In Vitro Evaluation of Antimicrobial Activities and Antibiotic Susceptibility Profiling of Culturable Actinobacteria from Fresh Water Streams

Indian Journal of Experimental Biology Vol. 56, September 2018, pp. 665-673

In vitro evaluation of antimicrobial activities and antibiotic susceptibility profiling of culturable actinobacteria from fresh water streams

Zothanpuia1, Ajit Kumar Passari1, Mukesh Kumar Yadav2 & Bhim Pratap Singh1* 1Molecular Microbiology and Systematics Laboratory, Department of Biotechnology, Aizawl, Mizoram University, Mizoram-796 004, India 2Korea University college of Medicine, Korea University, Seoul, South Korea

Received 01 June 2016; revised 25 April 2017

Actinobacteria are major producers of antibiotics, industrially significant enzymes and many pharmaceutically important biologically active compounds. Twenty two actinobacterial strains were isolated from fresh water stream sediment samples of Murlen National Park, Mizoram, India. The actinobacterial strains were screened against antifungal pathogens (Fusarium oxysporum, Fusarium udum, Fusarium proliferatum, Fusarium oxysporum ciceri and Fusarium graminearum), and antibacterial activities against five bacterial pathogens (Staphylococcus aureus, Pseudomonas aeruginosa, Micrococcus luteus, Bacillus subtilis and Escherichia coli) and a yeast pathogen Candida albicans. All strains showed antibacterial activity against E. coli and F. proliferatum. Based on the results of antagonistic, antibacterial and anti-yeast, two most potent strains Kocuria sp. and Streptomyces intermidus were further evaluated for their antibiotics susceptibility activity against 21 different antibiotics. Kocuria sp. showed resistance to 10 antibiotics whereas Streptomyces intermidus was resistance to 15 antibiotics. Modular genes Polyketide Synthase (PKS II) and Nonribosomal Peptide Synthetase (NRPS) were also detected in these two strains, which might be responsible for the production of secondary metabolites. Two volatile compounds, Di-N-octyl phthalate and 1-Bromo-3, 7-Dimethyloctane were identified from the extract of Streptomyces intermidus BPSWAC29 strain using Gas chromatography Mass spectrometry (GC-MS). This study highlights the promise of discovering novel actinobacteria with antimicrobial activity from underexplored niche biotopes such as fresh water stream sediments.

Keywords: 16S rRNA gene, Antibacterial, Antifungal, 1-bromo-3,7-Dimethyloctane, Fusarium spp., Kocuria sp., Nonribosomal peptide synthetase, Di-N-Octyl phthalate, Polyketide synthase, Streptomyces intermidus

Actinobacteria are a class of filamentous, Gram aquatic systems have been somewhat neglected positive bacteria, having high GC (guanine and compared to terrestrial ecosystems7,8. The profusion cytosine) content in their DNA and are widely of antibiotics produced by terrestrial actinobacteria is distributed in nature including terrestrial and aquatic known but, there has been a drop in unearthing of new habitats1. Actinobacteria has important role in carbon compounds from terrestrial sources9. cycle and in the breakdown of organic matter into Antimicrobial resistance is a grave challenge to easily accessible nutrients. Majority of them are found public health worldwide with the spread of multiple in soil, marine/fresh water and are an important antibiotic resistant strains. Therefore, there is need to source of biotechnologically, commercially and search novel antimicrobials compounds and their 2 therapeutically useful bioactive compounds . They are sources. It has been emphasized that new group of well-known antibiotic producer, producing more than microorganism from untapped habitats can assist as 3,4 two-thirds of all identified antibiotics . Among them, sources of novel pharmaceutical agents10. According genus Streptomyces accounts for the largest antibiotic to the review on actinobacteriological research in producer with more than 70% of all commercially India11, various habitats have been screened for the valuable antibiotics viz. tetracycline, streptomycin, study of actinobacteria, of which soil habitats have 5,6 nystatin, etc. . The study of actinobacteria and their been largely surveyed followed by marine water while potential to produce antimicrobials in general, from fresh water have been neglected and most of which —————— remains unexplored. Exploration of unexplored *Correspondence: Fax: +91 389 2330861 habitats such as fresh water in search of potential E-mail:[email protected] microbes in modern era is in advance interest to 666 INDIAN J EXP BIOL, SEPTEMBER 2018

