Antibacterial Activity of Marine Bioluminescent Bacteria

Indian Journal of Geo Marine Sciences Vol. 46 (10), October 2017, pp. 2063-2074

Antibacterial activity of marine bioluminescent bacteria

CH. Ramesh1,2* & R. Mohanraju1

1Department of Ocean Studies and Marine Biology, Pondicherry Central University, Brookshabad Campus, Port Blair-744102, Andaman & Nicobar Islands, India. 2Present address: Andaman Nicobar Center for Ocean Science and Technology (ANCOST), National Institute of Ocean Technology (NIOT), Port Blair-744103, Andaman & Nicobar Islands, India.

*[ E-mail : [email protected]]

Received 12 January 2017 ; revised 30 March 2017

The present study was undertaken to investigate the antibacterial activities of marine luminous bacteria against 17 human pathogenic bacteria. Fourteen strains isolated from coastal marine samples of South Andaman, India, have exhibited antibacterial activity against at least one human pathogenic bacteria. 16s rRNA gene sequence analysis identified them as Vibrio harveyi, V. campbellii, V. owensii, V. rotiferianus, V. alginolyticus, Photobacterium damselae and P. leiognathi. P. leiognathi strain SQEG2 and V. campbellii strain BSECU1 showed maximum inhibition zones of 25 and 24 mm against Escherichia coli and Bacillus subtilis respectively. Crude ethyl acetate extracts of all the strains were obtained and analysed by Gas Chromatography–Mass Spectrometry. Antibacterial compounds identified were indole, phenol, 2,4-bis(1,1-dimethylethyl)- ,dibutyl phthalate, and 1,2-Benzenedicarboxylic acid, butyl octyl ester.

[Keywords: antibacterial activity, 16s rRNA gene sequence, bioluminescent bacteria, GC-MS, Andaman Islands]

Introduction diverse antibiotics by terrestrial luminous Marine heterotrophic bacteria attached to bacteria are well known6. While, marine different marine samples are directly or luminous bacteria belonging to genera: indirectly known to be involved in chemical Allivibrio, Vibrio, and Photobacterium are not signalling with prokaryotic and eukarytotic well understood. organisms and in environmental balance1, and Marine luminous bacteria are attached on antagonistic activities2. In the past decade surface of several marine flora and faunal emergence of multidrug resistant bacteria have components7, and play a more important aroused a global issue that led research to focus ecological role in degradation of aggregates and on discovery of effective novel compounds from transportation of nutrients to food web. Since marine bacteria. Marine Gram-negative bacteria some of the luminous bacteria are pathogens in belonging to the order γ-proteobacteria, family aquaculture industry8, many studies have been Vibrionaceae are widespread in marine, estuary, undertaken to eradicate luminous bacteria and freshwater milieus. through emphasised practices such as addition of Some of the Vibrionaceae members are seaweed extracts in shrimp feed9,10, intracellular well known to produce potential novel secondary signal antagonists11, synthesized chemical metabolites with biological activities3. Recent compounds like furanones12, green water13, using review has highlighted the importance of poly-β-hydroxybutyrate (PHB) degrading production of antimicrobial secondary bacteria (Acidovorax spp., Acinetobacter spp. metabolites in certain Vibrio, Photobacterium and Ochrobactrum spp.)14, probiotics15,16, and Photorhabdus species4. About 93 antagonistic bacteria (Alteromonas sp.)17, and compounds have been isolated from members of bacteriophages18.Very few studies have Vibrionaceae family4. Nair and Simidu (1987) investigated the industrial applications of reported that Vibrios obtained from luminous bacteria for production of useful phytoplankton and zooplankton samples were enzymes such as bacteriocins, proteinase (Vibrio one among the dominant group of antibacterial harveyi), L-asparaginase (V. harveyi, V. fischeri isolates accounting for 16.3%5. Production of and Photobacterium leiognathi)19, 2064 INDIAN J. MAR. SCI., VOL. 46, NO. 10, OCTOBER 2017 exopolysaccharides, quorum sensing molecules4, sediment/99ml of sterile seawater) samples were and lux genes7. spread evenly on Luminescent agar (LA)25, with The ability to produce antimicrobial addition of 4% agar as suggested by Dunlap and compounds by luminous bacteria is not well Urbanczyk (2013) to prevent the growth of other understood. As a new insight, this study has been non-luminescent bacterial contaminants, using L- undertaken to examine the antibacterial glass spreader following the spread plate properties of luminous bacterial strains, method23. Swab samples detailed above were representing two different phylogenetic groups spread evenly on to surface of freshly prepared Vibrio and Photobacterium. LA media plates. These plates were incubated at 35⁰C for 24 hours and luminous colony counts Materials and Methods were made following incubation. Collection of samples After incubation, the petri plates were During the year 2012, fourteen active luminous examined in a dark room after straining the eyes bacterial isolates were isolated from different for about 10 to 15 minutes for luminous bacterial marine samples collected by handpick method colonies. High intense luminous colonies were during receding tide from different stations identified visually and picked up with sterile (Table 1). Sediment sample was collected using a tooth picks by adjusting red light26, and streaked sterile hand corer and transferred into a sterile onto fresh plates23. This was repeated twice or 20 plastic tube . Coastal surface seawater sample thrice to obtain pure isolated single colonies. was collected using a water sampler following Intense luminous pure isolates were obtained and 21 the procedure as detailed by Dutka (1989) . assigned with strain codes and stored as agar Zooplankton sample was collected using a slants on LA and as agar stabs in Marine agar zooplankton net as detailed by Agrawal and maintained at 4⁰C; and 10% glycerol stocks were 22 Gopal (2013) . For sediment sample, sediment maintained at -20⁰C. Further subcultures were aliquot was prepared by diluting 1 gm of 23 prepared from these stock cultures for further sediment in 9 ml of sterile seawater (w/v) . studies. Swab sampling technique was employed to obtain swab samples from surfaces of blue Genomic DNA isolation and PCR amplification starfish Linckia laevigata and sea anemone of 16s rRNA gene Cryptodendrum sp. in the field after washing Genomic DNA of fourteen cultures grown in their surfaces gently with sterile seawater2, 24. Marine broth at 35⁰C overnight were isolated Arabian red shrimp Aristeus alcocki was following CTAB method as described by collected from the Junglighat fish landing centre. Nishiguchi et al. (2002)27. Molecular analysis on Live specimens of both cuttlefish Sepia the basis of PCR amplification of 16s rRNA gene officinalis and leiognathid fish Leiognathus of these 14 strains was carried out according to brevirostris were collected from a fisherman. the method as described by Mohandas et al. The bottom part of a stilt root of the mangrove (2012) and sequenced28 (Table 1). Rhizophora apiculata was cut with a sterile knife and transferred to a sterile sample container. Extraction of crude metabolites from luminous Stone fish Synanceia verrucosa was collected bacteria using a hand net with caution. These were then All the fourteen strains were grown in 100 ml transferred into sterile test tubes. Other samples conical flasks containing30 ml Marine Broth such as seaweed Amphiroa anceps, seagrass incubated in a shaker at 220 rpm with continuous Halophila ovata, sponge Rhabdastrella agitation for 3 days at 28°C and examined for 29 globostellata, polychaete Neries sp., and sea growth . These were then centrifuged at 10000 squirt Clavinella moluccensis were handpicked rpm for 15 minutes, supernatants were using nitrile gloves and transferred to sterile transferred to fresh flasks, to which an equal plastic ziplock bags. All the samples were gently volume of ethyl acetate (EtOAc) was added. rinsed with sterile seawater to remove debris and These flasks were then placed on a shaker at bounded epibiotic organisms. Using sterile 220rpm with continuous agitation for an hour to cotton buds, swabs were obtained from surfaces obtain a homogenous mixture. Organic phases of the samples. were collected using a separating funnel and the solvent was evaporated under reduced pressure at Isolation of luminous bacteria 37° C in a Rota Evaporator to obtain pellets. To freshly prepared LA plates 100µl of seawater Pellets weighing 0.70µg were again redissolved (undiluted) and sediment aliquot (1gm of in 1 ml of EtOAc solvent for bioactivity assay. RAMESH & MOHANRAJU: ANTIBACTERIAL ACTIVITY OF MARINE BIOLUMINESCENT BACTERIA 2065

