Indian Journal of Geo Marine Sciences Vol. 46 (10), October 2017, pp. 2081-2087 Molecular characterization of marine pigmented bacteria showing antibacterial activity CH. Ramesh1, 2*, R. Mohanraju1, K. N. Murthy1 & P. Karthick1 1Department of Ocean Studies and Marine Biology, Pondicherry Central University, Port Blair-744102, Andaman & Nicobar Islands, India. 2Andaman and Nicobar Centre for Ocean Science and Technology, ESSO-NIOT, Dollygunj, Port Blair-744103, Andaman and Nicobar Islands, India. [*E-mail: [email protected]] Received 11 March 2016 ; revised 10 May 2016 In the present study 6 pigmented marine bacterial (PMB) strains BNO10, BNO20, BSO14, BNY11, CSP15 and BNB21 from marine sediment and 2 strains BWR18 and BWCY16 from seawater sample were isolated on Zobell Marine agar. These PMB appeared to be in blue, orange, pink, red, dark brownish yellow and yellowish green in colour. Phylogenetic analysis on the basis of small subunit ribosomal RNA (16s rRNA) gene partial sequence homologies of these isolates were identified as Bacillus flexus (BNY11, BWR18), Micrococcus luteus (BWCY16), Photobacterium ganghwense (CSP15), Stenotrophomonas maltophilia (BNO10, BNO20, BSO14), and Vibrio sp. (BNB21). Large and clear zones of inhibition formed by these bacteria against different human pathogenic bacteria were detailed. [Keywords: Pigmented marine bacteria, 16s rRNA analysis, antibacterial activity, Andaman Islands] Introduction however synthetic pigments and their biproducts Bacterial pigments are known to involved in are found to be toxic, carcinogenic, and several processes such as virulence1, cell teratogenic properties. Therefore exploration of signalling communication, photosynthesis and natural pigments from microbes is emerged in protection from radiation2, UV absorption3, recent years. In this context, the present study antibiotic activities4, antioxidant activities5, was focused to isolate pigmented marine bacteria membrane stabilization, and for taxonomic with antibacterial activity for biotechnological discrimination with other microbes6. Bacterial use, and their molecular identification based on pigments have several applications in cosmetics, 16s rRNA gene sequence analysis. food, pharmaceuticals, and textiles. Natural pigment molecules such as bacteriochlorophylls, Materials and Methods carotenoids, flavins, indigoids, melanins, Sample collection and isolation of PMB monascins, phenazines, prodigiosin, quinones, Samples of seawater and sediment were collected and violacein are produced as biproducts by during low tide from Burmanallah coast microbes have wide applications7. Significantly (Lat’ 11⁰33’36.24’N; long’ 92⁰43’49.73’E), the inhibitory activities caused by pigmented South Andaman. Sea water sample was collected bacteria are due to production of tropodithietic in a sterile water sampler bottle as per the 11 acid8, or other pigmented compounds such as procedure detailed by Dutka (1989) . Sediment violacein, prodigiosin, pycocyanin, glaukothalin, sample was collected with a sterile hand corer 12 xanthomonadin, pheomelanin, phenazostatin D, and transferred it into a sterile plastic tube . phenazine, canthaxanthin, astaxanthin, β- Both the samples were transported to laboratory carotene, etc.9, 10. within one hour of sampling for further There are many synthetic colorants being bacteriological studies. Serial dilution was not used in the above mentioned applications, performed for both the samples; only sediment 2082 INDIAN J. MAR. SCI., VOL. 46, NO. 10, OCTOBER 2017 aliquot was prepared by diluting 1 gm of Genomic DNA extraction and PCR amplification sediment in 99 ml of sterile filtered seawater of 16S rRNA gene (w/v)13. 100 µl of both seawater and sediment Genomic DNA of these eight pigmented aliquot were plated on separate petri plates bacterial isolates was extracted using DNA containing Zobell Marine agar using spread plate extraction kit procured from SIGMA. PCR method13. These plates were incubated in amplification of 16S rRNA gene was carried out bacteriological incubator at 35⁰C for 2 to 3days with the bacterial consensus primers 27F [5’- to observe pigmented colonies. Colonies AGA GTT TGA TCC TGG CTC AG-3’] and showing pigmentation were picked with sterile 1492R [5’-GGT TAC CTT GTT ACG ACT T- toothpicks and restreaked thrice to obtain pure 3’] universal forward and reverse 16S rDNA single isolated colonies. Purified isolates were primers16. The PCR of the genomic DNA isolate maintained on agar slants at 4⁰C for further were conducted in a final volume of 25μl. The analysis. reaction mixture contained 10x PCR buffer, 25 mM MgCl2, 10 μM DNTP’s, 1 U of Taq DNA Crude extractions preparation polymerase, 10 pmol of each forward and reverse Pigmented bacterial strains were grown in 30 ml