isolate novel biologically active compounds. However were incubated for around 15-30 days at 28°C. The scientists were becoming more aware of the potential colonies were purified by streaking and sub-culturing assessment of fresh water habitats as a source of on fresh culture media which were then stored and actinobacteria that yield useful metabolic products. maintained at 4C in slants of yeast extract malt All water bodies in Mizoram are fresh water which is extract agar (ISP2) and 30% glycerol at 80C for a promising habitat for the isolation of new and novel further studies. antimicrobials producing actinobacteria12. In this study, we isolated potential actinobacteria Preliminary identification of actinobacterial strains Preliminary identification of the isolates was producing biologically active compounds from the performed as depicted in the International sediments of fresh water, identified using 16S rRNA Streptomyces Project (ISP)15. All the morphological gene sequencing and screened them for the production and biochemical characters were recorded as of antimicrobial agents and evaluated their antibiotics described earlier16. susceptibility profiling. Determination of antimicrobial activity Materials and Methods In vitro antimicrobial activity of the isolates was Sample collection checked against five pathogenic bacterial strains Sediment samples were collected from fresh water [gram positive bacteria viz., Staphylococcus aureus streams inside Murlen National Park, located near (MTCC-96), Bacillus subtilis (NCIM-2097), and Myanmar border in Champhai District, India Micrococcus luteus (NCIM-2170); gram negative (23°37′N, 93°18″E). The park is situated about bacteria viz., Pseudomonas aeruginosa (MTCC-2453) 245 km East of Aizawl (Fig. 1). Several streams flow and Escherichia coli (MTCC-739)] and a yeast through this park providing copious water to its Candida albicans (MTCC-3017). Actinobacterial existing wildlife. Collection was done using sterile isolates were grown in tryptone yeast extract broth screw capped tubes and transported to laboratory and medium (ISP1), incubated at 28C, 150 rpm for stored at 4C until processed for isolation. Samples 7-15 days. The supernatant obtained after were subjected to pre-treatment method by keeping centrifugation of the grown cultures at 8000 rpm for the samples in water bath at 50°C for 6 min in order 5 min was used for determining the antimicrobial to simplify the isolation of actinobacteria and to avoid activity following agar well diffusion method17. The the growth of eubacteria13. testing bacteria were spread on LB modified agar plate, and 6 mm diameter cork borer was used to Isolation of actinobacteria prepare the wells, where 50 µL of the harvested cell Isolation was done using serial dilution method and free supernatant was added. The plates were kept at spread plate technique using starch casein agar 14 37C for 24 h and the antimicrobial activity of the media (SCA) . The culture media were supplemented isolates was recorded by measuring the inhibition with cyclohexamide (30 µg/mL) and nalidixic acid zone around each well. The experiments were done in (30 µg/mL) antibiotics to inhibit the growth of fungi triplicate and mean value was taken. and fast growing bacteria respectively. The plates Determination of antifungal activity The isolates were assessed for their antifungal activity against five pathogenic fungi viz. Fusarium oxysporum (CABI-293942), Fusarium udum (MTCC- 2755), Fusarium proliferatum (MTCC-286), Fusarium oxysporum ciceri (MTCC-2791), Fusarium graminearum (MTCC-1893) by dual culture in vitro assay18. Test was performed by keeping the fungal pathogens at the centre of potato dextrose agar (PDA) plates while actinobacterial isolates were streaked 3 cm away from the fungal strains on opposite sides. The pathogens grown in the absence of actinobacteria

Fig. 1 — Map showing the sample collecting site in Mizoram, is used as control. They were incubated at 28C for India 14 days and percentage of inhibition (%) were ZOTHANPUIA et al.: IN VITRO ANTIMICROBIAL POTENTIAL AND ANTIBIOTIC SUSCEPTIBILITY OF ACTINOBACTERIA 667

calculated using the formula: CT/C × 100 where C is Gas chromatography mass spectroscopy (GC-MS) analysis the control growth of fungal pathogens without The selected isolates were grown in ISP1 broth and actinobacteria and T is test in which fungal pathogens incubate at 28C in a shaker for 7 days, methanol and actinobacteria were grown together in a single extracts of the samples were prepared using rotary plate. All the experiments were performed in triplicate evaporator. The volatile compounds were detected and 24 and mean values was calculated. identified using GC-MS as described previously .