Human bacterial pathogens were compared with the spectrums available in Seventeen human pathogenic bacterial cultures the National Institute Standard and Technology of Bacillus subtilis MTCC 2616, Enterococcus (NIST) database. Chromatogram peak intensity faecalis MTCC 439, Escherichia coli MTCC heights were adjusted with ESI Compass Data 443, Klebsiella pneumonia MTCC 109, Analysis Version 4.0. Micrococcus luteus MTCC 106, Salmonella enterica serovar Typhimurium B12101, Results Salmonella enterica serovar Typhi C6953, Shiga Results showed that all these 14 strains exhibited toxin producing E. coli serotype O157:H7 VT3, significant antibacterial activity against at least a Shigella boydii type 1 NK2379, Shigella single pathogenic bacterium (Fig. 1-4). Negative dysenteriae type 5 NK2440,Shigella flexneri control did not show any inhibition zone. While type 2a 503004, Shigella sonnei NK4010, inhibition zones against pathogenic bacteria Staphylococcus aureus MTCC 96, Streptococcus ranged from 6 to 25mmwith variation in crude pneumonia MTCC 655, Vibrio cholera MTCC extract volumes (Table 2). Among all the crude 3905, Vibrio fluvialis IDH 02036 and Vibrio extracts, strains SQEG2 and BSECU1 showed parahaemolyticus serovar O3: K6 K5030 were maximum inhibition zones of 25 and 24mm used as test microorganisms to screen against E. coli and B. subtilis respectively. antibacterial activities of these fourteen strains. Significantly, when compared with other strains, STF3, CHSE4 and BSE4 showed inhibition Preparation of inoculums zones against most of the pathogens. Stringent aseptic conditions were adopted to GC-MS data showed the presence of prepare inoculums of the above mentioned similar compounds in most of these strains pathogenic bacterial cultures23. One loop full of (Table 3). Chromatograms of each strain were each human pathogenic bacterial culture was found to be similar with mixtures of compounds, inoculated into separate 5 ml glass tube containing with at least one or two major compounds (Table nutrient broth medium and incubated at 35 C for 3; Fig. 5-18). Compounds such as Phenol, 2,4- overnight (12 to 18 hours) and used as inoculums bis(1,1-dimethylethyl)-, 1,2-Benzenedicarboxylic suspension for antibacterial assay. acid, butyl octyl ester, and Benzoic acid, 4- ethoxy-, and ethyl ester were observed in the Antibacterial assay present study. Compositions of other compounds Antibacterial activity assay was performed were not determined due to their low level. following the ‗Kirby-Bauer well diffusion method30. Muller-Hinton agar plates were seeded Discussion with the inoculum suspensions of pathogenic Marine bacteria belonging to Vibrionaceae bacterial cultures by using spread plate technique family are widespread in diverse marine niches and wells were plugged with sterile cork borers. as free living, commensal, pathogenic, or as Different volumes of 10, 50, 100, 150 and 200 µl symbiotic associations, and can be isolated from of EtOAc extracts of all the fourteen strains were several marine flora, fauna and abiotic surfaces31. impregnated into these wells. EtOAc solvent was These biogeographically diverse bacteria are able used as negative control. After complete diffusion to produce potential secondary metabolites to of extracts, plates were incubated at 35C for 24 protect the host organism, defense against hours and clear zones of inhibition formed around pathogenic microorganisms, and for interspecies wells (including well size) were measured in signalling purposes. Observations on the diameters (mm). antimicrobial activities by nonluminous vibrios and Photobacterium species have been reported. GC-MS analysis and identification of Ritchie (2006) observed the coral Acropora antibacterial metabolites palmata to harbour different resident species of Crude extracts of active strains were analysed Photobacterium in their surface mucus, which with Gas Chromatography–Mass Spectrometry exhibited antimicrobial activity32. Both (GC-MS) to confirm the presence of compounds Photobacterium and Vibrio species utilizes in them. GC-MS analysis was performed with a nutrients such as dimethylsulfoniopropionate Thermo GC – Trace Ultra Ver: 5.0, Thermo MS (DMSP), dimethyl sulfide (DMS), and acrylic DSQ II, TR 5 – MS capillary standard non – acid from corals and renders in shaping bacterial polar column, injection volume 1 micro liter, community33. carrier gas Helium, with a flow rate of 1.0 µl/min. Spectrums of the crude extracts obtained 2066 INDIAN J. MAR. SCI., VOL. 46, NO. 10, OCTOBER 2017

