Background: Actinobacteria from freshwater habitats have been explored less than from other habitats in the search for compounds of pharmaceutical value. This study highlighted the abundance of actinobacteria from freshwater sediments of two rivers and one lake, and the isolates were studied for their ability to produce antimicrobial bioactive compounds. Results: 16S rRNA gene sequencing led to the identification of 84 actinobacterial isolates separated into a com- mon genus (Streptomyces) and eight rare genera (Nocardiopsis, Saccharopolyspora, Rhodococcus, Prauserella, Amyco- latopsis, Promicromonospora, Kocuria and Micrococcus). All strains that showed significant inhibition potentials were found against Gram-positive, Gram-negative and yeast pathogens. Further, three biosynthetic genes, polyketide synthases type II (PKS II), nonribosomal peptide synthetases (NRPS) and aminodeoxyisochorismate synthase (phzE), were detected in 38, 71 and 29% of the strains, respectively. Six isolates based on their antimicrobial potentials were selected for the detection and quantification of standard antibiotics using ultra performance liquid chromatography (UPLC–ESI–MS/MS) and volatile organic compounds ( VOCs) using gas chromatography mass spectrometry (GC/MS). Four antibiotics (fluconazole, trimethoprim, ketoconazole and rifampicin) and 35 VOCs were quantified and deter - mined from the methanolic crude extract of six selected Streptomyces strains. Conclusion: Infectious diseases still remain one of the leading causes of death globally and bacterial infections caused millions of deaths annually. Culturable actinobacteria associated with freshwater lake and river sediments has the prospects for the production of bioactive secondary metabolites. Keywords: Actinobacteria, UPLC–ESI–MS/MS, GC–MS, VOCs, PKSII, NRPS, phzE Antibiotic resistance against available drugs is one of Background the primary reasons to seek new and novel drugs such Actinobacteria are diverse group of Gram positive and fil - as antibiotics from a natural source to fight against mul - amentous bacteria that have high guanine–cytosine (GC) tidrug-resistant pathogens . The infections caused by content ranging from 50 to 70 mol% in their genome . globally emerging Gram-negative multidrug-resistant They are considered excellent elaborators of pharmaceu - pathogens are an important challenge. Vancomycin- tical products such as antibiotics and industrial enzymes resistant enterococci (VRE), Methicillin-resistant Staph- and are well known as a prominent source for finding ylococcus aureus (MRSA), extended-spectrum novel biologically active secondary metabolites [2, 3]. β-lactamase (ESBLs) that produce Gram-negative bac- teria, and Klebsiella pneumoniae carbapenemase (KPC) *Correspondence: email@example.com that produces Gram-negative bacteria are few of the Molecular Microbiology and Systematics Laboratory, Department of Biotechnology, Mizoram University, Aizawl, Mizoram 796004, India most significant cases that are gradually increasing in Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zothanpuia et al. Microb Cell Fact (2018) 17:68 Page 2 of 14 ubiquity and virulence . Due to this fact, there is an Methods incessant requirement for the search for new bioac- Sediment sampling tive compounds from unexplored/less explored envi- Samples were collected from two rivers [Tlawng River ronments . As a result, it is important to target such (24°52′N; 92°36′E), Tuirial River (24°21′N 92°53′)] and environments that could be highly potent sources of one lake [Tamdil Lake (23°44′N; 92°57′E)] (Additional novel and bioactive compounds. Among all living organ- file 1: Fig. S8). Samples were randomly collected from isms, the actinobacteria phylum currently represents five different stations of each river and lake at average the most prospective group of microorganisms for the depths of 2–5 m. The labeled samples were placed in ster - discovery of bioactive compounds such as antimicrobi- ile tubes (50 ml), transported to the laboratory and were als, antitumor agents, antiparasitics, anticancer agents processed immediately for the isolation of actinobacteria. and enzymes [7, 8]. It has been shown that 45% of all reported bioactive compounds of microbial origin are Isolation of freshwater actinobacterial strains produced by actinobacteria, more than 70% of which are The collected samples were subjected to physical pre - produced by the largest genus in the phylum Streptomy- treatment (55 °C for 6 min) to hinder the growth of fast- ces . growing bacteria and favor the growth of actinobacteria Since the discovery of the first antibiotic from act - . Actinobacteria were isolated using the serial dilution inobacteria in 1940, actinomycin, the exploration of method and the spread plate technique. The stock solu - these micro-organisms has resulted in the isolation tion of the sample was prepared with 1 ml of water sedi- of thousands of naturally occurring antibiotics to date ment (water + sediment suspension) and 9 ml of sterile . Several