Antibiotic sensitivity profiling Statistical analysis Twenty one standard antibiotics were tested against All experiments were performed in triplicate. One the actinobacterial strains to determine using Muller Way analysis of variance (ANOVA) was used to Hinton agar (MHA) medium. The isolates were analyzed significant differences (P = 0.05) between grown in ISP-1 broth and incubated at 28C, 150 rpm antimicrobial and antifungal activities of different for 10-15 days (depends on the growth). The isolates using SPSS software version 20.0. harvested cells were spread with a sterilized L-shaped spreader on MHA plates and different standard Results antibiotic discs were placed on the spread plates Isolation of actinobacteria which were then incubated at 37C for 24 h. A total of 22 pure actinobacteria strains were Sensitivity of the antibiotics against different recovered from the sediments samples. The pure isolates was observed by measuring zone of isolates were initially identified on the basis of inhibition19. Actinobacterial strains were either morphological characteristics after 2-3 weeks of considered as sensitive (S), intermediate (I) or incubation on SCA medium (Fig. 2 A1 & A2). resistant (R) to an antibiotic20. Majority of the isolated strains were observed with slow growth on solid culture media showing different Molecular identification using 16S rRNA gene amplification range of mycelium colors from yellow, white black, Genomic DNA of the selected strains was isolated brown to orange. Purple and blackish brown pigments using DNA extraction kit (Invitrogen, USA) were formed on the media by certain isolates. The according to the manufacturer’s protocol, DNA SEM results showed the presence of the aerial quantity was checked by measuring optical density mycelia with spiral spore chains (Fig. 2 B & B ), a (OD) at 260/280 nm. 16S rRNA gene was amplified by 1 2 21 typical characteristic of actinobacteria. using universal bacterial primers . PCR conditional 20 were used as described earlier . The obtained PCR Assessment of antimicrobial activity product was purified by quick PCR purification kit Antimicrobial assessment of the actinobacterial (Invitrogen) and sequenced commercially at strains was carried out against 5 pathogenic bacteria SciGenome Pvt. Ltd. Kochin, India. The spore chain morphology was examined using field emission gun scanning electron microscope (FEG-SEM).

Detection and PCR amplifications of biosynthetic gene fragments (PKSII and NRPS): The potential antagonistic isolates were subjected for the amplification of genes for KS domains of Polyketide synthase (PKS), the adenylation domains of non-ribosomal peptide synthetase (NRPS). The following degenerate primers were used for detection of PKS-II: KSF1 5'-TSGCSTGCTTGGAYGCSATC-3' as forward primer and KS1R – 5'TGGAANCCG 22 CCGAABCCTCT-3' as reverse primer . NRPS gene fragments were amplified using degenerate primers: Fig. 2 — Morphological appearance of Kocuria sp. strain A3F 5'-GCSTACSYSATSTACACSTCSGG-3' and BPSWAC27 (A1) and Streptomyces intermidus strain 23 A7R 5'-SASGTCVCCSGTSGCGTAS-3' . The PCR BPSWAC29 (A2), Scanning electron microscope showing their spore chain morphology of strain BPSWAC27 (B ) and amplification of PKSII and NRPS was carried out as 1 20 BPSWAC29 (B2) and antibiotics sensitivity pattern of selected described earlier . strains BPSWAC27 (C1) and BPSWAC29 (C2). 668 INDIAN J EXP BIOL, SEPTEMBER 2018