Fig. 2. Antibacterial activities of strain JPL2, CHSE4, Fig. 1. Antibacterial activities of strain STF3, SSBR3, BSECU1 and BSE1. SQEG2, PEVI1 and LB2.

Fig. 3. Antibacterial activities of strain BSECU1 and Fig. 4. Antibacterial activities of strain BSE5, BSE4 and CHSE2. AMPHI2. RAMESH & MOHANRAJU: ANTIBACTERIAL ACTIVITY OF MARINE BIOLUMINESCENT BACTERIA 2067

Table 1. Accession numbers of 21 luminous bacterial strains.

S. Strain GenBank Isolation source Identified Sequence No sequence species identity accession numbers 1. STF3 KR811229 Stonefish: Synanceia verrucosa V. harveyi 98% 2. SSBR3 KR811225 Blue starfish: Linckia laevigata V. owensii 99% 3. SQEG2 KR911956 Cuttlefish: Sepia officinalis Photobacterium 99% leiognathi 4. BSE1 KT354591 Sea Anemone: Cryptodendrum sp. V. campbellii 99% 5. BSE4 KT354589 Sea Anemone: Cryptodendrum sp. V. rotiferianus 99%

6. BSE5 KR811222 Sea Anemone: Cryptodendrum sp. V. campbellii 99% 7. BSECU1 KT354590 Sea Water V. campbellii 99% 8. BSECU3 KR811224 Sea Water V. campbellii 99% 9. JPL2 KR811220 Zooplankton sample V. rotiferianus 97%

10. LB2 KR811216 Shortnose ponyfish: Leiognathus brevirostris P. damselae 97% 11. AMPHI2 KR811219 Coralline red algae: Amphiroa anceps V. alginolyticus 99%

12. PEVI1 KT354592 Arabian red shrimp: Aristeus alcocki V. campbellii 99% 13. CHSE2 KR811223 Seagrass: Halophila ovata V. owensii 99%

14. CHSE4 KR811231 Mangrove: Rhizophora mucronata V. harveyi 99%

Gram et al. (2010) have reported the alginolyticus, P. damselae and P. leiognathi antibacterial activity of Vibrio and showed antibacterial activity against at least Photobacterium strains isolated from ocean one pathogenic bacteria with inhibition zone surface waters and surface swabs of different ranged between 7 to 25 mm. marine organisms2. Other studies also showed Different antimicrobial compounds the antimicrobial activities by nonluminous produced by vibrios and Photobacterium Vibrionaceae members such as Vibrio species have been reported. Compounds such comma34, V. ruber35, V. alginolyticus, V. as Unnarmicins A and C45, Holomycin29, parahaemolyticus, and V. anguillarum36, Ngercheumicin A, B, C, D, and E produced by V. fischeri37, V. tubiashiii38, V. coralliilyticus, Photobacterium were found to show V. neptunius, and Photobacterium antibacterial and antifungal activities4,46. halotolerans29, 39, P. jeanii40, V. Compounds produced by vibrios such as nigripulchritudo32, V. rhizosphaerae41 and aqabamycin A (1a), aqabamycin B (1b), other unidentified Vibrio sp.42. aqabamycin C (1c), aqabamycin D (1d), Apart from the antimicrobial properties aqabamycin E (1e and 1e'), aqabamycin F (1f) of nonluminous bacteria, antimicrobial and aqabamycin G (2), 3-nitro-1H-indazole (3), investigations on luminous bacterial members indazole-3-carbaldehyde (4), phenyl-2-bis- of Vibrionaceae are found to be scanty. indolylmethane (5a), turbomycin B (5b), Debnath et al. (2010) found that L-asparaginase vibrindole A (6), 1,4-dithiane (7), 3-(3-nitro-4- produced by V. harveyi, V. fischeri and P. hydroxyphenyl)-2-propenoic acid (8), 3-nitro- leiognathi showed anticancer, antileukaemic 4-hydroxybenzaldehyde (9), phenylacetic acid, properties, and good antimicrobial activity19. A benzoic acid, 3-hydroxybenzoic acid and 4- recent report revealed moderate antibacterial hydroxycinnamic acid3, depsipeptides, activity (2-5 mm) in phenotypically identified pseudopeptides47, indoles and bromodiphenyl luminous bacteria V. mediterranei against some ether48 showed antimicrobial activities. human pathogens43, and similarly a luminous Kahalalide F produced by V. mediterranei Vibrio sp. against Staphylococcus aureus and exhibited anticancer, antitumor, antifungal, Klebsiella pneumonia44. In the present study antiviral and antibacterial properties49. seven species of luminous bacteria, V. harveyi, Bisucaberin a compound produced by V. V. campbellii, V. owensii, V. rotiferianus, V. salmonicida showed anticancer activity50.