oligonucleotide primers and approximately 20 ng Marine Broth for 72 hours at 28°C in 100 ml of genomic DNA. The amplification profile 14 conical flasks , and extracted with an equal consisted of an initial denaturation at 94 ºC for 3 volume of ethyl acetate for 24 hours on shaker min, followed by 35 cycles at 94 ºC for 1 min, and the organic phases were collected using 55 ºC for 1 min and 72 ºC for 1 min. This was separating funnel and transferred into separate followed by a final extension step of 72 ºC for glass bottles. Solvent extracts were concentrated 5 min. The samples were held at 4 ºC until under Rotary Evaporator (Buchi) to remove further sequence analysis. solvent under pressure at 40⁰C and crude extracts were obtained in powder form. These were Sequencing and phylogenetic analysis of 16S redissolved in 1 ml of the same solvent and used rRNA gene for antibacterial assay. All the eight isolates were sequenced with an automatic sequencer Applied bio system, Foster Preparation of Human pathogenic bacterial City, USA. Raw sequences alignment and quality Inoculums were checked with MEGA 6 software17. The Stringent aseptic conditions were adopted for Black box chimera check (B2C2) software was preparation of pathogenic bacterial inoculums. A used to check chimera formations18, and no loop full of each human pathogenic bacterial anomalies were detected. The partial 16S rRNA cultures (Table 2) were transferred into separate gene sequences of these isolated strains were 5 ml glass tubes containing nutrient broth medium. compared with those available in the public The inoculated broth cultures were then incubated database GenBank. Identification to the species at 35C for 12 hours and used as inoculum level was determined by a 16S rDNA sequence suspensions for antibacterial assay. similarity of 99% with that of the prototype sequence in GenBank. Sequence alignment and Antibacterial assay comparison was performed using the multiple Antibacterial activity assay was determined 19 15 sequence alignment program ClustalW . following Kirby-Bauer well diffusion method . Sequences were edited manually to remove the Using sterile cotton swabs, the prepared inoculum gaps. Neighbor-joining method was followed to suspensions of pathogenic bacteria were seeded construct and to calculate the evolutionary onto the surface of Mueller-Hinton agar media distance of the phylogenetic tree using MEGA6 plates and lids of petri dishes were left ajar for 2 to software20. 5 minutes to remove surface moisture from media plates. On these media plates wells were made Results with cork borers and impregnated with 150 µl of Eight pigmented marine bacterial strains crude extracts. After complete diffusion of comprising of three orange, one yellow, one blue extractions into media, plates were incubated at and one pale pink from sediment sample; one red 35C for 24 hours and the results as inhibition and one yellowish green from seawater sample zones were measured in diameters (mm). were isolated (Fig 1). RAMESH et al.: ANTIBACTERIAL ACTIVITY OF MARINE PIGMENTED BACTERIA 2083 A total length of 1500 base pairs of 16s rRNA Phylogenetic analysis on the basis of gene of these PMB were successfully amplified Neighbor joining tree showed distinct clusters of by PCR and visualized under gel doc system (Fig each genus (Fig 3). Antibacterial activity of 2). 16s rRNA sequence analysis of these eight strains BNO10, BNO20, BSO14, CSP15, and bacterial strains demonstrated higher sequence BWCY16revealed maximum size of clear homology (98-100% similarity) with BLAST inhibition zones of 24 mm, 20 mm, 22 mm, hits. Strains BNO10, BNO20 and BSO14 were 20 mm and 18 mm against Vibrio cholera O139, identified as Stenotrophomonas maltophilia; Streptococcus pneumonia, V. cholera O139, BNY11 and BWR18 as Bacillus flexus; CSP15 Shigella flexneri type 2a, and V. cholera O139 as Photobacterium ganghwense, BNB21 as (Table 2) respectively. While strains BNY11, Vibrio sp. and BWCY16 as Micrococcus luteus. BWR18 and BNB21 did not show antibacterial Nucleotide sequences of the eight strains were activity against tested pathogens. deposited in GenBank and assigned under their respective accession numbers (Table 1). Fig. 1. Different pigments produced by pigmented bacterial strains. 1, BNO10 orange; 2, BNO20 orange; 3, BSO14 orange; 4, BNY11 Darkish Yellow; 5, BWR18 Red; 6, CSP15 Pale pink; 7, BNB21 Blue; 8, BWCY16 Yellowish green. Table 1. Details of pigmented bacterial strains identification based on 16s rRNA sequence analysis and their accession numbers. S. Strain Pigment colour Isolation Species identified GenBank No code source accession number 1. BNO10 Orange Sediment Stenotrophomonasmaltophilia KR091852 2. BNO20 Orange Sediment Stenotrophomonasmaltophilia KR091853 3. BSO14 Orange