Table 1 — In vitro antibacterial activity of selected 10 actinobacterial isolates against bacterial pathogens Isolate Antibacterial properties Yeast Code (zone in mm) (mm) E. coli P. aeruginosa S. aureus M. lutues B subtilis C. albicans BPSWAC1 09.10±0.16a 09.23±0.03a Nil Nil 12.42±0.02a Nil BPSWAC4 10.50±0.05bc Nil Nil 12.50±0.16a 8.37±0.02bc Nil BPSWAC9 09.50±0.01a 06.40±0.07bc Nil Nil Nil 12.00±0.09a BPSWAC10 10.70±0.02bc Nil 10.00±0.02a Nil Nil 09.50±0.10bc BPSWAC17 10.00±0.10b 09.65±0.02a Nil Nil 10.00±0.1bde Nil BPSWAC19 10.00±0.10bc 10.63±0.03bde Nil Nil Nil 10.35±0.05bde BPSWAC23 08.62±0.02bde Nil Nil 08.20±0.05bc 10.40±0.05bdfg 11.00±0.01bdfg BPSWAC27 10.06±0.05bc 10.43±0.05bdfg 09.00±0.10bc Nil Nil 10.00±0.10bdfhi BPSWAC29 14.5±0.1bdf 08.20±0.20bdfh 10.17±0.1bd Nil 09.80±0.04bdfh 09.25±0.05bdfhj BPSWAC30 10.62±0.02bc 10.62±0.02bde Nil 08.30±0.05bd Nil Nil [Mean (±SD) followed by the same letter(s) in each column are not significantly different at P <0.05 using Duncan’s new multiple range test] and one yeast pathogen. Out of 22 actinobacteria, 10 isolates (45.4%) inhibited the growth of at least three of the tested six pathogens. Two isolates (BPSWAC27 and BPSWAC29) exhibited positive activity against 4 and 5 pathogens, respectively (Table 1). E. coli was susceptible to all the isolated strains, 19 isolates (86.3%) showed positive activity against P. aeruginosa while no activity of the isolates found against S. aureus and M. luteus. Isolate BPSWAC29 showed highest antibacterial activity against E. coli (14.5 mm), S. aureus (10.1 mm), B. subtilis (9.8 mm) P. aeruginosa (8.2 mm) and C. albicans (9.2 mm). Isolates BPSWAC27 and 29 showed broad spectrum antimicrobial activities and Fig. 3 — Antibacterial activity of four different isolates against P. aeruginosa (A1) and E. coli (A2); C represents control. were selected for further investigations (Fig. 3 A1 & A2). B1. Control growth of F. udum and B2. Growth of pathogen (F. udum) inhibited by BPSWAC27. C1. Control growth of Evaluation of antifungal activity F. proliferatum and C2. Growth of pathogen (F. proliferatum) The antifungal activity of the isolates was inhibited by BPSWAC29. evaluated against five fungal pathogens viz. (67.68%) followed by isolate BPSWAC19 against F. oxysporum, F. udum, F. proliferatum, F. oxy. ciceri F. udum (52%) as shown in Table 2. and F. graminearum. A total of 10 strains, out of 22 isolates showed inhibition of at least two tested Antibiotic susceptibility profiling pathogens. All the isolates inhibited the growth of Isolates BPSWAC27 and BPSWAC 29 which F. proliferatum, with percentage of inhibition ranging showed antimicrobial activity against almost all the from 47-73%. Three actinobacterial strains tested bacterial and fungal pathogens were selected (BPSWAC19, BPSWAC27 and BPSWAC29) and screen their antibiotic sensitivity pattern against exhibited significant growth inhibition against 21 standard antibiotics viz. kanamycin (K5), ampicillin almost all the tested fungal pathogens except (Amp10), streptomycin (S10), erythromycin (E15), F. graminearum with percentage of inhibition ranging ketoconazole (KT50), norfloxacin (NX10), tetracycline from 31-73% whereas all the isolates were susceptible (TE30), gentamycin (Gen10), nystatin (NS50), penicillin to F. graminearum. Isolates BPSWAC27 and G (P2), fluconazole (FLC25), miconazole (MIC30), BPSWAC23 exhibited highest percentage of neomycin (N10), chloramphenicol (C10), polymixin B 50 1 10 inhibition against F. udum (Fig 3 B1 & B2) and (PB ), voriconazole (VRC ), ciprofloxacin (CIP ), 10 10 F. proliferatum (Fig 3 C1 & C2), respectively vancomycin (VA ), trimethoprim (TR ), ZOTHANPUIA et al.: IN VITRO ANTIMICROBIAL POTENTIAL AND ANTIBIOTIC SUSCEPTIBILITY OF ACTINOBACTERIA 669