2068 INDIAN J. MAR. SCI., VOL. 46, NO. 10, OCTOBER 2017

Table 2. Antibacterial activities of luminous bacterial strains.

) )

)

) )

)

) )

)

) )

ii )

)

V. campbellii V. campbellii

(

V. owensii V. harveyi V.

P. leiognathi

V. owens V.

V. campbellii

V. rotiferianus

V. campbellii V. rotiferianus V. campbellii V. harveyi V.

damselae P.

STF3 ( SSBR3 ( SQEG2 ( PEVI1 ( LB2 ( JPL2 ( CHSE2 ( CHSE4 ( BSECU1 ( BSECU3 ( BSE1 ( BSE4 ( BSE5 ( alginolyticus Bacterial AMPHI2

Pathogen µl) Extract (in volume Inhibition zone diameter (in mm) Vibrio 10 ------parahaemolyticus 50 ------serovar O3: K6 100 - - - 7 - - 10 - 10 11 13 9 13 - 150 - - - 10 - 11 12 - 12 13 15 11 14 - 200 13 - - 12 - 18 16 - 19 20 16 14 15 - Vibrio fluvialis 10 ------50 ------100 ------150 - - - - - 8 ------200 - - - - - 15 ------11 Vibrio cholerae 10 ------50 ------100 ------150 ------200 - - - - - 11 ------Escherichia coli 10 ------50 - - 6 ------100 - - 16 ------150 - - 20 ------200 - - 25 ------Enterococcus 10 ------faecalis 50 ------100 ------13 - - - 8 - - 150 ------18 - - - 9 - - 200 - - - - 12 - - 20 - - - 11 - - Shiga toxin 10 ------producing E.coli 50 ------6 - - serotype 100 ------10 - - - 16 - - O157:H7 150 ------12 - - - 17 - - 200 ------20 - - - 19 - - Klebsiella 10 ------pneumoniae 50 ------100 ------10 ------150 ------11 ------200 ------11 ------Bacillus subtilis 10 ------50 ------100 ------16 14 - - - - 150 ------20 16 - - - - 200 - - - - - 12 - - 24 18 - - - - Micrococcus 10 ------luteus 50 ------6 - 6 - - - 100 ------13 14 - 8 12 - - 150 ------16 18 - 19 13 - - 200 ------21 23 - 20 18 - - Salmonella 10 ------enterica serovar 50 ------Typhimurium 100 ------10 ------150 ------13 ------200 ------15 ------

RAMESH & MOHANRAJU: ANTIBACTERIAL ACTIVITY OF MARINE BIOLUMINESCENT BACTERIA 2069

Staphylococcus 10 ------aureus 50 ------8 - - 7 6 6 - - 100 - 9 - 12 - - 16 - - 13 10 8 - - 150 - 13 - 15 - - 17 - - 16 21 10 - - 200 - 19 - 19 - 12 20 - - 18 22 12 - -

Shigella flexneri 10 ------7 - - - - type 2a 50 ------9 - - 8 - 100 - - 7 ------10 - - 10 - 150 - 9 10 ------11 - - 12 - 200 - 12 12 ------15 - - 14 - Shigella sonnei 10 ------50 ------100 ------150 10 ------200 21 - - - 12 ------14 Streptococcus 10 ------pneumoniae 50 ------7 - 100 ------16 - 150 ------17 - 200 ------20 - Salmonella 10 ------enterica serovar 50 9 ------typhi 100 12 ------8 ------150 13 ------11 ------200 18 ------12 ------Shigella boydii 10 ------type 1 50 9 ------100 12 ------8 ------150 13 ------11 ------200 18 ------12 ------Shigella 10 ------dysenteriae type 50 9 ------5 100 12 ------8 ------150 13 ------11 ------200 18 ------12 ------* Denotes no activity.