novel species of Streptomyces have been distilled water in a test tube, and the solution was mixed reported worldwide as potential natural sources for the for 10 min. The suspension was serially diluted by trans - discovery of naturally occurring antibiotics [11–13]. ferring 1 ml aliquots to a series of test tubes; each con- Actinobacteria have been extensively reported from dif- taining 9 ml of sterile distilled water to prepare the final −1 −2 −3 ferent ecosystems such as soil, freshwater, marine and volumes of 10, 10 and 10 , and the diluted suspen- as endophytes from plants [14, 15] and have been inves- sion was spread over the surface of selected nutritional tigated for their potential contributions to the phar- media. Seven selective media, starch casein agar (SCA), maceutical industry by different researchers [16– 22]. yeast extract-malt extract agar (ISP2), Actinomycetes However, there has been a significant decline in the rate isolation agar (AIA), Streptomyces agar (SA), glycerol– of discovery of novel actinobacteria in recent years [23, asparagine agar (ISP5), tyrosine agar medium (ISP7), and 24]. Therefore, the exploration of potential actinobacte - tap water yeast extract agar (TWYE), were supplemented ria from unexplored habitats is an important approach with nalidixic acid (30 mg/ml) and cyclohexamide to discovering novel antibiotics to meet the current (30 mg/ml) to inhibit the growth of Gram-negative bac- needs [25, 26]. teria and fungi, respectively. The plates were incubated at Northeast India is a large bioprospecting area that was 28 ± 1 °C for 7–30 days, and the colonies were observed identified as the Indo-Burma mega-biodiversity hot - periodically. Pure cultures were obtained after two to spot by Conservation International, and the area is well three successive sub-culturing rounds and transferred known for its rich biodiversity and unexplored biological to fresh isolation media. The cultures were preserved in resources [27, 28]. Bioprospection studies on the actino- their respective slants at 4 °C and 30% glycerol at − 80 °C. bacteria phylum have mainly focused on terrestrial and marine ecosystems, and few have focused on freshwa- Morphological and microscopic characterization ter ecosystems . There are several reports that have of actinobacterial strains examined the diversity of actinobacteria in freshwater Pure cultures of the isolates were identified based on worldwide [29–31], but very few studies have reported their morphological and cultural characteristics follow- on their biosynthetic potential. Therefore, it will be of ing the International Streptomyces Project (ISP) ; great importance to characterize the various biologically the nature of the colony, the color of aerial and substrate active secondary metabolites produced by actinobacteria mycelium, the production of diffusible pigments and the obtained from freshwater sediments. The present study utilization of carbon source were studied . The spore intended to isolate actinobacterial cultures, screen them chain morphologies of the isolates were studied using for in vitro antimicrobial inhibitory activity, detect their a scanning electron microscope (SEM). The mycelium bioactive secondary metabolites and phylogenetically structures were observed using a phase contrast micro- identify the potential antibiotic-producing actinobacteria scope (Olympus), and the organisms were identified from freshwater sediments of selected freshwater lakes according to Bergey’s Manual of Determinative Bacteriol- and rivers in India. ogy 9th edition. Zothanpuia et al. Microb Cell Fact (2018) 17:68 Page 3 of 14 Molecular identification and phylogenetic analysis Antimicrobial assay using crude extract Genomic DNA was isolated and purified using a DNA The actinobacteria isolates that were selected based on extraction kit (Invitrogen) as described in previous stud- antimicrobial screening were grown in ISP1 broth using ies . Ribosomal RNA (16S rRNA) genes were ampli- a 500 ml conical flask at 28 °C in a shaker incubator for fied using universal bacterial primers . The reactions 30 days. The filtrates of the grown cultures were used for and conditions of the PCR were performed exactly as the extraction using methanol 1:1 ratio (v/v). The meth - reported in our previous studies , and sequencing anolic crude extracts of the isolates were prepared in was done commercially at Sci Genome Pvt. Ltd. Cochin, concentrations of 1, 2, 5, 20 mg and 40 mg/ml  with India. Sequences were compared with the reference sterile water and used for antimicrobial activity by the strains of actinobacteria from the NCBI genomic data- agar well diffusion method and disk diffusion assay [39, base using a BLASTn search to determine similarity per- 40]. centages. The strains with highest similarity percentages were retrieved from the EzTaxon database , and mul- Determination of MIC tiple sequence alignment was performed using Clustal W The minimum inhibitory concentration (MIC) of the software packaged in MEGA 6.0 . The evolutionary