Table 2 — In vitro antagonistic activity of selected 10 actinobacterial isolates against fungal pathogens Isolate Antifungal properties code (Percentage of inhibition) F. udum F. oxysporum F. graminearum F. proliferatum F. oxy ciceri BPSWAC1 Nil 60.50±0.01a Nil 55.26±0.01a Nil BPSWAC4 47.36±0.03a Nil Nil 50.00±00.00bc Nil BPSWAC9 Nil 47.36±0.12bc Nil 47.36±0.14bde Nil BPSWAC10 52.63±0.00bc Nil Nil 57.89±0.15bdfg Nil BPSWAC17 Nil 47.36±0.03bc Nil 67.12±0.18bdfh Nil BPSWAC19 71.79±0.47bde 31.83±0.3bde Nil 57.89±0.36bfg 61.64±0.02a BPSWAC23 Nil Nil Nil 73.68±0.12bdfhi 57.89±0.36bc BPSWAC27 73.68±0.01bdfg 45.20±0.09bdfg Nil 48.65±0.53bdfhjk 61.64±0.02a BPSWAC29 57.89±0.36bdfh 35.48±0.28bdfh Nil 52.63±0.00bdfhjlm 44.73±0.09bde BPSWAC30 52.63±0.00bc Nil Nil 61.64±0.02bdfhjln 45.20±0.02bdf [Mean (±SD) followed by the same letter(s) in each column are not significantly different at P <0.05 using Duncan’s new multiple range test]

Table 3 — Antibiotic sensitivity profile of actinobacteria isolates against 21 tested standard antibiotics Strain K5 Amp10 S10 E15 KT50 NX10 Te30 Gen10 NS50 FLC20 P2 Mic50 N10 C1O PB50 Cip10 VA10 Tr10 CD10 VRC1 LE5 Kocuria sp. 9(I) 2 (R) 0( R) 6(I) 0(R) 6(I) 6(I) 11(S) (0)R (0)R (0)R (0)R 5.4(I) 14(S) (0)R 10(S) 8.5(I) (0)R 13(S) (0)R 11(S) BPSWAC27

Streptomyce 3(R) 4 (R) 0(R) 0( R) 10(S) 0( R) 0(R) 0(R) (0)R (0)R (0)R 3(R) 3(R) 10(S) (0)R 10(S) 8(I) (0)R 10(S) (0)R 10(s) s intermidus BPSWAC29

[Degree of susceptibility: >10 mm: sensitive; 5.0-9.9 mm: intermediate; 0.0-4.9 mm: resistant. K5, kanamycin; Amp10, ampicillin; S10, streptomycin; E15, erythromycin; KT50, ketoconazole; NX10, norfloxacin; TE30, tetracycline; Gen10, gentamycin; NS50, nystatin; P2, penicillin G; FLC25, fluconazole; MIC30, miconazole; N10, neomycin; C10, chloramphenicol; PB50, polymixin B; VRC1, voriconazole; CIP10, ciprofloxacin; VA10, vancomycin; TR10, trimethoprim; CD10, clindamycin; and LE5, levofloxacin]

