Endophytic actinomycetes associated with Cinnamomum cassia Presl in Hoa Binh province, Vietnam: Distribution, antimicrobial activity and, genetic features.

Endophytic microbes associated with medicinal plants are considered to be potential producers of various bioactive secondary metabolites. The present study investigated the distribution, antimicrobial activity and genetic features of endophytic actinomycetes isolated from the medicinal plant Cinnamomum cassia Presl collected in Hoa Binh province of northern Vietnam. Based on phenotypic characteristics, 111 actinomycetes were isolated from roots, stems and leaves of the host plants by using nine selective media. The isolated actinomycetes were mainly recovered from stems (n = 67; 60.4%), followed by roots (n = 29; 26.1%) and leaves (n = 15; 13.5%). The isolates were accordingly assigned into 5 color categories of aerial mycelium, of which gray is the most dominant (n = 42; 37.8%), followed by white (n = 33; 29.7%), yellow (n = 25; 22,5%), red (n = 8; 7.2%) and green (n = 3; 2.7%). Of the total endophytic actinomycetes tested, 38 strains (occupying 34.2%) showed antimicrobial activity against at least one of nine tested microbes and, among them, 26 actinomycetes (68.4%) revealed anthracycline-like antibiotics production. Analysis of 16S rRNA gene sequences deposited on GenBank (NCBI) of the antibiotic-producing actinomycetes identified 3 distinct genera, including Streptomyces, Microbacterium, and Nocardia, among which Streptomyces genus was the most dominant and represented 25 different species. Further genetic investigation of the antibiotic-producing actinomycetes found that 28 (73.7%) and 11 (28.9%) strains possessed genes encoding polyketide synthase (pks) and nonribosomal peptide synthetase (nrps), respectively. The findings in the present study highlighted endophytic actinomycetes from C. cassia Presl which possessed broad-spectrum bioactivities with the potential for applications in the agricultural and pharmaceutical sectors.

Characterization of Endophytic Streptomyces griseorubens MPT42 and Assessment of Antimicrobial Synergistic Interactions of its Extract and Essential Oil from Host Plant Litsea cubeba.

The present study aimed to evaluate the synergistic effects of the crude ethyl acetate extract (CEAE) from endophytic actinomycete MPT42 and essential oil (EO) of the same host plant Litsea cubeba. The isolate MPT42, exhibiting broad-spectrum antimicrobial activities and harboring all three antibiotic-related biosynthetic genes pks-I, pks-II, and nrps, was identified as Streptomycete griseorubens based on an analysis of the morphology, physiology, and 16S rDNA sequence. Minimum inhibitory concentrations (MICs) and the fractional inhibitory concentration index were used to estimate the synergistic effects of various combined ratios between CEAE or antibiotics (erythromycin, vancomycin) and EO toward 13 microbial strains including pathogens. L. cubeba fruit EO, showing the main chemical constituents of 36.0% citral, 29.6% carveol, and 20.5% limonene, revealed an active-low against tested microbes (MICs ≥ 600 µg/mL). The CEAE of S. griseorubens culture exhibited moderate-strong antimicrobial activities against microbes (MICs = 80-600 µg/mL). Analysis of the mechanism of action of EO on Escherichia coli ATCC 25922 found that bacterial cells were dead after 7 h of the EO treatment at 1 MIC (5.5 mg/mL), where 62% cells were permeabilized after 2 h and 3% of them were filament (length ≥ 6 µm). Combinations of CEAE, erythromycin, or vancomycin with EO led to significant synergistic antimicrobial effects against microbes with 4-16 fold reduction in MIC values when compared to their single use. Interestingly, the vancomycin-EO combinations exhibited a strong synergistic effect against five Gram-negative bacterial species. This could assume that the synergy was possibly due to increasing the cell membrane permeability by the EO acting on the bacterial cells, which allows the uptake and diffusion of antimicrobial substances inside the cell easily. These findings in the present study therefore propose a possible alternative to combat the emergence of multidrug-resistant microbes in veterinary and clinics.

Paenibacillus esterisolvens sp. nov., isolated from soil.

