 (Kitani et al., 2018). More recently, two new rakicidin derivatives were isolated from M. chalcea FIM-R160609 and evaluated for their cytotoxic activities against human cancer cell lines (Chen et al., 2024). The detection of rakicidin-related compounds in strain KB-8 is therefore consistent with previous reports and highlights this strain as a potential producer of bioactive secondary metabolites. However, the crude extract of strain KB-8 did not exhibit antimicrobial activity against the tested microorganisms. Previous studies have demonstrated that the biological activities of rakicidins vary among individual analogs and target organisms, ranging from cytotoxicity to antibacterial activity against selected bacterial species (McBrien et al., 1995; Wiebke et al., 2016; Kitani et al., 2018; Chen et al., 2024). Therefore, the rakicidin-related metabolites detected in strain KB-8 may not be active against the microorganisms examined in the present study. Moreover, their identities remain tentative because the assignments were based only on LC-ESI-MS, UV spectra, and database comparisons. Therefore, purification of the compounds and structural confirmation using orthogonal methods such as NMR, together with comparison to authentic standards, will be required for definitive identification and more accurate evaluation of their biological activities.

CONCLUSION

The actinomycete strains SC3-16 and KB-8 were genetically related to S. glycovorans and M. chalcea, respectively, based on cultural and biochemical characteristics, as well as 16S rRNA gene sequence analysis. Strain SC3-16 exhibited clear antimicrobial activity against K. rhizophila ATCC 9341 and S. aureus ATCC 25923, both of which are Gram-positive bacteria. Chemical analysis of the culture broths suggested that strain SC3-16 contained compounds tentatively assigned as nocardamine, oxotomaymycin- and tomaymycin-related metabolites, whereas strain KB-8 contained compounds tentatively assigned as deferoxamine- and rakicidin-related metabolites. The observed antimicrobial activity of strain SC3-16 may be associated with the production of tomaymycin-related metabolites.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the Pharmaceutical Research Instrument Center, Faculty of Pharmaceutical Sciences, Chulalongkorn University, for the facilities.

AUTHOR CONTRIBUTIONS

Pawina Kanchanasin: Conceptualization (Equal), Methodology (Lead), Formal Analysis (Equal), Resource (Equal), Writing - Original Draft (Supporting), Investigation (Lead); Wongsakorn Phongsopitanun: Formal Analysis (Equal), Writing  - Review & Editing (Supporting); Nisachon Tedsree: Conceptualization (Equal), Methodology (Equal), Formal Analysis (Lead), Writing - Original Draft (Lead), Writing  - Review & Editing (Lead); Takuji Nakashima: Conceptualization (Lead), Methodology (Lead), Formal Analysis (Equal), Resource (Equal), Writing - Original Draft (Equal), Writing  - Review & Editing (Equal), Investigation (Equal); Somboon Tanasupawat: Conceptualization (Lead), Methodology (Lead), Formal Analysis (Equal), Resource (Equal), Writing - Original Draft (Lead), Writing  - Review & Editing (Lead), Project Administration (Lead).

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

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Supplementary

Figure S1. Antimicrobial activity of strain SC3-16 against K. rhizophila ATCC (A) and S. aureus ATCC 25923 (B) on agar plates.

Figure S2. UV spectra and MS/MS fragment of 2 compounds in group A.

Figure S3. MS/MS fragment of 1 compound in group B.

Figure S4. MS/MS fragment of 2 compounds in group C.

Figure S5. MS/MS fragmentations of 3 compounds in group D.

Figure S6. MS/MS fragmentation of 3 compounds in group E.

Table S1. Antimicrobial activities of the culture extract of strains SC3-16 and KB-8 cultivated in A16, 54 and ISP 2 broth.

Note: -, no activity. The inhibition-zone diameter was included the 8-mm paper disc. The data were expressed as mean values of triplicates ± standard deviation. Different superscript letters indicate significant differences (P < 0.05).