Table 4 — Morphological characteristics of actinobacterial isolates followed by identification using 16S rRNA gene sequencing and their accession no. Isolate name Closest relative species in % Similarity Gene bank Colony Aerial Substrate Pigmentation and code BLAST and accession no. Accession nature mycelium mycelium number Kocuria sp. strain Kocuria sp.(JF951262) 98% KM405312 sticky white yellow yellow BPSWAC27 Streptomyces Streptomyces intermedius 100% KM406396 hard white brown brown intermedius strain (KJ571077) BPSWAC29 clindamycin (CD10), levofloxacin (LE5) (Table 3). activity and antibiotic resistance capability were The two isolates showed high sensitivity against identified by the amplification of 16S rRNA gene as chloramphenicol, ciprofloxacin, clindamycin and Kocuria sp. and Streptomycetes intermidus levofloxacin. BPSWAC27 showed resistance activity respectively. Partial 16S rRNA gene sequences against 10 out of 21 antibiotics and showed (820 - 980 bp) of the two strains exhibited high level intermediate activity against 6 antibiotics and of sequence similarity (98 and 100%) with Genebank susceptible to only 5 antibiotics. BPSWAC29 showed sequences. The nucleotide sequences were deposited resistance to 15 antibiotics, susceptible to 5 and to NCBI GenBank and accession number was obtained as KM405312 and KM406396 (Table 4). intermediate to one antibiotic (Fig. 2 C1 & C2). 16S rRNA gene sequences were aligned by BLAST Amplification of 16S rRNA gene and phylogenetic analysis analysis with five type-strains obtained from Ez- The two potent isolates BPSWAC27 and Taxon database. Phylogenetic tree was constructed BPSWAC29 demonstrated good antimicrobial (Fig. 4) based on Maximum Likelihood method with 670 INDIAN J EXP BIOL, SEPTEMBER 2018

Fig. 4 — Maximum Likelihood phylogenetic tree based on Fig. 5 — Amplification of biosynthetic genes (A) PKS-II; and 16S rRNA gene of potential actinobacterial strains. (B) NRPS using degenerate primers

Table 5 — Chemical compounds detected in Streptomyces intermidus BPSWAC29 by GC-MS40-42 Compound Retention time (min) Area (%) Formula Molecular weight (MW) Quality (%) Activity

di-n-octyl phthalate 22.33 71.31 C24H3804 390 85% Antimicrobial

1-bromo-3,7- 25.28 28.69% C10H21Br 220 80% -- dimethyloctane Tamura 3-parameter model (R=1.36) according to Discussion lowest BIC values using Mega 5.05. Gaps were Actinobacteria remain one of the most potential treated by pair wise deletion and the estimated sources of useful bioactive products including Transition/Transversion bias (R) is 1.36. The secondary metabolites with an inconceivable variety topology of the phylogenetic tree shows that two of important biological activities, antimicrobials, and actinobacterial genera along with their type strains valuable different group of enzymes25,26. In the were separated into two different clades (clade I and present study, 22 actinobacterial strains were isolated clade II). The entire Kocuria genus forms a major from fresh water streams inside Murlen National clade I, and all the genus of Streptomyces forms a Park, India. The morphological identification showed separate clade II. The phylogenetic tree demonstrates strains specify characters, which were further verified that BPSWAC27 (Kocuria sp.) was clustered together by using FEG-SEM. The SEM analysis showed that with a type strain of Kocuria palustris and Kocuria the aerial mycelia of most of the actinobacterial assamensis under a bootstrap support value of 99%. strains displayed a long spore chains with smooth BPSWAC29 (Streptomyces intermidus) was very surface, similar character were reported earlier by close to the type strains of other Streptomyces genus 12 with a bootstrap value of 100%. Zothanpuia et al. . Among the 22 isolates, two isolates BPSAWAC27 and BPSWAC29 demonstrated

Detection of PKS-II and NRPS genes a significant antimicrobial activity against both The presence of PKS type II and NRPS genes were bacteria and fungi pathogen. The BPSAWAC27 and evaluated in two selected strains (Kocuria sp. and BPSWAC29 isolates were identified as Kocuria sp. Streptomyces intermidus). The PCR amplification and Streptomyces intermidus respectively using results showed a specific DNA band of 600 bp for 16S rRNA gene sequencing. These results showed that PKS type II and 750 bp for NRPS (Fig. 5). actinobacteria such as Kocuria sp. and Streptomyces