A Gram-stain-positive, aerobic, motile with peritrichous flagella, rod-shaped bacterium, designated CFH S0170T, was isolated from a soil sample collected from Catba island in Ha Long Bay, Hai Phong City, Vietnam. Comparison of the 16S rRNA gene sequences showed that strain CFH S0170T belonged to the genus Paenibacillus and showed closest relationship with Paenibacillus vulneris CCUG 53270T (98.1 % similarity). Phylogenetic analysis demonstrated that the novel candidate formed a coherent branch with P. vulneris CCUG 53270T and Paenibacillus yunnanensis YN2T. Furthermore, the novel strain shared 87.2 % rpoB gene sequence similarity with P. vulneris CCUG 53270T. Growth of strain CFH S0170T occurred at 10-40 °C, pH 6.0-8.0 and with 0-2.0 % (w/v) NaCl. Strain CFH S0170T contained mannose, glucose and rhamnose as the major whole-cell sugars. The cell-wall peptidoglycan contained meso-diaminopimelic acid, glutamic acid, lysine and aspartic acid. The polar lipid profile contained diphosphatidylglycerol, phosphatidylethanolamine, glycolipids and phospholipids. The dominant cellular fatty acids included anteiso-C15 : 0 and C15 : 0. The genomic DNA G+C content was 50.9 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic analysis, strain CFH S0170T is affiliated to the genus Paenibacillus, but could be distinguished from other valid species of this genus. It is concluded that strain CFH S0170T should be considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus esterisolvens sp. nov. is proposed. The type strain is CFH S0170T (=KCTC 33624T=BCRC 80802T).

Endophytic Actinobacteria Associated with Dracaena cochinchinensis Lour.: Isolation, Diversity, and Their Cytotoxic Activities.

Dracaena cochinchinensis Lour. is an ethnomedicinally important plant used in traditional Chinese medicine known as dragon's blood. Excessive utilization of the plant for extraction of dragon's blood had resulted in the destruction of the important niche. During a study to provide a sustainable way of utilizing the resources, the endophytic Actinobacteria associated with the plant were explored for potential utilization of their medicinal properties. Three hundred and four endophytic Actinobacteria belonging to the genera Streptomyces, Nocardiopsis, Brevibacterium, Microbacterium, Tsukamurella, Arthrobacter, Brachybacterium, Nocardia, Rhodococcus, Kocuria, Nocardioides, and Pseudonocardia were isolated from different tissues of D. cochinchinensis Lour. Of these, 17 strains having antimicrobial and anthracyclines-producing activities were further selected for screening of antifungal and cytotoxic activities against two human cancer cell lines, MCF-7 and Hep G2. Ten of these selected endophytic Actinobacteria showed antifungal activities against at least one of the fungal pathogens, of which three strains exhibited cytotoxic activities with IC50-values ranging between 3 and 33 µg·mL-1. Frequencies for the presence of biosynthetic genes, polyketide synthase- (PKS-) I, PKS-II, and nonribosomal peptide synthetase (NRPS) among these 17 selected bioactive Actinobacteria were 29.4%, 70.6%, and 23.5%, respectively. The results indicated that the medicinal plant D. cochinchinensis Lour. is a good niche of biologically important metabolites-producing Actinobacteria.

Agromyces insulae sp. nov., an actinobacterium isolated from a soil sample.

A novel Gram-reaction-positive, non-motile, aerobic bacterium, designated CFH S0483T, was isolated from a soil sample collected from Catba island in Halong Bay, Vietnam. 16S rRNA gene sequence analysis showed that the strain is a member of the genus Agromyces and has highest 16S rRNA gene sequence similarities with Agromyces humatus DSM 16389T (97.3 %) and Agromyces ramosus DSM 43045T (97.1 %), and similarities < 97.0 % with type strains of other species of the genus Agromyces. Strain CFH S0483T was able to grow at 10-37 °C, at pH 7.0-9.0 and tolerated NaCl up to 2.0 % (w/v). The whole-cell sugars were mannose, galactose, glucose and ribose. The isolate contained l-2,4-diaminobutyric acid, d-alanine, d-glutamic acid and glycine in the cell-wall peptidoglycan. Strain CFH S0483T exhibited a menaquinone system with MK-12, and the major fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The genomic DNA G+C content of strain CFH S0483T was 71.6 mol%. Based on the phylogenetic and phenotypic analysis, and low DNA-DNA hybridization values, strain CFH S0483T could not be classified into any recognized species of the genus Agromyces. Strain CFH S0483T is therefore considered to represent a novel species of the genus Agromyces, for which the name Agromyces insulae sp. nov. is proposed. The type strain is CFH S0483T ( = KCTC 39117T = CCTCC AB 2014301T).