However, the above mentioned metabolic rhythms in microbes may regulate compounds have not been observed in strains ecological and biogeochemical processes55. of Vibrio and Photobacterium species isolated Results of present study are in agreement with from this study. But compounds such as the earlier studies and infer that production of indole51, phenol, 2,4-bis(1,1-dimethylethyl)- similar compounds in these bacteria might be 52,dibutyl phthalate53, and 1,2- beneficial for their survival by means of intra Benzenedicarboxylic acid, butyl octyl ester54 and interspecies chemical communication. were observed, which were previously reported Based on this, we postulate a new hypothesis from other marine bacteria. So far these called ―Novice-Acquired Praxis hypothesis‖ in compounds have not been described from which microbes skill to produce bioactive vibrios and Photobacterium species. Most of compounds as produced by other bacteria (e.g. the isolated strains in this study showed this kind of activity is as like learning a new presence of these compounds, suggesting that language or habit from another person, in order presence of similar compounds may be to use it for self-benefit or community). Since involved in intercellular communication studies on production of secondary metabolites through chemical signalling network and by luminescent bacteria are scanty, therefore antagonistic activities against other microbes. efforts are yet to be extended on this aspect to Recently, Aylward et al. (2015) found understand microbial chemical signalling and that microbial communities in different regions to explore active metabolites for industrial of Pacific Ocean have displayed similar applications. metabolic processes, indicating that similar

2070 INDIAN J. MAR. SCI., VOL. 46, NO. 10, OCTOBER 2017

Table 3. GC-MS results of crude extracts of luminous strains.

S. Strain Compounds related to Antibacterial activity Molecular Peak No weight RT 1. AMPHI2 Indole 117 9.44 Phenol, 2,4-bis(1,1-dimethylethyl)- 206 12.32 Diethyl Phthalate (1,2-Benzenedicarboxylic acid, diethyl ester) 222 13.36 Phthalic acid, butyl cyclobutyl ester 276 17.79 2. BSE1 Indolizine 117 9.49 Dodecane, 1-iodo- 296 12.09 Phenol, 2,4-bis(1,1-dimethylethyl)- 206 12.33 Benzoic acid, 4-ethoxy-, ethyl ester 194 12.67 3. BSE4 Indole 117 9.47 Phenol, 2,4-bis(1,1-dimethylethyl)- 206 12.32 Benzoic acid, 4-ethoxy-, ethyl ester 194 12.71 Dibutyl phthalate 278 17.79 4. BSE5 Indolizine 117 9.44 Phenol, 2,4-bis(1,1-dimethylethyl)- 206 12.32 1,2-Benzenedicarboxylic acid, diisooctyl ester 390 27.93 5. BSECU1 Indole 117 9.44 Phenol, 2,4-bis(1,1-dimethylethyl)- 206 12.32 1,2-Benzenedicarboxylic acid, diisooctyl ester 390 17.15 Dibutyl phthalate 278 17.78 6. BSECU3 Indole 117 9.46 Phenol, 2,4-bis(1,1-dimethylethyl)- 206 12.33 Dibutyl phthalate 278 17.78 Phthalic acid, butyl isohexyl ester 306 18.03 7. CHSE2 Indole 117 9.46 Phenol, 2,4-bis(1,1-dimethylethyl)- 206 12.33 Phthalic acid, butyl 2-methoxyethyl ester 280 17.87 8. CHSE4 Indolizine 117 9.45 Phenol, 2,4-bis(1,1-dimethylethyl)- 206 12.32 Benzoic acid, 4-ethoxy-, ethyl ester 194 12.69 1,2-Benzenedicarboxylic acid, butyl octyl ester 334 17.86 9. JPL2 Indole 117 9.47 Phenol, 2,4-bis(1,1-dimethylethyl)- 206 12.33 Benzoic acid, 4-ethoxy-, ethyl ester 194 12.66 Ethaneperoxoic acid, 1-cyano-1-[2-(2-phenyl-1,3-dioxolan-2- 347 17.86 yl)ethyl]pentyl ester 10. LB2 Phenol, 2,4-bis(1,1-dimethylethyl)- 206 12.33 Dibutyl phthalate 278 17.78 Phthalic acid, butyl isohexyl ester 306 17.87 11. PEVI1 5H-1-Pyrindine 117 9.44 Phenol, 2,4-bis(1,1-dimethylethyl)- 206 12.32 Benzoic acid, 4-ethoxy-, ethyl ester 194 12.64 Dibutyl phthalate 278 17.79 12. SQEG2 Phenol, 2,4-bis(1,1-dimethylethyl)- 206 12.32 3-Phenyl-3-pentanol 164 12.33 1,2-Benzenedicarboxylic acid, butyl octyl ester 278 17.86 13. SSBR3 Indolizine 117 9.46 Phenol, 2,4-bis(1,1-dimethylethyl)- 206 12.33 Dibutyl phthalate 278 17.79 14. STF3 Indole 117 9.44 Phenol, 2,4-bis(1,1-dimethylethyl)- 206 12.32 1,2-Benzenedicarboxylic acid, diisooctyl ester 390 17.15

RAMESH & MOHANRAJU: ANTIBACTERIAL ACTIVITY OF MARINE BIOLUMINESCENT BACTERIA 2071

Intens. Intens.

106 106

12.3 min 17.8 min 105 105 12.3 min

9.4 min

18.0 min

9.5 min

104 104

103 103

6 8 10 12 14 16 18 20 Time [min] 6 8 10 12 14 16 18 20 Time [min] Amphi2.D: TIC + Bsecu3.D: TIC +

Fig. 5. GC-MS chromatogram of ethyl acetate extraction of Fig. 10. GC-MS chromatogram of ethyl acetate extraction of strain AMPHI2. strain BSECU3.

Intens. Intens.

106 106

12.3 min

5 5 10 10 12.3 min

9.4 min

12.6 min

9.5 min 4 10 104

103 103

6 8 10 12 14 16 18 20 22 24 Time [min] 6 8 10 12 14 16 18 20 Time [min] Bse1.D: AutoMS(n): TIC + Chse2.D: TIC + Chse2.D: TIC +

Fig. 6. GC-MS chromatogram of ethyl acetate extraction of Fig. 11. GC-MS chromatogram of ethyl acetate extraction of strain BSE1. strain CHSE2.