selected strains was determined using the broth micro models were selected based on the lowest Bayesian infor- dilution technique in a 96-well microtiter plate . The mation criterion (BIC) scores and the highest Akaike methanolic extracts of the strains were dissolved and information criterion (AIC) values using MEGA 6.0 diluted in different concentrations (0.025, 0.05, 0.1, 0.2, . Phylogenetic analysis was performed using MEGA 0.4, 0.8, 1.6 and 3.2 mg/ml) and were used to test the anti- 6 software using the maximum-likelihood method and microbial activity by growing them with bacterial culture using the Tamura Nei parameters algorithm taking E. coli in a 96-well microtiter plate. The ampicillin (1 mg/ml) as the outgroup . The significance of the branching amended bacterial culture was used as the positive con- order was determined by bootstrap analysis of 1000 alter- trol, and the bacterial cultures without treatment were native trees. The obtained nucleotide sequences of the used as the negative control. The plates were incubated at 16S rRNA gene fragments were deposited, and accession 37 °C for 36 h, and absorbance was taken at 700 nm in a numbers were acquired. Trees were viewed and edited UV–VIS spectrophotometer (MultiscanTM GO, Thermo using the FigTree 1.3.1 program. Scientific, MA, USA). EC50 was expressed and calculated as previously described . Screening for antimicrobial activity The antimicrobial activities of the actinobacterial iso - Amplifications of biosynthetic genes (PKS, phzE and NRPS) lates were tested against five bacterial pathogens [Gram- The presence of biosynthetic genes [Polyketide syn - positive bacteria: Staphylococcus aureus MTCC-96, thase type II (PKS II) non-ribosomal peptide synthetase Bacillus subtilis NCIM-2097, and Micrococcus luteus (NRPS) and aminodeoxyisochorismate synthase (phzE)] NCIM-2170; Gram-negative bacteria: Pseudomonas aer- was evaluated using degenerate primers for highly con- uginosa MTCC-2453 and Escherichia coli MTCC-739 served regions encoding enzymes associated with the and yeast: Candida albicans MTCC-3017]. The patho - biosynthesis of polyketides, peptides and phenazine, gens were obtained from the Microbial Type Culture respectively. The primers that were employed and the Collection (MTCC), Chandigarh and National Collec- PCR conditions for the amplification of PKS-II, phzE and tion of Industrial Microorganisms (NCIM), Pune, India. NRPS gene fragments were described in previous studies The crude extracts were prepared by inoculating a sin - [7, 21]. gle purified colony of actinobacteria in Tryptone yeast extract broth medium (ISP medium 1) and incubated at Phylogenetic analysis PKS II, NRPS and phzE gene 28 °C, 150 rpm for 7–20 days. The grown cultures after Biosynthetic gene sequences of PKS type II, NRPS and centrifugation were used to assess antimicrobial activity phzE from the selected seven strains were compared with by the agar well diffusion method . The test patho - the sequences from NCBI database using the BLASTn genic bacteria were spread on a nutrient agar plate, 6 mm search tool  and were aligned by Clustal W software diameter wells were prepared using a sterile cork borer, packaged in MEGA 6.0 . The evolutionary model for 70 µl of the clear supernatant of the actinobacteria was PKS II, NRPS and phzE gene was selected based on low- dispensed into individual wells, and the plates were incu- est BIC value and highest AIC value using MEGA 6.0 bated at 28 ± 2 °C for 24 h. The anti-microbial activity of . Phylogenetic tree was constructed by the maximum the isolates was evaluated as described by Zothanpuia likelihood method using MEGA 6.0 software with Gen- et al. . eral Time Reversible (GTR + G) model for PKS II and Zothanpuia et al. Microb Cell Fact (2018) 17:68 Page 4 of 14 of seven different media employed for isolation, 31 iso - Tamura 3-parameter (T92 + G) for NRPS and phzE gene lates were recovered from the SCA medium, 24 from [38, 39]. AIA, 3 from ISP5, 4 from SA, 1 from TWYE, 3 from ISP7, and 2 from ISP2. These results clearly indicated that SCA Detection of antibiotics using UPLC–ESI–MS/MS was the most suitable medium for the isolation of act- Ultra-performance liquid chromatography (UPLC– inobacteria from freshwater sediments and yielded 45% ESI–MS/MS) was employed to detect antibiotics in the of the total isolates followed by AIA (36%). After 30 days methanolic extracts of the selected strains. Four anti- of incubation, the cultures matured and the actinobacte- biotics (trimethoprim, fluconazole, ketoconazole and ria colony was observed to exhibit white, black, yellow, rifampicin) were selected, and a standard solution was orange, brownish white and pale yellow colors. Most prepared using methanol (Additional file 1: Table S4). of the actinobacterial strains analyzed by field emission The mixed standards were diluted in the ranges from 0.5 gun-scanning electron microscopy (FEG-SEM) showed to 500 ng/ml, and a standard calibration curve was pre- that the aerial mycelia produce spiral spore chains (Addi- pared. Instrumentation and analytical conditions were tional file 1: Fig. S1). performed using the standardized methods as described in our previous paper (Fig. 4) . Molecular characterization and phylogenetic affiliation According to the molecular identification using 16S GC–MS analysis rRNA gene sequencing, 68 actinobacteria isolates were Gas chromatography–mass spectroscopy (GC–MS) classified into six families and nine genera with similar - was used to determine the volatile organic compounds ity percentages ranging from 98 to 100%. The majority of (VOCs) present in the methanolic extracts of the selected the isolates were grouped under Streptomycetaceae fol- strains. For GC–MS, the Clarus 680 GC was used in the lowed by Pseudonocardiaceae, Nocardiopsaceae, Nocar- analysis employed with a fused silica column packed diaceae, Promicromonosporaceae and Micrococcaceae. with Elite-5MS (5% biphenyl 95% dimethylpolysiloxane, Streptomyces was the most dominant genus (n = 49, 72%), 30 m × 0.25 mm ID × 250 μm df), and the components and the other genera included Nocardiopsis (n = 6), Sac- were separated using helium as carrier gas at a constant charopolyspora (n = 4), Rhodococcus (n = 2), Prauserella flow of 1 ml/min. The injector temperature was set to (n = 1), Amycolatopsis (n = 1), Promicromonospora 260 °C during the chromatographic run. A total of 1 μl (n = 1) Kocuria (n = 1) and Micrococcus (n = 3) (Addi- of the extracted sample was injected into the instrument, tional file 1: Table S1). All sequences were deposited in the and the oven temperatures were as follows: 60 °C (2 min); NCBI GenBank database, and accession numbers were followed by 300 °C at a rate of 10 °C/min; and 300 °C given (KM243384, KM405296–KM405298, KM405300– for 6 min. The mass detector conditions were a transfer KM405304, KM405306–KM405307, KM405310, line temperature of 240 °C; an ion source temperature KM406395, KM406397, KM406398, KR703473– of 240 °C, an ionization mode electron impact at 70 eV, KR703475, KR857285, KR857286, KR857288, KR857290– a scan time of 0.2 s and a scan interval of 0.1 s. The frag - KR857296, KR857298–KR857318, KT232313–KT232316, ments from 40 to 600 Da were analyzed. The spectra of KT429605–KT429610, KT429612, KT429614–KT429616, the detected compounds were compared with their mass KY077681, MF536299–MF536302). The length of the spectra from the database of known components stored sequences was used for the construction of phylogenetic in the GC–MS NIST (2008) library. tree ranges from 500 to 1000 bp. The phylogenetic tree was constructed using the maximum-likelihood and Tamura Statistical analysis Nei parameters with the lowest BIC values (12,154.636) All experiments were conducted in triplicate, and the and highest AIC values (10,215.261) (Fig. 1). The topology readings were taken as the mean ± the standard devia- of the tree that was generated differentiated the isolates tion of the mean of three replicates, which were calcu- into 3 major clades. All genera of Streptomyces formed lated using Microsoft Excel XP 2010. One-way analysis major clade I with a bootstrap support value of 96%. Rare of variance (ANOVA) was performed to analyzed signifi - genera, such as Saccharopolyspora, Amycolatopsis and cant difference (P = 0.05) between antimicrobial activities Prauserella, which fell under the family Pseudonocardi- obtained isolates by using SPSS software version 20.0. aceae, were clustered together with Rhodococcus and had a bootstrap value of 87%. Micrococcus and Kocuria under Results the family Micrococcaceae formed a separate clade II with Isolation and distribution of freshwater actinobacteria Promicromonospora and had a bootstrap value of 83%. All A total of 68 isolates of actinobacteria were obtained strain types of Nocardiopsis formed a separate clade III from freshwater sediments; 30 strains from Tamdil Lake, with a bootstrap value of 100%. 19 from Tlawng River, 19 from Tuirial River. From a total Zothanpuia et al. Microb Cell Fact (2018) 17:68 Page 5 of 14 Fig. 1 Maximum likelihood phylogenetic tree constructed using Tamura-Nei model based on 16S rRNA gene sequences of actinobacteria showing the phylogenetic relationship between the isolates with closest type strain sequences. Numbers at branches indicate bootstrap values in 1000 replicates Relative abundance Streptomyces were obtained from all study sites (Fig. 2). The relative abundance of actinobacteria at the genus These results showed that the freshwater actinobacteria level revealed that Streptomyces was the most dominant population varies substantially between lakes and rivers. in Tamdil Lake (n = 20, 40.8%) followed by Tlawng River In comparison to the river ecosystem, the lake ecosystem (n = 18, 36.7%) and Tuirial River (n = 11, 22.4%) from a was observed to be more favorable for actinobacterial total of 49 isolates. However, some rare actinobacteria, growth, as indicated by the enhanced number of isolates such as Promicromonospora sp., Prauserella sp., Rho- obtained with greater diversity. dococcus