Characterization and evaluation of antimicrobial and cytotoxic effects of Streptomyces sp. HUST012 isolated from medicinal plant Dracaena cochinchinensis Lour.

A highly potent secondary metabolite producing endophytic strain, Streptomyces sp. HUST012 was isolated from the stems of the medicinal plant Dracaena cochinchinensis Lour. Strain HUST012 showed antimicrobial and antitumor activities which were significantly much higher than those of dragon's blood extracted from D. cochinchinensis Lour. On further analysis, the strain was found to produce two metabolites, SPE-B11.8 (elucidated to be a novel metabolite (Z)-tridec-7-ene-1,2,13-tricarboxylic acid) and SPE-B5.4 (elucidated as Actinomycin-D). The Minimum Inhibitory Concentration values of SPE-B11.8 against a set of test bacterial organisms (Methicillin-resistant Staphylococcus epidermis ATCC 35984, Methicillin-resistant Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Klebsiella pneumoniae ATCC 13883) ranged between 15.63 and 62.5 µg/ml while that for SPE-B5.4 ranged between 0.04 and 2.24 µg/ml. The compound SPE-B11.8 showed cytotoxic effect at 41.63 and 29.54 µg/ml IC 50-values against Hep G2 and MCF-7, respectively, while the compound SPE-B5.4 exhibited stronger activities against them at 0.23 and 0.18 µg/ml IC 50-values.

Marinactinospora endophytica sp. nov., isolated from a medicinal plant.

A novel endophytic actinobacterium, designated strain YIM 690053(T), was isolated from healthy stems of Salsola ferganica Drob, and was characterized using a polyphasic taxonomic approach. The strain formed well-branched substrate mycelia with no fragmentation, and abundant aerial mycelia that differentiated into long spores. The strain was found to grow at 10-55 °C, pH 6.0-10.0 and in the presence of 0-7 % (w/v) NaCl. The genomic DNA G+C content was determined to be 70.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 690053(T) belongs to the genus Marinactinospora, and it shared the highest 16S rRNA gene sequence similarity with Marinactinospora thermotolerans SCSIO 00652(T) (98.96 %). However, the DNA-DNA relatedness values between the two strains was 37.85 ± 2.77 %. Based on the differences in the molecular and biochemical characteristics from its closest relative, strain YIM 690053(T) is concluded to represent a novel species of the genus Marinactinospora, for which the name Marinactinospora endophytica sp. nov. is proposed. The type strain is YIM 690053(T)(=KCTC 29664(T) = DSM 46799(T)).

Streptomyces bohaiensis sp. nov., a novel actinomycete isolated from Scomberomorus niphonius in the Bohai Sea.

A novel actinomycete strain, designated 11A07(T), was isolated from young Scomberomorus niphonius in the Bohai Sea. Basic local alignment search tool analyses showed that this isolate had the highest 16S rRNA gene sequence similarity of 97.41% with Streptomyces rimosus subsp. paromomycinus DSM 41429(T). Phylogenetic tree revealed that strain 11A07(T) formed a distinct lineage clustered with Streptomyces panacagri Gsoil 519(T), Streptomyces sodiiphilus YIM 80305(T) and Streptomyces albus subsp. albus NRRL B-2365(T) having similarities of 97.30%, 97.10% and 96.83%, respectively. Multilocus sequence analysis further demonstrated that the new isolate was different from the selected representatives of Streptomyces as a separate phylogenetic line. Strain 11A07(T) produced straight or rectiflexibile spore chains with smooth surface, white aerial mycelia and brown diffusible pigments on international streptomyces project 2 medium. Maximum tolerated NaCl concentration for growth was 11.0%. Whole-cell sugars were mannose, ribose, glucose, galactose and xylose. The predominant menaquinones were MK-9(H2), MK-9(H4) and MK-9 (H6). The fatty-acid profile contained iso-C16:0, C18:0 10-methyl (tuberculostearic acid) and anteiso-C17:0 as the major compositions. The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and an unknown phospholipid. The G+C content of the genomic DNA was 71.4 mol%. These morphological, phenotypic and chemotaxonomic properties showed that strain 11A07(T) could be readily distinguished from the most closely related members of the genus Streptomyces. Thus, based on the polyphasic taxonomic data, strain 11A07(T) (=JCM 19630(T)=CCTCC AA 2013020(T)=KCTC 29263(T)) represents a novel species within the genus Streptomyces, for which the name Streptomyces bohaiensis sp. nov. is proposed.