Intens. Intens.

107

106

12.3 min

12.3 min 105

9.4 min 105

12.6 min

104

103

6 8 10 12 14 16 18 20 Time [min] 6 8 10 12 14 16 18 20 Time [min] Chse4.D: TIC + Bse1.D: TIC +

Fig. 7. GC-MS chromatogram of ethyl acetate extraction of Fig. 12. GC-MS chromatogram of ethyl acetate extraction of strain BSE4. strain CHSE4.

Intens. Intens.

106

12.3 min 105

9.4 min

104

103

6 8 10 12 14 16 18 20 Time [min] 6 8 10 12 14 16 18 20 Time [min] Jpl2.D: TIC + Bse5.D: TIC + Bse5.D: AutoMS(n): TIC +

Fig. 8. GC-MS chromatogram of ethyl acetate extraction of Fig. 13. GC-MS chromatogram of ethyl acetate extraction of strain BSE5. strain JPL2.

Intens. Intens.

106

105 12.3 min

12.3 min 105 17.8 min

9.4 min

18.0 min 104

104

103

6 8 10 12 14 16 18 20 Time [min] 6 8 10 12 14 16 18 20 Time [min] LB2.D: TIC + Bsecu1.D: TIC +

Fig. 9. GC-MS chromatogram of ethyl acetate extraction of Fig. 14. GC-MS chromatogram of ethyl acetate extraction of strain BSECU1. strain LB2. 2072 INDIAN J. MAR. SCI., VOL. 46, NO. 10, OCTOBER 2017

Intens. Intens.

106

12.3 min 105 12.3 min 105 9.4 min 17.8 min

17.8 min

104

103

6 8 10 12 14 16 18 20 Time [min] 6 8 10 12 14 16 18 20 Time [min] Pevi1.D: TIC + Ssbr3.D: TIC +

Fig. 15. GC-MS chromatogram of ethyl acetate extraction of Fig. 17. GC-MS chromatogram of ethyl acetate extraction of strain PEVI1. strain SSBR3.

Intens. Intens.

6 10 106

12.3 min 12.3 min 5 10 105 17.8 min

9.4 min

18.0 min

4 10 104

103 103

6 8 10 12 14 16 18 20 Time [min] 6 8 10 12 14 16 18 20 Time [min] Sqeg2.D: TIC + Bsecu1.D: TIC +

Fig. 16. GC-MS chromatogram of ethyl acetate extraction of Fig. 18. GC-MS chromatogram of ethyl acetate extraction of strain SQEG2. strain STF3.

Acknowledgements aquatic organisms, Microbiology., 150(2004) 1769- CH. Ramesh thanks the Department of Science 1777. 9. Kanjana, K., Radtanatip, T., Asuvapongpatana, S., and Technology for providing INSPIRE Withyachumnarnkul, B. & Wongprasert, K., Solvent fellowship DST/120230/280. extracts of the red seaweed Gracilaria fisheri prevent Vibrio harveyi infections in the black tiger References shrimp Penaeus monodon, Fish. Shellfish. Immun., 1. Nemecek-marshall, M., Wojciechowski, C., 30(2011) 389-396. Wagner, W.P. & Fall, R., Acetone formation in the 10. Sivakumar, K., Kannappan, S., Dineshkumar, M. & Vibrio family: a new pathway for bacterial leucine Patil, P.K., Antagonism of marine macro alga catabolism, J. Bacteriol., 181(1999) 7493-7499. Kappaphycus alvarezii extract against luminescence 2. Gram, L., Melchiorsen, J. & Bruhn, J.B., disease causing Vibrio harveyi during Penaeus Antibacterial activity of marine culturable bacteria monodon larviculture, Afr. J. Microbiol. Res., collected from a global sampling of ocean surface 8(2014) 458-469. waters and surface swabs of marine organisms, Mar. 11. Manefield, M., Harris, L., Rice, S.A., Nys, R. & Biotechnol., 12(2010) 439-451. Kjelleberg, S., Inhibition of luminescence and 3. Al-Zereini, W., Yao, C.B.F.F., Laatsch, H. & Anke, virulence in the black tiger prawn (Penaeus H., Aqabamycins A-G: novel nitro maleimides from monodon) pathogen Vibrio harveyi by intercellular a marine Vibrio species. I. Taxonomy, fermentation, signal antagonists, Appl. Environ. Microbiol., isolation and biological activities, J. Antibiot., 66(2000) 2079-2084. 63(2010) 297-301. 12. Defoirdt, T., Halet, D., Vervaeren, H., Boon, N., 4. Mansson, M., Gram, L. & Larsen, T.O., Production Wiele, T.V., Sorgeloos, P., Bossier, P. & of bioactive secondary metabolites by marine Verstraete, W., The bacterial storage compound Vibrionaceae, Mar. Drugs., 9(2011) 1440-1468. poly-b-hydroxybutyrate protects Artemia 5. Nair, A.G. & Simidu, U., Distribution and franciscana from pathogenic Vibrio campbellii, significance of heterotrophic marine bacteria with Environ. Microbiol., 9(2007) 445-452. antibacterial activity, Appl. Environ. Microbiol., 13. Lio-Po, G. D., Leano, E.M., Penaranda, M.M.D., 53(1987) 2957-2962. Villa-Franco, A.U., Sombito, C.D. & Guanzon, 6. Forst, S. & Nealson, K., Molecular biology of the N.G., Anti-luminous Vibrio factors associated with symbiotic-pathogenic bacteria Xenorhabdus spp. the ‗green water‘ grow-out culture of the tiger and Photorhabdus spp. Microbiol. Rev., 60(1996) shrimp Penaeus monodon, Aquaculture., 250(2005) 21-43. 1-7. 7. Dunlap, P.V. & Urbanczyk, H., Luminous Bacteria, 14. Liu, Y., Schryver, P.D., Delsen, B. V., Maignien, L., in: Prokaryotes 4thedn, edited by E. Rosenberg, E.F. Boon, N., Sorgeloos, P., Verstraete, W., Bossier, P. DeLong, F. Thompson, S. Lory & E. Stackebrandt, & Defoirdt, T., PHB-degrading bacteria isolated (Springer-Verlag, Berlin) 2013, pp. 495-528. from the gastrointestinal tract of aquatic animals as 8. Gomez-Gil, B., Soto-Rodríguez, S., García-Gasca, protective actors against luminescent vibriosis, A., Roque, A., Vazquez-Juarez, R., Thompson, F.L. FEMS. Microbiol. Ecol., 74(2010) 196-204. & Swings, J., Molecular identification of Vibrio 15. Moriarty, D.J.W., Control of luminous Vibrio harveyi-related isolates associated with diseased species in penaeid aquaculture ponds, Aquaculture., 164(1998) 351-358. RAMESH & MOHANRAJU: ANTIBACTERIAL ACTIVITY OF MARINE BIOLUMINESCENT BACTERIA 2073