sp., and Kocuria sp., were obtained from only Tamdil Lake, while Amycolatopsis sp. was found in only Evaluation of antimicrobial activity Tlawng River. Saccharopolyspora sp., Nocardiopsis sp. Initially, all isolates (n = 68) were subjected to prelimi- and Micrococcus sp. were obtained from Tamdil Lake nary screening against five bacterial pathogens (S. aureus, and Tlawng River, whereas several different species of Zothanpuia et al. Microb Cell Fact (2018) 17:68 Page 6 of 14 broad-spectrum antimicrobial activities (Table 1), inhib- iting all six tested pathogens, and these strains were selected as potential candidates for further investigation. Mean (± SD) followed by the same letter(s) in each col- umn are not significantly different at P < 0.05 using Dun- can’s new multiple range test. Antimicrobial activity using methanol crude extract The methanolic crude extracts of the six selected strains that were tested for their antimicrobial activity showed adequate inhibition zones at 20 and 40 mg/ml (Addi- tional file 1: Fig. S1) for all six samples, while all the iso- lates showed no activity in 1 and 2 mg/ml. The agar well diffusion assay showed better results compared to the fil - ter paper disk diffusion assay. MIC of selected strains Fig. 2 Abundance of actinobacterial isolates in three fresh water The methanolic crude extracts of the six strains were systems subjected to antimicrobial activity quantification by determining the MIC of each strain against six patho- gens. Streptomyces sp. DST116 showed maximum activ- ity against M. luteus (EC50 = 0.05103 mg/ml) among B. subtilis, M. luteus, P. aeruginosa, E. coli) and yeast all tested pathogens. Streptomyces cellulosae DST28 (C. albicans). All isolates exhibited antagonistic activ- (EC50 = 0.3371 mg/ml) and Streptomyces flavogriseus ity against at least three of the six tested pathogens. E. DST52 (EC50 = 0.003 mg/ml) also showed the highest coli was found to be the most susceptible pathogen and antimicrobial activities against M. luteus. Streptomyces all isolates (100%) showed activity against it within the sp. DST25 showed the highest activity against B. subti- inhibition range of 7.4 mm to 15.5 mm diameter (Addi- lis (EC50 = 0.009 mg/ml), and Streptomyces albidoflavus tional file 1: Table S2). This was followed by P. aerugi - DST71 showed the highest activity against P. aeruginosa nosa (95.65%), C. albicans (79.71%), B. subtilis (78.26%) (EC50 = 0.05042 mg/ml) (Table 2). and S. aureus (63.76%). Only 26 (37%) isolates showed positive activity against M. luteus. The maximum activity Biosynthetic gene analysis was recorded by Streptomyces flavogriseus strain DST30 Out of the 68 isolates screened for a biosynthetic gene, (18.8 mm) followed by Streptomyces cyaneofuscatus NRPS was detected in 71% (n = 49) of the isolates, PKS strain DST57 (15.95 mm) and Streptomyces albidoflavus type II was detected in 26 isolates (38%), and phzE was DST71 (15.9 mm) against M. luteus, C. albicans and B. detected in 28% (n = 19) of the isolates (Additional file 1: subtilis, respectively. Six strains (Streptomyces sp. DST25, Table S2). A total of 11 isolates (DST45, DST47, DST54, Streptomyces cellulosae DST28, Streptomyces flavogriseus DST56, DST57, DST58, DST74, DST76, DST77, DST99, DST52, Streptomyces albidoflavus DST71, Streptomy - DST101) were found to have all three genes. ces sp. DST116 and Streptomyces sp. DST119) showed Table 1 Antimicrobial activity of selected strains of actinobacteria Strain Antibacterial properties Yeast Biosynthetic genes E. coli P. aeruginosa S. aureus M. luteus B subtilis C. albicans PKS-II NRPS phzE a a a a a a Streptomyces sp. DST25 9.40 ± 0.03 12.0 ± 0.06 9.00 ± 0.06 13.2 ± 0.10 12.5 ± 0.2 12.5 ± 0.20 + + − a bc bc a a bc Streptomyces cellulosae DST28 9.50 ± 0.01 10.0 ± 0.10 10.0 ± 0.10 12.7 ± 0.10 12.5 ± 0.1 11.5 ± 0.50 − − − bc a bde bc bc bde Streptomyces flavogriseus DST52 15.0 ± 0.01 11.5 ± 0.15 8.00 ± 0.05 10.8 ± 0.10 8.4 ± 0.00 15.5 ± 0.05 + + − bde bc bdf bde bde bdfg Streptomyces albidoflavus DST71 13.0 ± 0.30 10.0 ± 0.04 6.60 ± 0.40 6.20 ± 0.20 15.9 ± 0.2 13.8 ± 0.10 − + + a bde a bdfg bdfg a Streptomyces sp. DST116 9.00 ± 0.30 8.50 ± 0.05 9.20 ± 0.25 18.8 ± 0.10 14.4 ± 0.2 12.9 ± 0.05 + + − bdf bdf bde bdfh bdfg bdfh Streptomyces sp. DST119 8.10 ± 0.10 7.00 ± 0.10 8.00 ± 0.10 14.3 ± 0.10 14.2 ± 0.1 13.1 ± 0.10 + + + Mean (± SD) followed by the same letter(s) in each column are not significant different at P < 0.05 using Duncan’s new multiple range test Zothanpuia et al. Microb Cell Fact (2018) 17:68 Page 7 of 14 Table 2 EC of six Streptomyces strains against six pathogens Strain EC50 mg/ml E. coli P. aeuginosa S. aureus B. subtilis M. luteus C. albicans Streptomyces sp. 0.235 0.231 0.110 0.227 0.051 0.069 DST116 Streptomyces cellulosae 1.673 2.353 1.085 0.804 0.331 0.900 DST28 Streptomyces sp. 0.086 0.144 0.070 0.009 0.286 0.070 DST25 Streptomyces sp. 0.260 0.015 0.015 0.278 0.950 1.195 DST119 Streptomyces flavogriseus 0.056 0.267 0.040 0.170 0.003 1.600 DST52 Streptomyces albidoflavus 0.102 0.050 