Vitellibacter nionensis sp. nov., isolated from a shallow water hydrothermal vent.

A novel, Gram-stain-negative, non-motile, rod-shaped yellow bacterium, designated VBW088(T) was isolated from a shallow water hydrothermal vent in Espalamaca in the Azores. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain VBW088(T) clustered with three type strains of species of the genus Vitellibacter and exhibited a sequence similarity of 97.3 % with Vitellibacter soesokkakensis RSSK-12(T). However, strain VBW088(T) and V. soesokkakensis RSSK-12(T) exhibited low DNA-DNA relatedness (12.7±3.5 %). Strain VBW088(T) was positive for catalase and oxidase. Growth occurred at 10-37 °C, with the optimum at 30 °C, and at pH 6.0-8.0 (optimum pH 6.0) and in up to 5 % (w/v) NaCl with optimum growth at 1-2 % (w/v) NaCl. The major fatty acids (>10 %) were iso-C15 : 0 (33.5 %) and iso-C17 : 0 3-OH (32.0 %). The polar lipids detected in strain VBW088(T) consisted of phosphatidylethanolamine, one unidentified aminolipid and three unidentified phospholipids. The DNA G+C content of strain VBW088(T) was 36.7 mol%. On the basis of phylogenetic inference, DNA-DNA relatedness, chemotaxonomic analysis and physiological data, the isolate represents a novel species of the genus Vitellibacter, for which the name Vitellibacter nionensis sp. nov. is proposed, with the type strain as VBW088(T) ( = KCTC 32420(T) = MCC 2354(T)).

Nioella nitratireducens gen. nov., sp. nov., a novel member of the family Rhodobacteraceae isolated from Azorean Island.

A novel Gram-negative, non-spore forming, rod-shaped aerobic bacterium, designated SSW136(T), was isolated from a surface seawater sample collected at Espalamaca (in Faial Island), Azores. Growth was found to occur from 10 to 37 °C, pH 6.0-8.0, and with 2-11 % of NaCl. 16S rRNA gene sequence indicated that the strain SSW136(T) belongs to the family Rhodobacteraceae. Strain SSW136(T) exhibited 96.3, 95.9, 95.7 and 95.5 sequence similarity to the type strains Oceanicola litoreus M-M22(T), Roseovarius aestuarii SMK-122(T), Marivita geojedonensis DPG-138(T), and Pseudoruegeria aquimaris SW-255(T) respectively. Neighbour-joining and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences revealed that strain SSW136(T) was affiliated to the family Rhodobacteraceae and formed a separate branch. The G+C content was 63.5 mol%. The major respiratory quinone was found to be Q-10. The polar lipids of strain SSW136(T) consisted of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminolipids and three unidentified phospholipids. The major fatty acids were C18:1 ω7c (46.5 %), Cyclo-C19:0 ω8c (16.0 %) and C16:0 (12.8 %). On the basis of the morphological, genotypic, chemotaxonomic characteristics and low DNA-DNA relatedness, strain SSW136(T) is proposed to represent a novel genus and novel species, Nioella nitratireducens gen. nov., sp. nov., in the family Rhodobacteraceae. The type strain is SSW136(T) (=KCTC 32417(T) = NCIM 5499(T)).

Streptomyces canchipurensis sp. nov., isolated from a limestone habitat.