Gas chromatography-mass spectrometry (GC-MS) analysis intermidus isolated from sediments possess potential Methanolic crude extract of the potential strain for the production of antimicrobial compounds 27 BPSWAC29 was further analyzed for their chemical supported by the findings of Baskaran et al . 28 constituents and two volatile compounds were Similarly, Sibanda et al. also described the determined by comparison of their mass spectra antimicrobial potential of freshwater actinomycetes. with the NIST library. The two compounds are Our results showed that fresh water actinobacteria of (1) di-n-octyl phthalate (2) 1-bromo-3, 7-dimethyl- streams at Murlen National Park possess a significant octane (Table 5). The peak area and the quantity of antifungal activity by inhibiting the growth of four out compound present in the sample are directly of five fungal phytopathogens tested, several species proportional to each other. of Streptomyces have earlier been reported to have ZOTHANPUIA et al.: IN VITRO ANTIMICROBIAL POTENTIAL AND ANTIBIOTIC SUSCEPTIBILITY OF ACTINOBACTERIA 671

antifungal activity29-31. These findings are also in Gas chromatography mass spectrometry is one agreement with the results that demonstrated the of the most consistent techniques for determination of antifungal activity of fresh water actinobacteria volatile compounds described by a different scientists isolated from river Nile7. Furthermore, the recent worldwide24,39. The GC-MS chromatogram of findings of Saravanan et al.32 and Zothanpuia et al.12 Streptomyces intermidus BPSWAC29 extract exhibited also demonstrated the antimicrobial activity of the two major peaks with different retention time. Di-n-octyl fresh water isolates of Pudukkottai and Tuichang phthalate (DOP) was detected in maximum amount river, India. Here, the two potent strains were constituted 71.31% of the total compounds present in the confirmed as Kocuria sp. and Streptomyces sample. DOP has already been reported by several 40-42 intermidus using 16S rRNA gene sequencing. And it researchers in plants for its antimicrobial activity . To is evident that the Kocuria assamensis, a novel best of our knowledge, 1-bromo-3, 7-dimethyloctane has actinobacterium was isolated from the river not been reported for its antimicrobial activity. Brahmaputra, India33 and various species of This study revealed that freshwater actinobacteria Streptomyces have been reported earlier from possess enormous potential as a source of antimicrobial freshwater34,35. Therefore, our result of this study compounds which was supported by the isolation of clearly indicates that fresh water streams at Murlen several novel species and other potential actinobacteria from fresh water as previously described32,43-46. National Park habits actinobacteria with potential antimicrobial activity. Acknowledgement The Kocuria sp. and Streptomyces intermidus This work was supported by grants received from strains were evaluated for their antibiotic resistance the Department of Science and Technology (DST), pattern using 21 standard antibiotic discs. Kocuria sp. Government of India, New Delhi as young scientist showed resistance activity against 10 out of project to BPS (No:SERB/F/2501/2013-14). Authors 21 antibiotics and were susceptible to 5 antibiotics, are also thankful to the Department of Biotechnology, while Streptomyces intermidus showed resistance for for supporting establishment of DBT-BIF centre and 15 antibiotics and found susceptible for 5 antibiotics. DBT state Biotech Hub in the Department, which The two isolates were susceptible to tetracycline (85%) hasbeen used for the present study. Authors are thankful followed by gentamicin (50%) and ciprofloxacin to North Eastern Hill University, Shillong, for SEM (45%). All the isolates showed resistance to two types analysis. Authors are also thankful to DST/VIT-SIF Lab, of antibiotics viz. penicillin G and ampicillin (100% SAS, VIT University, Vellore for GC-MS Analysis. each). Gousterova et al.36 also reported the biosynthetic References potential of twenty six actinobacteria and their 1 Jiang Z, Tuo L, Huang D, Osterman IA, Tyurin AP, Liu S, antibiotic sensitivity profiling against 12 antibiotics Lukyanov DA, Sergiev PV, Dontsova OA, Korshun VA, were also described which is in accordance with the Li FN & Sun CH, Diversity, Novelty, and antimicrobial present study. These results according to previous activity of endophytic actinobacteria from mangrove plants reports indicate that these two isolates could be a in Beilun estuary national nature reserve of Guangxi, China. 12,37 Front Microbiol, 9 (2018) 868. http://doi.org/10.3389/fmicb. good candidate for the discovery of antibiotics . 2018.0086. 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