16. Hai, N.V., The use of probiotics in aquaculture. J. 32. Ritchie, K.B., Regulation of microbial populations Appl. Microbiol., 119(2015) 917-935. by coral surface mucus and mucus-associated 17. Abraham, T.J., Antibacterial marine bacterium deter bacteria, Mar. Ecol. Prog. Ser., 322(2006) 1-14. luminous vibriosis in shrimp larvae, NAGA, 33. Raina, J., Tapiolas, D., Willis, B.L. & Bourne, D.G., WorldFish Center Quart., 27(2004) 28-31. Coral associated bacteria and their role in the 18. Vinod, M. G., Shivu, M.M., Umesha, K.R., Rajeeva, biogeochemical cycling of Sulfur, Appl. Environ. B.C., Krohne, G., Karunasagar, I. & Karunasagar, I., Microbiol., 75(2009) 3492-3501. Isolation of Vibrio harveyi bacteriophage with a 34. Farkas-Himsley, H. & Seyfried, L., Lethal potential for biocontrol of luminous vibriosis in biosynthesis of a new antibacterial principle: hatchery environments, Aquaculture., 255(2006) Vibriocin, Nature., 193(1962) 1193-1194. 117-124. 35. Wan Norhana, N. & Darah, I., Vibrio ruber (S2A1), 19. Debnath, M., Paul, A.K. & Bisen, P.S., Natural a marine bacterium that exhibits significant bioactive compounds and biotechnological potential antimicrobial activity, Malays. J. Microbiol., of marine bacteria, Curr. Pharm. Biotechnol., 1(2005) 25-30. 8(2010) 253-260. 36. Long, R. A., Rowley, D.C., Zamora, E., Liu, J., 20. Cavanagh, N.S., Swain, L.G., Nordin, R.N. & Bartlett, D.H. & Azam, F., Antagonistic Interactions Pommen, L.W., Lake and stream bottom sediment among Marine Bacteria Impede the Proliferation of sampling manual. BC Ministry of Environment, Vibrio cholerae, Appl. Environ. Microbiol., Lands and Parks, Resources Information Standards 71(2005) 8531-8536. Committee, Victoria BC (1994). 37. Baranova, N.A. & Egorov, N.S., Production of 21. Dutka, B. J., Methods for Microbiological and antibiotic substances by natural variants of the Toxicological Analysis of Waters, Wastewaters and marine bacterium Vibrio fischeri, Antibiot. Sediments, Rivers Research Branch, National Water Khimioter., 34(1989) 7-10. Research Institute, Burlington, Ontario, Canada 38. Castro, D., Pujalte, M.J., Lopez-Cortes, L., Garay, (1989). E. & Borrego, J.J., Vibrios isolated from the 22. Agrawal A & Gopal K, Analysis of Phytoplankton cultured manila clam (Ruditapes philippinarum): and Zooplankton: Qualitative and Quantitative, in: Numerical taxonomy and antibacterial activities, J. Biomonitoring of Water and Waste, (Springer, New Appl. Microbiol., 93(2002) 438-447. Delhi) 2013, pp. 33-40. 39. Ngeiywa, M., Edwin, M., Liti, D. & Mutai, R., 23. Benson.,Microbiological Applications Lab Manual, Isolation of Antibiotic Vibrionaceae Bacteria from a 8thedn, (The McGraw−Hill Companies) 2001, pp. Community Marine Silvo-Fishery Farm along 478. Mtwapa Creek, Kenya, Open Access Library 24. DEHP., Department of Environment and Heritage Journal., 2(2015) e1313. Protection. Monitoring and Sampling Manual, 40. Dufourcq, R., Chalkiadakis, E., Fauchon, M., Version 2, 2009. Deslandes, E., Kerjean, V., Chanteau, S., Petit, E., 25. West, P.A. & Colwell, R.R., Identification and Guezennec, J. & Dupont-Rouzeyrol, M., Isolation classification of Vibrionaceae an overview, in: and partial characterization of bacteria Vibrios in the Environment, edited by R.R. Colwell, (Pseudoalteromonas sp.) with potential antibacterial (Wiley, New York) 1984, pp. 285-363. activity from a marine costal environment from New 26. Dunlap, P.V. & Kita-Tsukamoto, K., Luminous Caledonia, Lett. Appl. Microbiol., 58(2014) 102- Bacteria, in: Prokaryotes vol 2, edited by M. 108. Dworkin, (Springer, New York) 2006, pp. 863-892. 41. Rameshkumar, N. & Nair, S., Isolation and 27. Nishiguchi, M. K., Doukakis, P., Egan, M., Kizirian, molecular characterization of genetically diverse D., Phillips, A., Prendini, L., Rosenbaum, H.C., antagonistic, diazotrophic red-pigmented vibrios Torres, E., Wyner, Y., DeSalle, R. & Giribet, G., from different mangrove rhizospheres, FEMS DNA Isolation Procedures, in:Methods and Tools in Microbiol. Ecol., 67(2009) 455-467. Biosciences and Medicine Techniques in molecular 42. Towse, S. J., Antimicrobial activity of marine Vibrio systematics and evolution, edited by R. DeSalle et sp., isolate NI-22. Honours Thesis, University of al.(BirkhäuserVerlag, Basel/Switzerland) 2002, pp. Winnipeg (2005). 249-287. 43. Arulmoorthy, M.P., Karunakaran, K., Vignesh, R., 28. Mohandass, C.,Rajasabapathy, R., Ravindran, C., Vasudevan, S. & Srinivasan, M., Identification and Colaco, A., Santos, R.S. & Meena,R. M.,Bacterial antimicrobial potential of bioluminescent bacteria diversity and their adaptations in the shallow water isolated from the mangrove ecosystem of the Roach hydrothermal vent at D. Joao de Castro Seamount Park, Tuticorin, South east coast of India, BMR (DJCS), Azores, Portugal. Cah. Biol. Mar., Microbiology., 1(2014) 1-15. 53(2012) 65-76. 44. Molina, A.J., Abisado, R.G. & Calugay, R.J., 29. Wietz, M., Mansson, M., Gotfredsen, C.H., Larsen, Bioluminescent Vibrio spp. with antibacterial T.O. & Gram, L., Antibacterial compounds from activity against the nosocomial pathogens marine Vibrionaceae isolated on a global Staphylococcus aureus and Klebsiella pneumoniae, expedition, Mar. Drugs., 8(2010) 2946-2960. AACL Bioflux., 9(2016)185-194. 30. Bauer, A.W., Kirby, W.M.M., Sherris, J. S. & Turk, 45. Oku, N., Kawabata, K., Adachi, K., Katsuta, A. & M., Antibiotic susceptibility by standardized single Shizuri, Y., Unnarmicins A and C, new antibacterial disc method, Am. J. Clinical. Pathol., 45(1966) depsipeptides produced by marine bacterium 493-496. Photobacterium sp. MBIC06485, J. Antibiot., 31. Thompson, F. L., Iida, T. & Swings, J., Biodiversity 61(2008) 11-17. of Vibrios, Microbiol. Mol. Biol. Rev., 68(2004) 46. Adachi, K., Kawabata, Y., Kasai, H, Katsuta, M. & 403-431. Shizuri, Y., Novel Ngercheumicin or its salt useful 2074 INDIAN J. MAR. SCI., VOL. 46, NO. 10, OCTOBER 2017