0.138 0.650 0.190 0.075 DST71 Phylogenetic analysis of biosynthetic genes Streptomyces sp. DST52 and Streptomyces sp. DST119 The nucleotide sequences of three biosynthetic genes each formed a separate clade with a bootstrap support (PKS II, NRPS and phzE) showed 82–92% similar- value of 99–100% (Fig. 3a). Similarly the NRPS gene ity with the type strain from NCBI-BLASTn database. sequences of Streptomyces sp. DST116 Streptomyces sp. The transition and transversion bias ratio of PKSII, DST25, Streptomyces sp. DST71 and Streptomyces sp. NRPS and phzE gene was 0.55, 0.33 and 0.17 respec- DST119 formed separate clade with Streptomyces sp. tively whereas the maximum log likelihood for the CAH29-18, Streptomyces albidus NBRC14052, Strepto- substitution computation was − 2765.453, − 501.484 myces cyaneofuscatus DST103, Streptomyces bamensis and − 801.607 respectively. The phylogenetic tree NBRC14727 and Streptomyces sp. BSH50-42 respec- constructed using PKS II sequences revealed that tively with a bootstrap value of 84–89% (Fig. 3b). Streptomyces sp. DST29 formed separate clade with Similarly Streptomyces sp. DST119 and Streptomy- Streptomyces sp. MM48 Streptomyces gobitricini with ces sp. DST71 were clustered separately in phzE gene bootstrap values of 99% while Streptomyces sp. DST116, sequences forming same clade with Streptomyces sp. Fig. 3 Maximum likelihood (ML) phylogenetic tree constructed using amino acid sequences for a PKS type II gene; b NRPS gene and c phzE gene. The scale bar represents the amino acid changes Zothanpuia et al. Microb Cell Fact (2018) 17:68 Page 8 of 14 HB291 and Streptomyces sp. 13–33–9 respectively with the selected isolates showed that rifamycin was present a bootstrap value of 100% (Fig. 3c). in the highest amount in all samples followed by keto- conazole, trimethoprim and fluconazole. Trimethoprim GC–MS analysis was found to be present in higher amounts (39 μg/g) in The methanolic crude extracts of the six selected strains extracts of Streptomyces flavogriseus DST52 compared were investigated to determine their volatile organic to the other samples. Extracts of Streptomyces cellulosae compounds using GC–MS, which revealed thirty-five DST28 contained more fluconazole (17 μg/g), ketocona - VOCs (Additional file 1: Table S3). Fourteen compounds zole (50 μg/g) and rifamycin in (74 μg/g) compared to were detected from the extract of Streptomyces albido- other samples (Table 3 and Figs. 4, 5). MS/MS Spectra of flavus DST71 within the retention time of 15–29 min standard reference analytes i.e. trimethoprim, flucona - (Additional file 1: Fig. S2). Among the compounds, zole, ketoconazole and rifampicin showed as Fig. 5 was hexanal constituted the maximum amount, which used from our earlier publication . accounted for 23.2% of the total volume. Six VOCs, valine, glutaraldehyde, d-leucine, 3,3-dimethyl-4-meth - Discussion ylamino-butan-2-one, pentadecylamine, cyclopropane The bio-resources in freshwater ecosystems are largely and 1-butyl-2-(2-methylpropyl)-, were detected from unexplored, especially in the field of microbiology. Fresh - the extract of Streptomyces sp. DST25, and glutaralde- water ecosystems are becoming a promising area for the hyde was the most abundant followed by an amino acid, isolation of bioactive compounds of pharmaceutical and valine (Additional file 1: Fig. S3). Only one compound biotechnological importance . In the present investi- (di-n-octyl phthalate) was detected in extracts of Strepto- gation, 68 actinobacterial strains were isolated from three myces cellulosae DST28 (Additional file 1: Fig. S4). Seven freshwater systems, and maximum strains were obtained compounds were determined from the extract of Strep- from the lake sediment compared to the sediments from tomyces flavogriseus DST52, of which carbonic acid, 2, the two rivers. This could be because sediments contain - 2, 2-trichloroethyl undec-10-enyl ester alone constituted ing actinobacteria in rivers are continuously removed 49.78% (Additional file 1: Fig. S5). Only 2-methoxy-4,5- by running water and get deposited in different areas diphenyl-6-(2′-phenylethyl)pyrimidine was detected throughout the river. At the same time, lake sediments in the extract of Streptomyces sp. DST116 (Additional are concentrated in particular areas that are not drasti- file 1: Fig. S6), while six compounds were detected in cally affected by running water. Different nutritional the extract of Streptomyces sp. DST119 in which 2-ben- media were employed to achieve maximum diversities zylthio-8-methyl-7-phenylpyrano [2,3-f]benzoxazol- of actinobacteria, since nutrient uptake differed between 6(h)-one constituted the maximum amount (42.66%) organisms. The results indicated that SCA was the best (Additional file 1: Fig. S7). medium for the isolation of the maximum number of act- inobacteria strains, which was in accordance with earlier Detection and quantification of antibiotics using studies [42–45]. the UPLC-MRM method Streptomyces represents the largest genus under the The UPLC–ESI–MS/MS