Hundung Limestone habitat, Manipur, India is an unexplored site for microbial diversity studies. Using polyphasic taxonomy, a Streptomyces strain, MBRL 172(T), has been characterized. The strain was found to show highest 16S rRNA gene sequence similarity with Streptomyces coeruleofuscus NBRC 12757(T) (99.2 %). The DNA relatedness between MBRL 172(T) and S. coeruleofuscus NBRC 12757(T), and between MBRL 172(T) and Streptomyces nogalater NBRC 13445(T), were 36.8 ± 4.4 and 52.5 ± 2.7 %, respectively. Strain MBRL 172(T) was found to contain LL-diaminopimelic acid as the diagnostic diamino acid and glucose, mannose and xylose as the major sugars in whole cell hydrolysates. The polar lipids in the cell membrane were identified as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositolmannoside. The predominant menaquinones detected were MK-9(H6) and MK-9(H8). The cellular fatty acids identified were mainly saturated fatty acids: anteiso-C15:0, iso-C16:0 and iso-C15:0. Based on differences in the biochemical and molecular characteristics from its closest relatives, the strain can be proposed to represent a novel taxon in the genus Streptomyces, for which the name Streptomyces canchipurensis is proposed, with the type strain MBRL 172(T) (=JCM 17575(T) = KCTC 29105(T)).

Bacillus tianshenii sp. nov., isolated from a marine sediment sample.

A novel Gram-stain-positive, motile, catalase- and oxidase-positive, aerobic, endospore-forming, peritrichous, rod-shaped bacterium, designated YIM M13235(T), was isolated from a marine sediment sample collected from the South China Sea. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM M13235(T) belonged to the genus Bacillus. The strain grew optimally at 30 °C, pH 7.0 and in the presence of 2-4% (w/v) NaCl. meso-Diaminopimelic acid was present in the cell-wall peptidoglycan. Strain YIM M13235(T) exhibited a menaquinone system with MK-7, and the major polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, four unknown phospholipids and one unknown glycolipid. The major fatty acids (>5%) were iso-C(15 : 0), anteiso-C(15 : 0), anteiso-C(17 : 0), iso-C(17 : 1)ω10c and summed feature 4 (anteiso-C(17 : 1) and/or iso-C(17 : 1)). The genomic DNA G+C content was 42.1 mol%. The DNA-DNA relatedness values between strain YIM M13235(T) and its close relatives (16S rRNA gene sequence similarities >97%) including Bacillus halmapalus DSM 8723(T), Bacillus horikoshii DSM 8719(T) and Bacillus zhanjiangensis JSM 099021(T) were 41%, 44% and 44%, respectively. On the basis of genotypic, phenotypic and DNA-DNA relatedness data, it is apparent that strain YIM M13235(T) represents a novel species of the genus Bacillus, for which the name Bacillus tianshenii sp. nov. is proposed. The type strain is YIM M13235(T) ( = DSM 25879(T) = KCTC 33044(T)).

Roseovarius azorensis sp. nov., isolated from seawater at Espalamaca, Azores.

A Gram-negative, motile, non-spore forming, rod shaped aerobic bacterium, designated strain SSW084(T), was isolated from a surface seawater sample collected at Espalamaca (38°33'N; 28°39'W), Azores. Growth was found to occur from 15 to 40 °C (optimum 30 °C), at pH 7.0-9.0 (optimum pH 7.0) and with 25-100 % seawater or 0.5-7.0 % NaCl in the presence of Mg(2+) and Ca(2+); no growth was found with NaCl alone. Colonies on seawater nutrient agar were observed to be punctiform, white, convex, circular, smooth, and translucent. Strain SSW084(T) did not grow on Zobell marine agar and tryptic soy agar even when seawater supplemented. The major respiratory quinone was found to be Q-10 and the G + C content was determined to be 61.9 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain SSW084(T) belongs to the genus Roseovarius and that its closest neighbours are Roseovarius tolerans EL-172(T), Roseovarius mucosus DFL-24(T) and Roseovarius lutimaris 112(T) with 95.7, 95.4 and 95.3 % sequence similarity respectively. The remaining species of Roseovarius showed <95 % similarity. The polar lipids of strain SSW084(T) were determined to be phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, an unidentified lipid and one unidentified aminolipid. The major fatty acids identified were identified as C18:1 ω7c (52.5 %) and C16:0 (13.8 %). On the basis of phenotypic, molecular and chemotaxonomic characteristics, strain SSW084(T) is considered to represent a novel species of the genus Roseovarius, for which Roseovarius azorensis sp. nov is proposed. The type strain is SSW084(T) (=KCTC 32421(T) = MTCC 11812(T)).