for treating infection caused by Pseudovibrio 52. Padmavathi, A., Abinaya, A. & Pandian, S.K., denitrificans, Patent JP2007230911-A, 2007. Phenol, 2, 4- bis(1,1-dimethylethyl) of marine 47. Desriac, F., Jégou, C., Balnois, E., Brillet, B., bacterial origin inhibits quorum sensing mediated Chevalier, P.L. & Fleury, Y., Antimicrobial peptides biofilm formation in an uropathogen Serratia from marine Proteobacteria, Mar. Drugs., 11(2013) marcescens, Biofouling., 30(2014) 1111-1122. 3632-3660. 53. Bhatnagar, I. & Kim, S., Immense essence of 48. Kelecom, A., Secondary metabolites from marine excellence: Marine microbial bioactive compounds, microorganisms, An. Acad. Bras. Cienc., 74(2002) Mar. Drugs., 8(2010) 2673-2701. 151-170. 54. Pandey, A., Naik, M.M., Dubey, S.K. & Kumar, S., 49. Hill, R.T., Hamann, M. T., Enticknap, J. J. & Rao, Biological characterization of marine fish K.V., Kahalalide-Producing Bacteria. Patent pathogen Acinetobacter sp. strain An 2 producing PCT/US2004/036201 (2005). antibacterial metabolites, J. Sci. Ind. Res., 70(2011) 50. Winkelmann, G., Schmid, D.G., Nicholson, G., 135-141. Jung, G. & Colquhoun, D.J., Bisucaberin—A 55. Aylward, F.O., Eppley, J.M., Smith, J.M., Chavez, dihydroxamatesiderophore isolated from Vibrio F.P., Scholin, C.A. & DeLong, E.F., Microbial salmonicida, an important pathogen of farmed community transcriptional networks are conserved Atlantic salmon (Salmosalar). Biometals, 15(2002) in three domains at ocean basin scales, PNAS., 153-160. 112(2015) 5443-5448. 51. Oimomi, M., Hamada, M. & Hara, T., Antimicrobial activities of indole, J. Antibiot., 27(1974) 987-988.