analysis for detection of certain bacteria domain  and the actinobacteria phylum . standard antibiotics of the methanolic crude extracts of The present investigation also showed that Streptomyces Table 3 Antibiotics content of six selected strains (μg/g) Strain no. Trimethoprim Fluconazole Ketoconazole Rifamycin Streptomyces sp. 17.0 8.0 29.0 51.0 DST25 Streptomyces cellulosae 21.0 17.0 50.0 74.0 DST28 Streptomyces flavogriseus 39.0 5.0 28.0 78.0 DST52 Streptomyces albidoflavus 27.0 16.0 35.0 68.0 DST71 Streptomyces sp. 26.0 10.0 49.0 86.0 DST116 Streptomyces sp. 28.0 6.0 32.0 64.0 DST119 Zothanpuia et al. Microb Cell Fact (2018) 17:68 Page 9 of 14 Fig. 4 MRM extracted ion chromatogram of reference analyte: a trimethoprim, b fluconazole, c ketoconazole, d rifampicin was the most dominant genus in freshwater sediments, 20, 21, 40]. The present study reports the antimicrobial which is in accordance with the findings of Wohl and activity of sixty-eight actinobacterial isolates that showed McArthur. ; Deshmukh and Sridhar. ; Ningth- activity against at least three of the tested pathogens. oujam et al. ; Sanasam et al. ; Jami et al.  and Some rare genera of actinobacteria, such as Kocuria, Zothanpuia et al. . There are also several genera of Nocardiopsis, Amycolatopsis, Saccharopolyspora, Rhodo- actinobacteria other than Streptomyces that are called coccus, Prauserella, Promicromonospora and Micrococ- rare genera whose isolation frequencies were lower cus, were also evaluated for their antimicrobial activities compared to Streptomyces . Only 12% of the actino- in the present study. Among them, Saccharopolyspora bacterial isolates that were recovered were rare genera, sp. DST31, Nocardiopsis DST32, Rhodococcus sp. DST38 which included Kocuria, Nocardiopsis, Amycolatopsis, and Nocardiopsis DST95 showed activity against five of Saccharopolyspora, Rhodococcus, Prauserella, Promi- the six tested pathogens. Sibanda et al.  isolated act- cromonospora and Micrococcus, and these genera have inobacteria belonging to Saccharopolyspora and Actino- been previously reported from freshwater habitats [29, synnema from the Tyume River, South Africa, and found 49, 50, 52]. To the best of our knowledge, Amycolatopsis, antibacterial activity against the tested pathogens, which Prauserella and Promicromonospora have not yet been supports the present investigation. Several other rare reported from freshwater sediments and were isolated genera of actinobacteria from freshwater habitats, except for the first time in the present study. However, halophilic for Amycolatopsis, Prauserella and Promicromonospora, actinobacteria, Amycolatopsis halophila , Prauserella have been previously reported for their antimicrobial salsuginis, Prauserella flava, Prauserella aidingensis, and activity, as discussed earlier. Prauserella sediminis , were reported to be isolated Six potential Streptomyces strains that showed a from a saline lake in Xinjiang Province, northwest China, broad spectrum of antimicrobial activities against all while Promicromonospora thailandica has also been tested pathogens were further selected, and the metha- reported from marine sediment . nolic extracts of the strains showed better activity using Actinobacteria are a potential candidate to fight against the agar well diffusion method compared to the fil - multidrug-resistant organisms and are well-known pro- ter paper disk diffusion assay, which was supported by ducers of antimicrobial compounds, and actinobacte- the findings of Gebreyohannes et al. . Recently, we ria have been found in different habitats worldwide [4, recorded the potential microbial activity of Streptomyces Zothanpuia et al. Microb Cell Fact (2018) 17:68 Page 10 of 14 Fig. 5 MS/MS Spectra of reference analytes; a trimethoprim, b fluconazole, c ketoconazole, d rifampicin (as per ) Zothanpuia et al. Microb Cell Fact (2018) 17:68 Page 11 of 14 cyaneofuscatus from freshwater sediments of Tamdil Secondary metabolite profiling based on GC–MS Lake . Broad-spectrum antimicrobial activity was is becoming a foundation in the field of biological sci - also measured in Streptomyces sp. AZ-NIOFD1 from the ences and has been successfully employed to determine Nile River . Various potential strains of Streptomy- VOCs from various samples [20, 21]. The actinobac - ces have also been identified in freshwater habitats [21, teria phylum has been reported as prolific producers 57–59]. The methanolic crude extract of Streptomyces of thousands of bioactive secondary metabolites. The flavogriseus DST52 showed antimicrobial activity with present investigations measured 35 VOCs from six an MIC value of 0.003 mg/ml, which was lower than the methanolic extracts of Streptomyces strains, of which actinobacterial strain SMS_SU21 from a mangrove eco- maximum compounds were retrieved from Strep- system that showed antimicrobial activity with an MIC tomyces albidoflavus DST71. Among the identified value of 0.05 mg/ml . The MIC of Streptomyces sp. compounds from extracts of Streptomyces albidofla - DST119 extract was 0.015 mg/ml against S. aureus, while vus DST71, all except oxirane, 2-butyl-3-methyl-, cis, that of Streptomyces flavogriseus DST52 was 0.056 mg/ azacyclodecan-5-ol, N-(4-chlorobenzenesulfonyl)aze- ml, which was far lower than those of the actinobacterial tidin-3-one and 1,3,5-triazaadamantane detected com- crude extract (1.65 and 1.84 mg/ml) against S. aureus and pounds that have the antimicrobial activity, as reported E. coli, respectively . by earlier researchers [65–73]. In the present study, the PKS-II, phzE and NRPS have been extensively amount of hexanal in the methanol extract of Strepto- described as responsible for the synthesis of a broad myces albidoflavus DST71 was found to be maximum range of structurally diverse secondary metabolites in (23.2%), and this compound was reported to be one actinobacteria [7, 21, 61]. PKS and NRPS are responsible of the constituents of the crude extract of the roots of for the synthesis of bioactive polyketides and peptides, Leonurus sibiricus for its antibacterial, anti-inflamma - while phenazine is an antibiotic that has been reported tory, antioxidant, and antiproliferative properties . to be derived from phzE, and all three genes are all Antimicrobial activities of 2-thiophenecarboxylic acid, renowned for playing vital roles in biological control [7, 5-(1, 1-dimethylethoxy)- and heptanal have also been 21, 62]. The present study also correlated antimicrobial observed in the extracts of Phormidium autumnale and compounds with reference to their biosynthetic genes in Chlorella vulgaris, respectively . The antimicrobial some of the selected strains. Among the selected strains activity of glutaraldehyde was also discussed earlier [66, that showed antimicrobial activity against all tested path- 74] and was also measured in the extract of Streptomy- ogens, the biosynthetic genes PKS-II, phzE and NRPS ces sp. DST25. All compounds extracted from the crude were all detected and amplified with the expected size in extract of Streptomyces sp. DST25 except cyclopropane, Streptomyces sp. DST116 and DST119. However, none 1-butyl-2-(2-methylpropyl)-were previously reported in of the genes were detected in Streptomyces cellulosae antimicrobial studies [71, 75, 76]. The amino acid valine DST28, which clearly showed that the strains that show was also determined as a major compound next to glu- antimicrobial activity do not necessarily contain PKS-II, taraldehyde in the present study, and this compound phzE or NRPS genes, and these findings are in agreement increases the production of the glycopeptide antibi- with previous studies [63, 64]. The biosynthetic genes otic, as reported by Beltrametti et al.  in the actino- for six selected Streptomyces strains were sequenced bacteria strain Nonomuraea sp. Only pyrrolo [1, 2-a] and deposited in NCBI database and Genbank acces- pyrazine-1, 4-dione, hexahydro-3-(2-methylpropyl) sion number were given as MG200184–MG200188 for out of the six compounds detected from the extract of NRPS; MG200189–MG200192 for PKSII; MG200193– Streptomyces sp. DST119 has been previously reported MG200194 for phzE. for its antimicrobial activity [78, 79]. Two of the In the present study, four antibiotics were detected seven compounds, carbonic acid, 2,2,2-trichloroethyl and quantified using the UPLC–ESI–MS/MS method. undec-10-enyl ester and 1-butanol, 2-methyl-acetate This method has been successfully employed to quan - from the extract of Streptomyces flavogriseus DST52 tify bioactive compounds such as antibiotics and phe- were reported earlier for their antimicrobial activities nolic compounds [15, 21]. Antibiotics such as rifamycin [80–82]. Only one compound was determined in the and trimethoprim were detected and quantified from extracts of Streptomyces cellulosae DST28 and Strepto- the crude methanol extract of six Streptomyces strains, myces sp. DST116 with a single peak. Di-n-octyl phtha- which was supported by the findings of Passari et al. . late obtained from Streptomyces cellulosae DST28 was Fluconazole and ketoconazole were also quantified in all reported earlier by various researchers for its antimi- selected extracts of Streptomyces strains, which were also crobial activity [83, 84], while no activity was reported recently reported from Streptomyces cyaneofuscatus iso- for 2-methoxy-4,5-diphenyl-6-(2′-phenylethyl)- lated from Tamdil Lake, Northeast India . pyrimidine obtained from the extract of Streptomyces Zothanpuia et al. Microb Cell Fact (2018) 17:68 Page 12 of 14 References sp. DST116 for its antimicrobial activity. Thus, from 1. Ventura M, Canchaya C, Tauch A, Chandra G, Fitzgerald GF, Chater KF, van this study, we conclude that further investigation of the Sinderen D. Genomics of actinobacteria: tracing the evolutionary history purification of these potent compounds will certainly of an ancient phylum. 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Microbial Cell Factories – Springer Journals
Published: May 5, 2018
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